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Sample records for enzyme prodrug therapy

  1. Advances in antibody-directed enzyme prodrug therapy.

    PubMed

    Sharma, Surinder K; Bagshawe, Kenneth D; Begent, Richard H J

    2005-06-01

    Antibody-directed enzyme prodrug therapy has demonstrated feasibility as a treatment for cancer. Numerous prodrug/drug systems have been developed for activation by a variety of enzymes and although many have shown potential in preclinical studies, so far only one system has progressed to the clinic. Clinical studies have identified issues that were not readily apparent in xenograft models, however, these have not been addressed in the development and testing of new prodrugs. The issue of immunogenicity arising from the use of non-human enzymes has also been a major hurdle. The development of recombinant fusion proteins provides reproducible and effective antibody-enzyme products that retain the necessary specificity for prodrug activation. Advances in molecular, structural and systems biology, in combination with bioinformatics, have allowed these molecules to be readily manipulated to provide the desired characteristics.

  2. Lectin-directed enzyme activated prodrug therapy (LEAPT): Synthesis and evaluation of rhamnose-capped prodrugs.

    PubMed

    Garnier, Philippe; Wang, Xiang-Tao; Robinson, Mark A; van Kasteren, Sander; Perkins, Alan C; Frier, Malcolm; Fairbanks, Antony J; Davis, Benjamin G

    2010-12-01

    The lectin-directed enzyme activated prodrug therapy (LEAPT) bipartite drug delivery system utilizes glycosylated enzyme, localized according to its sugar pattern, and capped prodrugs released by that enzyme. In this way, the sugar coat of a synthetic enzyme determines the site of release of a given drug. Here, prodrugs of doxorubicin and 5-fluorouracil capped by the nonmammalian l-rhamnosyl sugar unit have been efficiently synthesized and evaluated for use in the LEAPT system. Both are stable in blood, released by synthetically d-galactosylated rhamnosidase enzyme, and do not inhibit the uptake of the synthetic enzyme to its liver target. These results are consistent with their proposed mode of action and efficacy in models of liver cancer, and confirm modular flexibility in the drugs that may be used in LEAPT.

  3. Macrophage mediated PCI enhanced gene-directed enzyme prodrug therapy

    NASA Astrophysics Data System (ADS)

    Christie, Catherine E.; Zamora, Genesis; Kwon, Young J.; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

    2015-03-01

    Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. Prodrug activating gene therapy (suicide gene therapy) employing the transduction of the E. coli cytosine deaminase (CD) gene into tumor cells, is a promising method. Expression of this gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-FC, to the toxic metabolite, 5-fluorouracil (5-FU). 5-FC may be particularly suitable for brain tumors, because it can readily cross the bloodbrain barrier (BBB). In addition the bystander effect, where activated drug is exported from the transfected cancer cells into the tumor microenvironment, plays an important role by inhibiting growth of adjacent tumor cells. Tumor-associated macrophages (TAMs) are frequently found in and around glioblastomas. Monocytes or macrophages (Ma) loaded with drugs, nanoparticles or photosensitizers could therefore be used to target tumors by local synthesis of chemo attractive factors. The basic concept is to combine PCI, to enhance the ex vivo transfection of a suicide gene into Ma, employing specially designed core/shell NP as gene carrier.

  4. Synthesis and biological studies of different duocarmycin based glycosidic prodrugs for their use in the antibody-directed enzyme prodrug therapy.

    PubMed

    Tietze, Lutz F; Schuster, Heiko J; Krewer, Birgit; Schuberth, Ingrid

    2009-01-22

    The synthesis and biological evaluation of novel prodrugs for use in the antibody directed enzyme prodrug therapy (ADEPT) of cancer based on the cytotoxic antibiotic duocarmycin SA (1) are described. In this approach, we investigated the influence of the sugar moiety of the glycosidic prodrug on the QIC(50) values as well as on the stability and the water solubility. The best result was found for prodrug 22 containing an alpha-mannoside moiety with a QIC(50) value of 4500.

  5. Annexin V-targeted enzyme prodrug therapy using cytosine deaminase in combination with 5-fluorocytosine.

    PubMed

    Van Rite, Brent D; Harrison, Roger G

    2011-08-01

    A fusion protein, consisting of cytosine deaminase (CD) linked to human annexin V, was created for use in an enzyme prodrug therapy targeted to the tumor vasculature and associated cancer cells in the primary tumor and distant metastases. The major finding of this study is that the CD-annexin V fusion protein in combination with the prodrug 5-fluorocytosine has significant cytotoxic activity against endothelial cells and two breast cancer cells lines in vitro that expose phosphatidylserine on their surface. The cytotoxicity experiments verified this novel enzyme prodrug system has the ability to produce therapeutic levels of 5-fluorouracil and thus appears promising.

  6. Enzyme prodrug therapy designed to target L-methioninase to the tumor vasculature.

    PubMed

    Van Rite, Brent D; Lazrak, Yahya A; Pagnon, Magali L; Palwai, Naveen R; Neves, Luís F F; McFetridge, Peter S; Harrison, Roger G

    2011-02-28

    A new approach for enzyme prodrug therapy for cancer was tested using human endothelial cells and two breast cancer cell lines in vitro. The concept is to use the human annexin V protein to selectively target the enzyme L-methioninase to the tumor vasculature. The major finding was that enzyme prodrug treatment using the L-methioninase-annexin V fusion protein and selenomethionine as the prodrug over 3 days was shown to be lethal to the endothelial cells and the cancer cells, while having little or no effect with the prodrug but with no fusion protein present. Thus, this new approach appears promising. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  7. Antitumor activity of an enzyme prodrug therapy targeted to the breast tumor vasculature.

    PubMed

    Van Rite, Brent D; Krais, John J; Cherry, Mohamad; Sikavitsas, Vassilios I; Kurkjian, Carla; Harrison, Roger G

    2013-10-01

    The L-methioninase-annexin V/selenomethionine enzyme prodrug system, designed to target the tumor vasculature and release the methylselenol anticancer drug in the tumor, was tested in mice with implanted MBA-MB-231 breast tumors. This therapy was able to cause a reduction in the size of the tumors during the treatment period. It was shown that L-methioninase-annexin V was uniformly bound at the blood vessel surface in the tumor and also that there was a substantial cutoff of blood flowing through the treated tumor, consistent with the therapy's design. This new approach for enzyme prodrug therapy of breast cancer appears promising.

  8. Antibody-directed enzyme prodrug therapy: a promising approach for a selective treatment of cancer based on prodrugs and monoclonal antibodies.

    PubMed

    Tietze, Lutz F; Krewer, Birgit

    2009-09-01

    The antibody-directed enzyme prodrug therapy allows a selective liberation of cytotoxic agents from non-toxic prodrugs in cancerous tissue by targeted antibody-enzyme conjugates. We have developed a series of novel glycosidic prodrugs based on the natural antibiotic CC-1065 and the duocarmycins, which are up to 4800 times less toxic than the drugs liberated from these prodrugs in the presence of the activating enzyme (e.g., beta-D-galactosidase). Furthermore, the drugs show very high cytotoxicities with IC(50) values of as low as 4.5 pm. In this report, we summarize our recent results on the development and biological evaluation of these novel third-generation prodrugs with higher water solubility, higher difference in cytotoxicity between the prodrugs and the corresponding drugs and improved cytotoxicity of the drugs as compared with previous compounds.

  9. Annexin V-Directed Enzyme Prodrug Therapy Plus Docetaxel for the Targeted Treatment of Pancreatic Cancer.

    PubMed

    Guillen, Katrin P; Restuccia, Antonietta; Kurkjian, Carla; Harrison, Roger G

    2015-08-01

    The bleak prognosis associated with pancreatic cancer (PDAC) drives the need for the development of novel treatment methodologies. Here, we evaluate the applicability of 3 enzyme prodrug therapies for PDAC, which are simultaneously targeted to the tumor, tumor vasculature, and metastases via annexin V. In these therapies, annexin V is fused to an enzyme, creating a fusion protein that converts nontoxic drug precursors, prodrugs, into anticancer compounds while bound to the tumor, therefore mitigating the risk of side effects. The binding strength of fusion proteins to the human PDAC cell lines Panc-1 and Capan-1 was measured via streptavidin-horseradish peroxidase binding to biotinylated fusion proteins. Cytotoxic efficacy was evaluated by treatment with saturating concentrations of fusion protein followed by varying concentrations of the corresponding prodrug plus docetaxel. All fusion proteins exhibited strong binding to PDAC cells, with dissociation constants between 0.02 and 1.15 nM. Cytotoxic efficacy was determined to be very good for 2 of the systems, both of which achieved complete cell death on at least 1 cell line at physiologically attainable prodrug concentrations. Strong binding of fusion proteins to PDAC cells and effective cytotoxicity demonstrate the potential applicability of enzyme prodrug therapy to the treatment of PDAC.

  10. Beta-galactoside prodrugs of doxorubicin for application in antibody directed enzyme prodrug therapy/prodrug monotherapy.

    PubMed

    Devalapally, HariKrishna; Navath, Raghavendra Swamy; Yenamandra, Venkateshwarlu; Akkinepally, RaghuRam Rao; Devarakonda, Rama Krishna

    2007-06-01

    Anthracycline antibiotics, particularly doxorubicin and daunorubicin, have been used exten sively in the treatment of human malignancies. However cardiotoxicity and multidrug resistance are significant problems that limit the clinical efficacy of such agents. Rational design to avoid these side effects includes strategies such as drug targeting and prodrug synthesis. Described here are the synthesis and preliminary biological evaluation of the enzymatically activated two new prodrugs (6 & 11) of doxorubicin. These prodrugs were designed as potential candidates for selective chemotherapy in ADEPT or PMT strategies. They are constituted of a galactose moiety, a spacer and the cytotoxic drug and they differ by the type of spacer. The prodrugs were stable in a buffer, and the in vitro studies showed good detoxification and hydrolysis kinetics. As prodrug 11 was readily hydrolyzed, this could be a valuable candidate for further development.

  11. Optimization of alkylating agent prodrugs derived from phenol and aniline mustards: a new clinical candidate prodrug (ZD2767) for antibody-directed enzyme prodrug therapy (ADEPT).

    PubMed

    Springer, C J; Dowell, R; Burke, P J; Hadley, E; Davis, D H; Blakey, D C; Melton, R G; Niculescu-Duvaz, I

    1995-12-22

    Sixteen novel potential prodrugs derived from phenol or aniline mustards and their 16 corresponding drugs with ring substitution and/or different alkylating functionalities were designed. The [[[4-]bis(2-bromoethyl)-(1a), [[[4-[bis(2-iodoethyl)-(1b), and [[[4-[(2-chloroethyl)-[2-(mesyloxy)ethyl]amino]phenyl]oxy] carbonyl]-L-glutamic acids (1c), their [[[2- and 3-substituted-4-[bis(2-chloroethyl)amino]phenyl]oxy]carbonyl]-L- glutamic acids (1e-1), and the [[3-substituted-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl]-L- glutamic acids (1o-r) were synthesized. They are bifunctional alkylating agents in which the activating effect of the phenolic hydroxyl or amino function is masked through an oxycarbonyl or a carbamoyl bond to a glutamic acid. These prodrugs were designed to be activated to their corresponding phenol and aniline nitrogen mustard drugs at a tumor site by prior administration of a monoclonal antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2) in antibody-directed enzyme prodrug therapy (ADEPT). The synthesis of the analogous novel parent drugs (2a-r) is also described. The viability of a colorectal cell line (LoVo) was monitored with the potential prodrugs and the parent drugs. The differential in the cytotoxicity between the potential prodrugs and their corresponding active drugs ranged between 12 and > 195 fold. Compounds 1b-d,f,o exhibited substantial prodrug activity, since a cytotoxicity differential of > 100 was achieved compared to 2b-d,f,o respectively. The ability of the potential prodrugs to act as substrates for CPG2 was determined (kinetic parameters KM and kcat), and the chemical stability was measured for all the compounds. The unsubstituted phenols with different alkylating functionalities (1a-c) proved to have the highest ratio of the substrates kcat:KM. From these studies [[[4-[bis(2-iodoethyl)amino]phenyl]oxy]carbonyl]-L-glutamic acid (1b) emerges as a new ADEPT clinical trial candidate due to its physicochemical and

  12. Carboxypeptidase-G2-based gene-directed enzyme-prodrug therapy: a new weapon in the GDEPT armoury.

    PubMed

    Hedley, Douglas; Ogilvie, Lesley; Springer, Caroline

    2007-11-01

    Gene-directed enzyme-prodrug therapy (GDEPT) aims to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. A gene encoding a 'suicide' enzyme is introduced into the tumour to convert a subsequently administered non-toxic prodrug into an active drug selectively in the tumour, but not in normal tissues. Significant effects can now be achieved in vitro and in targeted experimental models, and GDEPT therapies are entering the clinic. Our group has developed a GDEPT system that uses the bacterial enzyme carboxypeptidase G2 to convert nitrogen mustard prodrugs into potent DNA crosslinking agents, and a clinical trial of this system is pending.

  13. Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy.

    PubMed

    Nemani, Krishnamurthy V; Ennis, Riley C; Griswold, Karl E; Gimi, Barjor

    2015-06-10

    Engineered bacterial cells that are designed to express therapeutic enzymes under the transcriptional control of remotely inducible promoters can mediate the de novo conversion of non-toxic prodrugs to their cytotoxic forms. In situ cellular expression of enzymes provides increased stability and control of enzyme activity as compared to isolated enzymes. We have engineered Escherichia coli (E. coli), designed to express cytosine deaminase at elevated temperatures, under the transcriptional control of thermo-regulatory λpL-cI857 promoter cassette which provides a thermal switch to trigger enzyme synthesis. Enhanced cytosine deaminase expression was observed in cultures incubated at 42°C as compared to 30°C, and enzyme expression was further substantiated by spectrophotometric assays indicating enhanced conversion of 5-fluorocytosine to 5-fluorouracil. The engineered cells were subsequently co-encapsulated with magnetic iron oxide nanoparticles in immunoprotective alginate microcapsules, and cytosine deaminase expression was triggered remotely by alternating magnetic field-induced hyperthermia. The combination of 5-fluorocytosine with AMF-activated microcapsules demonstrated tumor cell cytotoxicity comparable to direct treatment with 5-fluorouracil chemotherapy. Such enzyme-prodrug therapy, based on engineered and immunoisolated E. coli, may ultimately yield an improved therapeutic index relative to monotherapy, as AMF mediated hyperthermia might be expected to pre-sensitize tumors to chemotherapy under appropriate conditions.

  14. Synthesis and characterization of a novel chitosan based E. coli cytosine deaminase nanocomposite for potential application in prodrug enzyme therapy.

    PubMed

    Yata, Vinod Kumar; Ghosh, Siddhartha Sankar

    2011-01-01

    Cytosine deaminase is a non-mammalian enzyme of widespread interest for prodrug enzyme therapy due to its ability to convert prodrug 5-fluorocytosine into anticancer drug 5-fluorouracil. Cytosine deaminase enzyme has been purified to homogeneity from E. coli K-12 MTCC 1302 strain. K(m) values for cytosine and 5-fluorocytosine were found to be 0.26 mM and 1.82 mM, respectively. We developed a chitosan-entrapped cytosine deaminase nanocomposite. Atomic force microscopy and transmission electron microscopy images showed an elongated sphere shape nanocomposite with an average size of 80 nm diameter. Fourier transform infrared spectroscopy and X-ray diffraction results confirmed gel formation and entrapment of cytosine deaminase within the nanocomposite. Sustained release of cytosine deaminase from the nanocomposite up to one week depicted its potential implication in prodrug inducted enzyme therapy.

  15. Clinical potential of gene-directed enzyme prodrug therapy to improve radiation therapy in prostate cancer patients.

    PubMed

    Vajda, Alice; Marignol, Laure; Foley, Ruth; Lynch, Thomas H; Lawler, Mark; Hollywood, Donal

    2011-12-01

    Despite the advances in prostate cancer diagnosis and treatment, current therapies are not curative in a significant proportion of patients. Gene-directed enzyme prodrug therapy (GDEPT), when combined with radiation therapy, could improve the outcome of treatment for prostate cancer, the second leading cause of cancer death in the western world. GDEPT involves the introduction of a therapeutic transgene, which can be targeted to the tumour cells. A prodrug is administered systemically and is converted to its toxic form only in those cells containing the transgene, resulting in cell kill. This review will discuss the clinical trials which have investigated the potential of GDEPT at various stages of prostate cancer progression. The advantages of using GDEPT in combination with radiotherapy will be examined, as well as some of the recent advances which enhance the potential utility of GDEPT.

  16. Identification of novel enzyme-prodrug combinations for use in cytochrome P450-based gene therapy for cancer.

    PubMed

    Baldwin, Alex; Huang, Zeqi; Jounaidi, Youssef; Waxman, David J

    2003-01-01

    Gene-directed enzyme prodrug therapy can be used to increase the therapeutic activity of anti-cancer prodrugs that undergo liver cytochrome P450 (CYP)-catalyzed prodrug to active drug conversion. The present report describes a cell-culture-based assay to identify CYP gene-CYP prodrug combinations that generate bystander cytotoxic metabolites and that may potentially be useful for CYP-based gene therapy for cancer. A panel of rat liver microsomes, comprising distinct subsets of drug-inducible hepatic CYPs, was evaluated for prodrug activation in a four-day 9L gliosarcoma cell growth inhibition assay. A strong NADPH- and liver microsome-dependent increase in 9L cytotoxicity was observed for the CYP prodrugs cyclophosphamide, ifosfamide, and methoxymorpholinyl doxorubicin (MMDX) but not with three other CYP prodrugs, procarbazine, dacarbazine, and tamoxifen. MMDX activation was potentiated approximately 250-fold by liver microsomes from dexamethasone-induced rats (IC(50) (MMDX) approximately 0.1nM), suggesting that dexamethasone-inducible CYP3A enzymes contribute to activation of this novel anthracycline anti-tumor agent. This CYP3A dependence was verified in studies using liver microsomes from uninduced male and female rats and by using the CYP3A-selective inhibitors troleandomycin and ketoconazole. These findings highlight the advantages of using cell culture assays to identify novel CYP prodrug-CYP gene combinations that are characterized by production of cell-permeable, cytotoxic metabolites and that may potentially be incorporated into CYP-based gene therapies for cancer treatment.

  17. Development of inhibitor-directed enzyme prodrug therapy (IDEPT) for prostate cancer.

    PubMed

    Martin, Stacy E; Ganguly, Tanushree; Munske, Gerhard R; Fulton, Melody D; Hopkins, Mark R; Berkman, Clifford E; Black, Margaret E

    2014-10-15

    Prostate cancer (PCa) is the second most common cause of cancer death among American men after lung cancer. Unfortunately, current therapies do not provide effective treatments for patients with advanced, metastatic, or hormone refractory disease. Therefore, we seek to generate therapeutic agents for a novel PCa treatment strategy by delivering a suicide enzyme (yCDtriple) to a cell membrane bound biomarker found on PCa cells (prostate-specific membrane antigen (PSMA)). This approach has resulted in a new PCa treatment strategy reported here as inhibitor-directed enzyme prodrug therapy (IDEPT). The therapeutic agents described were generated using a click chemistry reaction between the unnatural amino acid (p-azidophenylalanine (pAzF)) incorporated into yCDtriple and the dibenzylcyclooctyne moiety of our PSMA targeting agent (DBCO-PEG4-AH2-TG97). After characterization of the therapeutic agents, we demonstrate significant PCa cell killing of PSMA-positive cells. Importantly, we demonstrate that this click chemistry approach can be used to efficiently couple a therapeutic protein to a targeting agent and may be applicable to the ablation of other types of cancers and/or malignancies.

  18. Prodrug applications for targeted cancer therapy.

    PubMed

    Giang, Irene; Boland, Erin L; Poon, Gregory M K

    2014-09-01

    Prodrugs are widely used in the targeted delivery of cytotoxic compounds to cancer cells. To date, targeted prodrugs for cancer therapy have achieved great diversity in terms of target selection, activation chemistry, as well as size and physicochemical nature of the prodrug. Macromolecular prodrugs such as antibody-drug conjugates, targeted polymer-drug conjugates and other conjugates that self-assemble to form liposomal and micellar nanoparticles currently represent a major trend in prodrug development for cancer therapy. In this review, we explore a unified view of cancer-targeted prodrugs and highlight several examples from recombinant technology that exemplify the prodrug concept but are not identified as such. Recombinant "prodrugs" such as engineered anthrax toxin show promise in biological specificity through the conditionally targeting of multiple cellular markers. Conditional targeting is achieved by structural complementation, the spontaneous assembly of engineered inactive subunits or fragments to reconstitute functional activity. These complementing systems can be readily adapted to achieve conditionally bispecific targeting of enzymes that are used to activate low-molecular weight prodrugs. By leveraging strengths from medicinal chemistry, polymer science, and recombinant technology, prodrugs are poised to remain a core component of highly focused and tailored strategies aimed at conditionally attacking complex molecular phenotypes in clinically relevant cancer.

  19. Combination cancer therapy by hapten-targeted prodrug-activating enzymes and cytokines.

    PubMed

    Chuang, Kuo-Hsiang; Cheng, Chiu-Min; Roffler, Steve R; Lu, Yu-Lin; Lin, Shiu-Ru; Wang, Jaw-Yuan; Tzou, Wen-Shyong; Su, Yu-Cheng; Chen, Bing-Mae; Cheng, Tian-Lu

    2006-01-01

    Combination therapy can help overcome limitations in the treatment of heterogeneous tumors. In the current study, we examined whether multiple therapeutic agents could be targeted to anti-dansyl single-chain antibodies (DNS scFv) that were anchored on the plasma membrane of cancer cells. Functional DNS scFv could be stably expressed on CT-26 colon cancer cells both in vitro and in vivo. Dansyl moieties were covalently attached to recombinant beta-glucuronidase (betaG) and interleukin 2 (IL-2) via a flexible poly(ethylene glycol) linker to form DNS-PEG-betaG and DNS-PEG-IL-2 conjugates. The conjugates displayed enzymatic and splenocyte-stimulatory activities, respectively, that were similar to those of the unmodified proteins. The conjugates selectively bound CT-26 cells that expressed anti-DNS scFv (CT-26/DNS cells) but not CT-26 cells that expressed control scFv (CT-26/phOx cells). DNS-PEG-betaG preferentially activated a glucuronide prodrug (BHAMG) of p-hydroxy aniline mustard at CT-26/DNS cells in culture and accumulated in subcutaneous CT-26/DNS tumors after intravenous administration. Systemic administration of DNS-PEG-IL-2 or DNS-PEG-betaG and BHAMG significantly delayed the growth of CT-26/DNS but not control CT-26/phOx tumors. Combination treatment with DNS-PEG-betaG and BHAMG followed by DNS-PEG-IL-2 therapy significantly suppressed the growth of CT-26/DNS tumors as compared to either single-agent regimen. These results show that at least two DNS-modified therapeutic agents can be selectively delivered to DNS scFv receptors in vitro and in vivo, allowing combination therapy of DNS scFv-modified tumors.

  20. A phase I trial of antibody directed enzyme prodrug therapy (ADEPT) in patients with advanced colorectal carcinoma or other CEA producing tumours.

    PubMed

    Francis, R J; Sharma, S K; Springer, C; Green, A J; Hope-Stone, L D; Sena, L; Martin, J; Adamson, K L; Robbins, A; Gumbrell, L; O'Malley, D; Tsiompanou, E; Shahbakhti, H; Webley, S; Hochhauser, D; Hilson, A J; Blakey, D; Begent, R H J

    2002-09-09

    Antibody-directed enzyme prodrug therapy is a targeted therapy in which a prodrug is activated selectively at the tumour site by an enzyme, which has been targeted to the tumour by an antibody (antibody-enzyme conjugate). Previous clinical trials have shown evidence of tumour response, however, the activated drug had a long half-life, which resulted in dose-limiting myelosuppression. Also, the targeting system, although giving high tumour to blood ratios of antibody-enzyme conjugate (10 000 : 1) required administration of a clearing antibody in addition to the antibody-enzyme conjugate. The purpose of this current study therefore was to attempt tumour targeting of the antibody-enzyme conjugate without the clearing antibody, and to investigate a new prodrug (bis-iodo phenol mustard, ZD2767P) whose activated form is highly potent and has a short half-life. Twenty-seven patients were treated with antibody-directed enzyme prodrug therapy using A5CP antibody-enzyme conjugate and ZD2767P prodrug, in a dose-escalating phase I trial. The maximum tolerated dose of ZD2767P was reached at 15.5 mg m(-2)x three administrations with a serum carboxypeptidase G2 level of 0.05 U ml(-1). Myelosuppression limited dose escalation. Other toxicities were mild. Patients' quality of life was not adversely affected during the trial as assessed by the measures used. There were no clinical or radiological responses seen in the study, but three patients had stable disease at day 56. Human anti-mouse antibody and human anti-carboxypeptidase G2 antibody were produced in response to the antibody enzyme conjugate (A5CP). The antibody-enzyme conjugate localisation data (carboxypeptidase G2 enzyme levels by HPLC on tumour and normal tissue samples, and gamma camera analysis of I-131 radiolabelled conjugate) are consistent with inadequate tumour localisation (median tumour: normal tissue ratios of antibody-enzyme conjugate of less than 1). A clearance system is therefore desirable with this antibody-enzyme

  1. Antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1: in vitro and in vivo studies with prodrugs of methotrexate and the thymidylate synthase inhibitors GW1031 and GW1843.

    PubMed

    Wolfe, L A; Mullin, R J; Laethem, R; Blumenkopf, T A; Cory, M; Miller, J F; Keith, B R; Humphreys, J; Smith, G K

    1999-01-01

    Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technology has been to design a mutant of human carboxypeptidase A (hCPA1-T268G) which is capable of hydrolyzing in vivo stable prodrugs of MTX and targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the use of this >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hope to increase ADEPT selectivity while decreasing overall immunogenicity of the enzyme-antibody conjugate. In the current report, prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase inhibitor methotrexate were studied for their wild-type and mutant hCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high kcat/Km ratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in human pancreatic juice, and in vivo for their stability in mouse plasma and tissues. In addition, targeting and in vivo enzyme activity studies were performed with an ING1 antibody conjugate of the mutant enzyme (ING1-hCPA1-T268G). Finally, in vivo therapy studies were performed with LS174T tumors to demonstrate proof of principle. Results indicate that prodrugs can be synthesized that are selective and efficient substrates of hCPA1-T268G and not substrates of the endogenous CPA activities; this leads to excellent in vivo stability for these compounds. In vivo conjugate targeting studies showed that the antibody-enzyme conjugate was targeted to the tumor and enzyme was initially active in vivo at the site. Unfortunately therapeutic studies did not demonstrate tumor reduction. Experiments to determine reasons for the lack of antitumor activity showed that the enzyme activity decreased as a result of enzyme instability. The results offer encouragement for additional novel mutant enzyme improvements and additional in vivo studies

  2. Cellular glutathione as a determinant of the sensitivity of colorectal tumour cell-lines to ZD2767 antibody-directed enzyme prodrug therapy (ADEPT)

    PubMed Central

    Monks, N R; Calvete, J A; Curtin, N J; Blakey, D C; East, S J; Newell, D R

    2000-01-01

    ZD2767P, a nitrogen mustard glutamate prodrug, is currently being evaluated in Phase 1 clinical trials of antibody directed enzyme prodrug therapy (ADEPT). There was no significant relationship between basal glutathione (GSH) concentration and sensitivity to ZD2767P + carboxpeptidase G2 (CPG2) in colorectal tumour cell-lines. Depletion of intracellular GSH using buthionine sulfoximine (BSO) resulted in only a modest potentiation of ZD2767P + CPG2 activity and hence BSO is unlikely to markedly enhance the activity of this ADEPT treatment. © 2000 Cancer Research Campaign PMID:10901381

  3. Silk-Elastinlike Hydrogel Improves the Safety of Adenovirus-Mediated Gene-Directed Enzyme-Prodrug Therapy

    PubMed Central

    Gustafson, Joshua A.; Price, Robert A.; Greish, Khaled; Cappello, Joseph; Ghandehari, Hamidreza

    2010-01-01

    Recombinant Silk-Elastinlike Protein polymers (SELPs) are well-known for their highly tunable properties on both the molecular and macroscopic hydrogel level. One specific structure of these polymers, SELP-815K, has been investigated as an injectable controlled delivery system for the treatment of head and neck cancer via a gene-directed enzyme prodrug therapy (GDEPT) approach. Due to its pore size and gelation properties in vivo, SELP restricts the distribution and controls the release of therapeutic viruses for up to one month. It has been shown that SELP-mediated delivery significantly improves therapeutic outcome of the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system in xenograft models of human head and neck cancer. However little is known about potential benefits of this approach with regard to toxicity in the presence of a fully intact immune system. The studies presented here were designed to assess the change in toxicity of the SELP mediated viral delivery compared to free viral injection in a non-tumor bearing immune competent mouse model. Toxicity was assessed at 1, 2, 4, and 12 weeks via body weight monitoring, complete blood count (CBC), and blood chemistry. It was found that in the acute and subacute phases (weeks 1-4) there is significant toxicity in groups combining the virus and the prodrug, and matrix-mediated gene delivery with SELP demonstrates a reduction in toxicity from the 2 week time point through the 4 week time point. At the end of the subchronic phase (12 weeks), signs of toxicity had subsided in both groups. Based on these results, recombinant SELPs offer a significant reduction in toxicity of virus-mediated GDEPT treatment compared to free virus injection in the acute and subacute phases. PMID:20586469

  4. New Enzyme Prodrug and Methionine-Depletion Combination Therapy of Breast Cancer Designed for Effective Delivery to the Tumor

    DTIC Science & Technology

    2012-10-01

    methylselenol , was tested in vivo in SCID mice with implanted MDA-MB-231/GFP cancer cells using i.p. injection of the FP and the prodrug. The result was that...indicate that the use of an enzyme prodrug system targeted to the tumor vasculature that yields a drug with a longer half-life than methylselenol may be...conversion of selenomethionine (SeMet) to toxic methylselenol , α-ketobutyrate, and ammonia. Methylselenol has been shown to be cytotoxic to various cancer

  5. Formulated Delivery of Enzyme/Prodrug and Cytokine Gene Therapy to Promote Immune Reduction of Treated and Remote Tumors in Mouse Models of Prostate Cancer

    DTIC Science & Technology

    2007-01-01

    nitroreductase (NTR) and the human enzyme, cytochrome P450 (CYP) [69-71]. The use of other systems, such as human thymidine phosphorylase [72] and...Enhanced efficacy of CYP–prodrug therapy is achieved by coexpression of the flavoenzyme, NADPH –CYP reductase , by metronomic (antiangiogenic) scheduling of...shock protein (Hsp-70 and -25; preclinical) GM-CSF (preclinical) Oncolytic Ad (preclinical) Chemotherapy (5FU; in vitro) Cytochrome P450

  6. HDAC inhibition amplifies gap junction communication in neural progenitors: Potential for cell-mediated enzyme prodrug therapy

    SciTech Connect

    Khan, Zahidul . E-mail: Zahidul.Khan@ki.se; Akhtar, Monira; Asklund, Thomas; Juliusson, Bengt . E-mail: Tomas.Ekstrom@ki.se

    2007-08-01

    Enzyme prodrug therapy using neural progenitor cells (NPCs) as delivery vehicles has been applied in animal models of gliomas and relies on gap junction communication (GJC) between delivery and target cells. This study investigated the effects of histone deacetylase (HDAC) inhibitors on GJC for the purpose of facilitating transfer of therapeutic molecules from recombinant NPCs. We studied a novel immortalized midbrain cell line, NGC-407 of embryonic human origin having neural precursor characteristics, as a potential delivery vehicle. The expression of gap junction protein connexin 43 (C x 43) was analyzed by western blot and immunocytochemistry. While C x 43 levels were decreased in untreated differentiating NGC-407 cells, the HDAC inhibitor 4-phenylbutyrate (4-PB) increased C x 43 expression along with increased membranous deposition in both proliferating and differentiating cells. Simultaneously, Ser 279/282-phosphorylated form of C x 43 was declined in both culture conditions by 4-PB. The 4-PB effect in NGC-407 cells was verified by using HNSC.100 human neural progenitors and Trichostatin A. Improved functional GJC is of imperative importance for therapeutic strategies involving intercellular transport of low molecular-weight compounds. We show here an enhancement by 4-PB, of the functional GJC among NGC-407 cells, as well as between NGC-407 and human glioma cells, as indicated by increased fluorescent dye transfer.

  7. Strong enhancement of recombinant cytosine deaminase activity in Bifidobacterium longum for tumor-targeting enzyme/prodrug therapy.

    PubMed

    Hamaji, Yoshinori; Fujimori, Minoru; Sasaki, Takayuki; Matsuhashi, Hitomi; Matsui-Seki, Keiichi; Shimatani-Shibata, Yuko; Kano, Yasunobu; Amano, Jun; Taniguchi, Shun'ichiro

    2007-04-01

    In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity. In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy.

  8. Prodrugs in photodynamic anticancer therapy.

    PubMed

    Musiol, Robert; Serda, Maciej; Polanski, Jaroslaw

    2011-01-01

    Photodynamic therapy (PDT), the concept of cancer treatment through the selective uptake of a light-sensitive agent followed by exposure to a specific wavelength, is limited by the transport of a photosensitizer (PS) to the tumor tissue. Porphyrin, an important PS class, can be used in PDT in the form of its prodrug molecule 5-aminolevulinic acid (5-ALA). Unfortunately, its poor pharmacokinetic properties make this compound difficult to administer. Two different methods for eliminating this problem can be distinguished. The first approach is to play with its formulation in order to improve the drug's applicability. The second approach, which is to find possible 5- ALA prodrugs, is an example of the double-prodrug method, a strategy often used in modern drug design. In this approach, the biological mechanisms in a long biosynthetic pathway involving several steps must be completed before the active drug appears. Recently, an idea of enhancing PDT sensitization using the so-called iron chelators seemed to increase the accumulation of protoporphyrin in cells. At the same time, iron chelators can destroy tumor cells by producing active oxygen after the formation of an active drug by chelating iron in the cancer cells. Thus, in the latter case, the therapy resembles a prodrug strategy. The mechanism can be explained by the Fenton reaction. Vitamin C is another example of a potential anticancer agent of this type.

  9. Rational design of an AKR1C3-resistant analog of PR-104 for enzyme-prodrug therapy.

    PubMed

    Mowday, Alexandra M; Ashoorzadeh, Amir; Williams, Elsie M; Copp, Janine N; Silva, Shevan; Bull, Matthew R; Abbattista, Maria R; Anderson, Robert F; Flanagan, Jack U; Guise, Christopher P; Ackerley, David F; Smaill, Jeff B; Patterson, Adam V

    2016-09-15

    The clinical stage anti-cancer agent PR-104 has potential utility as a cytotoxic prodrug for exogenous bacterial nitroreductases expressed from replicating vector platforms. However substrate selectivity is compromised due to metabolism by the human one- and two-electron oxidoreductases cytochrome P450 oxidoreductase (POR) and aldo-keto reductase 1C3 (AKR1C3). Using rational drug design we developed a novel mono-nitro analog of PR-104A that is essentially free of this off-target activity in vitro and in vivo. Unlike PR-104A, there was no biologically relevant cytotoxicity in cells engineered to express AKR1C3 or POR, under aerobic or anoxic conditions, respectively. We screened this inert prodrug analog, SN34507, against a type I bacterial nitroreductase library and identified E. coli NfsA as an efficient bioactivator using a DNA damage response assay and recombinant enzyme kinetics. Expression of E. coli NfsA in human colorectal cancer cells led to selective cytotoxicity to SN34507 that was associated with cell cycle arrest and generated a robust 'bystander effect' at tissue-like cell densities when only 3% of cells were NfsA positive. Anti-tumor activity of SN35539, the phosphate pre-prodrug of SN34507, was established in 'mixed' tumors harboring a minority of NfsA-positive cells and demonstrated marked tumor control following heterogeneous suicide gene expression. These experiments demonstrate that off-target metabolism of PR-104 can be avoided and identify the suicide gene/prodrug partnership of E. coli NfsA/SN35539 as a promising combination for development in armed vectors. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Enterolactone glucuronide and β-glucuronidase in antibody directed enzyme prodrug therapy for targeted prostate cancer cell treatment.

    PubMed

    Di, Yunyun; Ji, Shaoping; Wolf, Philipp; Krol, Ed S; Alcorn, Jane

    2017-08-01

    Evidence from preclinical and animal studies demonstrated an anticancer effect of flaxseed lignans, particularly enterolactone (ENL), against prostate cancer. However, extensive first-pass metabolism following oral lignan consumption results in their systemic availability primarily as glucuronic acid conjugates (ENL-Gluc) and their modest in vivo effects. To overcome the unfavorable pharmacokinetics and improve their effectiveness in prostate cancer, antibody-directed enzyme prodrug therapy (ADEPT) might offer a novel strategy to allow for restricted activation of ENL from circulating ENL-Gluc within the tumor environment. The anti-prostate-specific membrane antigen (PSMA) antibody D7 was fused with human β-glucuronidase (hβG) via a flexible linker. The binding property of the fusion construct, D7-hβG, against purified or cell surface PSMA was determined by flow cytometry and Octet Red 384 system, respectively, with a binding rate constant, K d, of 2.5 nM. The enzymatic activity of D7-hβG was first tested using the probe, 4-methylumbelliferone glucuronide. A 3.8-fold greater fluorescence intensity was observed at pH 4.5 at 2 h compared with pH 7.4. The ability of D7-hβG to activate ENL from ENL-Gluc was tested and detected using LC-MS/MS. Enhanced generation of ENL was observed with increasing ENL-Gluc concentrations and reached 3613.2 ng/mL following incubation with 100 μM ENL-Gluc at pH 4.5 for 0.5 h. D7-hβG also decreased docetaxel IC50 value from 23 nM to 14.9 nM in C4-2 cells. These results confirmed the binding and activity of D7-hβG and additional in vitro investigation is needed to support the future possibility of introducing this ADEPT system to animal models.

  11. The high-affinity maltose switch MBP317-347 has low affinity for glucose: implications for targeting tumors with metabolically directed enzyme prodrug therapy.

    PubMed

    Valdes, Gilmer; Schulte, Reinhard W; Ostermeier, Marc; Iwamoto, Keisuke S

    2014-03-01

    Development of agents with high affinity and specificity for tumor-specific markers is an important goal of molecular-targeted therapy. Here, we propose a shift in paradigm using a strategy that relies on low affinity for fundamental metabolites found in different concentrations in cancerous and non-cancerous tissues: glucose and lactate. A molecular switch, MBP317-347, originally designed to be a high-affinity switch for maltose and maltose-like polysaccharides, was demonstrated to be a low-affinity switch for glucose, that is, able to be activated by high concentrations (tens of millimolar) of glucose. We propose that such a low-affinity glucose switch could be used as a proof of concept for a new prodrug therapy strategy denominated metabolically directed enzyme prodrug therapy (MDEPT) where glucose or, preferably, lactate serves as the activator. Accordingly, considering the typical differential concentrations of lactate found in tumors and in healthy tissues, a low-affinity lactate-binding switch analogous to the low-affinity glucose-binding switch MBP317-347 would be an order of magnitude more active in tumors than in normal tissues and therefore can work as a differential activator of anticancer drugs in tumors.

  12. Sustained tumor regression of human colorectal cancer xenografts using a multifunctional mannosylated fusion protein in antibody-directed enzyme prodrug therapy.

    PubMed

    Sharma, Surinder K; Pedley, R Barbara; Bhatia, Jeetendra; Boxer, Geoffrey M; El-Emir, Ethaar; Qureshi, Uzma; Tolner, Berend; Lowe, Helen; Michael, N Paul; Minton, Nigel; Begent, Richard H J; Chester, Kerry A

    2005-01-15

    Antibody-directed enzyme prodrug therapy (ADEPT) requires highly selective antibody-mediated delivery of enzyme to tumor. MFE-CP, a multifunctional genetic fusion protein of antibody and enzyme, was designed to achieve this by two mechanisms. First by using a high affinity and high specificity single chain Fv antibody directed to carcinoembryonic antigen. Second by rapid removal of antibody-enzyme from normal tissues by virtue of post-translational mannosylation. The purpose of this paper is to investigate these dual functions in an animal model of pharmacokinetics, pharmacodynamics, toxicity, and efficacy. MFE-CP was expressed in the yeast Pichia pastoris and purified via an engineered hexahistidine tag. Biodistribution and therapeutic effect of a single ADEPT cycle (1,000 units/kg MFE-CP followed by 70 mg/kg ZD2767P prodrug at 6, 7, and 8 hours) and multiple ADEPT cycles (9-10 cycles within 21-24 days) was studied in established human colon carcinoma xenografts, LS174T, and SW1222. Selective localization of functional enzyme in tumors and rapid clearance from plasma was observed within 6 hours, resulting in tumor to plasma ratios of 1,400:1 and 339:1, respectively for the LS174T and SW1222 models. A single ADEPT cycle produced reproducible tumor growth delay in both models. Multiple ADEPT cycles significantly enhanced the therapeutic effect of a single cycle in the LS174T xenografts (P = 0.001) and produced regressions in the SW1222 xenografts (P = 0.0001), with minimal toxicity. MFE-CP fusion protein, in combination with ZD2767P, provides a new and successful ADEPT system, which offers the potential for multiple cycles and antitumor efficacy. These results provide a basis for the next stage in clinical development of ADEPT.

  13. The engineered thymidylate kinase (TMPK)/AZT enzyme-prodrug axis offers efficient bystander cell killing for suicide gene therapy of cancer.

    PubMed

    Sato, Takeya; Neschadim, Anton; Lavie, Arnon; Yanagisawa, Teruyuki; Medin, Jeffrey A

    2013-01-01

    We previously described a novel suicide (or 'cell fate control') gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK) that potentiates azidothymidine (AZT) activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander cell killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander cell killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional intercellular communications (GJICs). Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer cell line, PC-3, resulted in effective bystander killing of PC-3 cells lacking TMPK expression--an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 cells expressed both connexin43 (Cx43) and Pannexin1 (Panx1), but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 cell line was evaluated in an animal model. We demonstrate the contribution of bystander cell killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumoral injection of recombinant therapeutic lentivirus. Taken together, our data underscore that the TMPK/AZT enzyme-prodrug axis can be effectively utilized in suicide gene therapy of solid tumors, wherein significant tumor regression can be achieved via bystander effects mediated by GJICs.

  14. Enzymatic stability of 2'-ethylcarbonate-linked paclitaxel in serum and conversion to paclitaxel by rabbit liver carboxylesterase for use in prodrug/enzyme therapy.

    PubMed

    Tanino, Tadatoshi; Nawa, Akihiro; Miki, Yasuyoshi; Iwaki, Masahiro

    2008-07-01

    In prodrug/enzyme therapy for cancer, information on the sensitivity of hydrolytic enzymes to prodrug is required to reduce adverse effects of the parental drug and to find the activating enzyme. The aim of this study was to characterize the enzymatic stability of 2'-ethylcarbonate-linked paclitaxel (TAX-2'-Et) in the sera of several different species including humans. TAX-2'-Et disposition in serum was kinetically analysed using models with hydrolytic and/or degradation processes. To further evaluate the capability of liver carboxylesterases (CESs) in TAX-2'-Et hydrolysis, a CES isolated from rabbit liver (Ra-CES) was utilized as a model enzyme. Rat serum provided rapid enzymatic hydrolysis of TAX-2'-Et with a half-life of 4 min. The degradation of paclitaxel (TAX) (degradation rate constant, 0.16 h(-1)) was accompanied by the formation of an unknown compound. The conversion to TAX was almost completely inhibited by phenylmethyl sulfonylfluoride (PMSF) and bis(p-nitrophenyl) phosphate (BNPP). In human and rabbit sera, the degradation rate constant of TAX-2'-Et was 5.1 x 10(-2) and 0.15 h(-1), respectively, when excepting hydrolysis. The degradation products had the same molecular weight as TAX-2'-Et. The amount of TAX produced accounted for only 8-11% of the decrease in TAX-2'-Et after a 9 h exposure to rabbit or human serum. PMSF, but not BNPP, inhibited more than 90% of the TAX production in a 1.5 h incubation with human or rabbit serum. Ra-CES enzyme converted TAX-2'-Et to TAX with V(max) and K(m) of 74.7+/-13.8 nmol/min/mg protein and 8.8+/-2.8 microM, respectively. These results indicate that TAX-2'-Et is sensitive to serum CESs, but not cholinesterases. However, serum CESs show species-dependent hydrolysis of TAX-2'-Et. Although human serum allows the slow release of TAX, TAX-2'-Et is expected to reduce the side-effects of TAX. The Ra-CES enzyme is capable of hydrolysing TAX-2'-Et, which may be beneficial for the development of a TAX-2'-Et/enzyme therapy

  15. Evaluation of Bystander Cell Killing Effects in Suicide Gene Therapy of Cancer: Engineered Thymidylate Kinase (TMPK)/AZT Enzyme-Prodrug Axis.

    PubMed

    Sato, Takeya; Neschadim, Anton; Nakagawa, Ryo; Yanagisawa, Teruyuki; Medin, Jeffrey A

    2015-01-01

    Suicide gene therapy of cancer (SGTC) entails the introduction of a cDNA sequence into tumor cells whose polypeptide product is capable of either directly activating apoptotic pathways itself or facilitating the activation of pharmacologic agents that do so. The latter class of SGTC approaches is of the greater utility in cancer therapy owing to the ability of some small, activated cytotoxic compounds to diffuse from their site of activation into neighboring malignant cells, where they can also mediate destruction. This phenomenon, termed "bystander killing", can be highly advantageous in driving significant tumor regression in vivo without the requirement of transduction of each and every tumor cell with the suicide gene. We have developed a robust suicide gene therapy enzyme/prodrug system based on an engineered variant of the human thymidylate kinase (TMPK), which has been endowed with the ability to drive azidothymidine (AZT) activation. Delivery of this suicide gene sequence into tumors by means of recombinant lentivirus-mediated transduction embodies an SGTC strategy that successfully employs bystander cell killing as a mechanism to achieve significant ablation of solid tumors in vivo. Thus, this engineered TMPK/AZT suicide gene therapy axis holds great promise for clinical application in the treatment of inoperable solid tumors in the neoadjuvant setting. Here we present detailed procedures for the preparation of recombinant TMPK-based lentivirus, transduction of target cells, and various approaches for the evaluation of bystander cell killing effects in SGCT in both in vitro and in vivo models.

  16. Adenovirally delivered enzyme prodrug therapy with herpes simplex virus-thymidine kinase in composite tissue free flaps shows therapeutic efficacy in rat models of glioma

    PubMed Central

    2016-01-01

    Structured Abstract Introduction Free flap gene therapy exploits a novel therapeutic window when viral vectors can be delivered to the flap ex vivo. We investigated the therapeutic potential of the thymidine kinase (TK)/ganciclovir pro-drug system in treating residual disease when delivered into a free flap by intra-arterial injection of an adenoviral vector (Ad.TK). Methods We demonstrated direct in vitro efficacy of the Ad.TK/ganciclovir system by treating a panel of malignant cell lines with Ad.TK/ganciclovir to show significant cell kill proportional to the multiplicity of infection (MOI) of Ad.TK. Indirect (bystander) cytotoxicity was demonstrated by transferring conditioned medium from Ad.TK-infected malignant, or non-malignant, producer cells to uninfected tumour cells. We investigated the effect of Ad.TK/ganciclovir therapy in vivo, using models of microscopic (MiRD) and macroscopic (MaRD) residual disease in a rodent superficial inferior epigastric artery flap model. Results We observed retardation of tumour volume growth in both MiRD and MaRD models (p<0.05) and improvements in animal survival (MiRD median survival: MOI10 = 28 days, MOI 50 = 25 days, control = 18.5 days, p=0.0004; MaRD median survival: MOI 50 = 30 days, control = 18 days, p=0.0005). Gene expression studies demonstrated that viral genomic material was found predominantly in flap tissues but declined over time. Conclusion In summary, we describe the utility of virally-delivered enzyme/pro-drug therapy (VDEPT), using a free flap as a vehicle for delivery. We discuss the merits and limitations of this approach and the unique role of therapeutic free flaps in the reconstructive armamentarium. PMID:25626794

  17. Radiosynthesis of 4-[(2-chloroethyl)(2-[(11)C]ethyl)amino]-phenoxycarbonyl-l-glutamic acid a half mustard prodrug as a potential probe for imaging antibody- and gene-directed enzyme prodrug therapy with positron emission tomography.

    PubMed

    Malik, Nazreen; Luthra, S K Sajinder K; Burke, Phil; Price, P M Patrica M; Aboagye, E O Eric O; Latigo, John; Zhao, Yongjun; Brady, Frank

    2004-06-01

    The potential antibody directed prodrug therapy half-mustard prodrug 4-[(2-chloroethyl)(2-ethyl)amino]-phenoxycarbonyl-L-glutamic acid was synthesised by reductive alkylation of 4-[(2-chloroethyl)amino]-phenoxycarbonyl-L-glutamic acid using acetaldehyde. 4-[(2-chloroethyl)[(11)C](2-ethyl)amino]phenoxycarbonyl-L-glutamic acid was synthesized with 18-22% decay corrected radiochemical yield in 45 min from EOB by reductive alkylation of 4-[(2-chloroethyl)amino]-phenoxycarbonyl-L-glutamic acid using [(11)C]acetaldehyde. [(11)C]Acetaldehyde was prepared in 60% decay corrected radiochemical yield by oxidation of [(11)C]ethanol over heated copper oxide. The radiosynthesis of [(11)C]ethanol was re-examined and optimized. 4-[(2-chloroethyl)(2-ethyl)amino]-phenoxycarbonyl-L-glutamic acid was found to have affinity for carboxypeptidase G2; the K(m) and V(max) were 99.4-115.9 microM (n=3) and 3.6-5.0 microM/min, respectively, at a carboxypeptidase G2 concentration of 0.0247 U/ml.

  18. Ligand-conjugated mesoporous silica nanorattles based on enzyme targeted prodrug delivery system for effective lung cancer therapy

    SciTech Connect

    Sundarraj, Shenbagamoorthy; Thangam, Ramar; Sujitha, Mohanan V.; Vimala, Karuppaiya; Kannan, Soundarapandian

    2014-03-15

    Epidermal growth factor receptor antibody (EGFRAb) conjugated silica nanorattles (SNs) were synthesized and used to develop receptor mediated endocytosis for targeted drug delivery strategies for cancer therapy. The present study determined that the rate of internalization of silica nanorattles was found to be high in lung cancer cells when compared with the normal lung cells. EGFRAb can specifically bind to EGFR, a receptor that is highly expressed in lung cancer cells, but is expressed at low levels in other normal cells. Furthermore, in vitro studies clearly substantiated that the cPLA{sub 2}α activity, arachidonic acid release and cell proliferation were considerably reduced by pyrrolidine-2 loaded EGFRAb-SN in H460 cells. The cytotoxicity, cell cycle arrest and apoptosis were significantly induced by the treatment of pyrrolidine-2 loaded EGFRAb-SN when compared with free pyrrolidine-2 and pyrrolidine-2 loaded SNs in human non-small cell lung cancer cells. An in vivo toxicity assessment showed that silica nanorattles and EGFRAb-SN-pyrrolidine-2 exhibited low systemic toxicity in healthy Balb/c mice. The EGFRAb-SN-pyrrolidine-2 showed a much better antitumor activity (38%) with enhanced tumor inhibition rate than the pyrrolidine-2 on the non-small cell lung carcinoma subcutaneous model. Thus, the present findings validated the low toxicity and high therapeutic potentials of EGFRAb-SN-pyrrolidine-2, which may provide a convincing evidence of the silica nanorattles as new potential carriers for targeted drug delivery systems. - Highlights: • EGFRAb-SN developed for receptor-mediated Drug delivery system (DDS). • EGFRAb-SN-pyrrolidine-2 targeted DDS for cPLA2α inhibition in NSLC. • Study indicates EGFRAb-SN-pyrrolidine-2 as an efficient in target dug delivery carrier. • Study explains entire efficiency of EGFRAb-SN-pyrrolidine-2 in vitro and in vivo models.

  19. Prodrugs for targeted tumor therapies: recent developments in ADEPT, GDEPT and PMT.

    PubMed

    Tietze, Lutz F; Schmuck, Kianga

    2011-01-01

    The treatment of cancer with common anti-proliferative agents generally suffers from an insufficient differentiation between normal and malignant cells which results in extensive side effects. To enhance the efficacy and reduce the normal tissue toxicity of anticancer drugs, numerous selective tumor therapies have emerged including the highly promising approaches ADEPT (Antibody-Directed Enzyme Prodrug Therapy), GDEPT (Gene-Directed Enzyme Prodrug Therapy) and PMT (Prodrug Monotherapy). These allow a selective release of cytotoxic agents from non-toxic prodrugs at the tumor site either by targeted antibody-enzyme conjugates, enzyme encoding genes or by exploiting physiological and metabolic aberrations in cancerous tissue. Herein, recent developments in the design and biological evaluation of prodrugs for use in ADEPT, GDEPT and PMT are reviewed. As a highlight, a series of novel glycosidic prodrugs based on the natural antibiotics CC-1065 and the duocarmycins will be discussed which show a therapeutic window of up to one million. Notably, the corresponding drugs have tremendously high cytotoxicities with IC(50) values of down to 110 fM.

  20. Utilization of Enzyme-Immobilized Mesoporous Silica Nanocontainers (IBN-4) in Prodrug-Activated Cancer Theranostics

    PubMed Central

    Hung, Bau-Yen; Kuthati, Yaswanth; Kankala, Ranjith Kumar; Kankala, Shravankumar; Deng, Jin-Pei; Liu, Chen-Lun; Lee, Chia-Hung

    2015-01-01

    To develop a carrier for use in enzyme prodrug therapy, Horseradish peroxidase (HRP) was immobilized onto mesoporous silica nanoparticles (IBN-4: Institute of Bioengineering and Nanotechnology), where the nanoparticle surfaces were functionalized with 3-aminopropyltrimethoxysilane and further conjugated with glutaraldehyde. Consequently, the enzymes could be stabilized in nanochannels through the formation of covalent imine bonds. This strategy was used to protect HRP from immune exclusion, degradation and denaturation under biological conditions. Furthermore, immobilization of HRP in the nanochannels of IBN-4 nanomaterials exhibited good functional stability upon repetitive use and long-term storage (60 days) at 4 °C. The generation of functionalized and HRP-immobilized nanomaterials was further verified using various characterization techniques. The possibility of using HRP-encapsulated IBN-4 materials in prodrug cancer therapy was also demonstrated by evaluating their ability to convert a prodrug (indole-3-acetic acid (IAA)) into cytotoxic radicals, which triggered tumor cell apoptosis in human colon carcinoma (HT-29 cell line) cells. A lactate dehydrogenase (LDH) assay revealed that cells could be exposed to the IBN-4 nanocomposites without damaging their membranes, confirming apoptotic cell death. In summary, we demonstrated the potential of utilizing large porous mesoporous silica nanomaterials (IBN-4) as enzyme carriers for prodrug therapy. PMID:28347114

  1. Methioninase cancer gene therapy with selenomethionine as suicide prodrug substrate.

    PubMed

    Miki, K; Xu, M; Gupta, A; Ba, Y; Tan, Y; Al-Refaie, W; Bouvet, M; Makuuchi, M; Moossa, A R; Hoffman, R M

    2001-09-15

    In this study, we report a novel approach to gene-directed enzyme prodrug therapy for cancer. This gene therapy strategy exploits the toxic pro-oxidant property of methylselenol, which is released from selenomethionine (SeMET) by cancer cells with the adenoviral-delivered methionine alpha,gamma-lyase (MET) gene cloned from Pseudomonas putida. In MET-transduced tumor cells, the cytotoxicity of SeMET is increased up to 1000-fold compared with nontransduced cells. A strong bystander effect occurred because of methylselenol release from MET gene-transduced cells and uptake by surrounding tumor cells. Methylselenol damaged the mitochondria via oxidative stress and caused cytochrome c release into the cytosol, thereby activating the caspase cascade and apoptosis. Adenoviral MET-gene/SeMET treatment also inhibited tumor growth in rodents and significantly prolonged their survival. Recombinant adenovirus-encoding MET gene-SeMET treatment thereby offers a new paradigm for cancer gene therapy.

  2. Prodrug-based nanoparticulate drug delivery strategies for cancer therapy.

    PubMed

    Luo, Cong; Sun, Jin; Sun, Bingjun; He, Zhonggui

    2014-11-01

    Despite the rapid developments in nanotechnology and biomaterials, the efficient delivery of chemotherapeutic agents is still challenging. Prodrug-based nanoassemblies have many advantages as a potent platform for anticancer drug delivery, such as improved drug availability, high drug loading efficiency, resistance to recrystallization upon encapsulation, and spatially and temporally controllable drug release. In this review, we discuss prodrug-based nanocarriers for cancer therapy, including nanosystems based on polymer-drug conjugates, self-assembling small molecular weight prodrugs and prodrug-encapsulated nanoparticles (NPs). In addition, we discuss new trends in the field of prodrug-based nanoassemblies that enhance the delivery efficiency of anticancer drugs, with special emphasis on smart stimuli-triggered drug release, hybrid nanoassemblies, and combination drug therapy.

  3. Formulated Delivery of Enzyme/Prodrug and Cytokine Gene Therapy to Promote Immune Reduction of Treated and Remote Tumors in Mouse Models of Prostate Cancer

    DTIC Science & Technology

    2006-01-01

    Figure 1). pVITRO2-GFP/LacZ, which also contains the genes for the jelly fish green fluorescent protein, GFP, and for the bacterial enzyme... Royal Prince Alfred Hospital to set up a GLP facility for Gene Therapy trials. She was the successful candidate for this position because she was a DOD...Wales, Randwick, NSW, 2031, Australia; 2Cell and Molecular Therapy Unit, Royal Prince Alfred Hospital; Gene and Stem Cell Therapy Group

  4. Prodrug thiamine analogs as inhibitors of the enzyme transketolase.

    PubMed

    Le Huerou, Yvan; Gunawardana, Indrani; Thomas, Allen A; Boyd, Steven A; de Meese, Jason; Dewolf, Walter; Gonzales, Steven S; Han, May; Hayter, Laura; Kaplan, Tomas; Lemieux, Christine; Lee, Patrice; Pheneger, Jed; Poch, Gregory; Romoff, Todd T; Sullivan, Francis; Weiler, Solly; Wright, S Kirk; Lin, Jie

    2008-01-15

    Transketolase, a key enzyme in the pentose phosphate pathway, has been suggested as a target for inhibition in the treatment of cancer. Compound 5a ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of the transketolase cofactor thiamine, is a potent transketolase inhibitor but suffers from poor pharmacokinetics due to high clearance and C(max) linked toxicity. An efficient way of improving the pharmacokinetic profile of 5a is to prepare oxidized prodrugs which are slowly reduced in vivo yielding longer, sustained blood levels of the drug. The synthesis of such prodrugs and their evaluation in rodent models is reported.

  5. Genetically engineered theranostic mesenchymal stem cells for the evaluation of the anticancer efficacy of enzyme/prodrug systems.

    PubMed

    Nouri, Faranak Salman; Wang, Xing; Hatefi, Arash

    2015-02-28

    Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TK(SR39) mutants), yeast cytosine deaminase:uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs' bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems.

  6. Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes.

    PubMed

    Xu, Hao; Majmudar, Jaimeen D; Davda, Dahvid; Ghanakota, Phani; Kim, Ki H; Carlson, Heather A; Showalter, Hollis D; Martin, Brent R; Amidon, Gordon L

    2015-09-08

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating preclinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporter-linked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a four-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substrate-competitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design, and

  7. Lipid prodrug nanocarriers in cancer therapy.

    PubMed

    Mura, Simona; Bui, Duc Trung; Couvreur, Patrick; Nicolas, Julien

    2015-06-28

    Application of nanotechnology in the medical field (i.e., nanomedicine) plays an important role in the development of novel drug delivery methods. Nanoscale drug delivery systems can indeed be customized with specific functionalities in order to improve the efficacy of the treatments. However, despite the progresses of the last decades, nanomedicines still face important obstacles related to: (i) the physico-chemical properties of the drug moieties which may reduce the total amount of loaded drug; (ii) the rapid and uncontrolled release (i.e., burst release) of the encapsulated drug after administration and (iii) the instability of the drug in biological media where a fast transformation into inactive metabolites can occur. As an alternative strategy to alleviate these drawbacks, the prodrug approach has found wide application. The covalent modification of a drug molecule into an inactive precursor from which the drug will be freed after administration offers several benefits such as: (i) a sustained drug release (mediated by chemical or enzymatic hydrolysis of the linkage between the drug-moiety and its promoiety); (ii) an increase of the drug chemical stability and solubility and, (iii) a reduced toxicity before the metabolization occurs. Lipids have been widely used as building blocks for the design of various prodrugs. Interestingly enough, these lipid-derivatized drugs can be delivered through a nanoparticulate form due to their ability to self-assemble and/or to be incorporated into lipid/polymer matrices. Among the several prodrugs developed so far, this review will focus on the main achievements in the field of lipid-based prodrug nanocarriers designed to improve the efficacy of anticancer drugs. Gemcitabine (Pubchem CID: 60750); 5-fluorouracil (Pubchem CID: 3385); Doxorubicin (Pubchem CID: 31703); Docetaxel (Pubchem CID: 148124); Methotrexate (Pubchem CID: 126941); Paclitaxel (Pubchem CID: 36314).

  8. 3D multicellular models reflect the efficiency of MSC-directed enzyme/prodrug treatment.

    PubMed

    Bohovic, R; Demkova, L; Cihova, M; Skolekova, S; Durinikova, E; Toro, L; Tyciakova, S; Kozovska, Z; Matuskova, M; Kucerova, L

    2015-01-01

    Mesenchymal stromal cells (MSC) exhibit beneficial properties to serve as cellular vehicles for enzyme/prodrug cancer gene therapy approaches. We have previously shown that engineered human adipose tissue-derived MSC in combination with non-toxic prodrug mediated substantial cytotoxic and antitumor effect. The aim of this study was to develop advanced 3D cultivation method to serve for modelling of the therapeutic outcome in vitro. We have used engineered MSC expressing fusion transgene cytosine deaminase::uracilphosphoribosyltransferase (CD-MSC) in combination with prodrug 5-fluorocytosine (5FC). This therapeutic regimen designated CD-MSC/5FC was combined with the human melanoma cells A375 or EGFP-A375 in both standard monolayer culture and 3-dimensional (3D) multicellular nodules. The extent of cytotoxicity was evaluated by standard viability assay MTS, ATP-based luminescence assay, fluorimetric test, measurement of Caspase-3/7 activation and DNA laddering. The data have shown that the extent of cytotoxic bystander effect mediated by CD-MSC/5FC is significantly lower in 3D culture conditions. However, these data better recapitulate the therapeutic efficiency as observed previously in vivo. We suggest here to use the 3D multicellular culture conditions for better prediction of the therapeutic outcome in mouse xenograft models.

  9. Pyrrolo[2,1-c][1,4]benzodiazepine-beta-glucuronide prodrugs with a potential for selective therapy of solid tumors by PMT and ADEPT strategies.

    PubMed

    Kamal, Ahmed; Tekumalla, Venkatesh; Raju, P; Naidu, V G M; Diwan, Prakash V; Sistla, Ramakrishna

    2008-07-01

    Pyrrolo[2,1-c][1,4]benzodiazepine-beta-glucuronide prodrugs 15a-b, with a potential for selective therapy of solid tumors by PMT and ADEPT have been designed, synthesized and evaluated for selective cytotoxicity in the presence of the enzyme beta-glucuronidase. The prodrugs have been found to possess reduced cytotoxicity compared to the parent moieties, and are excellent substrates for the enzyme, exhibiting cytotoxicity selectively in the presence of the enzyme. Enhanced water solubility and improved stability are the other important outcomes upon modifying these molecules as their prodrugs.

  10. Combined enzyme/prodrug treatment by genetically engineered AT-MSC exerts synergy and inhibits growth of MDA-MB-231 induced lung metastases.

    PubMed

    Matuskova, Miroslava; Kozovska, Zuzana; Toro, Lenka; Durinikova, Erika; Tyciakova, Silvia; Cierna, Zuzana; Bohovic, Roman; Kucerova, Lucia

    2015-04-09

    Metastatic spread of tumor cells remains a serious problem in cancer treatment. Gene-directed enzyme/prodrug therapy mediated by tumor-homing genetically engineered mesenchymal stromal cells (MSC) represents a promising therapeutic modality for elimination of disseminated cells. Efficacy of gene-directed enzyme/prodrug therapy can be improved by combination of individual systems. We aimed to define the combination effect of two systems of gene therapy mediated by MSC, and evaluate the ability of systemically administered genetically engineered mesenchymal stromal cells to inhibit the growth of experimental metastases derived from human breast adenocarcinoma cells MDA-MB-231/EGFP. Human adipose tissue-derived mesenchymal stromal cells (AT-MSC) were retrovirally transduced with fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT) or with Herpes simplex virus thymidine kinase (HSVtk). Engineered MSC were cocultured with tumor cells in the presence of prodrugs 5-fluorocytosin (5-FC) and ganciclovir (GCV). Combination effect of these enzyme/prodrug approaches was calculated. SCID/bg mice bearing experimental lung metastases were treated with CD::UPRT-MSC, HSVtk-MSC or both in combination in the presence of respective prodrug(s). Treatment efficiency was evaluated by EGFP-positive cell detection by flow cytometry combined with real-time PCR quantification of human cells in mouse organs. Results were confirmed by histological and immunohistochemical examination. We demonstrated various extent of synergy depending on tested cell line and experimental setup. The strongest synergism was observed on breast cancer-derived cell line MDA-MB-231/EGFP. Systemic administration of CD::UPRT-MSC and HSVtk-MSC in combination with 5-FC and GCV inhibited growth of MDA-MB-231 induced lung metastases. Combined gene-directed enzyme/prodrug therapy mediated by MSC exerted synergic cytotoxic effect and resulted in high therapeutic efficacy in vivo.

  11. Enzyme-functionalized vascular grafts catalyze in-situ release of nitric oxide from exogenous NO prodrug.

    PubMed

    Wang, Zhihong; Lu, Yaxin; Qin, Kang; Wu, Yifan; Tian, Yingping; Wang, Jianing; Zhang, Jimin; Hou, Jingli; Cui, Yun; Wang, Kai; Shen, Jie; Xu, Qingbo; Kong, Deling; Zhao, Qiang

    2015-07-28

    Nitric oxide (NO) is an important signaling molecule in cardiovascular system, and the sustained release of NO by endothelial cells plays a vital role in maintaining patency and homeostasis. In contrast, lack of endogenous NO in artificial blood vessel is believed to be the main cause of thrombus formation. In this study, enzyme prodrug therapy (EPT) technique was employed to construct a functional vascular graft by immobilization of galactosidase on the graft surface. The enzyme-functionalized grafts exhibited excellent catalytic property in decomposition of the exogenously administrated NO prodrug. Localized and on-demand release of NO was demonstrated by in vitro release assay and fluorescent probe tracing in an ex vivo model. The immobilized enzyme retained catalytic property even after subcutaneous implantation of the grafts for one month. The functional vascular grafts were implanted into the rat abdominal aorta with a 1-month monitoring period. Results showed effective inhibition of thrombus formation in vivo and enhancement of vascular tissue regeneration and remodeling on the grafts. Thus, we create an enzyme-functionalized vascular graft that can catalyze prodrug to release NO locally and sustainably, indicating that this approach may be useful to develop new cell-free vascular grafts for treatment of vascular diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Antitumor Synergism and Enhanced Survival with a Tumor Vasculature-Targeted Enzyme Prodrug System, Rapamycin, and Cyclophosphamide.

    PubMed

    Krais, John J; Virani, Needa; McKernan, Patrick H; Nguyen, Quang; Fung, Kar-Ming; Sikavitsas, Vassilios I; Kurkjian, Carla; Harrison, Roger G

    2017-09-01

    Mutant cystathionine gamma-lyase was targeted to phosphatidylserine exposed on tumor vasculature through fusion with Annexin A1 or Annexin A5. Cystathionine gamma-lyase E58N, R118L, and E338N mutations impart nonnative methionine gamma-lyase activity, resulting in tumor-localized generation of highly toxic methylselenol upon systemic administration of nontoxic selenomethionine. The described therapeutic system circumvents systemic toxicity issues using a novel drug delivery/generation approach and avoids the administration of nonnative proteins and/or DNA required with other enzyme prodrug systems. The enzyme fusion exhibits strong and stable in vitro binding with dissociation constants in the nanomolar range for both human and mouse breast cancer cells and in a cell model of tumor vascular endothelium. Daily administration of the therapy suppressed growth of highly aggressive triple-negative murine 4T1 mammary tumors in immunocompetent BALB/cJ mice and MDA-MB-231 tumors in SCID mice. Treatment did not result in the occurrence of negative side effects or the elicitation of neutralizing antibodies. On the basis of the vasculature-targeted nature of the therapy, combinations with rapamycin and cyclophosphamide were evaluated. Rapamycin, an mTOR inhibitor, reduces the prosurvival signaling of cells in a hypoxic environment potentially exacerbated by a vasculature-targeted therapy. IHC revealed, unsurprisingly, a significant hypoxic response (increase in hypoxia-inducible factor 1 α subunit, HIF1A) in the enzyme prodrug-treated tumors and a dramatic reduction of HIF1A upon rapamycin treatment. Cyclophosphamide, an immunomodulator at low doses, was combined with the enzyme prodrug therapy and rapamycin; this combination synergistically reduced tumor volumes, inhibited metastatic progression, and enhanced survival. Mol Cancer Ther; 16(9); 1855-65. ©2017 AACR. ©2017 American Association for Cancer Research.

  13. Theranostic reduction-sensitive gemcitabine prodrug micelles for near-infrared imaging and pancreatic cancer therapy

    NASA Astrophysics Data System (ADS)

    Han, Haijie; Wang, Haibo; Chen, Yangjun; Li, Zuhong; Wang, Yin; Jin, Qiao; Ji, Jian

    2015-12-01

    A biodegradable and reduction-cleavable gemcitabine (GEM) polymeric prodrug with in vivo near-infrared (NIR) imaging ability was reported. This theranostic GEM prodrug PEG-b-[PLA-co-PMAC-graft-(IR820-co-GEM)] was synthesized by ring-opening polymerization and ``click'' reaction. The as-prepared reduction-sensitive prodrug could self-assemble into prodrug micelles in aqueous solution confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In vitro drug release studies showed that these prodrug micelles were able to release GEM in an intracellular-mimicking reductive environment. These prodrug micelles could be effectively internalized by BxPC-3 pancreatic cancer cells, which were observed by confocal laser scanning microscopy (CLSM). Meanwhile, a methyl thiazolyl tetrazolium (MTT) assay demonstrated that this prodrug exhibited high cytotoxicity against BxPC-3 cells. The in vivo whole-animal near-infrared (NIR) imaging results showed that these prodrug micelles could be effectively accumulated in tumor tissue and had a longer blood circulation time than IR820-COOH. The endogenous reduction-sensitive gemcitabine prodrug micelles with the in vivo NIR imaging ability might have great potential in image-guided pancreatic cancer therapy.A biodegradable and reduction-cleavable gemcitabine (GEM) polymeric prodrug with in vivo near-infrared (NIR) imaging ability was reported. This theranostic GEM prodrug PEG-b-[PLA-co-PMAC-graft-(IR820-co-GEM)] was synthesized by ring-opening polymerization and ``click'' reaction. The as-prepared reduction-sensitive prodrug could self-assemble into prodrug micelles in aqueous solution confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In vitro drug release studies showed that these prodrug micelles were able to release GEM in an intracellular-mimicking reductive environment. These prodrug micelles could be effectively internalized by BxPC-3 pancreatic cancer cells, which

  14. Enzyme-Sensitive and Amphiphilic PEGylated Dendrimer-Paclitaxel Prodrug-Based Nanoparticles for Enhanced Stability and Anticancer Efficacy.

    PubMed

    Li, Ning; Cai, Hao; Jiang, Lei; Hu, Jiani; Bains, Ashika; Hu, Jesse; Gong, Qiyong; Luo, Kui; Gu, Zhongwei

    2017-03-01

    In this study, we prepared a smart polymeric vehicle for the hydrophobic drug paclitaxel (PTX) that allowed a maximum steady-state circulation and a fast intracellular release in tumors. PTX was linked to the Janus PEGylated (PEG = poly(ethylene glycol)) peptide dendrimer via an enzyme-sensitive linker glycylphenylalanylleucylglycine tetrapeptide by efficient click reaction, resulting in Janus dendritic prodrug with 20.9% PTX content. The prodrug self-assembled into nanoscale particles with appropriate nanosizes, compact morphology, and negative surface charge. In addition to high stability during circulation, as demonstrated by protein adsorption assays and drug release studies in the cancer's intracellular environment, the nanoparticles were able to quickly release the drug intact in its original molecular structure, as verified via high-performance liquid chromatography and mass spectrometry analyses. Compared to free PTX, the enzyme-responsive feature of nanoparticles promoted higher cytotoxicity against 4T1 cancer cells and much lower cytotoxicity against normal cells. The nanoparticles accumulated in the tumor and were retained for an extended period of time, as confirmed by fluorescence imaging. Therefore, these nanoparticles exhibited significantly enhanced antitumor efficiency in the 4T1 breast cancer model as indicated by the observed inhibition of angiogenesis and proliferation as well as induction of apoptosis. Moreover, the nanoparticles reduced the occurrence of side effects, particularly dose-limited toxicities, as monitored by body weight and hematological features. Hence, our Janus PEGylated dendrimer-PTX prodrug-based nanoparticles may potentially serve as nanoscale vehicles for breast cancer therapy.

  15. Molecular Pathways: Hypoxia-activated prodrugs in cancer therapy.

    PubMed

    Baran, Natalia; Konopleva, Marina

    2017-01-30

    Hypoxia is a known feature of aggressive solid tumors as well as a critical hallmark of the niche in aggressive hematologic malignances. Hypoxia is associated with insufficient response to standard therapy, resulting in disease progression and curtailed patients' survival through maintenance of noncycling cancer stem-like cells. A better understanding of the mechanisms and signaling pathways induced by hypoxia is essential to overcoming these effects. Recent findings demonstrate that bone marrow in the setting of hematologic malignancies is highly hypoxic and that progression of the disease is associated with expansion of hypoxic niches and stabilization of the oncogenic hypoxia-inducible factor-1alpha (HIF-1α). Solid tumors have also been shown to harbor hypoxic areas, maintaining survival of cancer cells via the HIF-1α pathway. Developing new strategies for targeting hypoxia has become a crucial approach in modern cancer therapy. The number of preclinical and clinical trials targeting low-oxygen tumor compartments or the hypoxic bone marrow niche via hypoxia-activated prodrugs is increasing. This review discusses the development of the hypoxia-activated prodrugs and their applicability in treating both hematologic malignancies and solid tumors.

  16. Prodrug approaches for CNS delivery.

    PubMed

    Rautio, Jarkko; Laine, Krista; Gynther, Mikko; Savolainen, Jouko

    2008-01-01

    Central nervous system (CNS) drug delivery remains a major challenge, despite extensive efforts that have been made to develop novel strategies to overcome obstacles. Prodrugs are bioreversible derivatives of drug molecules that must undergo an enzymatic and/or chemical transformation in vivo to release the active parent drug, which subsequently exerts the desired pharmacological effect. In both drug discovery and drug development, prodrugs have become an established tool for improving physicochemical, biopharmaceutical or pharmacokinetic properties of pharmacologically active agents that overcome barriers to a drug's usefulness. This review provides insight into various prodrug strategies explored to date for CNS drug delivery, including lipophilic prodrugs, carrier- and receptor-mediated prodrug delivery systems, and gene-directed enzyme prodrug therapy.

  17. Cancer chemotherapy: a SN-38 (7-ethyl-10-hydroxycamptothecin) glucuronide prodrug for treatment by a PMT (Prodrug MonoTherapy) strategy.

    PubMed

    Angenault, Stéphane; Thirot, Sylvie; Schmidt, Frédéric; Monneret, Claude; Pfeiffer, Bruno; Renard, Pierre

    2003-03-10

    A glucuronide-based prodrug of SN-38 (7-ethyl-10-hydroxycamptothecin) has been synthesized for use in a Prodrug MonoTherapy Strategy (PMT). Since this prodrug is significantly less cytotoxic than SN-38 itself and efficiently releases the drug in vitro in the presence of beta-D-glucuronidase, it can be considered as an appropriate candidate for cancer treatment by a PMT strategy.

  18. Boronic Prodrug of Endoxifen as an Effective Hormone Therapy for Breast Cancer

    PubMed Central

    Zhang, Changde; Zhong, Qiu; Zhang, Qiang; Zheng, Shilong; Miele, Lucio; Wang, Guangdi

    2015-01-01

    As a prodrug, tamoxifen is activated by the P450 enzyme CYP2D6 that is responsible for converting it to the active metabolites, 4-hydroxytamoxifen and endoxifen. Patients with genetic polymorphisms of CYP2D6 may not receive the full benefit of tamoxifen therapy. There is increasing evidence that poor metabolizer patients have lower plasma concentrations of endoxifen and suffer worse disease outcome, although some clinical studies reported no correlation between CYP2D6 polymorphism and tamoxifen therapy outcome. Endoxifen is currently undergoing clinical trials as a potentially improved and more potent SERM (Selective Estrogen Receptor Modulator) for endocrine therapy that is independent of CYP2D6 status in patients. However, direct administration of endoxifen may present the problem of low bioavailability due to its rapid first-pass metabolism via O-glucuronidation. We have designed and synthesized ZB483, a boronic prodrug of endoxifen suitable for oral administration with greatly enhanced bioavailability by increasing the concentration of endoxifen in mouse blood. Our study demonstrated that ZB483 potently inhibited growth of ER+ breast cancer cells in vitro and was efficiently converted to endoxifen in cell culture media by oxidative deboronation. In vivo this metabolic conversion is equally efficient as indicated in the pharmacokinetic study. Moreover, at the same dose, ZB483 afforded a 30-40 fold higher level endoxifen in mouse blood compared to unconjugated endoxifen administration. The significantly enhanced bioavailability of endoxifen conferred by the boronic prodrug was further validated in an in vivo efficacy study. ZB483 was demonstrated to be more efficacious than endoxifen in inhibiting xenograft tumor growth in mice at equal dosage but more so at lower dosage. Together, these preclinical studies demonstrate that ZB483 is a promising endocrine therapy agent with markedly enhanced bioavailability in systemic circulation and superior efficacy compared to

  19. Use of enzyme inhibitors to evaluate the conversion pathways of ester and amide prodrugs: a case study example with the prodrug ceftobiprole medocaril.

    PubMed

    Eichenbaum, Gary; Skibbe, Jennifer; Parkinson, Andrew; Johnson, Mark D; Baumgardner, Dawn; Ogilvie, Brian; Usuki, Etsuko; Tonelli, Fred; Holsapple, Jeff; Schmitt-Hoffmann, Anne

    2012-03-01

    An approach was developed that uses enzyme inhibitors to support the assessment of the pathways that are responsible for the conversion of intravenously administered ester and amide prodrugs in different biological matrices. The methodology was applied to ceftobiprole medocaril (BAL5788), the prodrug of the cephalosporin antibiotic, ceftobiprole. The prodrug was incubated in plasma, postmitochondrial supernatant fractions from human liver (impaired and nonimpaired), kidney, and intestine as well as erythrocytes, in the presence and absence of different enzyme inhibitors (acetylcholinesterase, pseudocholinesterase, retinyl palmitoyl hydrolase, serine esterases, amidases, and cholinesterase). Hydrolysis was rapid, extensive, and not dependent on the presence of β-nicotinamide-adenine dinucleotide phosphate (reduced form) in all matrices tested, suggesting the involvement of carboxylesterases but not P450 enzymes. Hydrolysis in healthy human plasma was rapid and complete and only partially inhibited in the presence of paraoxonase inhibitors or in liver from hepatic impaired patients, suggesting involvement of nonparaoxonase pathways. The results demonstrate the utility of this approach in confirming the presence of multiple conversion pathways of intravenously administered prodrugs and in the case of BAL5788 demonstrated that this prodrug is unlikely to be affected by genetic polymorphisms, drug interactions, or other environmental factors that might inhibit or induce the enzymes involved in its conversion.

  20. Theranostic reduction-sensitive gemcitabine prodrug micelles for near-infrared imaging and pancreatic cancer therapy.

    PubMed

    Han, Haijie; Wang, Haibo; Chen, Yangjun; Li, Zuhong; Wang, Yin; Jin, Qiao; Ji, Jian

    2016-01-07

    A biodegradable and reduction-cleavable gemcitabine (GEM) polymeric prodrug with in vivo near-infrared (NIR) imaging ability was reported. This theranostic GEM prodrug PEG-b-[PLA-co-PMAC-graft-(IR820-co-GEM)] was synthesized by ring-opening polymerization and "click" reaction. The as-prepared reduction-sensitive prodrug could self-assemble into prodrug micelles in aqueous solution confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In vitro drug release studies showed that these prodrug micelles were able to release GEM in an intracellular-mimicking reductive environment. These prodrug micelles could be effectively internalized by BxPC-3 pancreatic cancer cells, which were observed by confocal laser scanning microscopy (CLSM). Meanwhile, a methyl thiazolyl tetrazolium (MTT) assay demonstrated that this prodrug exhibited high cytotoxicity against BxPC-3 cells. The in vivo whole-animal near-infrared (NIR) imaging results showed that these prodrug micelles could be effectively accumulated in tumor tissue and had a longer blood circulation time than IR820-COOH. The endogenous reduction-sensitive gemcitabine prodrug micelles with the in vivo NIR imaging ability might have great potential in image-guided pancreatic cancer therapy.

  1. Gelatin-encapsulated iron oxide nanoparticles for platinum (IV) prodrug delivery, enzyme-stimulated release and MRI.

    PubMed

    Cheng, Ziyong; Dai, Yunlu; Kang, Xiaojiao; Li, Chunxia; Huang, Shanshan; Lian, Hongzhou; Hou, Zhiyao; Ma, Pingan; Lin, Jun

    2014-08-01

    A facile method for transferring hydrophobic iron oxide nanoparticles (IONPs) from chloroform to aqueous solution via encapsulation of FITC-modified gelatin based on the hydrophobic-hydrophobic interaction is described in this report. Due to the existence of large amount of active groups such as amine groups in gelatin, the fluorescent labeling molecules of fluorescein isothiocyanate (FITC) and platinum (IV) prodrug functionalized with carboxylic groups can be conveniently conjugated on the IONPs. The nanoparticles carrying Pt(IV) prodrug exhibit good anticancer activities when the Pt(IV) complexes are reduced to Pt(II) in the intracellular environment, while the pure Pt(IV) prodrug only presents lower cytotoxicity on cancer cells. Meanwhile, fluorescence of FITC on the surface of nanoparticles was completely quenched due to the possible Förster Resonance Energy Transfer (FRET) mechanism and showed a fluorescence recovery after gelatin release and detachment from IONPs. Therefore FITC as a fluorescence probe can be used for identification, tracking and monitoring the drug release. In addition, adding pancreatic enzyme can effectively promote the gelatin release from IONPs owing to the degradation of gelatin. Noticeable darkening in magnetic resonance image (MRI) was observed at the tumor site after in situ injection of nanoparticles, indicating the IONPs-enhanced T2-weighted imaging. Our results suggest that the gelatin encapsulated Fe3O4 nanoparticles have potential applications in multi-functional drug delivery system for disease therapy, MR imaging and fluorescence sensor. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Enzymes To Die For: Exploiting Nucleotide Metabolizing Enzymes for Cancer Gene Therapy

    PubMed Central

    Ardiani, Andressa; Johnson, Adam J.; Ruan, Hongmei; Sanchez-Bonilla, Marilyn; Serve, Kinta; Black, Margaret E.

    2012-01-01

    Suicide gene therapy is an attractive strategy to selectively destroy cancer cells while minimizing unnecessary toxicity to normal cells. Since this idea was first introduced more than two decades ago, numerous studies have been conducted and significant developments have been made to further its application for mainstream cancer therapy. Major limitations of the suicide gene therapy strategy that have hindered its clinical application include inefficient directed delivery to cancer cells and the poor prodrug activation capacity of suicide enzymes. This review is focused on efforts that have been and are currently being pursued to improve the activity of individual suicide enzymes towards their respective prodrugs with particular attention to the application of nucleotide metabolizing enzymes in suicide cancer gene therapy. A number of protein engineering strategies have been employed and our discussion here will center on the use of mutagenesis approaches to create and evaluate nucleotide metabolizing enzymes with enhanced prodrug activation capacity and increased thermostability. Several of these studies have yielded clinically important enzyme variants that are relevant for cancer gene therapy applications because their utilization can serve to maximize cancer cell killing while minimizing the prodrug dose, thereby limiting undesirable side effects. PMID:22384805

  3. PSMA-Specific Theranostic Nanoplex for Combination of TRAIL Gene and 5-FC Prodrug Therapy of Prostate Cancer

    PubMed Central

    Chen, Zhihang; Penet, Marie-France; Krishnamachary, Balaji; Banerjee, Sangeeta R.; Pomper, Martin G.; Bhujwalla, Zaver M.

    2015-01-01

    Metastatic prostate cancer causes significant morbidity and mortality and there is a critical unmet need for effective treatments. We have developed a theranostic nanoplex platform for combined imaging and therapy of prostate cancer. Our prostate-specific membrane antigen (PSMA) targeted nanoplex is designed to deliver plasmid DNA encoding tumor necrosis factor related apoptosis-inducing ligand (TRAIL), together with bacterial cytosine deaminase (bCD) as a prodrug enzyme. Nanoplex specificity was tested using two variants of human PC3 prostate cancer cells in culture and in tumor xenografts, one with high PSMA expression and the other with negligible expression levels. The expression of EGFP-TRAIL was demonstrated by fluorescence optical imaging and real-time PCR. Noninvasive 19F MR spectroscopy detected the conversion of the nontoxic prodrug 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU) by bCD. The combination strategy of TRAIL gene and 5-FC/bCD therapy showed significant inhibition of the growth of prostate cancer cells and tumors. These data demonstrate that the PSMA-specific theranostic nanoplex can deliver gene therapy and prodrug enzyme therapy concurrently for precision medicine in metastatic prostate cancer. PMID:26706476

  4. Mesenchymal stem cells as cellular vehicles for prodrug gene therapy against tumors.

    PubMed

    Amara, Ikrame; Touati, Walid; Beaune, Philippe; de Waziers, Isabelle

    2014-10-01

    Gene-directed enzyme prodrug therapy (GDEPT) consists of targeted delivery to tumor cells of a suicide gene responsible for the in situ conversion of a prodrug into cytotoxic metabolites. One of the major impediments of GDEPT is to target specifically the tumor cells with the suicide gene. Among gene delivery methods, mesenchymal stem cells (MSCs) have emerged recently as potential cellular vehicles for gene delivery. MSCs are particularly suited for gene transduction. They exhibit remarkable migratory property towards tumors and their metastases and they are weakly immunogenic. This review will summarize the current knowledge about MSCs engineered to express different suicide genes (cytosine deaminase, thymidine kinase, carboxylesterase, cytochrome P450) to elicit a significant antitumor response against brain tumors, ovarian, hepatocellular, pancreatic, renal or medullary thyroid carcinomas, breast or prostate cancer and pulmonary metastases. The potential side effects of these MSC-based tumor therapies will also be considered to highlight certain aspects that need to be improved prior to clinical use. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  5. Delayed Progression of Lung Metastases Following Delivery of a Prodrug-activating Enzyme.

    PubMed

    Dore-Savard, Louis; Chen, Zhihang; Winnard, Paul T; Krishnamachary, Balaji; Raman, Venu; Black, Margaret E; Bhujwalla, Zaver M

    2017-05-01

    Chemotherapy is an effective option to treat recurrent or metastatic cancer but its debilitating side-effects limit the dose and time of exposure. Prodrugs that can be activated locally by an activating enzyme can minimize collateral damage from chemotherapy. We previously demonstrated the efficacy of a poly-L-lysine-based theranostic nanoplex containing bacterial cytosine deaminase (bCD) that locally converted 5-fluorocytosine (5-FC) to the chemotherapeutic agent 5-fluorouracil in MDA-MB-231 primary tumor xenografts. Here we used a more effective variant of bCD to target metastatic red fluorescence protein expressing MDA-MB-435 cells in the lungs. We used an intravenous injection of tumor cells and monitored tumor growth in the lungs for 5 weeks by which time metastatic nodules were detected with optical imaging. The animals were then treated with the bCD-nanoplex and 5-FC. We observed a significant decrease in metastatic burden with a single dose of the enzyme-nanoplex and two consecutive prodrug injections. These results are a first step towards the longitudinal evaluation of such a strategy with multiple doses. Additionally, the enzyme can be directly coupled to imaging reporters to time prodrug administration for the detection and treatment of aggressive metastatic cancer. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  6. A polymeric colchicinoid prodrug with reduced toxicity and improved efficacy for vascular disruption in cancer therapy

    PubMed Central

    Crielaard, Bart J; van der Wal, Steffen; Lammers, Twan; Le, Huong Thu; Hennink, Wim E; Schiffelers, Raymond M; Storm, Gert; Fens, Marcel HAM

    2011-01-01

    Colchicinoids are very potent tubulin-binding compounds, which interfere with microtubule formation, giving them strong cytotoxic properties, such as cell mitosis inhibition and induction of microcytoskeleton depolymerization. While this makes them promising vascular disrupting agents (VDAs) in cancer therapy, their dose-limiting toxicity has prevented any clinical application for this purpose. Therefore, colchicinoids are considered attractive lead molecules for the development of novel vascular disrupting nanomedicine. In a previous study, a polymeric colchicinoid prodrug that showed favorable hydrolysis characteristics at physiological conditions was developed. In the current study, this polymeric colchicinoid prodrug was evaluated in vitro and in vivo for its toxicity and vascular disrupting potential. Cell viability studies with human umbilical vein endothelial cells, as an in vitro measure for colchicine activity, reflected the degradation kinetics of the prodrug accordingly. Upon intravenous treatment, in vivo, of B16F10 melanoma-bearing mice with colchicine or with the polymeric colchicinoid prodrug, apparent vascular disruption and consequent tumor necrosis was observed for the prodrug but not for free colchicine at an equivalent dose. Moreover, a five-times-higher dose of the prodrug was well tolerated, indicating reduced toxicity. These findings demonstrate that the polymeric colchicinoid prodrug has a substantially improved efficacy/toxicity ratio compared with that of colchicine, making it a promising VDA for cancer therapy. PMID:22114500

  7. Combination efficacy of doxorubicin and adenoviral methioninase gene therapy with prodrug selenomethionine.

    PubMed

    Gupta, Anshu; Miki, Kenji; Xu, Mingxu; Yamamoto, Norio; Moossa, A R; Hoffman, R M

    2003-01-01

    We have previously demonstrated an enzyme activation prodrug gene therapy strategy using the methionine alpha,gamma-lyase gene (MET) cloned from Pseudomonas putida, in combination with selenomethionine (SeMET) as a prodrug. MET gene transfer via a recombinant adenovirus (Ad-MET) converts the physiologic compound SeMET to highly toxic methylselenol. In this study, we have developed a combination therapy approach using Ad-MET/SeMET gene therapy and doxorubicin (DOX). The combination significantly delayed the growth of H460, an aggressively-growing human lung cancer cell line, in nude mice. H460 cells were injected intra-dermally in nude mice. Tumor-bearing mice were divided into 12 groups [Control (Ctrl), DOX, SeMET, SeMET + DOX, Ad-Ctrl, Ad-Ctrl + SeMET, Ad-Ctrl + DOX, Ad-Ctrl + SeMET + DOX, Ad-MET, Ad-MET + DOX, Ad-MET + SeMET, and Ad-MET + SeMET + DOX]. DOX (2 mg/kg body weight) was given intra-peritoneally twice at 7-day intervals. SeMET (1 microM/mouse) was given by intra-tumor injection everyday, starting the following day after transfection with adenovirus. Tumor growth in the untreated group showed a 10-fold increase in tumor volume after two weeks. In contrast, the increase was only 2.5-fold in the DOX + Ad-MET/SeMET group. The treatment with DOX alone at the low-dose used showed no effect compared to the control group. There was a 5.8-fold increase in tumor volume in mice treated with Ad-MET/SeMET gene therapy alone. The tumor doubling-time was increased to approximately 10 days with the combination therapy of Ad-MET + SeMET + DOX as opposed to 2-3 days in all other treatment groups.

  8. Nanoassemblies from amphiphilic cytarabine prodrug for leukemia targeted therapy.

    PubMed

    Liu, Jing; Zhao, Dujuan; He, Wenxiu; Zhang, Huiyuan; Li, Zhonghao; Luan, Yuxia

    2017-02-01

    The anti-leukemia effect of cytarabine (Ara-C) is severely restricted by its high hydrophilic properties and rapid plasma degradation. Herein, a novel amphiphilic small molecular prodrug of Ara-C was developed by coupling a short aliphatic chain, hexanoic acid (HA) to 4-NH2 of the parent drug. Based on the amphiphilic nature, the resulting bioconjugate (HA-Ara) could spontaneously self-assemble into stable spherical nanoassemblies (NAs) with an extremely high drug loading (∼71wt%). Moreover, folate receptor (FR)-targeting NAs with high grafting efficient folic acid - bovine serum albumin (FA-BSA) conjugate immobilized on the surface (NAs/FA-BSA) was prepared. The results of MTT assays on FR-positive K562 cells and FR-negative A549 cells demonstrated higher cytotoxicity of HA-Ara NAs than the native drug. Especially, the IC50 values revealed that NAs/FA-BSA was 3 and 2-fold effective than non-targeted NAs after 24 and 48h treatment with K562 cells, respectively indicating FR-mediated enhanced anti-tumor efficacy. In vitro cellular uptake, larger accumulation of HA-Ara NAs were observed in comparative with the free FITC and the results further confirmed the selective uptake of NAs/FA-BSA in folate receptor enriched cancer cells. Above all, self-assembled HA-Ara NAs exhibited potential superiority for Ara-C delivery and FA-modified NAs would be an excellent candidate for targeting leukemia therapy.

  9. Rapid delivery of diazepam from supersaturated solutions prepared using prodrug/enzyme mixtures: toward intranasal treatment of seizure emergencies.

    PubMed

    Kapoor, Mamta; Winter, Tate; Lis, Lev; Georg, Gunda I; Siegel, Ronald A

    2014-05-01

    Current treatments for seizure emergencies, such as status epilepticus, include intravenous or rectal administration of benzodiazepines. While intranasal delivery of these drugs is desirable, the small volume of the nasal cavity and low drug solubility pose significant difficulties. Here, we prepared supersaturated diazepam solutions under physiological conditions and without precipitation, using a prodrug/enzyme system. Avizafone, a peptide prodrug of diazepam, was delivered with--Aspergillus oryzae (A.O.) protease, an enzyme identified from a pool of hydrolytic enzymes in assay buffer, pH 7.4 at 32°C. This enzyme converted avizafone to diazepam at supersaturated concentrations. In vitro permeability studies were performed at various prodrug/enzyme ratios using Madin-Darby canine kidney II-wild type (MDCKII-wt) monolayers, a representative model of the nasal epithelium. Monolayer integrity was examined using TEER measurement and the lucifer yellow permeability assay. Prodrug/drug concentrations were measured using HPLC. Enzyme kinetics with avizafone-protease mixtures revealed K(M) = 1,501 ± 232 μM and V(max) = 1,369 ± 94 μM/s. Prodrug-protease mixtures, when co-delivered apically onto MDCKII-wt monolayers, showed 2-17.6-fold greater diazepam flux (S = 1.3-15.3) compared to near-saturated diazepam (S = 0.7). Data for prodrug conversion upstream (apical side) and drug permeability downstream (basolateral side) fitted reasonably well to a previously developed in vitro two compartment pharmacokinetic model. Avizafone-protease mixtures resulted in supersaturated diazepam in less than 5 min, with the rate and extent of supersaturation determined by the prodrug/enzyme ratio. Together, these results suggest that an intranasal avizafone-protease system may provide a rapid and alternative means of diazepam delivery.

  10. Synthesis and biological evaluation of prodrugs based on the natural antibiotic duocarmycin for use in ADEPT and PMT.

    PubMed

    Tietze, Lutz F; Schmuck, Kianga; Schuster, Heiko J; Müller, Michael; Schuberth, Ingrid

    2011-02-07

    Chemotherapy of malign tumors is usually associated with serious side effects as common anticancer drugs lack selectivity. An approach to deal with this problem is the antibody-directed enzyme prodrug therapy (ADEPT) and the prodrug monotherapy (PMT). Herein, the synthesis and biological evaluation of new glycosidic prodrugs suitable for both concepts are described. All prodrugs but one are stable in human serum and show QIC(50) values (IC(50) of prodrug/IC(50) of prodrug in the presence of the appropriate glycohydrolase) of up to 6500. This is the best value found so far for compounds interacting with DNA.

  11. Identification of activating enzymes of a novel FBPase inhibitor prodrug, CS-917.

    PubMed

    Kubota, Kazuishi; Inaba, Shin-Ichi; Nakano, Rika; Watanabe, Mihoko; Sakurai, Hidetaka; Fukushima, Yumiko; Ichikawa, Kimihisa; Takahashi, Tohru; Izumi, Takashi; Shinagawa, Akira

    2015-06-01

    CS-917 (MB06322) is a selective small compound inhibitor of fructose 1,6-bisphosphatase (FBPase), which is expected to be a novel drug for the treatment of type 2 diabetes by inhibiting gluconeogenesis. CS-917 is a bisamidate prodrug and activation of CS-917 requires a two-step enzyme catalyzed reaction. The first-step enzyme, esterase, catalyzes the conversion of CS-917 into the intermediate form (R-134450) and the second-step enzyme, phosphoramidase, catalyzes the conversion of R-134450 into the active form (R-125338). In this study, we biochemically purified the CS-917 esterase activity in monkey small intestine and liver. We identified cathepsin A (CTSA) and elastase 3B (ELA3B) as CS-917 esterases in the small intestine by mass spectrometry, whereas we found CTSA and carboxylesterase 1 (CES1) in monkey liver. We also purified R-134450 phosphoramidase activity in monkey liver and identified sphingomyelin phosphodiesterase, acid-like 3A (SMPADL3A), as an R-134450 phosphoramidase, which has not been reported to have any enzyme activity. Recombinant human CTSA, ELA3B, and CES1 showed CS-917 esterase activity and recombinant human SMPDL3A showed R-134450 phosphoramidase activity, which confirmed the identification of those enzymes. Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS-917 at the molecular level. This is the first identification of a phosphoramidase other than histidine triad nucleotide-binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs.

  12. Identification of activating enzymes of a novel FBPase inhibitor prodrug, CS-917

    PubMed Central

    Kubota, Kazuishi; Inaba, Shin-ichi; Nakano, Rika; Watanabe, Mihoko; Sakurai, Hidetaka; Fukushima, Yumiko; Ichikawa, Kimihisa; Takahashi, Tohru; Izumi, Takashi; Shinagawa, Akira

    2015-01-01

    CS-917 (MB06322) is a selective small compound inhibitor of fructose 1,6-bisphosphatase (FBPase), which is expected to be a novel drug for the treatment of type 2 diabetes by inhibiting gluconeogenesis. CS-917 is a bisamidate prodrug and activation of CS-917 requires a two-step enzyme catalyzed reaction. The first-step enzyme, esterase, catalyzes the conversion of CS-917 into the intermediate form (R-134450) and the second-step enzyme, phosphoramidase, catalyzes the conversion of R-134450 into the active form (R-125338). In this study, we biochemically purified the CS-917 esterase activity in monkey small intestine and liver. We identified cathepsin A (CTSA) and elastase 3B (ELA3B) as CS-917 esterases in the small intestine by mass spectrometry, whereas we found CTSA and carboxylesterase 1 (CES1) in monkey liver. We also purified R-134450 phosphoramidase activity in monkey liver and identified sphingomyelin phosphodiesterase, acid-like 3A (SMPADL3A), as an R-134450 phosphoramidase, which has not been reported to have any enzyme activity. Recombinant human CTSA, ELA3B, and CES1 showed CS-917 esterase activity and recombinant human SMPDL3A showed R-134450 phosphoramidase activity, which confirmed the identification of those enzymes. Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS-917 at the molecular level. This is the first identification of a phosphoramidase other than histidine triad nucleotide-binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs. PMID:26171222

  13. Chirally Pure Prodrugs and Their Converting Enzymes Lead to High Supersaturation and Rapid Transcellular Permeation of Benzodiazepines.

    PubMed

    Kapoor, Mamta; Cheryala, Narsihmulu; Rautiola, Davin; Georg, Gunda I; Cloyd, James C; Siegel, Ronald A

    2016-08-01

    Water-soluble prodrugs can be rapidly converted by enzymes to hydrophobic drugs, whose aqueous thermodynamic solubilities are low, but are maintained in aqueous solution at supersaturated concentrations due to slow precipitation kinetics. Recently, we investigated avizafone (AVF) in combination with Aspergillus oryzae protease as a prodrug/enzyme system intended to produce supersaturated diazepam (DZP). Several fold enhancement of permeation of supersaturated DZP across Madin-Darby canine kidney II-wild type (MDCKII-wt) monolayers was observed, compared to saturated DZP solutions. However, prodrug conversion was incomplete, putatively due to partial racemization of AVF and stereoselectivity of A oryzae protease. Here we report synthesis of chirally pure AVF, and demonstrate complete conversion to supersaturated DZP followed by complete DZP permeation at enhanced rates across MDCKII-wt cell monolayers. We also synthesized, for the first time, a chirally pure prodrug of midazolam (MDZ-pro) and carried out the same sequence of studies. A oryzae protease was identified as a benign and efficient activating enzyme for MDZ-pro. The MDZ-pro/A oryzae protease system showed greater than 25-fold increase in absorption rate of MDZ across MDCKII-wt monolayers, compared to saturated MDZ. Such chirally pure prodrug/enzyme systems are promising candidates for efficient intranasal delivery of benzodiazepine drugs used in the treatment of seizure emergencies. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  14. Legubicin a Tumor-activated Prodrug for Breast Cancer Therapy

    DTIC Science & Technology

    2008-04-01

    labels. For example, when cytotoxic drugs are part of the prodrug, a hydrophilic group is preferably used for R1 to limit cell uptake by non-target... myeloma , non-small cell lung cancer, retinoblastoma, or tumors in the ovaries. The invention also provides a method for inhibiting cancer metastasis and...

  15. A DT-diaphorase responsive theranostic prodrug for diagnosis, drug release monitoring and therapy.

    PubMed

    Liu, Peilian; Xu, Jiangsheng; Yan, Donghang; Zhang, Peisheng; Zeng, Fang; Li, Bowen; Wu, Shuizhu

    2015-06-11

    A DT-diaphorase-activatable theranostic prodrug, which contains camptothecin, a self-immolative linker and a trigger group, has been developed for the detection of DT-diaphorase, tracking of drug release and selectively killing cancer cells over-expressed with DT-diaphorase. This strategy may offer a new approach for the development of enzyme-catalyzed theranostic anticancer therapeutics.

  16. Somatostatin receptor-mediated specific delivery of paclitaxel prodrugs for efficient cancer therapy.

    PubMed

    Huo, Meirong; Zhu, Qinnv; Wu, Qu; Yin, Tingjie; Wang, Lei; Yin, Lifang; Zhou, Jianping

    2015-06-01

    In this study, a novel PTX prodrug, octreotide(Phe)-polyethene glycol-paclitaxel [OCT(Phe)-PEG-PTX], was successfully synthesized and used for targeted cancer therapy. A nontargeting conjugate, mPEG-PTX, was also synthesized and used as a control. Chemical structures of OCT(Phe)-PEG-PTX and mPEG-PTX were confirmed using (1) H nuclear magnetic resonance and circular dichroism. The drug contents in both the conjugates were 12.0% and 14.0%, respectively. Compared with the parent drug (PTX), OCT(Phe)-PEG-PTX, and mPEG-PTX prodrugs showed a 20,000- and 30,000-fold increase in water solubility, respectively. PTX release from mPEG-PTX and OCT(Phe)-PEG-PTX exhibited a pH-dependent profile. Moreover, compared with mPEG-PTX, OCT(Phe)-PEG-PTX exhibited significantly stronger cytotoxicity against NCI-H446 cells (SSTR overexpression) but comparable cytotoxicity against WI-38 cells (no SSTR expression). Results of confocal laser scanning microscopy revealed that the targeting prodrug labeled with fluorescence probe was selectively taken into tumor cells via SSTR-mediated endocytosis. In vivo investigation of prodrugs in nude mice bearing NCI-H446 cancer xenografts confirmed that OCT(Phe)-PEG-PTX prodrug exhibited stronger antitumor efficacy and lower systemic toxicity than mPEG-PTX and commercial Taxol. These results suggested that OCT(Phe)-PEG-PTX is a promising anticancer drug delivery system for targeted cancer therapy.

  17. Acetal-linked paclitaxel prodrug micellar nanoparticles as a versatile and potent platform for cancer therapy.

    PubMed

    Gu, Yudan; Zhong, Yinan; Meng, Fenghua; Cheng, Ru; Deng, Chao; Zhong, Zhiyuan

    2013-08-12

    Endosomal pH-activatable paclitaxel (PTX) prodrug micellar nanoparticles were designed and prepared by conjugating PTX onto water-soluble poly(ethylene glycol)-b-poly(acrylic acid) (PEG-PAA) block copolymers via an acid-labile acetal bond to the PAA block and investigated for potent growth inhibition of human cancer cells in vitro. PTX was readily conjugated to PEG-PAA with high drug contents of 21.6, 27.0, and 42.8 wt % (denoted as PTX prodrugs 1, 2, and 3, respectively) using ethyl glycol vinyl ether (EGVE) as a linker. The resulting PTX conjugates had defined molecular weights and self-assembled in phosphate buffer (PB, pH 7.4, 10 mM) into monodisperse micellar nanoparticles with average sizes of 158.3-180.3 nm depending on PTX contents. The in vitro release studies showed that drug release from PTX prodrug nanoparticles was highly pH-dependent, in which ca. 86.9%, 66.4% and 29.0% of PTX was released from PTX prodrug 3 at 37 °C in 48 h at pH 5.0, 6.0, and pH 7.4, respectively. MTT assays showed that these pH-sensitive PTX prodrug nanoparticles exhibited high antitumor effect to KB and HeLa cells (IC(50) = 0.18 and 0.9 μg PTX equiv/mL, respectively) as well as PTX-resistant A549 cells. Notably, folate-decorated PTX prodrug micellar nanoparticles based on PTX prodrug 3 and 20 wt % folate-poly(ethylene glycol)-b-poly(D,L-lactide) (FA-PEG-PLA) displayed apparent targetability to folate receptor-overexpressing KB cells with IC(50) over 12 times lower than nontargeting PTX prodrug 3 under otherwise the same conditions. Furthermore, PTX prodrug nanoparticles could also load doxorubicin (DOX) to simultaneously release PTX and DOX under mildly acidic pH. These acetal-linked PTX prodrug micellar nanoparticles have appeared as a highly versatile and potent platform for cancer therapy.

  18. Glutathione- and pH-responsive nonporous silica prodrug nanoparticles for controlled release and cancer therapy

    NASA Astrophysics Data System (ADS)

    Xu, Zhigang; Liu, Shiying; Kang, Yuejun; Wang, Mingfeng

    2015-03-01

    A myriad of drug delivery systems such as liposomes, micelles, polymers and inorganic nanoparticles (NPs) have been developed for cancer therapy. Very few of them, however, have the ability to integrate multiple functionalities such as specific delivery, high circulation stability, controllable release and good biocompatibility and biodegradability in a single system to improve the therapeutic efficacy. Herein, we report two types of stimuli-responsive nonporous silica prodrug NPs towards this goal for controlled release of anticancer drugs and efficient combinatorial cancer therapy. As a proof of concept, anticancer drugs camptothecin (CPT) and doxorubicin (DOX) were covalently encapsulated into silica matrices through glutathione (GSH)-responsive disulfide and pH-responsive hydrazone bonds, respectively, resulting in NPs with sizes tunable in the range of 50-200 nm. Both silica prodrug NPs showed stimuli-responsive controlled release upon exposure to a GSH-rich or acidic environment, resulting in improved anticancer efficacy. Notably, two prodrug NPs simultaneously taken up by HeLa cells showed a remarkable combinatorial efficacy compared to free drug pairs. These results suggest that the stimuli-responsive silica prodrug NPs are promising anticancer drug carriers for efficient cancer therapy.A myriad of drug delivery systems such as liposomes, micelles, polymers and inorganic nanoparticles (NPs) have been developed for cancer therapy. Very few of them, however, have the ability to integrate multiple functionalities such as specific delivery, high circulation stability, controllable release and good biocompatibility and biodegradability in a single system to improve the therapeutic efficacy. Herein, we report two types of stimuli-responsive nonporous silica prodrug NPs towards this goal for controlled release of anticancer drugs and efficient combinatorial cancer therapy. As a proof of concept, anticancer drugs camptothecin (CPT) and doxorubicin (DOX) were

  19. Combination therapy with bioengineered miR-34a prodrug and doxorubicin synergistically suppresses osteosarcoma growth

    PubMed Central

    Zhao, Yong; Tu, Mei-Juan; Yu, Yi-Feng; Wang, Wei-Peng; Chen, Qiu-Xia; Qiu, Jing-Xin; Yu, Ai-Xi; Yu, Ai-Ming

    2016-01-01

    Osteosarcoma (OS) is the most common form of primary malignant bone tumor and prevalent among children and young adults. Recently we have established a novel approach to bioengineering large quantity of microRNA-34a (miR-34a) prodrug for miRNA replacement therapy. This study is to evaluate combination treatment with miR-34a prodrug and doxorubicin, which may synergistically suppress human OS cell growth via RNA interference and DNA intercalation. Synergistic effects were indeed obvious between miR-34a prodrug and doxorubicin for the suppression of OS cell proliferation, as defined by Chou-Talalay method. The strongest antiproliferative synergism was achieved when both agents were administered simultaneously to the cells at early stage, which was associated with much greater degrees of late apoptosis, necrosis, and G2 cell cycle arrest. Alteration of OS cellular processes and invasion capacity was linked to the reduction of protein levels of miR-34a targeted (proto-)oncogenes including SIRT1, c-MET, and CDK6. Moreover, orthotopic OS xenograft tumor growth was repressed to a significantly greater degree in mouse models when miR-34a prodrug and doxorubicin were co-administered intravenously. In addition, multiple doses of miR-34a prodrug and doxorubicin had no or minimal effects on mouse blood chemistry profiles. The results demonstrate that combination of doxorubicin chemotherapy and miR-34a replacement therapy produces synergistic antiproliferative effects and it is more effective than monotherapy in suppressing OS xenograft tumor growth. These findings support the development of mechanism-based combination therapy to combat OS and bioengineered miR-34a prodrug represents a new natural miRNA agent. PMID:26518752

  20. Combination therapy with bioengineered miR-34a prodrug and doxorubicin synergistically suppresses osteosarcoma growth.

    PubMed

    Zhao, Yong; Tu, Mei-Juan; Yu, Yi-Feng; Wang, Wei-Peng; Chen, Qiu-Xia; Qiu, Jing-Xin; Yu, Ai-Xi; Yu, Ai-Ming

    2015-12-15

    Osteosarcoma (OS) is the most common form of primary malignant bone tumor and prevalent among children and young adults. Recently we have established a novel approach to bioengineering large quantity of microRNA-34a (miR-34a) prodrug for miRNA replacement therapy. This study is to evaluate combination treatment with miR-34a prodrug and doxorubicin, which may synergistically suppress human OS cell growth via RNA interference and DNA intercalation. Synergistic effects were indeed obvious between miR-34a prodrug and doxorubicin for the suppression of OS cell proliferation, as defined by Chou-Talalay method. The strongest antiproliferative synergism was achieved when both agents were administered simultaneously to the cells at early stage, which was associated with much greater degrees of late apoptosis, necrosis, and G2 cell cycle arrest. Alteration of OS cellular processes and invasion capacity was linked to the reduction of protein levels of miR-34a targeted (proto-)oncogenes including SIRT1, c-MET, and CDK6. Moreover, orthotopic OS xenograft tumor growth was repressed to a significantly greater degree in mouse models when miR-34a prodrug and doxorubicin were co-administered intravenously. In addition, multiple doses of miR-34a prodrug and doxorubicin had no or minimal effects on mouse blood chemistry profiles. The results demonstrate that combination of doxorubicin chemotherapy and miR-34a replacement therapy produces synergistic antiproliferative effects and it is more effective than monotherapy in suppressing OS xenograft tumor growth. These findings support the development of mechanism-based combination therapy to combat OS and bioengineered miR-34a prodrug represents a new natural miRNA agent. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Simultaneous modulation of transport and metabolism of acyclovir prodrugs across rabbit cornea: An approach involving enzyme inhibitors.

    PubMed

    Katragadda, Suresh; Talluri, Ravi S; Mitra, Ashim K

    2006-08-31

    The aim of this study is to identify the class of enzymes responsible for the hydrolysis of amino acid and dipeptide prodrugs of acyclovir (ACV) and to modulate transport and metabolism of amino acid and dipeptide prodrugs of acyclovir by enzyme inhibitors across rabbit cornea. l-Valine ester of acyclovir, valacyclovir (VACV) and l-glycine-valine ester of acyclovir, gly-val-acyclovir (GVACV) were used as model compounds. Hydrolysis studies of VACV and GVACV in corneal homogenate were conducted in presence of various enzyme inhibitors. IC(50) values were determined for the enzyme inhibitors. Transport studies were conducted with isolated rabbit corneas at 34 degrees C. Complete inhibition of VACV hydrolysis was observed in the presence of Pefabloc SC (4-(2-aminoethyl)-benzenesulfonyl-fluoride) and PCMB (p-chloromercuribenzoic acid). Similar trend was also observed with GVACV in the presence of bestatin. IC(50) values of PCMB and bestatin for VACV and GVACV were found to be 3.81+/-0.94 and 0.34+/-0.08muM respectively. Eserine, tetraethyl pyrophosphate (TEPP) and diisopropyl fluorophosphate (DFP) also produced significant inhibition of VACV hydrolysis. Transport of VACV and GVACV across cornea showed decreased metabolic rate and modulation of transport in presence of PCMB and bestain respectively. The principle enzyme classes responsible for the hydrolysis of VACV and GVACV were carboxylesterases and aminopeptidases respectively. Enzyme inhibitors modulated the transport and metabolism of prodrugs simultaneously even though their affinity towards prodrugs was distinct. In conclusion, utility of enzyme inhibitors to modulate transport and metabolism of prodrugs appears to be promising strategy for enhancing drug transport across cornea.

  2. Legubicin, a Tumor-Activated Prodrug for Breast Cancer Therapy

    DTIC Science & Technology

    2007-04-01

    Thr-Asn-Leu, Ala - Ala - Asn-Leu (SEQ ID NO: 5 ), Ala -Thr-Asn-Leu (SEQ ID NO:6), and Boc- Ala - Ala -Asn-Leu (SEQ ID NO:4). Examples of prodrugs provided by...O N AEPI-4 AEPI- 5 is N-acetyl- Ala - Ala -AzaAsn-(S,S)-EPCOOEt, a compound of the structure: Annual Report...Cheng Liu, M.D., Ph.D. 31 H N H N O O HN O N H2N O O O O O O AEPI- 5 AEPI-6 is N-succinyl- Ala - Ala -AzaAsn-(S,S)-EPCOOEt, a compound of the

  3. Targeted prodrug approaches for hormone refractory prostate cancer.

    PubMed

    Aloysius, Herve; Hu, Longqin

    2015-05-01

    Due to the propensity of relapse and resistance with prolonged androgen deprivation therapy (ADT), there is a growing interest in developing non-hormonal therapeutic approaches as alternative treatment modalities for hormone refractory prostate cancer (HRPC). Although the standard treatment for HRPC consists of a combination of ADT with taxanes and anthracyclines, the clinical use of chemotherapeutics is limited by systemic toxicity stemming from nondiscriminatory drug exposure to normal tissues. In order to improve the tumor selectivity of chemotherapeutics, various targeted prodrug approaches have been explored. Antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT) strategies leverage tumor-specific antigens and transcription factors for the specific delivery of cytotoxic anticancer agents using various prodrug-activating enzymes. In prostate cancer, overexpression of tumor-specific proteases such as prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) is being exploited for selective activation of anticancer prodrugs designed to be activated through proteolysis by these prostate cancer-specific enzymes. PSMA- and PSA-activated prodrugs typically comprise an engineered high-specificity protease peptide substrate coupled to a potent cytotoxic agent via a linker for rapid release of cytotoxic species in the vicinity of prostate cancer cells following proteolytic cleavage. Over the past two decades, various such prodrugs have been developed and they were effective at inhibiting prostate tumor growth in rodent models; several of these prodrug approaches have been advanced to clinical trials and may be developed into effective therapies for HRPC.

  4. Utilization of peptide carrier system to improve intestinal absorption: targeting prolidase as a prodrug-converting enzyme

    NASA Technical Reports Server (NTRS)

    Bai, J. P.; Hu, M.; Subramanian, P.; Mosberg, H. I.; Amidon, G. L.

    1992-01-01

    The feasibility of targeting prolidase as a peptide prodrug-converting enzyme has been examined. The enzymatic hydrolysis by prolidase of substrates for the peptide transporter L-alpha-methyldopa-pro and several dipeptide analogues without an N-terminal alpha-amino group (phenylpropionylproline, phenylacetylproline, N-benzoylproline, and N-acetylproline) was investigated. The Michaelis-Menten parameters Km and Vmax for L-alpha-methyldopa-pro are 0.09 +/- 0.02 mM and 3.98 +/- 0.25 mumol/min/mg protein, respectively. However, no hydrolysis of the dipeptide analogues without an N-terminal alpha-amino group is observed, suggesting that an N-terminal alpha-amino group is required for prolidase activity. These results demonstrate that prolidase may serve as a prodrug-converting enzyme for the dipeptide-type prodrugs, utilizing the peptide carrier for transport of prodrugs into the mucosal cells and prolidase, a cytosolic enzyme, to release the drug. However, a free alpha-amino group appears to be necessary for prolidase hydrolysis.

  5. Insight into Prodrugs of Quinolones and Fluoroquinolones.

    PubMed

    Sharma, Prabodh Chander; Piplani, Mona; Mittal, Monika; Pahwa, Rakesh

    2016-01-01

    Quinolones and fluoroquinolones are principal weapons against variety of bacterial infections and exert their antibacterial potential by interfering the activities of bacterial enzymes. As these agents are associated with some limitations, an important approach to overcome these major constraints is to prepare covalent derivatives, i.e. prodrugs. Prodrug design has been employed to improve the limitations of these drugs such as less aqueous solubility, poor absorption and distribution, toxicity, disagreeable taste, poor lipophilicity etc and for improving their pharmacological profile. This paper highlights the utility of various prodrug strategies in optimizing the therapeutic index of these antibacterial agents and their recent patents. Some of their prodrugs being utilized at preclinical and clinical levels have also been discussed. Hence, this paper has been prepared to present the significant findings of various research papers that would be helpful in motivating scientific researchers to forward the research in direction of utilization of prodrugs in clinical therapy.

  6. Biocompatible polymeric nanocomplexes as an intracellular stimuli-sensitive prodrug for type-2 diabetes combination therapy.

    PubMed

    Wang, Feng-Zhen; Xie, Zhi-Shen; Xing, Lei; Zhang, Bing-Feng; Zhang, Jia-Liang; Cui, Peng-Fei; Qiao, Jian-Bin; Shi, Kun; Cho, Chong-Su; Cho, Myung-Haing; Xu, Xiaojun; Li, Ping; Jiang, Hu-Lin

    2015-12-01

    Combination therapy is usually considered as a promising strategy owing to its advantages such as reduced doses, minimized side effects and improved therapeutic efficiency in a variety of diseases including diabetes. Here we synthesized a new highly intracellular stimuli-sensitive chitosan-graft-metformin (CS-MET) prodrug by imine reaction between oxidative chitosan and metformin for type 2 diabetes (T2D) therapy. Hypothetically, CS-MET functions dually as an anti-diabetes prodrug as well as a gene delivery vector without superfluous materials. CS-MET formed nanocomplexes with therapeutic gene through electrostatic interactions and entered cells by Organic Cation Transporter (OCT)-independent endocytosis. The incorporation of metformin into chitosan has been found to increase endosomal escape via the proton sponge effect. When vector carrying a short-hairpin RNA (shRNA) silencing sterol regulatory element-binding protein (SREBP), a major transcription factor involved in de novo lipogenisis, it reduced the SREBP mRNA and proteins efficiently. Furthermore, by intraperitoneal injection, CS-MET/shSREBP nanocomplexes effectively knocked down SREBP in livers of western-type diet (WD)-induced obese C57BL/6J mice, markedly reversed insulin resistance and alleviated the fatty liver phenotype without obvious toxic effects. Thus we were able to show that the intracellular stimuli-sensitive CS-MET prodrug renders a potential platform to increase the anti-diabetes activity with synergistic enhancement of gene therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Complete regression of glioblastoma by mesenchymal stem cells mediated prodrug gene therapy simulating clinical therapeutic scenario.

    PubMed

    Altaner, Cestmir; Altanerova, Veronika; Cihova, Marina; Ondicova, Katarina; Rychly, Boris; Baciak, Ladislav; Mravec, Boris

    2014-03-15

    Suicide gene therapy mediated by mesenchymal stem cells with their ability to engraft into tumors makes these therapeutic stem cells an attractive tool to activate prodrugs directly within the tumor mass. In this study, we evaluated the therapeutic efficacy of human mesenchymal stem cells derived from bone marrow and from adipose tissue, engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase to treat intracerebral rat C6 glioblastoma in a simulated clinical therapeutic scenario. Intracerebrally grown glioblastoma was treated by resection and subsequently with single or repeated intracerebral inoculations of therapeutic stem cells followed by a continuous intracerebroventricular delivery of 5-fluorocytosine using an osmotic pump. Kaplan-Meier survival curves revealed that surgical resection of the tumor increased the survival time of the resected animals depending on the extent of surgical intervention. However, direct injections of therapeutic stem cells into the brain tissue surrounding the postoperative resection cavity led to a curative outcome in a significant number of treated animals. Moreover, the continuous supply of therapeutic stem cells into the brain with growing glioblastoma by osmotic pumps together with continuous prodrug delivery also proved to be therapeutically efficient. We assume that observed curative therapy of glioblastoma by stem cell-mediated prodrug gene therapy might be caused by the destruction of both tumor cells and the niche where glioblastoma initiating cells reside. © 2013 UICC.

  8. Tumor-targeted gene therapy using Adv-AFP-HRPC/IAA prodrug system suppresses growth of hepatoma xenografted in mice.

    PubMed

    Dai, M; Liu, J; Chen, D-E; Rao, Y; Tang, Z-J; Ho, W-Z; Dong, C-Y

    2012-02-01

    Clinical efficacy of current therapies for hepatocellular carcinoma (HCC) treatment is limited. Indole-3-acetic acid (IAA) is non-toxic for mammalian cells. Oxidative decarboxylation of IAA by horseradish peroxidase (HRP) leads to toxic effects of IAA. The purpose of this study was to investigate the effects of a novel gene-targeted enzyme prodrug therapy with IAA on hepatoma growth in vitro and in vivo mouse hepatoma models. We generated a plasmid using adenovirus to express HRP isoenzyme C (HRPC) with the HCC marker, alpha-fetoprotein (AFP), as the promoter (pAdv-AFP-HRPC). Hepatocellular cells were infected with pAdv-AFP-HRPC and treated with IAA. Cell death was detected using MTT assay. Hepatoma xenografts were developed in mice by injection of mouse hepatoma cells. The size and weight of tumors and organs were evaluated. Cell death in tumors was assessed using hematoxylin and eosin-stained tissue sections. HRPC expression in tissues was detected using Reverse Transcriptase-Polymerase Chain Reaction. IAA stimulated death of hepatocellular cells infected with pAdv-AFP-HRPC, in a dose- and time-dependent manner, but not in control cells. Growth of hepatoma xenografts, including the size and weight, was inhibited in mice treated with pAdv-AFP-HRPC and IAA, compared with that in control group. pAdv-AFP-HRPC/IAA treatment induced cell death in hepatoma xenografts in mice. HRPC gene expressed only in hepatoma, but not in other normal organs of mice. pAdv-AFP-HRPC/IAA treatment did not cause any side effects on normal organs. These findings suggest that pAdv-AFP-HRPC/IAA enzyme/prodrug system may serve as a strategy for HCC therapy.

  9. Platinum(IV) prodrug conjugated Pd@Au nanoplates for chemotherapy and photothermal therapy.

    PubMed

    Shi, Saige; Chen, Xiaolan; Wei, Jingping; Huang, Yizhuan; Weng, Jian; Zheng, Nanfeng

    2016-03-14

    Owing to the excellent near infrared (NIR) light absorption and efficient passive targeting toward tumor tissue, two-dimensional (2D) core-shell PEGylated Pd@Au nanoplates have great potential in both photothermal therapy and drug delivery systems. In this work, we successfully conjugate Pd@Au nanoplates with a platinum(IV) prodrug c,c,t-[Pt(NH3)2Cl2(O2CCH2CH2CO2H)2] to obtain a nanocomposite (Pd@Au-PEG-Pt) for combined photothermal-chemotherapy. The prepared Pd@Au-PEG-Pt nanocomposite showed excellent stability in physiological solutions and efficient Pt(IV) prodrug loading. Once injected into biological tissue, the Pt(IV) prodrug was easily reduced by physiological reductants (e.g. ascorbic acid or glutathione) into its cytotoxic and hydrophilic Pt(II) form and released from the original nanocomposite, and the NIR laser irradiation could accelerate the release of Pt(II) species. More importantly, Pd@Au-PEG-Pt has high tumor accumulation (29%ID per g), which makes excellent therapeutic efficiency at relatively low power density possible. The in vivo results suggested that, compared with single therapy the combined thermo-chemotherapy treatment with Pd@Au-PEG-Pt resulted in complete destruction of the tumor tissue without recurrence, while chemotherapy using Pd@Au-PEG-Pt without irradiation or photothermal treatment using Pd@Au-PEG alone did not. Our work highlights the prospects of a feasible drug delivery strategy of the Pt prodrug by using 2D Pd@Au nanoplates as drug delivery carriers for multimode cancer treatment.

  10. Platinum(iv) prodrug conjugated Pd@Au nanoplates for chemotherapy and photothermal therapy

    NASA Astrophysics Data System (ADS)

    Shi, Saige; Chen, Xiaolan; Wei, Jingping; Huang, Yizhuan; Weng, Jian; Zheng, Nanfeng

    2016-03-01

    Owing to the excellent near infrared (NIR) light absorption and efficient passive targeting toward tumor tissue, two-dimensional (2D) core-shell PEGylated Pd@Au nanoplates have great potential in both photothermal therapy and drug delivery systems. In this work, we successfully conjugate Pd@Au nanoplates with a platinum(iv) prodrug c,c,t-[Pt(NH3)2Cl2(O2CCH2CH2CO2H)2] to obtain a nanocomposite (Pd@Au-PEG-Pt) for combined photothermal-chemotherapy. The prepared Pd@Au-PEG-Pt nanocomposite showed excellent stability in physiological solutions and efficient Pt(iv) prodrug loading. Once injected into biological tissue, the Pt(iv) prodrug was easily reduced by physiological reductants (e.g. ascorbic acid or glutathione) into its cytotoxic and hydrophilic Pt(ii) form and released from the original nanocomposite, and the NIR laser irradiation could accelerate the release of Pt(ii) species. More importantly, Pd@Au-PEG-Pt has high tumor accumulation (29%ID per g), which makes excellent therapeutic efficiency at relatively low power density possible. The in vivo results suggested that, compared with single therapy the combined thermo-chemotherapy treatment with Pd@Au-PEG-Pt resulted in complete destruction of the tumor tissue without recurrence, while chemotherapy using Pd@Au-PEG-Pt without irradiation or photothermal treatment using Pd@Au-PEG alone did not. Our work highlights the prospects of a feasible drug delivery strategy of the Pt prodrug by using 2D Pd@Au nanoplates as drug delivery carriers for multimode cancer treatment.Owing to the excellent near infrared (NIR) light absorption and efficient passive targeting toward tumor tissue, two-dimensional (2D) core-shell PEGylated Pd@Au nanoplates have great potential in both photothermal therapy and drug delivery systems. In this work, we successfully conjugate Pd@Au nanoplates with a platinum(iv) prodrug c,c,t-[Pt(NH3)2Cl2(O2CCH2CH2CO2H)2] to obtain a nanocomposite (Pd@Au-PEG-Pt) for combined photothermal-chemotherapy. The

  11. Pharmacokinetic analysis of the microscopic distribution of enzyme-conjugated antibodies and prodrugs: comparison with experimental data.

    PubMed Central

    Baxter, L. T.; Jain, R. K.

    1996-01-01

    A mathematical model was developed to improve understanding of the biodistribution and microscopic profiles of drugs and prodrugs in a system using enzyme-conjugated antibodies as part of a two-step method for cancer treatment. The use of monoclonal antibodies alone may lead to heterogeneous uptake within the tumour tissue; the use of a second, low molecular weight agent may provide greater penetration into tumour tissue. This mathematical model was used to describe concentration profiles surrounding individual blood vessels within a tumour. From these profiles the area under the curve and specificity ratios were determined. By integrating these results spatially, average tissue concentrations were determined and compared with experimental results from three different systems in the literature; two using murine antibodies and one using humanised fusion proteins. The maximum enzyme conversion rate (Vmax) and the residual antibody concentration in the plasma and normal tissue were seen to be key determinants of drug concentration and drug-prodrug ratios in the tumour and other organs. Thus, longer time delays between the two injections, clearing the antibody from the blood stream and the use of 'weaker' enzymes (lower Vmax) will be important factors in improving this prodrug approach. Of these, the model found the effective clearance of the antibody outside of the tumour to be the most effective. The use of enzyme-conjugated antibodies may offer the following advantages over the bifunctional antibody-hapten system: (i) more uniform distribution of the active agent; (ii) higher concentrations possible for the active agent; and (iii) greater specificity (therapeutic index). PMID:8595158

  12. Effect of alginate microencapsulation on the catalytic efficiency and in vitro enzyme-prodrug therapeutic efficacy of cytosine deaminase and of recombinant E. coli expressing cytosine deaminase.

    PubMed

    Funaro, Michael G; Nemani, Krishnamurthy V; Chen, Zhihang; Bhujwalla, Zaver M; Griswold, Karl E; Gimi, Barjor

    2016-02-01

    Cytosine deaminase (CD) catalyses the enzymatic conversion of the non-toxic prodrug 5-fluorocytosine (5-FC) to the potent chemotherapeutic form, 5-fluorouracil (5-FU). Intratumoral delivery of CD localises chemotherapy dose while reducing systemic toxicity. Encapsulation in biocompatible microcapsules immunoisolates CD and protects it from degradation. We report on the effect of alginate encapsulation on the catalytic and functional activity of isolated CD and recombinant E. coli engineered to express CD (E. coli(CD)). Alginate microcapsules containing either CD or Escherichia coli(CD) were prepared using ionotropic gelation. Conversion of 5-FC to 5-FU was quantitated in unencapsulated and encapsulated CD/E. coli(CD) using spectrophotometry, with a slower rate of conversion observed following encapsulation. Both encapsulated CD/5-FC and E. coli(CD)/5-FC resulted in cell kill and reduced proliferation of 9 L rat glioma cells, which was comparable to direct 5-FU treatment. Our results show that encapsulation preserves the therapeutic potential of CD and E. coli(CD) is equally effective for enzyme-prodrug therapy.

  13. Esterase-activatable β-lapachone prodrug micelles for NQO1-targeted lung cancer therapy

    PubMed Central

    Ma, Xinpeng; Huang, Xiumei; Moore, Zachary; Huang, Gang; Kilgore, Jessica A.; Wang, Yiguang; Hammer, Suntrea; Williams, Noelle S.; Boothman, David A.; Gao, Jinming

    2016-01-01

    Lung cancer is one of the most lethal forms of cancer and current chemotherapeutic strategies lack broad specificity and efficacy. Recently, β-lapachone (β-lap) was shown to be highly efficacious in killing non-small cell lung cancer (NSCLC) cells regardless of their p53, cell cycle and caspase status. Pre-clinical and clinical use of β-lap (clinical form, ARQ501 or 761) is hampered by poor pharmacokinetics and toxicity due to hemolytic anemia. Here, we report the development and preclinical evaluation of β-lap prodrug nanotherapeutics consisting of diester derivatives of β-lap encapsulated in biocompatible and biodegradable poly(ethylene glycol)-b-poly(d,l-lactic acid) (PEG-b-PLA) micelles. Compared to the parent drug, diester derivatives of β-lap showed higher drug loading densities inside PEG-b-PLA micelles. After esterase treatment, micelle-delivered β-lap-dC3 and -dC6 prodrugs were converted to β-lap. Cytotoxicity assays using A549 and H596 lung cancer cells showed that both micelle formulations maintained NAD(P)H:quinone oxidoreductase 1 (NQO1)-dependent cytotoxicity. However, antitumor efficacy study of β-lap-dC3 micelles against orthotopic A549 NSCLC xenograft-bearing mice showed significantly greater long-term survival over β-lap-dC6 micelles or β-lap-HPβCD complexes. Improved therapeutic efficacy of β-lap-dC3 micelles correlated with higher area under the concentration-time curves of β-lap in tumors, and enhanced pharmacodynamic endpoints (e.g., PARP1 hyperactivation, γH2AX, and ATP depletion). β-Lap-dC3 prodrug micelles provide a promising strategy for NQO1-targeted therapy of lung cancer with improved safety and antitumor efficacy. PMID:25542645

  14. Noninvasive theranostic imaging of HSV1-sr39TK-NTR/GCV-CB1954 dual-prodrug therapy in metastatic lung lesions of MDA-MB-231 triple negative breast cancer in mice.

    PubMed

    Sekar, Thillai V; Foygel, Kira; Ilovich, Ohad; Paulmurugan, Ramasamy

    2014-01-01

    Metastatic breast cancer is an obdurate cancer type that is not amenable to chemotherapy regimens currently used in clinic. There is a desperate need for alternative therapies to treat this resistant cancer type. Gene-Directed Enzyme Prodrug Therapy (GDEPT) is a superior gene therapy method when compared to chemotherapy and radiotherapy procedures, proven to be effective against many types of cancer in pre-clinical evaluations and clinical trials. Gene therapy that utilizes a single enzyme/prodrug combination targeting a single cellular mechanism needs significant overexpression of delivered therapeutic gene in order to achieve therapy response. Hence, to overcome this obstacle we recently developed a dual therapeutic reporter gene fusion that uses two different prodrugs, targeting two distinct cellular mechanisms in order to achieve effective therapy with a limited expression of delivered transgenes. In addition, imaging therapeutic reporter genes offers additional information that indirectly correlates gene delivery, expression, and functional effectiveness as a theranostic approach. In the present study, we evaluate the therapeutic potential of HSV1-sr39TK-NTR fusion dual suicide gene therapy system that we recently developed, in MDA-MB-231 triple negative breast cancer lung-metastatic lesions in a mouse model. We compared the therapeutic potential of HSV1-sr39TK-NTR fusion with respective dual prodrugs GCV-CB1954 with HSV1-sr39TK/GCV and NTR/CB1954 single enzyme prodrug system in this highly resistant metastatic lesion of the lungs. In vitro optimization of dose and duration of exposure to GCV and CB1954 was performed in MDA-MB-231 cells. Drug combinations of 1 μg/ml GCV and 10 μM CB1954 for 3 days was found to be optimal regimen for induction of significant cell death, as assessed by FACS analysis. In vivo therapeutic evaluation in animal models showed a complete ablation of lung metastatic nodules of MDA-MB-231 triple negative breast cancer cells following

  15. Noninvasive Theranostic Imaging of HSV1-sr39TK-NTR/GCV-CB1954 Dual-Prodrug Therapy in Metastatic Lung Lesions of MDA-MB-231 Triple Negative Breast Cancer in Mice

    PubMed Central

    Sekar, Thillai V.; Foygel, Kira; Ilovich, Ohad; Paulmurugan, Ramasamy

    2014-01-01

    Metastatic breast cancer is an obdurate cancer type that is not amenable to chemotherapy regimens currently used in clinic. There is a desperate need for alternative therapies to treat this resistant cancer type. Gene-Directed Enzyme Prodrug Therapy (GDEPT) is a superior gene therapy method when compared to chemotherapy and radiotherapy procedures, proven to be effective against many types of cancer in pre-clinical evaluations and clinical trials. Gene therapy that utilizes a single enzyme/prodrug combination targeting a single cellular mechanism needs significant overexpression of delivered therapeutic gene in order to achieve therapy response. Hence, to overcome this obstacle we recently developed a dual therapeutic reporter gene fusion that uses two different prodrugs, targeting two distinct cellular mechanisms in order to achieve effective therapy with a limited expression of delivered transgenes. In addition, imaging therapeutic reporter genes offers additional information that indirectly correlates gene delivery, expression, and functional effectiveness as a theranostic approach. In the present study, we evaluate the therapeutic potential of HSV1-sr39TK-NTR fusion dual suicide gene therapy system that we recently developed, in MDA-MB-231 triple negative breast cancer lung-metastatic lesions in a mouse model. We compared the therapeutic potential of HSV1-sr39TK-NTR fusion with respective dual prodrugs GCV-CB1954 with HSV1-sr39TK/GCV and NTR/CB1954 single enzyme prodrug system in this highly resistant metastatic lesion of the lungs. In vitro optimization of dose and duration of exposure to GCV and CB1954 was performed in MDA-MB-231 cells. Drug combinations of 1 μg/ml GCV and 10 μM CB1954 for 3 days was found to be optimal regimen for induction of significant cell death, as assessed by FACS analysis. In vivo therapeutic evaluation in animal models showed a complete ablation of lung metastatic nodules of MDA-MB-231 triple negative breast cancer cells following

  16. Identification of novel nitroreductases from Bacillus cereus and their interaction with the CB1954 prodrug.

    PubMed

    Gwenin, Vanessa V; Poornima, Paramasivan; Halliwell, Jennifer; Ball, Patrick; Robinson, George; Gwenin, Chris D

    2015-12-01

    Directed enzyme prodrug therapy is a form of cancer chemotherapy in which bacterial prodrug-activating enzymes, or their encoding genes, are directed to the tumour before administration of a prodrug. The prodrug can then be activated into a toxic drug at the tumour site, reducing off-target effects. The bacterial nitroreductases are a class of enzymes used in this therapeutic approach and although very promising, the low turnover rate of prodrug by the most studied nitroreductase enzyme, NfnB from Escherichia coli (NfnB_Ec), is a major limit to this technology. There is a continual search for enzymes with greater efficiency, and as part of the search for more efficient bacterial nitroreductase enzymes, two novel enzymes from Bacillus cereus (strain ATCC 14579) have been identified and shown to reduce the CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) prodrug to its respective 2'-and 4'-hydroxylamine products. Both enzymes shared features characteristic of the nitro-FMN-reductase superfamily including non-covalently associated FMN, requirement for the NAD(P)H cofactor, homodimeric, could be inhibited by Dicoumarol (3,3'-methylenebis(4-hydroxy-2H-chromen-2-one)), and displayed ping pong bi bi kinetics. Based on the biochemical characteristics and nucleotide alignment with other nitroreductase enzymes, one enzyme was named YdgI_Bc and the other YfkO_Bc. Both B. cereus enzymes had greater turnover for the CB1954 prodrug compared with NfnB_Ec, and in the presence of added NADPH cofactor, YfkO_Bc had superior cell killing ability, and produced mainly the 4'-hydroxylamine product at low prodrug concentration. The YfkO_Bc was identified as a promising candidate for future enzyme prodrug therapy.

  17. Controllable synthesis of polymerizable ester and amide prodrugs of acyclovir by enzyme in organic solvent.

    PubMed

    Li, Xia; Wu, Qi; Lv, De-shui; Lin, Xian-fu

    2006-05-15

    A facile control of the acylation position at the primary hydroxyl and amino of acyclovir, respectively, was achieved and five polymerizable acyclovir prodrugs were synthesized. Various reaction conditions were studied in detail. Thus, lipase acrylic resin from Candida antarctica (CAL-B) in pyridine or acetone showed high chemo-selectivity toward the primary hydroxyl of acyclovir. However, lipase PS 'Amano' (PS) in DMSO selectively acylated the amino group. The selectivity of PS could be adjusted by changing reaction solvents. The acyclovir vinyl derivatives obtained would be important monomers used for the preparation of macromolecular nucleoside drugs.

  18. "Mixed inhibitor-prodrug" as a new approach toward systemically active inhibitors of enkephalin-degrading enzymes.

    PubMed

    Fournié-Zaluski, M C; Coric, P; Turcaud, S; Lucas, E; Noble, F; Maldonado, R; Roques, B P

    1992-06-26

    In order to evaluate the possible advantages of potentiating the effects of the endogenous enkephalins, to obtain analgesia without the serious drawbacks of morphine, it was essential to design systemically active compounds which inhibit the two metabolizing enzymes, aminopeptidase N (APN) and neutral endopeptidase 24.11 (NEP). A new concept combining the idea of "prodrug" and "mixed inhibitor" was therefore developed. Given the high efficiency of beta-mercaptoalkylamines as APN inhibitors and of N-(mercaptoacyl) amino acids as NEP inhibitors, compounds associating these molecules through disulfide or thioester bonds, which are known to increase lipophilicity and to favor passage across the blood-brain barrier, have been synthesized. An HPLC study indicated that the disulfide bridge was resistant to serum enzymes but was cleaved by brain membrane homogenates, suggesting that the active inhibitors were released in the central nervous system. The validity of the approach was verified by the efficient antinociceptive responses obtained in the hot plate test in mice after iv administration of disulfide-containing inhibitors (ED50s of from 4 to 26 mg/kg on the jump latency time). The analgesic potencies of the "mixed inhibitor-prodrug" RB 101 [H2NCH(CH2CH2SCH3)CH2SSCH2CH(CH2Ph)CONHCH( CH2Ph)COOCH2Ph] after iv administration were three times greater than those of a similar combined dose of its two constitutive moieties. The separation of the two diastereoisomers constituting RB 101 showed that the analgesia has a stereochemical dependence, the (S,S,S)-isomer being more active than the (S,R,S)-isomer. Furthermore, in the tail flick test in the rat, RB 101 gave 38% analgesia at a dose of 80 mg/kg. Due to its high efficiency and its longer pharmacological effect, RB 101 was selected for a complete study of its analgesic properties.

  19. Augmentation of a Novel Enzyme/Pro-Drug Gene Therapy "Distant Bystander Effect" to Target Prostate Cancer Metastasis

    DTIC Science & Technology

    2005-09-01

    enzyme/prodrug therapy(GDEPT)_ mIL-12; mIL-18; cytosine deaminase and uracil phopho-ribosyl transferase (CDUPRT) 16. SECURITY CLASSIFICATION OF: 17...therapy, a gene (a fusion of cytosine deaminase and uracil phosphoribosyltransferase (CD/UPRT)) is delivered to a cancer cell so that harmless bacterial...reduction both at the treatment site and at remote locations. In this therapy, a gene (a fusion of cytosine deaminase and uracil phosphoribosyltransferase

  20. Co-delivery of doxorubicin and curcumin by pH-sensitive prodrug nanoparticle for combination therapy of cancer

    NASA Astrophysics Data System (ADS)

    Zhang, Yumin; Yang, Cuihong; Wang, Weiwei; Liu, Jinjian; Liu, Qiang; Huang, Fan; Chu, Liping; Gao, Honglin; Li, Chen; Kong, Deling; Liu, Qian; Liu, Jianfeng

    2016-02-01

    Ample attention has focused on cancer drug delivery via prodrug nanoparticles due to their high drug loading property and comparatively lower side effects. In this study, we designed a PEG-DOX-Cur prodrug nanoparticle for simultaneous delivery of doxorubicin (DOX) and curcumin (Cur) as a combination therapy to treat cancer. DOX was conjugated to PEG by Schiff’s base reaction. The obtained prodrug conjugate could self-assemble in water at pH 7.4 into nanoparticles (PEG-DOX NPs) and encapsulate Cur into the core through hydrophobic interaction (PEG-DOX-Cur NPs). When the PEG-DOX-Cur NPs are internalized by tumor cells, the Schiff’s base linker between PEG and DOX would break in the acidic environment that is often observed in tumors, causing disassembling of the PEG-DOX-Cur NPs and releasing both DOX and Cur into the nuclei and cytoplasma of the tumor cells, respectively. Compared with free DOX, free Cur, free DOX-Cur combination, or PEG-DOX NPs, PEG-DOX-Cur NPs exhibited higher anti-tumor activity in vitro. In addition, the PEG-DOX-Cur NPs also showed prolonged blood circulation time, elevated local drug accumulation and increased tumor penetration. Enhanced anti-tumor activity was also observed from the PEG-DOX-Cur-treated animals, demonstrating better tumor inhibitory property of the NPs. Thus, the PEG-DOX-Cur prodrug nanoparticle system provides a simple yet efficient approach of drug delivery for chemotherapy.

  1. Co-delivery of doxorubicin and curcumin by pH-sensitive prodrug nanoparticle for combination therapy of cancer

    PubMed Central

    Zhang, Yumin; Yang, Cuihong; Wang, Weiwei; Liu, Jinjian; Liu, Qiang; Huang, Fan; Chu, Liping; Gao, Honglin; Li, Chen; Kong, Deling; Liu, Qian; Liu, Jianfeng

    2016-01-01

    Ample attention has focused on cancer drug delivery via prodrug nanoparticles due to their high drug loading property and comparatively lower side effects. In this study, we designed a PEG-DOX-Cur prodrug nanoparticle for simultaneous delivery of doxorubicin (DOX) and curcumin (Cur) as a combination therapy to treat cancer. DOX was conjugated to PEG by Schiff’s base reaction. The obtained prodrug conjugate could self-assemble in water at pH 7.4 into nanoparticles (PEG-DOX NPs) and encapsulate Cur into the core through hydrophobic interaction (PEG-DOX-Cur NPs). When the PEG-DOX-Cur NPs are internalized by tumor cells, the Schiff’s base linker between PEG and DOX would break in the acidic environment that is often observed in tumors, causing disassembling of the PEG-DOX-Cur NPs and releasing both DOX and Cur into the nuclei and cytoplasma of the tumor cells, respectively. Compared with free DOX, free Cur, free DOX-Cur combination, or PEG-DOX NPs, PEG-DOX-Cur NPs exhibited higher anti-tumor activity in vitro. In addition, the PEG-DOX-Cur NPs also showed prolonged blood circulation time, elevated local drug accumulation and increased tumor penetration. Enhanced anti-tumor activity was also observed from the PEG-DOX-Cur-treated animals, demonstrating better tumor inhibitory property of the NPs. Thus, the PEG-DOX-Cur prodrug nanoparticle system provides a simple yet efficient approach of drug delivery for chemotherapy. PMID:26876480

  2. A self-immolative prodrug nanosystem capable of releasing a drug and a NIR reporter for in vivo imaging and therapy.

    PubMed

    Wang, Ziqian; Wu, Hao; Liu, Peilian; Zeng, Fang; Wu, Shuizhu

    2017-09-01

    In vivo monitoring of the biodistribution and activation of prodrugs is highly attractive, and the self-immolative dendritic architecture is deemed as a promising approach for constructing theranostic prodrug in which the release/activation of different payloads is needed. Herein, A GSH-triggered and self-immolative dendritic platform comprising an anticancer drug camptothecin (CPT), a cleavable linker and a two-photon NIR fluorophore (dicyanomethylene-4H-pyran, DCM) has been developed for in situ tracking of drug release and antitumour therapy. In vitro experiments demonstrate that, the presence of glutathione (GSH) induces the cleavage of the self-immolative linker, resulting in comitant release of the drug and the dye. Upon cell internalization and under one- or two-photon excitation, prominent intracellular fluorescence can be observed, indicating the release of the payloads in live cells. Upon loaded in phospholipid vesicles, the prodrug has also been successfully utilized for in vivo and in situ tracking of drug release and cancer therapy in a mouse model. Several hours post injection, the prodrug generates strong fluorescence on tumour sites, demonstrating the prodrug's capability of monitoring the on-site drug release. Moreover, the prodrug shows considerable high activity and exerts obvious inhibition towards tumour growth. This work suggests that the prodrug with self-immolative dendritic structure can work well in vivo and this strategy may provide an alternative approach for designing theranostic drug delivery systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas.

    PubMed

    Chung, Taemoon; Na, Juri; Kim, Young-Il; Chang, Da-Young; Kim, Young Il; Kim, Hyeonjin; Moon, Ho Eun; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key; Kim, Sung-Soo; Suh-Kim, Haeyoung; Paek, Sun Ha; Youn, Hyewon

    2016-01-01

    We investigated a therapeutic strategy for recurrent malignant gliomas using mesenchymal stem cells (MSC), expressing cytosine deaminase (CD), and prodrug 5-Fluorocytosine (5-FC) as a more specific and less toxic option. MSCs are emerging as a novel cell therapeutic agent with a cancer-targeting property, and CD is considered a promising enzyme in cancer gene therapy which can convert non-toxic 5-FC to toxic 5-Fluorouracil (5-FU). Therefore, use of prodrug 5-FC can minimize normal cell toxicity. Analyses of microarrays revealed that targeting DNA damage and its repair is a selectable option for gliomas after the standard chemo/radio-therapy. 5-FU is the most frequently used anti-cancer drug, which induces DNA breaks. Because dihydropyrimidine dehydrogenase (DPD) was reported to be involved in 5-FU metabolism to block DNA damage, we compared the survival rate with 5-FU treatment and the level of DPD expression in 15 different glioma cell lines. DPD-deficient cells showed higher sensitivity to 5-FU, and the regulation of DPD level by either siRNA or overexpression was directly related to the 5-FU sensitivity. For MSC/CD with 5-FC therapy, DPD-deficient cells such as U87MG, GBM28, and GBM37 showed higher sensitivity compared to DPD-high U373 cells. Effective inhibition of tumor growth was also observed in an orthotopic mouse model using DPD- deficient U87MG, indicating that DPD gene expression is indeed closely related to the efficacy of MSC/CD-mediated 5-FC therapy. Our results suggested that DPD can be used as a biomarker for selecting glioma patients who may possibly benefit from this therapy.

  4. Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas

    PubMed Central

    Chung, Taemoon; Na, Juri; Kim, Young-il; Chang, Da-Young; Kim, Young Il; Kim, Hyeonjin; Moon, Ho Eun; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key; Kim, Sung-Soo; Suh-Kim, Haeyoung; Paek, Sun Ha; Youn, Hyewon

    2016-01-01

    We investigated a therapeutic strategy for recurrent malignant gliomas using mesenchymal stem cells (MSC), expressing cytosine deaminase (CD), and prodrug 5-Fluorocytosine (5-FC) as a more specific and less toxic option. MSCs are emerging as a novel cell therapeutic agent with a cancer-targeting property, and CD is considered a promising enzyme in cancer gene therapy which can convert non-toxic 5-FC to toxic 5-Fluorouracil (5-FU). Therefore, use of prodrug 5-FC can minimize normal cell toxicity. Analyses of microarrays revealed that targeting DNA damage and its repair is a selectable option for gliomas after the standard chemo/radio-therapy. 5-FU is the most frequently used anti-cancer drug, which induces DNA breaks. Because dihydropyrimidine dehydrogenase (DPD) was reported to be involved in 5-FU metabolism to block DNA damage, we compared the survival rate with 5-FU treatment and the level of DPD expression in 15 different glioma cell lines. DPD-deficient cells showed higher sensitivity to 5-FU, and the regulation of DPD level by either siRNA or overexpression was directly related to the 5-FU sensitivity. For MSC/CD with 5-FC therapy, DPD-deficient cells such as U87MG, GBM28, and GBM37 showed higher sensitivity compared to DPD-high U373 cells. Effective inhibition of tumor growth was also observed in an orthotopic mouse model using DPD- deficient U87MG, indicating that DPD gene expression is indeed closely related to the efficacy of MSC/CD-mediated 5-FC therapy. Our results suggested that DPD can be used as a biomarker for selecting glioma patients who may possibly benefit from this therapy. PMID:27446484

  5. Targeted Chemo-Photodynamic Combination Platform Based on the DOX Prodrug Nanoparticles for Enhanced Cancer Therapy.

    PubMed

    Zhang, Yumin; Huang, Fan; Ren, Chunhua; Yang, Lijun; Liu, Jianfeng; Cheng, Zhen; Chu, Liping; Liu, Jinjian

    2017-04-19

    Chemo-photodynamic combination therapy has been received widespread attention in cancer treatment due to its excellent characteristics, such as reducing the adverse side effects of chemo-drugs and improving the therapeutic effects for various cancers. In this study, RGD and DOX was conjugated to PEG by thiol-ene addition and Schiff's base reaction, respectively, to prepare the targeted and pH-sensitive antitumor prodrug nanoparticles (RGD-PEG-DOX NPs, RGD-NPs). Subsequently, the photosensitizer chlorin e6 (Ce6) was encapsulated into RGD-NPs, thus obtaining a simple and efficient chemo-photodynamic combination platform (RGD-PEG-DOX/Ce6 NPs, RGD-NPs/Ce6). This nanoparticle possessed high drug loading property of both the chemo-drug and photosensitizer and could simultaneously release them under the mild acidic microenvironment of cancer cells, which was expected to realize the synchronization therapy of chemotherapy and photodynamic therapy (PDT). Compared with free DOX and Ce6, RGD-NPs/Ce6 could significantly improve the cellular uptake capacities of DOX and Ce6, resulting in the increased contents of ROS in cancer cells and effective cytotoxicity for tumor cells (MDA-MB-231 cells and MCF-7 cells) upon a laser radiation. The in vivo experiment showed that RGD-NPs/Ce6 displayed superior tumor targeting, accumulation, and retention ability than the other groups (free DOX, free Ce6 and NPs/Ce6), and thus significantly enhancing the antitumor effect in vivo with a laser radiation. In addition, the cardiotoxicity induced by DOX was thoroughly wiped out after being loaded and delivered by the nanoparticles according to the pathological analysis. Therefore, the targeted chemo-photodynamic combination therapeutic platform may be a promising candidate for enhanced cancer therapy.

  6. Cisplatin Prodrug-Conjugated Gold Nanocluster for Fluorescence Imaging and Targeted Therapy of the Breast Cancer

    PubMed Central

    Zhou, Fangyuan; Feng, Bing; Yu, Haijun; Wang, Dangge; Wang, Tingting; Liu, Jianping; Meng, Qingshuo; Wang, Siling; Zhang, Pengcheng; Zhang, Zhiwen; Li, Yaping

    2016-01-01

    Theranostic nanomedicine has emerged as a promising modality for cancer diagnosis and treatment. In this study, we report the fabrication of fluorescence gold nanoclusters (GNC) conjugated with a cisplatin prodrug and folic acid (FA) (FA-GNC-Pt) for fluorescence imaging and targeted chemotherapy of breast cancer. The physio-chemical properties of FA-GNC-Pt nanoparticles are thoroughly characterized by fluorescence/UV-Vis spectroscopic measurement, particle size and zeta-potential examination. We find that FA-modification significantly accelerated the cellular uptake and increased the cytotoxicity of GNC-Pt nanoparticles in murine 4T1 breast cancer cells. Fluorescence imaging in vivo using 4T1 tumor bearing nude mouse model shows that FA-GNC-Pt nanoparticles selectively accumulate in the orthotopic 4T1 tumor and generate strong fluorescence signal due to the tumor targeting effect of FA. Moreover, we demonstrate that FA-GNC-Pt nanoparticles significantly inhibit the growth and lung metastasis of the orthotopically implanted 4T1 breast tumors. All these data imply a good potential of the GNC-based theranostic nanoplatform for fluorescence tumor imaging and cancer therapy. PMID:27022415

  7. Cisplatin Prodrug-Conjugated Gold Nanocluster for Fluorescence Imaging and Targeted Therapy of the Breast Cancer.

    PubMed

    Zhou, Fangyuan; Feng, Bing; Yu, Haijun; Wang, Dangge; Wang, Tingting; Liu, Jianping; Meng, Qingshuo; Wang, Siling; Zhang, Pengcheng; Zhang, Zhiwen; Li, Yaping

    2016-01-01

    Theranostic nanomedicine has emerged as a promising modality for cancer diagnosis and treatment. In this study, we report the fabrication of fluorescence gold nanoclusters (GNC) conjugated with a cisplatin prodrug and folic acid (FA) (FA-GNC-Pt) for fluorescence imaging and targeted chemotherapy of breast cancer. The physio-chemical properties of FA-GNC-Pt nanoparticles are thoroughly characterized by fluorescence/UV-Vis spectroscopic measurement, particle size and zeta-potential examination. We find that FA-modification significantly accelerated the cellular uptake and increased the cytotoxicity of GNC-Pt nanoparticles in murine 4T1 breast cancer cells. Fluorescence imaging in vivo using 4T1 tumor bearing nude mouse model shows that FA-GNC-Pt nanoparticles selectively accumulate in the orthotopic 4T1 tumor and generate strong fluorescence signal due to the tumor targeting effect of FA. Moreover, we demonstrate that FA-GNC-Pt nanoparticles significantly inhibit the growth and lung metastasis of the orthotopically implanted 4T1 breast tumors. All these data imply a good potential of the GNC-based theranostic nanoplatform for fluorescence tumor imaging and cancer therapy.

  8. Synthesis, Chemical and Enzymatic Hydrolysis, and Aqueous Solubility of Amino Acid Ester Prodrugs of 3-Carboranyl Thymidine Analogues for Boron Neutron Capture Therapy of Brain Tumors

    PubMed Central

    Hasabelnaby, Sherifa; Goudah, Ayman; Agarwal, Hitesh K.; Abd alla, Mosaad S. M.; Tjarks, Werner

    2012-01-01

    Various water-soluble L-valine-, L-glutamate-, and glycine ester prodrugs of two 3-Carboranyl Thymidine Analogues (3-CTAs), designated N5 and N5-2OH, were synthesized for Boron Neutron Capture Therapy (BNCT) of brain tumors since the water solubilities of the parental compounds proved to be insufficient in preclinical studies. The amino acid ester prodrugs were prepared and stored as hydrochloride salts. The water solubilities of these amino acid ester prodrugs, evaluated in phosphate buffered saline (PBS) at pH 5, pH 6 and pH 7.4, improved 48 to 6600 times compared with parental N5 and N5-2OH. The stability of the amino acid ester prodrugs was evaluated in PBS at pH 7.4, Bovine serum, and Bovine cerebrospinal fluid (CSF). The rate of the hydrolysis in all three incubation media depended primarily on the amino acid promoiety and, to a lesser extend, on the site of esterification at the deoxyribose portion of the 3-CTAs. In general, 3'-amino acid ester prodrugs were less sensitive to chemical and enzymatic hydrolysis than 5'-amino acid ester prodrugs and the stabilities of the latter decreased in the following order: 5'-valine > 5'-glutamate > 5'-glycine. The rate of the hydrolysis of the 5'-amino acid ester prodrugs in Bovine CSF was overall higher than in PBS and somewhat lower than in Bovine serum. Overall, 5'-glutamate ester prodrug of N5 and the 5'-glycine ester prodrugs of N5 and N5-2OH appeared to be the most promising candidates for preclinical BNCT studies. PMID:22889558

  9. Far-Red Light-Activatable Prodrug of Paclitaxel for the Combined Effects of Photodynamic Therapy and Site-Specific Paclitaxel Chemotherapy.

    PubMed

    Thapa, Pritam; Li, Mengjie; Bio, Moses; Rajaputra, Pallavi; Nkepang, Gregory; Sun, Yajing; Woo, Sukyung; You, Youngjae

    2016-04-14

    Paclitaxel (PTX) is one of the most useful chemotherapeutic agents approved for several cancers, including ovarian, breast, pancreatic, and nonsmall cell lung cancer. However, it causes systemic side effects when administered parenterally. Photodynamic therapy (PDT) is a new strategy for treating local cancers using light and photosensitizer. Unfortunately, PDT is often followed by recurrence due to incomplete ablation of tumors. To overcome these problems, we prepared the far-red light-activatable prodrug of PTX by conjugating photosensitizer via singlet oxygen-cleavable aminoacrylate linker. Tubulin polymerization enhancement and cytotoxicity of prodrugs were dramatically reduced. However, once illuminated with far-red light, the prodrug effectively killed SKOV-3 ovarian cancer cells through the combined effects of PDT and locally released PTX. Ours is the first PTX prodrug that can be activated by singlet oxygen using tissue penetrable and clinically useful far-red light, which kills the cancer cells through the combined effects of PDT and site-specific PTX chemotherapy.

  10. Novel water-soluble prodrugs of acyclovir cleavable by the dipeptidyl-peptidase IV (DPP IV/CD26) enzyme.

    PubMed

    Diez-Torrubia, Alberto; Cabrera, Silvia; de Castro, Sonia; García-Aparicio, Carlos; Mulder, Gwenn; De Meester, Ingrid; Camarasa, María-José; Balzarini, Jan; Velázquez, Sonsoles

    2013-01-01

    We herein report for the first time the successful use of the dipeptidyl peptidase IV (DPPIV/CD26) prodrug approach to guanine derivatives such as the antiviral acyclovir (ACV). The solution- and solid-phase synthesis of the tetrapeptide amide prodrug 3 and the tripeptide ester conjugate 4 of acyclovir are reported. The synthesis of the demanding tetrapeptide amide prodrug of ACV 3 was first established in solution and successfully transferred onto solid support by using Ellman's dihydropyran (DHP) resin. In contrast with the valyl ester prodrug (valacyclovir, VACV), the tetrapeptide amide prodrug 3 and the tripeptide ester conjugate 4 of ACV proved fully stable in PBS. Both prodrugs converted to VACV (for 4) or ACV (for 3) upon exposure to purified DPPIV/CD26 or human or bovine serum. Vildagliptin, a potent inhibitor of DPPIV/CD26 efficiently inhibited the DPPIV/CD26-catalysed hydrolysis reaction. Both amide and ester prodrugs of ACV showed pronounced anti-herpetic activity in cell culture and significantly improved the water solubility in comparison with the parent drug. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  11. The Roles of Macrophages and Nitric Oxide in Interleukin-3-Enhanced HSV-Sr39tk-Mediated Prodrug Therapy

    PubMed Central

    Yu, Ching-Fang; Hong, Ji-Hong; Chiang, Chi-Shiun

    2013-01-01

    The herpes simplex virus thymidine kinase/ganciclovir (HSV-sr39tk/GCV) system is a well-established prodrug system used in cancer gene therapy. However, this system is currently not effective enough to eradicate malignant tumors completely. This study aimed to evaluate whether co-expression of interleukin-3 (IL-3) could enhance the anti-tumor activity of HSV-sr39tk/GCV prodrug gene therapy using a murine TRAMP-C1 prostate tumor model. In vitro results demonstrated that HSV-sr39tk-transfected cells exhibited enhanced sensitivity to the GCV prodrug, which was not affected by co-expression of the mIL-3 gene. However, in vivo studies showed that co-expression of the mIL-3 gene significantly increased the HSV-sr39tk/GCV-induced tumor growth delay and even cured the tumor. The TRAMP-C1-specific immune response of spleen lymphocytes from mice bearing HSV-sr39tk- and IL-3-expressing TRAMP-C1 tumors was measured by ELISA. Results showed that IL-3-activated IL-4-dominant lymphocytes became IFN-γ- dominant lymphocytes after combined HSV-sr39tk/GCV therapy. The efficacy of combined therapies on tumor regression was reduced when macrophages populations were depleted by carrageenan or NO production was inhibited by administration of the iNOS inhibitor, L-NAME. These results suggest that utilizing a bicistronic vector to express HSV-sr39tk and the IL-3 gene induced an enhanced macrophage- or NO-dependent anti-tumor effect. PMID:23441198

  12. Mesenchymal stromal cells retrovirally transduced with prodrug-converting genes are suitable vehicles for cancer gene therapy.

    PubMed

    Ďuriniková, E; Kučerová, L; Matúšková, M

    2014-01-01

    Mesenchymal stem/stromal cells (MSC) possess a set of several fairly unique properties which make them ideally suitable both for cellular therapies and regenerative medicine. These include: relative ease of isolation, the ability to differentiate along mesenchymal and non-mesenchymal lineages in vitro and the ability to be extensively expanded in culture without a loss of differentiative capacity. MSC are not only hypoimmunogenic, but they mediate immunosuppression upon transplantation, and possess pronounced anti-inflammatory properties. They are able to home to damaged tissues, tumors, and metastases following systemic administration. The ability of homing holds big promise for tumor-targeted delivery of therapeutic agents. Viruses are naturally evolved vehicles efficiently transferring their genes into host cells. This ability made them suitable for engineering vector systems for the delivery of genes of interest. MSC can be retrovirally transduced with genes encoding prodrug-converting genes (suicide genes), which are not toxic per se, but catalyze the formation of highly toxic metabolites following the application of a nontoxic prodrug. The homing ability of MSC holds advantages compared to virus vehicles which display many shortcomings in effective delivery of the therapeutic agents. Gene therapies mediated by viruses are limited by their restricted ability to track cancer cells infiltrating into the surrounding tissue, and by their low migratory capacity towards tumor. Thus combination of cellular therapy and gene delivery is an attractive option - it protects the vector from immune surveillance, and supports targeted delivery of a therapeutic gene/protein to the tumor site.

  13. Spiral assembly of amphiphilic cytarabine prodrug assisted by probe sonication: Enhanced therapy index for leukemia.

    PubMed

    Liu, Jing; Ma, Naxin; Zhao, Dujuan; Li, Zhonghao; Luan, Yuxia

    2015-12-01

    In order to overcome the drawbacks of cytarabine (Ara-C), such as low lipophilicity as well as short plasma half-life and rapid inactivation, a new derivative of Ara-C was designed by incorporation into the non-toxic material, oleic acid (OA), obtaining an amphiphilic small molecular weight prodrug (OA-Ara). By a simple amidation reaction, OA-Ara was synthesized successfully with a yield up to 61.32%. It was for the first time to see that the novel prodrug molecules could assemble into the unexpectedly spiral assembly under probe ultrasonication in aqueous solution. The oil/water partition coefficient (Ko/w) and the permeability of cell membrane of the prodrug were significantly increased compared with Ara-C molecules. In addition, OA-Ara molecules were stable in various pH solutions and artificial digestives, which indicated that it could be administrated orally. Cell viability assay showed that the prodrug displayed much higher antiproliferative effect against K562 and HL60 cells due to its improvement of the lipophilicity and penetrability of cell membrane. These findings demonstrate the feasibility of utilizing structural modification to broaden the clinic application of Ara-C and thus provide an effective new therapeutic alternative for leukemia.

  14. Poly(L-histidine)-tagged 5-aminolevulinic acid prodrugs: new photosensitizing precursors of protoporphyrin IX for photodynamic colon cancer therapy

    PubMed Central

    Johnson, Renjith P; Chung, Chung-Wook; Jeong, Young-Il; Kang, Dae Hwan; Suh, Hongsuk; Kim, Il

    2012-01-01

    Background 5-Aminolevulinic acid (ALA) and its derivatives have been widely used in photodynamic therapy. The main drawback associated with ALA-based photodynamic therapy (ALA-PDT) and ALA fluorescence diagnosis results from the hydrophilic nature of ALA and lack of selectivity for tumor versus nontumor cells. The application of certain triggers, such as pH, into conventional sensitizers for controllable 1O2 release is a promising strategy for tumor-targeted treatment. Methods A series of pH-sensitive ALA-poly(L-histidine) [p(L-His)n] prodrugs were synthesized via ring opening polymerization of 1-benzyl-N-carboxy-L-histidine anhydride initiated by the amine hydrochloride group of ALA itself. As an alternative to ALA for PDT, the synthesized prodrugs were used to treat a cultured human colon cancer HCT116 cell line under different pH conditions. The effect of ALA-p(L-His)n derivatives was evaluated by monitoring the fluorescence intensity of protoporphyrin IX, and measuring the cell survival rate after suitable light irradiation. Results The cytotoxicity and dark toxicity of ALA and synthesized ALA-p(L-His) derivatives in HEK293T and HCT116 cells in the absence of light at pH 7.4 and 6.8 shows that the cell viability was relatively higher than 100%. ALA-p(L-His)n showed high phototoxicity and selectivity in different pH conditions compared with ALA alone. Because the length of the histidine chain increases in the ALA-p(L-His)n prodrugs, the PDT effect was found to be more powerful. In particular, high phototoxicity was observed when the cells were treated with ALA-p(L-His)15, compared with treatment using ALA alone. Conclusion The newly synthesized ALA-p(L-His)n derivatives are an effective alternative to ALA for enhancing protoporphyrin IX production and the selectivity of the phototoxic effect in tumor cells. PMID:22679363

  15. Digestive Enzyme Replacement Therapy: Pancreatic Enzymes and Lactase.

    PubMed

    Felicilda-Reynaldo, Rhea Faye D; Kenneally, Maria

    2016-01-01

    Maldigestion occurs when digestive enzymes are lacking to help break complex food components into absorbable nutrients within the gastrointestinal tract. Education is needed to help patients manage the intricacies of digestive enzyme replacement therapies and ensure their effectiveness in reducing symptoms of maldigestion.

  16. Bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by human NAD(P)H quinone oxidoreductase 2: a novel co-substrate-mediated antitumor prodrug therapy.

    PubMed

    Knox, R J; Jenkins, T C; Hobbs, S M; Chen, S; Melton, R G; Burke, P J

    2000-08-01

    accessible and suitable for further pharmaceutical development. NQO2 activity appears to be related to expression of NQO1 (DT-diaphorase), an enzyme that is known to have a favorable distribution toward certain human cancers. NQO2 is a novel target for prodrug therapy and has a unique activation mechanism that relies on a synthetic co-substrate to activate an apparently latent enzyme. Our findings may reopen the use of CB 1954 for the direct therapy of human malignant disease.

  17. Polymer prodrug nanoparticles based on naturally occurring isoprenoid for anticancer therapy.

    PubMed

    Trung Bui, Duc; Maksimenko, Andrei; Desmaële, Didier; Harrisson, Simon; Vauthier, Christine; Couvreur, Patrick; Nicolas, Julien

    2013-08-12

    The synthesis of a novel class of polymer prodrug nanoparticles with anticancer activity is reported by using squalene, a naturally occurring isoprenoid, as a building block by the reversible addition-fragmentation (RAFT) technique. The RAFT agent was functionalized by gemcitabine (Gem) as anticancer drug, and the polymerization of squalenyl-methacrylate (SqMA) led to well-defined macromolecular prodrugs comprising one Gem at the extremity of each polymer chain. The amphiphilic nature of the resulting Gem-PSqMA conjugates allowed them to self-assemble into long-term stable and narrowly dispersed nanoparticles with significant anticancer activity in vitro on various cancer cell lines. To confer stealth properties on these nanoparticles, their PEGylation was successfully performed, as confirmed by X-ray photoelectron spectroscopy (XPS) and complement activation assay. It was also shown that the PEGylated nanoparticles could be internalized in cancer cells to a greater extent than their non-PEGylated counterparts.

  18. Amplifying the Red-Emission of Upconverting Nanoparticles for Biocompatible Clinically Used Prodrug-Induced Photodynamic Therapy

    SciTech Connect

    Punjabi, Amol; Wu, Xiang; Tokatli-Apollon, Amira; El-Rifai, Mahmoud; Lee, Hyungseok; Zhang, Yuanwei; Wang, Chao; Liu, Zhuang; Chan, Emory M.; Duan, Chunying; Han, Gang

    2014-09-25

    A class of biocompatible upconverting nanoparticles (UCNPs) with largely amplified red-emissions was developed. The optimal UCNP shows a high absolute upconversion quantum yield of 3.2% in red-emission, which is 15-fold stronger than the known optimal β-phase core/shell UCNPs. When conjugated to aminolevulinic acid, a clinically used photodynamic therapy (PDT) prodrug, significant PDT effect in tumor was demonstrated in a deep-tissue (>1.2 cm) setting in vivo at a biocompatible laser power density. Furthermore, we show that our UCNP–PDT system with NIR irradiation outperforms clinically used red light irradiation in a deep tumor setting in vivo. This study marks a major step forward in photodynamic therapy utilizing UCNPs to effectively access deep-set tumors.Lastly, it also provides an opportunity for the wide application of upconverting red radiation in photonics and biophotonics.

  19. Amplifying the Red-Emission of Upconverting Nanoparticles for Biocompatible Clinically Used Prodrug-Induced Photodynamic Therapy

    DOE PAGES

    Punjabi, Amol; Wu, Xiang; Tokatli-Apollon, Amira; ...

    2014-09-25

    A class of biocompatible upconverting nanoparticles (UCNPs) with largely amplified red-emissions was developed. The optimal UCNP shows a high absolute upconversion quantum yield of 3.2% in red-emission, which is 15-fold stronger than the known optimal β-phase core/shell UCNPs. When conjugated to aminolevulinic acid, a clinically used photodynamic therapy (PDT) prodrug, significant PDT effect in tumor was demonstrated in a deep-tissue (>1.2 cm) setting in vivo at a biocompatible laser power density. Furthermore, we show that our UCNP–PDT system with NIR irradiation outperforms clinically used red light irradiation in a deep tumor setting in vivo. This study marks a major stepmore » forward in photodynamic therapy utilizing UCNPs to effectively access deep-set tumors.Lastly, it also provides an opportunity for the wide application of upconverting red radiation in photonics and biophotonics.« less

  20. In vitro evaluation of cancer-specific NF-kappaB-CEA enhancer-promoter system for 5-fluorouracil prodrug gene therapy in colon cancer cell lines.

    PubMed

    Guo, X; Evans, T R J; Somanath, S; Armesilla, A L; Darling, J L; Schatzlein, A; Cassidy, J; Wang, W

    2007-09-17

    Nuclear factor-kappa B (NF-kappaB) is a transcription factor with high transcriptional activity in cancer cells. In this study, we developed a novel enhancer-promoter system, kappaB4-CEA205, in which the basal carcinoembryonic antigen (CEA) promoter sequence (CEA205) was placed downstream of the four tandem-linked NF-kappaB DNA-binding sites (kappaB4). In combination with a kappaB4 enhancer, the transcriptional activity of the CEA promoter was significantly enhanced (three- to eight-fold) in cancer cell lines but not in normal cells. In cancer cell lines, the transcriptional activity of kappaB4-CEA205 was comparable with that of the SV40 promoter. We also constructed vectors in which the thymidine phosphorylase (TP) cDNA was under the control of CEA205, kappaB4, kappaB4-CEA205 and CMV promoters, respectively. TP protein and enzyme activity were detected at comparable levels in kappaB4-CEA205- and CMV-driven TP cDNA-transfected cancer cell lines (H630 and RKO). The kappaB4-TP and CEA205-TP-transfected cell lines, respectively, only demonstrated negligible and low levels of TP protein and enzyme activity. Both CMV- and kappaB4-CEA205-driven TP cDNA transiently transfected cells were 8- to 10-fold sensitised to 5-fluorouracil (5-FU) prodrug, 5'-deoxy-5-fluorouradine (5'-DFUR), in contrast to only 1.5- to 2-fold sensitised by the kappaB4- and CEA205-driven TP cDNA-transfected cells. The bystander killing effect of CMV- and kappaB4-CEA205-driven TP cDNA-transfected cells was comparable. This is the first report that indicates that the NF-kappaB DNA-binding site could be used as a novel cancer-specific enhancer to improve cancer-specific promoter activity in gene-directed enzyme prodrug therapy.

  1. iPS-derived MSCs from an expandable bank to deliver a prodrug-converting enzyme that limits growth and metastases of human breast cancers.

    PubMed

    Ullah, M; Kuroda, Y; Bartosh, T J; Liu, F; Zhao, Q; Gregory, C; Reger, R; Xu, J; Lee, R H; Prockop, D J

    2017-01-01

    One attractive strategy to treat cancers is to deliver an exogenous enzyme that will convert a non-toxic compound to a highly toxic derivative. The strategy was tested with viral vectors but was disappointing because the efficiency of transduction into tumor cells was too low. Recent reports demonstrated that the limitation can be addressed by using tissue-derived mesenchymal stromal cells (MSCs) to deliver enzyme/prodrug systems that kill adjacent cancer cells through bystander effects. Here we addressed the limitation that tissue-derived MSCs vary in their properties and are difficult to generate in the large numbers needed for clinical applications. We prepared a Feeder Stock of MSCs from induced pluripotent stem cells (iPSs) that provided an extensively expandable source of standardized cells. We then transduced the iPS-derived MSCs to express cytosine deaminase and injected them locally into a mouse xenogeneic model of human breast cancer. After administration of the prodrug (5-fluorocytosine), the transduced iPS-MSCs both limited growth of preformed tumors and decreased lung metastases.

  2. iPS-derived MSCs from an expandable bank to deliver a prodrug-converting enzyme that limits growth and metastases of human breast cancers

    PubMed Central

    Ullah, M; Kuroda, Y; Bartosh, T J; Liu, F; Zhao, Q; Gregory, C; Reger, R; Xu, J; Lee, R H; Prockop, D J

    2017-01-01

    One attractive strategy to treat cancers is to deliver an exogenous enzyme that will convert a non-toxic compound to a highly toxic derivative. The strategy was tested with viral vectors but was disappointing because the efficiency of transduction into tumor cells was too low. Recent reports demonstrated that the limitation can be addressed by using tissue-derived mesenchymal stromal cells (MSCs) to deliver enzyme/prodrug systems that kill adjacent cancer cells through bystander effects. Here we addressed the limitation that tissue-derived MSCs vary in their properties and are difficult to generate in the large numbers needed for clinical applications. We prepared a Feeder Stock of MSCs from induced pluripotent stem cells (iPSs) that provided an extensively expandable source of standardized cells. We then transduced the iPS-derived MSCs to express cytosine deaminase and injected them locally into a mouse xenogeneic model of human breast cancer. After administration of the prodrug (5-fluorocytosine), the transduced iPS-MSCs both limited growth of preformed tumors and decreased lung metastases. PMID:28179988

  3. Prodrugs of peptides. 6. Bioreversible derivatives of thyrotropin-releasing hormone (TRH) with increased lipophilicity and resistance to cleavage by the TRH-specific serum enzyme.

    PubMed

    Bundgaard, H; Møss, J

    1990-09-01

    Bioreversible derivatization of TRH (pGlu-His-Pro-NH2) to protect the tripeptide against rapid enzymatic inactivation in the systemic circulation and to improve the lipophilicity of this highly hydrophilic peptide was performed by N-acylation of the imidazole group of the histidine residue with various chloroformates. Whereas TRH was rapidly hydrolyzed at its pGlu-His bond in human plasma by a TRH-specific pyroglutamyl aminopeptidase serum enzyme, the N-alkoxycarbonyl derivatives were resistant to cleavage by the enzyme. On the other hand, these derivatives are readily bioreversible as the parent TRH is formed quantitatively from the derivatives by spontaneous hydrolysis or by plasma esterase-catalyzed hydrolysis. In addition to protecting the parent TRH against rapid inactivation in the circulation and hence potentially prolonging the duration of action of TRH in vivo, the N-alkoxycarbonyl prodrug derivatives were much more lipophilic than TRH as assessed by octanol-buffer partitioning. This property may enhance prodrug penetration of the blood-brain barrier and various other biomembranes compared to the parent peptide.

  4. Development of pyrrolo[2,1-c][1,4]benzodiazepine beta-galactoside prodrugs for selective therapy of cancer by ADEPT and PMT.

    PubMed

    Kamal, Ahmed; Tekumalla, Venkatesh; Krishnan, Anita; Pal-Bhadra, Manika; Bhadra, Utpal

    2008-05-01

    The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a class of well-studied DNA-interactive agents with a potential for use in the treatment of cancer. The clinical utility of these molecules is limited because of the lack of selectivity for tumor tissues, high reactivity of the pharmacophoric imine functionality, low water solubility, and stability. To address the shortcomings, especially the lack of selectivity, associated with the molecules, two new beta-galactoside prodrugs of PBDs have been synthesized and evaluated for their potential use in selective therapy of solid tumors by ADEPT and PMT protocols. The preliminary studies reveal the prodrugs to be much less toxic compared to the parent moieties. These prodrugs are activated by E. coli beta-galactosidase (EC 3.2.1.23) to form the active cytotoxic moiety signifying their utility in ADEPT of cancer. One of the significant outcomes of the present study is the toxification of the prodrug 1 a by the endogenous beta-galactosidase of human liver cancer cells (Hep G2) to form the cytotoxic moiety, enabling selective therapy of hepatocellular carcinoma. Another important property of these molecules is their enhanced water solubility and stability, which are essential for a molecule to be an effective drug.

  5. Recent Trends in Targeted Anticancer Prodrug and Conjugate Design

    PubMed Central

    Singh, Yashveer; Palombo, Matthew; Sinko, Patrick J.

    2009-01-01

    Anticancer drugs are often nonselective antiproliferative agents (cytotoxins) that preferentially kill dividing cells by attacking their DNA at some level. The lack of selectivity results in significant toxicity to noncancerous proliferating cells. These toxicities along with drug resistance exhibited by the solid tumors are major therapy limiting factors that results into poor prognosis for patients. Prodrug and conjugate design involves the synthesis of inactive drug derivatives that are converted to an active form inside the body and preferably at the site of action. Classical prodrug and conjugate design has focused on the development of prodrugs that can overcome physicochemical (e.g., solubility, chemical instability) or biopharmaceutical problems (e.g., bioavailability, toxicity) associated with common anticancer drugs. The recent targeted prodrug and conjugate design, on the other hand, hinges on the selective delivery of anticancer agents to tumor tissues thereby avoiding their cytotoxic effects on noncancerous cells. Targeting strategies have attempted to take advantage of low extracellular pH, elevated enzymes in tumor tissues, the hypoxic environment inside the tumor core, and tumor-specific antigens expressed on tumor cell surfaces. The present review highlights recent trends in prodrug and conjugate rationale and design for cancer treatment. The various approaches that are currently being explored are critically analyzed and a comparative account of the advantages and disadvantages associated with each approach is presented. PMID:18691040

  6. Targeted delivery of a cisplatin prodrug for safer and more effective prostate cancer therapy in vivo

    PubMed Central

    Dhar, Shanta; Kolishetti, Nagesh; Lippard, Stephen J.; Farokhzad, Omid C.

    2011-01-01

    Targeted delivery and controlled release of inactive platinum (Pt) prodrugs may offer a new approach to improve the efficacy and tolerability of the Pt family of drugs, which are used to treat 50% of all cancers today. Using prostate cancer (PCa) as a model disease, we previously described the engineering of aptamer (Apt)-targeted poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-b-PEG) nanoparticles (NPs) encapsulating a Pt(IV) prodrug c,t,c[Pt(NH3)2-(O2CCH2CH2CH2CH2CH3)2Cl2] (1) (Pt-PLGA-b-PEG-Apt-NP), which target the extracellular domain of the prostate specific membrane antigen (PSMA), for enhanced in vitro cytotoxicity. Here we demonstrate enhanced in vivo pharmacokinetics (PK), biodistribution, tolerability, and efficacy of Pt-PLGA-b-PEG-Apt-NP (150±15 nm encapsulating ∼5% wt/wt Pt(IV) prodrug) when compared to cisplatin administered in its conventional form in normal Sprague Dawley rats, Swiss Albino mice, and the PSMA-expressing LNCaP subcutaneous xenograft mouse model of PCa, respectively. The 10-d maximum tolerated dose following a single i.v. injection of Pt-PLGA-b-PEG-NP in rats and mice was determined at 40 mg/kg and 5 mg/kg, respectively. PK studies with Pt-PLGA-b-PEG-NP revealed prolonged Pt persistence in systemic blood circulation and decreased accumulation of Pt in the kidneys, a major target site of cisplatin toxicity. Pt-PLGA-b-PEG-Apt-NPs further displayed the significant dose-sparing characteristics of the drug, with equivalent antitumor efficacy in LNCaP xenografts at 1/3 the dose of cisplatin administered in its conventional form (0.3 mg/kg vs. 1 mg/kg). When considering the simultaneous improvement in tolerability and efficacy, the Pt-PLGA-b-PEG-Apt NP provides a remarkable improvement in the drug therapeutic index. PMID:21233423

  7. Prodrug encapsulated albumin nanoparticles as an alternative approach to manifest anti-proliferative effects of suicide gene therapy.

    PubMed

    Tirkey, Bulbul; Bhushan, Bharat; Uday Kumar, S; Gopinath, P

    2017-04-01

    Conventional anticancer agents are associated with limited therapeutic efficacy and substantial nonspecific cytotoxicity. Thus, there is an imminent need for an alternative approach that can specifically annihilate the cancer cells with minimal side effects. Among such alternative approaches, CD::UPRT (cytosine deaminase uracil phosphoribosyl transferase) suicide gene therapy has tremendous potential due to its high efficacy. Prodrug 5-Fluorocytosine (5-FC) used in combination with CD::UPRT suicide gene suffers from limited solubility which subsequently leads to decline in therapeutic efficacy. In order to overcome this, 5-FC encapsulated bovine serum albumin nanoparticles (BSA-5-FC NPs) were prepared in this work by desolvation method. Physico-chemical characterizations studies revealed amorphous nature of BSA-5-FC NPs with uniform spherical morphology. Apart from increase in solubility, encapsulated 5-FC followed slow and sustained release profile. Suicide gene expressing stable clone of L-132 cells were adapted for investigating therapeutic potential of BSA-5-FC NPs. These nanoparticles were readily taken up by the cells in a concentration dependent manner and subsequently manifested apoptosis, which was further confirmed by morphological examination and gene expression analysis. These findings clearly illustrate that CD::UPRT suicide gene therapy can be efficiently utilized in combination with this nanosystem for improved suicide gene therapy and tumor eradication. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Enzyme replacement therapy: conception, chaos and culmination.

    PubMed Central

    Brady, Roscoe O

    2003-01-01

    Soon after the enzymatic defects in Gaucher disease and in Niemann-Pick disease were discovered, enzyme replacement or enzyme supplementation was proposed as specific treatment for patients with these and related metabolic storage disorders. While relatively straightforward in concept, successful implementation of this approach required many years of intensive effort to bring it to fruition. Procedures were eventually developed to produce sufficient quantities of the requisite enzymes for clinical trials and to target therapeutic enzymes to lipid-storing cells. These achievements led to the development of effective enzyme replacement therapy for patients with Gaucher disease and for Fabry disease. These demonstrations provide strong incentive for the application of this strategy for the treatment of many human disorders of metabolism. PMID:12803925

  9. Oral enzyme therapy for celiac sprue

    PubMed Central

    Bethune, Michael T; Khosla, Chaitan

    2012-01-01

    Celiac sprue is an inflammatory disease of the small intestine caused by dietary gluten and treated by adherence to a lifelong gluten-free diet. The recent identification of immunodominant gluten peptides, the discovery of their cogent properties, and the elucidation of the mechanisms by which they engender immunopathology in genetically-susceptible individuals have advanced our understanding of the molecular pathogenesis of this complex disease, enabling the rational design of new therapeutic strategies. The most clinically advanced of these is oral enzyme therapy, in which enzymes capable of proteolyzing gluten (i.e. glutenases) are delivered to the alimentary tract of a celiac sprue patient to detoxify ingested gluten in situ. In this chapter, we discuss the key challenges for discovery and preclinical development of oral enzyme therapies for celiac sprue. Methods for lead identification, assay development, gram-scale production and formulation, and lead optimization for next-generation proteases are described and critically assessed. PMID:22208988

  10. Seneca Valley Virus 3Cpro Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy

    PubMed Central

    Miles, Linde A.; Brennen, W. Nathaniel; Rudin, Charles M.; Poirier, John T.

    2015-01-01

    The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3Cpro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3Cpro, we measured cleavage kinetics of predicted SVV-001 3Cpro substrates. An efficient substrate, L/VP4 (kcat/KM = 1932 ± 183 M-1s-1), was further optimized by a P2’ N→P substitution yielding L/VP4.1 (kcat/KM = 17446 ± 2203 M-1s-1). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3Cpro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3Cpro, with direct implications for anti-cancer therapeutic development. PMID:26069962

  11. Seneca Valley Virus 3Cpro Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy.

    PubMed

    Miles, Linde A; Brennen, W Nathaniel; Rudin, Charles M; Poirier, John T

    2015-01-01

    The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3Cpro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3Cpro, we measured cleavage kinetics of predicted SVV-001 3Cpro substrates. An efficient substrate, L/VP4 (kcat/KM = 1932 ± 183 M(-1)s(-1)), was further optimized by a P2' N→P substitution yielding L/VP4.1 (kcat/KM = 17446 ± 2203 M(-1)s(-1)). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3Cpro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3Cpro, with direct implications for anti-cancer therapeutic development.

  12. PET imaging of β-glucuronidase activity by an activity-based 124I-trapping probe for the personalized glucuronide prodrug targeted therapy.

    PubMed

    Su, Yu-Cheng; Cheng, Ta-Chun; Leu, Yu-Ling; Roffler, Steve R; Wang, Jaw-Yuan; Chuang, Chih-Hung; Kao, Chien-Han; Chen, Kai-Chuan; Wang, Hsin-Ell; Cheng, Tian-Lu

    2014-12-01

    Beta-glucuronidase (βG) is a potential biomarker for cancer diagnosis and prodrug therapy. The ability to image βG activity in patients would assist in personalized glucuronide prodrug cancer therapy. However, whole-body imaging of βG activity for medical usage is not yet available. Here, we developed a radioactive βG activity-based trapping probe for positron emission tomography (PET). We generated a (124)I-tyramine-conjugated difluoromethylphenol beta-glucuronide probe (TrapG) to form (124)I-TrapG that could be selectively activated by βG for subsequent attachment of (124)I-tyramine to nucleophilic moieties near βG-expressing sites. We estimated the specificity of a fluorescent FITC-TrapG, the cytotoxicity of tyramine-TrapG, and the serum half-life of (124)I-TrapG. βG targeting of (124)I-TrapG in vivo was examined by micro-PET. The biodistribution of (131)I-TrapG was investigated in different organs. Finally, we imaged the endogenous βG activity and assessed its correlation with therapeutic efficacy of 9-aminocamptothecin glucuronide (9ACG) prodrug in native tumors. FITC-TrapG showed specific trapping at βG-expressing CT26 (CT26/mβG) cells but not in CT26 cells. The native TrapG probe possessed low cytotoxicity. (124)I-TrapG preferentially accumulated in CT26/mβG but not CT26 cells. Meanwhile, micro-PET and whole-body autoradiography results demonstrated that (124)I-TrapG signals in CT26/mβG tumors were 141.4-fold greater than in CT26 tumors. Importantly, Colo205 xenografts in nude mice that express elevated endogenous βG can be monitored by using infrared glucuronide trapping probes (NIR-TrapG) and suppressed by 9ACG prodrug treatment. (124)I-TrapG exhibited low cytotoxicity allowing long-term monitoring of βG activity in vivo to aid in the optimization of prodrug targeted therapy.

  13. Enzyme replacement therapy and Fabry nephropathy.

    PubMed

    Warnock, David G; Daina, Erica; Remuzzi, Giuseppe; West, Michael

    2010-02-01

    Involvement of the kidneys in Fabry disease ("nephropathy") occurs in male and female individuals. The majority of patients with progressive nephropathy will have significant proteinuria and develop progressive loss of kidney function, leading to ESRD. All too often, treating physicians may ignore "normal" serum creatinine levels or "minimal" proteinuria and fail to assess properly the severity of kidney involvement and institute appropriate management. Fabry nephropathy is treatable, even in patients with fairly advanced disease. Although the cornerstone of therapy remains enzyme replacement therapy with agalsidase, this treatment alone does not reduce urine protein excretion. Treatment with angiotensin receptor blockers or angiotensin-converting enzyme inhibitors must be added to enzyme replacement therapy to reduce urine protein excretion with the hope that this will stabilize kidney function. Kidney function, with at least estimated GFR based on serum creatinine and measurements of urinary protein, should be measured at every clinic visit, and the rate of change of the estimated GFR should be followed over time. Antiproteinuric therapy can be dosed to a prespecified urine protein target rather than a specific BP goal, with the proviso that successful therapy will usually lower the BP below the goal of 130/80 mmHg that is used for other forms of kidney disease. The overall goal for treating Fabry nephropathy is to reduce the rate of loss of GFR to -1 ml/min per 1.73 m(2)/yr, which is that seen in the normal adult population. A systematic approach is presented for reaching this goal in the individual patient.

  14. Prodrugs design based on inter- and intramolecular chemical processes.

    PubMed

    Karaman, Rafik

    2013-12-01

    This review provides the reader a concise overview of the majority of prodrug approaches with the emphasis on the modern approaches to prodrug design. The chemical approach catalyzed by metabolic enzymes which is considered as widely used among all other approaches to minimize the undesirable drug physicochemical properties is discussed. Part of this review will shed light on the use of molecular orbital methods such as DFT, semiempirical and ab initio for the design of novel prodrugs. This novel prodrug approach implies prodrug design based on enzyme models that were utilized for mimicking enzyme catalysis. The computational approach exploited for the prodrug design involves molecular orbital and molecular mechanics (DFT, ab initio, and MM2) calculations and correlations between experimental and calculated values of intramolecular processes that were experimentally studied to assign the factors determining the reaction rates in certain processes for better understanding on how enzymes might exert their extraordinary catalysis.

  15. Targeting therapy of hepatocellular carcinoma with doxorubicin prodrug PDOX increases anti-metastatic effect and reduces toxicity: a preclinical study

    PubMed Central

    2013-01-01

    Background This study was to investigate the effects and safety of cathepsin B-cleavable doxorubicin (DOX)-prodrug (PDOX) for targeting therapy of metastatic human hepatocellular carcinoma (HCC) using DOX as a positive control drug. Methods The orthotopic nude mice model of highly metastatic HCC was established and the animals were randomized and treated with PDOX, DOX and saline, respectively. Hematology, biochemistry and tumor markers were studied. At autopsy, liver tumor weight and size, ascites, abdominal lymph nodes metastases, experimental peritoneal carcinomatosis index (ePCI), and tumor-host body weight ratio were investigated. Immunohistochemical studies and western blotting were done to investigate key molecules involved in the mechanism of action. Results Compared with Control, both PDOX and DOX could similarly and significantly reduce liver tumor weight and tumor volume by over 40%, ePCI values, retroperitoneal lymph node metastases and lung metastases and serum AFP levels (P < 0.05). The PDOX group had significantly higher WBC than the DOX group (P < 0.05), and higher PLT than Control (P < 0.05). Serum BUN and Cr levels were lower in the PDOX group than DOX and Control groups (P < 0.05). Compared with Control, DOX increased CK and CK-MB; while PDOX decreased CK compared with DOX (P < 0.05). Multiple spotty degenerative changes of the myocardium were observed in DOX-treated mice, but not in the Control and PDOX groups. PDOX could significantly reduce the Ki-67 positive rate of tumor cells, compared with DOX and Control groups. PDOX produced the effects at least via the ERK pathway. Conclusion Compared with DOX, PDOX may have better anti-metastatic efficacy and reduced side effects especially cardio-toxicities in this HCC model. PMID:23961994

  16. Modern prodrug design for targeted oral drug delivery.

    PubMed

    Dahan, Arik; Zimmermann, Ellen M; Ben-Shabat, Shimon

    2014-10-14

    The molecular information that became available over the past two decades significantly influenced the field of drug design and delivery at large, and the prodrug approach in particular. While the traditional prodrug approach was aimed at altering various physiochemical parameters, e.g., lipophilicity and charge state, the modern approach to prodrug design considers molecular/cellular factors, e.g., membrane influx/efflux transporters and cellular protein expression and distribution. This novel targeted-prodrug approach is aimed to exploit carrier-mediated transport for enhanced intestinal permeability, as well as specific enzymes to promote activation of the prodrug and liberation of the free parent drug. The purpose of this article is to provide a concise overview of this modern prodrug approach, with useful successful examples for its utilization. In the past the prodrug approach used to be viewed as a last option strategy, after all other possible solutions were exhausted; nowadays this is no longer the case, and in fact, the prodrug approach should be considered already in the very earliest development stages. Indeed, the prodrug approach becomes more and more popular and successful. A mechanistic prodrug design that aims to allow intestinal permeability by specific transporters, as well as activation by specific enzymes, may greatly improve the prodrug efficiency, and allow for novel oral treatment options.

  17. GR-891: a novel 5-fluorouracil acyclonucleoside prodrug for differentiation therapy in rhabdomyosarcoma cells

    PubMed Central

    Marchal, J A; Prados, J; Melguizo, C; Gómez, J A; Campos, J; Gallo, M A; Espinosa, A; Arena, N; Aránega, A

    1999-01-01

    Differentiation therapy provides an alternative treatment of cancer that overcomes the undesirable effects of classical chemotherapy, i.e. cytotoxicity and resistance to drugs. This new approach to cancer therapy focuses on the development of specific agents designed to selectively engage the process of terminal differentiation, leading to the elimination of tumorigenic cells and recovery of normal cell homeostasis. A series of new anti-cancer pyrimidine acyclonucleoside-like compounds were designed and synthesized by structural modifications of 5-fluorouracil, a drug which causes considerable cell toxicity and morbidity, and we evaluated their applicability for differentiation therapy in human rhabdomyosarcoma cells. We tested the pyrimidine derivative GR-891, (RS)-1-{[3-(2-hydroxyethoxy)-1-isopropoxy]propyl}-5-fluorouracil, an active drug which shows low toxicity in vivo and releases acrolein which is an aldehyde with anti-tumour activity. Both GR-891 and 5-fluorouracil caused time- and dose-dependent growth inhibition in vitro; however, GR-891 showed no cytotoxicity at low doses (22.5 μmol l−1 and 45 μmol l−1) and induced terminal myogenic differentiation in RD cells (a rhabdomyosarcoma cell line) treated for 6 days. Changes in morphological features and in protein organization indicated re-entry in the pathway of muscular maturation. Moreover, GR-891 increased adhesion capability mediated by the expression of fibronectin, and did not induce overexpression of P-glycoprotein, the mdr1 gene product, implicated in multidrug resistance. New acyclonucleoside-like compounds such as GR-891 have important potential advantages over 5-fluorouracil because of their lower toxicity and their ability to induce myogenic differentiation in rhabdomyosarcoma cells. Our results suggest that this drug may be useful for differentiation therapy in this type of tumour. 1999 Cancer Research Campaign PMID:10070873

  18. [Perioperative enzyme therapy. A significant supplement to postoperative pain therapy?].

    PubMed

    Hoernecke, R; Doenicke, A

    1993-12-01

    Following ethics committee approval and with written informed consent, 80 patients (61 female, 19 male) were randomly allocated in this double-blind, placebo-controlled, parallel group study to two groups of 40 patients each. The treatment group received a compound of proteolytic, glycolytic, and lipolytic enzymes and rutoside (Wobenzym), while the control-group received placebo. Efficacy and tolerance of the study medication was examined before and after day case surgery for the carpal tunnel syndrome. Medication was given 10 tabs t.i.d. for 15 days, starting 7 days before surgery. Pain intensity (visual analogue scale), intake of analgesics, and tolerance of the study medication were recorded daily and treatment differences between the two groups were statistically evaluated. Comparison of demographic data of all enrolled patients showed no significant difference between the two groups. In the enzyme group, pain scores were lower on the day of operation and for the following 5 days but did not differ significantly from the control group; however, patients of the enzyme group took significantly less analgesics and experienced significantly earlier pain relief than those of the control group, thus demonstrating a pain-reducing effect of the enzyme compound. Only few side effects were reported in both groups, none of them serious. Enzyme therapy is an effective and safe adjuvant treatment for the control of postoperative pain.

  19. Effect of ion pairing on in vitro transcorneal permeability of a Δ(9) -tetrahydrocannabinol prodrug: potential in glaucoma therapy.

    PubMed

    Hingorani, Tushar; Gul, Waseem; Elsohly, Mahmoud; Repka, Michael A; Majumdar, Soumyajit

    2012-02-01

    The aim of the present study was to evaluate and improve the in vitro transcorneal permeability characteristics of Δ(9) -tetrahydrocannabinol (THC) through prodrug derivatization and formulation approaches. In vitro corneal permeability of THC and its hemisuccinate (THC-HS) and hemiglutarate (THC-HG) ester prodrugs and WIN 55-212-2 (WIN), a synthetic cannabinoid, was determined using isolated rabbit cornea. The formulations studied included hydroxypropyl beta cyclodextrin (HPβCD) or randomly methylated beta cyclodextrin (RMβCD), as well as prodrug-ion-pair complexes with l-arginine or tromethamine. Corneal permeability of WIN was found to be two-fold higher than THC in the presence of HPβCD. THC-HS and THC-HG exhibited pH-dependent permeability. In the presence of HPβCD, at pH 5 (donor solution pH), both prodrugs exhibited six-fold higher permeability compared with THC. However, permeability of the prodrugs was about three-fold lower than that of THC at pH 7.4. RMβCD, at pH 7.4, led to a significant improvement in permeability. Formation of ion-pair complexes markedly improved the solubility and permeability of THC-HG (sevenfold and threefold greater permeability compared with THC and WIN, respectively) at pH 7.4. The in vitro results demonstrate that the use of an ion-pair complex of THC-HG could be an effective strategy for topical delivery of THC.

  20. Why Prodrugs and Propesticides Succeed.

    PubMed

    Casida, John E

    2017-05-15

    What are the advantages of bioactivation in optimizing drugs and pesticides? Why are there so many prodrugs and propesticides? These questions are examined here by considering compounds selected on the basis of economic value or market success in 2015. The 100 major drugs and 90 major pesticides are divided into ones acting directly and those definitely or possibly requiring bioactivation. Established or candidate prodrugs accounted for 19% of the total drug sales, with corresponding values of 20, 37, and 17% for proinsecticides, proherbicides, and profungicides. The 19 prodrugs acting in humans generally had better pharmacodynamic/pharmacokinetic properties for target enzyme, receptor, tissue, or organ specificity due to their physical properties (lipophilicity and stabilization). Bioactivation usually involved hydrolases or cytochrome P450 oxidation or reduction. Prodrugs considered are neuroactive aripiprazole, eletriptan, desvenlafaxin, lisdexamfetamine, quetiapine, and fesoterodine; cholesterol-lowering atorvastatin, ezetimibe, and fenofibrate; various prodrugs activated by esterases or sulfatases, ciclesonide, oseltamivir, dabigatran; omega-3 fatty acid ethyl esters and esterone sulfate; and five others with various targets (sofosbuvir, fingolimod, clopidogrel, dapsone, and sildenafil). The proinsecticides are the neuroactive chlorpyrifos, thiamethoxam, and indoxacarb, two spiro enol ester inhibitors of acetyl CoA carboxylase (ACCase), and the bacterial protein delta-endotoxin. The proherbicides considered are five ACCase inhibitors including pinoxaden and clethodim, three protox inhibitors (saflufenacil, flumioxazin, and canfentrazone-ethyl), and three with various targets (fluroxypyr, isoxaflutole, and clomazone). The profungicides are prothioconazole, mancozeb, thiophanate-methyl, dazomet, and fosetyl-aluminum. The prodrug and propesticide concept is broadly applicable and has created some of the most selective pharmaceutical and pest control agents

  1. Intravenous enzyme replacement therapy: hospital vs home.

    PubMed

    Parini, Rossella; Pozzi, Katia; Di Mauro, Stefania; Furlan, Francesca; Rigoldi, Miriam

    Two surveys were carried out to establish the status of enzyme replacement therapy (ERT) for lysosomal storage diseases in Italy. The first was a national survey covering the regional reference centres (RRCs) for these diseases; replies disclosed that 57.7% of patients are on ERT, administered almost exclusively in hospital settings (local hospital 60.7%, RRC 34.8%, home 2.6%); Italian health service procedures do not support ERT at home. The second survey was a regional survey in Lombardy, involving 48 patients (six of whom were on ERT at home). According to 40% of the patients, hospital-based ERT is disruptive, causing loss of days at school/work, stress and family issues. The patients on home therapy did not have these problems. However, 93% of patients receiving ERT in hospital perceived the advantages of greater safety, closer monitoring and more support from health professionals and experts. A total of 55% were willing to receive ERT at home, but 33% were against it. This may be the result of a lack of experience with ERT at home in Italy, or because of different opinions between family members and physicians. As international experience shows that ERT at home saves healthcare resources and improves quality of life, the issue should be raised with Italian healthcare policy makers, who should ensure nursing support for home-based ERT.

  2. New prodrugs against tuberculosis.

    PubMed

    Mori, Giorgia; Chiarelli, Laurent Roberto; Riccardi, Giovanna; Pasca, Maria Rosalia

    2017-03-01

    The term 'prodrug' was first introduced by Albert in 1958. Generally, prodrugs can be utilized for improving active drug solubility and bioavailability, increasing drug permeability and absorption, modifying the distribution profile, preventing fast metabolism and excretion, and reducing toxicity. Previously, the prodrug approach was a final resort during the drug discovery process only after all other approaches had been exhausted. However, this strategy is now considered during the early stages of the drug development process. Most antitubercular agents are defined as 'prodrugs', including isoniazid and ethionamide. Thus, the prodrug approach could provide novel targets for the rational design of more effective treatments for tuberculosis (TB).

  3. Comparative analysis of enzyme and pathway engineering strategies for 5FC-mediated suicide gene therapy applications.

    PubMed

    Johnson, A J; Ardiani, A; Sanchez-Bonilla, M; Black, M E

    2011-08-01

    Bacterial- and yeast- encoded cytosine deaminases (bCD and yCD, respectively) are widely investigated suicide enzymes used in combination with the prodrug 5-fluorocytosine (5FC) to achieve localized cytotoxicity. Yet characteristics such as poor turnover rates of 5FC (bCD) and enzyme thermolability (yCD) preclude their full therapeutic potential. We previously applied regio-specific random mutagenesis and computational design to create novel bCD and yCD variants with altered substrate preference (bCD(1525)) or increased thermostability (yCD(double), yCD(triple)) to aid in overcoming these limitations. Others have utilized pathway engineering in which the microbial enzyme uracil phosphoribosyltransferase (UPRT) is fused with its respective CD, creating bCD/bUPRT or yCD/yUPRT. In this study, we evaluated whether the overlay of CD mutants onto their respective CD/UPRT fusion construct would further enhance 5FC activation, cancer cell prodrug sensitivity and bystander activity in vitro and in vivo. We show that all mutant fusion enzymes allowed for significant reductions in IC(50) values relative to their mutant CD counterparts. However, in vivo the CD mutants displayed enhanced tumor growth inhibition capacity relative to the mutant fusions, with bCD(1525) displaying the greatest tumor growth inhibition and bystander activity. In summary, mutant bCD(1525) appears to be the most effective of all bacterial or yeast CD or CD/UPRT enzymes examined and as such is likely to be the best choice to significantly improve the clinical outcome of CD/5FC suicide gene therapy applications.

  4. The future of prodrugs - design by quantum mechanics methods.

    PubMed

    Karaman, Rafik; Fattash, Beesan; Qtait, Alaa

    2013-05-01

    The revolution in computational chemistry greatly impacted the drug design and delivery fields, in general, and recently the utilization of the prodrug approach in particular. The use of ab initio, semiempirical and molecular mechanics methods to understand organic reaction mechanisms of certain processes, especially intramolecular reactions, has opened the door to design and to rapidly produce safe and efficacious delivery of a wide range of active small molecule and biotherapeutics such as prodrugs. This article provides the readers with a concise overview of this modern approach to prodrug design. The use of computational approaches, such as density functional theory (DFT), semiempirical and ab initio molecular orbital methods, in modern prodrugs design will be discussed. The novel prodrug approach to be reported in this review implies prodrug design based on enzyme model (mimicking enzyme catalysis) that has been utilized to understand how enzymes work. The tool used in the design is a computational approach consisting of calculations using molecular orbital and molecular mechanics methods (DFT, ab initio and MM2) and correlations between experimental and calculated values of intramolecular processes that were used to understand the mechanism by which enzymes might exert their high rates catalysis. The future of prodrug technology is exciting yet extremely challenging. Advances must be made in understanding the chemistry of many organic reactions that can be effectively utilized to enable the development of even more types of prodrugs. Despite the increase in the number of marketed prodrugs, we have only started to appreciate the potential of the prodrug approach in modern drug development, and the coming years will witness many novel prodrug innovations.

  5. Self-immolative nitrogen mustards prodrugs cleavable by carboxypeptidase G2 (CPG2) showing large cytotoxicity differentials in GDEPT.

    PubMed

    Niculescu-Duvaz, Dan; Niculescu-Duvaz, Ion; Friedlos, Frank; Martin, Jan; Lehouritis, Panos; Marais, Richard; Springer, Caroline J

    2003-04-24

    Nineteen novel potential self-immolative prodrugs and their corresponding drugs have been synthesized for gene-directed enzyme prodrug therapy (GDEPT) with carboxypeptidase G2 (CPG2) as the activating enzyme. The compounds are derived from o- and p-amino and p-methylamino aniline nitrogen mustards. Their aqueous stability, kinetics of drug release by CPG2, and cytotoxicity in the colon carcinoma cell line WiDr, expressing either surface-tethered CPG2 (stCPG2(Q)3) or control beta-galactosidase, are assessed. The effect of various structural features on stability, kinetics of activation, and biological activity is discussed. The p-methylamino prodrugs are the most stable compounds from this series, with the largest cytotoxicity differentials between CPG2-expressing and nonexpressing cells. The most potent compounds in all series are prodrugs of bis-iodo nitrogen mustards. 4-[N-[4'-Bis(2' '-iodoethyl)aminophenyl]-N'-methylcarbamoyloxymethyl]phenylcarbamoyl-l-glutamic acid, compound 39b, is 124-fold more cytotoxic to WiDr cells expressing CPG2 than to cells expressing beta-galactosidase. An additional six compounds show better cytotoxicity differential than the published N-[4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl]-l-glutamic acid (CMDA) prodrug.

  6. Prodrug Strategies in Ocular Drug Delivery

    PubMed Central

    Barot, Megha; Bagui, Mahuya; Gokulgandhi, Mitan R.; Mitra, Ashim K.

    2015-01-01

    Poor bioavailability of topically instilled drug is the major concern in the field of ocular drug delivery. Efflux transporters, static and dynamic ocular barriers often possess rate limiting factors for ocular drug therapy. Different formulation strategies like suspension, ointment, gels, nanoparticles, implants, dendrimers and liposomes have been employed in order to improve drug permeation and retention by evading rate limiting factors at the site of absorption. Chemical modification such as prodrug targeting various nutrient transporters (amino acids, peptide and vitamin) has evolved a great deal ofintereSt to improve ocular drug delivery. In this review, we have discussed various prodrug strategies which have been widely applied for enhancing therapeutic efficacy of ophthalmic drugs. The purpose of this review is to provide an update on the utilization of prodrug concept in ocular drug delivery. In addition, this review will highlight ongoing academic and industrial research and development in terms of ocular prodrug design and delivery. PMID:22530907

  7. Towards antibody-drug conjugates and prodrug strategies with extracellular stimuli-responsive drug delivery in the tumor microenvironment for cancer therapy.

    PubMed

    Joubert, Nicolas; Denevault-Sabourin, Caroline; Bryden, Francesca; Viaud-Massuard, Marie-Claude

    2017-08-23

    The design of innovative anticancer chemotherapies with superior antitumor efficacy and reduced toxicity continues to be a challenging endeavor. Recently, the success of Adcetris(®) and Kadcyla(®) made antibody-drug conjugates (ADCs) serious contenders to reach the envied status of Paul Ehrlich's "magic bullet". However, ADCs classically target overexpressed and internalizing antigens at the surface of cancer cells, and in solid tumors are associated with poor tumor penetration, insufficient targeting in heterogeneous tumors, and appearance of several resistance mechanisms. In this context, alternative non-internalizing ADCs and prodrugs have been developed to circumvent these limitations, in which the drug can be selectively released by an extracellular stimulus in the tumor microenvironment. Each strategy and method of activation will be discussed as potential alternatives to internalizing ADCs for cancer therapy. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  8. Enzymes approved for human therapy: indications, mechanisms and adverse effects.

    PubMed

    Baldo, Brian A

    2015-02-01

    Research and drug developments fostered under orphan drug product development programs have greatly assisted the introduction of efficient and safe enzyme-based therapies for a range of rare disorders. The introduction and regulatory approval of 20 different recombinant enzymes has enabled, often for the first time, effective enzyme-replacement therapy for some lysosomal storage disorders, including Gaucher (imiglucerase, taliglucerase, and velaglucerase), Fabry (agalsidase alfa and beta), and Pompe (alglucosidase alfa) diseases and mucopolysaccharidoses I (laronidase), II (idursulfase), IVA (elosulfase), and VI (galsulfase). Approved recombinant enzymes are also now used as therapy for myocardial infarction (alteplase, reteplase, and tenecteplase), cystic fibrosis (dornase alfa), chronic gout (pegloticase), tumor lysis syndrome (rasburicase), leukemia (L-asparaginase), some collagen-based disorders such as Dupuytren's contracture (collagenase), severe combined immunodeficiency disease (pegademase bovine), detoxification of methotrexate (glucarpidase), and vitreomacular adhesion (ocriplasmin). The development of these efficacious and safe enzyme-based therapies has occurred hand in hand with some remarkable advances in the preparation of the often specifically designed recombinant enzymes; the manufacturing expertise necessary for commercial production; our understanding of underlying mechanisms operative in the different diseases; and the mechanisms of action of the relevant recombinant enzymes. Together with information on these mechanisms, safety findings recorded so far on the various adverse events and problems of immunogenicity of the recombinant enzymes used for therapy are presented.

  9. [Clinical and pharmacological aspects of pancreatic enzyme substitution therapy].

    PubMed

    Löser, C; Fölsch, U R

    1991-03-01

    The adequate therapy of pancreatic enzyme replacement in patients with exocrine pancreatic insufficiency is still a difficult clinical problem especially in patients following pancreatectomys, with chronic alcoholic pancreatitis or cystic fibrosis. The substitution of lipase to eliminate steatorrhoea is the most important aim but due to its acid lability even the most serious problem in pancreatic enzyme replacement therapy. Various different medications are meanwhile available: conventional preparations from porcine pancreatin or fungal enzymes as rizolipase, enteric-coated tablets or even enteric-coated microspheres or adjunctive therapy with H2-receptor antagonists. While dosage requirements vary widely and therefore have to be tried out individually, the choice of the adequate preparation should be influenced by the realization of the physiological and pathophysiological characteristics of the individual patient and the pharmaceutical characteristics of the different supplements. The advantages and disadvantages of the various medications for enzyme replacement therapy in patients with exocrine pancreatic insufficiency are reviewed in this article.

  10. Metabolic Enzyme Considerations in Cancer Therapy

    PubMed Central

    Jain, Amit K.; Jain, Sweta; Rana, A.C

    2007-01-01

    The clinical application of new antineoplastic drugs has been limited because of low therapeutic index and lack of efficacy in humans. Thus, improvement in efficacy of old and new anticancer drugs has been attempted by manipulating their pharmacokinetic properties. Four inter-related factors, which determine the pharmacokinetic behavior of a drug include absorption, distribution, metabolism and excretion. The drug-metabolizing enzymes have been classified in two major groups: phase I and phase II enzymes. Phase I enzymes comprise the oxidases, dehydrogenases, deaminases, hydrolases. Phase II enzymes include primarily UDP-glucuronosyltransferases (UGTs), glutathionetransferases (GSTs), sulfotransferases (SULTs), N-acetyl transferases (NATs), methyltransferases and aminoacid transferases that conjugate products of phase I reactions and parent compounds with appropriate functional groups to generate more water soluble compounds which are more readily eliminated. The importance of these enzymes in the metabolism of specific drugs varies according to the chemical nature of the drug, Drug metabolism is modulated by factors that change among species and even among individuals in a population. Such factors can be environmental or genetic in origin, and influence how a drug is metabolized and to what extent. An awareness of these variables is invaluable when the safety and efficacy of new anticancer drugs are evaluated 1 PMID:22593646

  11. [Systemic enzyme therapy of experimental gout glomerulonephritis].

    PubMed

    Ignatenko, G A; Mukhin, I V

    2004-01-01

    Renal lesion deteriorates the course and prognosis of gouty glomerulonephritis. Current pathogenetic therapy is not sufficiently effective. Effects of different treatments on morphological and functional manifestations of renal disorders in experimental gouty glomerulonephritis are reviewed.

  12. Pharmacological screening of glycine amino acid prodrug of acetaminophen

    PubMed Central

    Parashar, Arun

    2015-01-01

    Objective: To develop an amino acid prodrug of acetaminophen with comparable therapeutic profile and less hepatotoxicity than acetaminophen. Materials and Methods: Acetaminophen prodrug was synthesized by esterification between the carboxyl group of amino acid glycine and hydroxyl group of acetaminophen. Analgesic, antipyretic, ulcer healing, and hepatotoxic activities were performed on Wistar rats in this study. Results: Prodrug showed a 44% inhibition in writhings as compared to 53.3% of acetaminophen. Acetaminophen also offered highest antipyretic activity. Prodrug showed gastroprotective and hepatoprotective effects as it reduced the gastric lesions by 32.1% (P < 0.01) and significantly prevented the rise in liver enzymes (serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase and bilirubin). The most notable effect of prodrug was in preventing the depletion of hepatic glutathione (GSH), which is reduced by acetaminophen. Conclusion: Prodrug showed hepatoprotective and gastroprotective effects, although the therapeutic efficacy was compromised. Prodrug was successful in preventing a decrease in GSH, thereby exhibiting promising results in the field of prodrug designing to avoid the toxic effects of acetaminophen. PMID:25878383

  13. Pharmacological screening of glycine amino acid prodrug of acetaminophen.

    PubMed

    Parashar, Arun

    2015-01-01

    To develop an amino acid prodrug of acetaminophen with comparable therapeutic profile and less hepatotoxicity than acetaminophen. Acetaminophen prodrug was synthesized by esterification between the carboxyl group of amino acid glycine and hydroxyl group of acetaminophen. Analgesic, antipyretic, ulcer healing, and hepatotoxic activities were performed on Wistar rats in this study. Prodrug showed a 44% inhibition in writhings as compared to 53.3% of acetaminophen. Acetaminophen also offered highest antipyretic activity. Prodrug showed gastroprotective and hepatoprotective effects as it reduced the gastric lesions by 32.1% (P < 0.01) and significantly prevented the rise in liver enzymes (serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase and bilirubin). The most notable effect of prodrug was in preventing the depletion of hepatic glutathione (GSH), which is reduced by acetaminophen. Prodrug showed hepatoprotective and gastroprotective effects, although the therapeutic efficacy was compromised. Prodrug was successful in preventing a decrease in GSH, thereby exhibiting promising results in the field of prodrug designing to avoid the toxic effects of acetaminophen.

  14. Layer-by-layer nanoparticles co-loading gemcitabine and platinum (IV) prodrugs for synergistic combination therapy of lung cancer

    PubMed Central

    Zhang, Rongrong; Ru, Yun; Gao, Yiping; Li, Jinyin; Mao, Shilong

    2017-01-01

    Purpose Cisplatin plus gemcitabine (GEM) is a standard regimen for the first-line treatment of advanced non-small cell lung cancer. The aim of this study was to prepare biocompatible and biodegradable polymeric prodrugs and construct nanoparticles (NPs) with layer-by-layer (LbL) technique. Methods Platinum (Pt) (IV) complex with a carboxyl group was conjugated to the amino group of chitosan (CH), resulting in a CH-Pt conjugation with positive charge. GEM with amino group was conjugated to the carboxyl group of hyaluronic acid (HA), resulting in a HA-GEM conjugation with negative charge. Novel LbL NPs consisting of the CH-Pt core and the HA-GEM layer, named as HA-GEM/CH-Pt NPs, were constructed. The physicochemical properties of the HA-GEM/CH-Pt NPs were investigated. In vitro cytotoxicity against human non-small lung cancer cells (NCl-H460 cells) was investigated, and in vivo antitumor efficiency was evaluated on mice bearing NCl-H460 cells xenografts. Results HA-GEM/CH-Pt NPs have a size of about 187 nm, a zeta potential value of −21 mV and high drug encapsulation efficiency of 90%. The drug release of HA-GEM/CH-Pt NPs exhibited a sustained behavior. HA-GEM/CH-Pt NPs could significantly enhance in vitro cytotoxicity and in vivo antitumor effect against lung cancer animal model compared to the single-drug-loaded NPs and free drug solutions. Conclusion The results demonstrated that the HA-GEM/CH-Pt NPs might be a promising system for the synergetic treatment of lung carcinoma. PMID:28919713

  15. Developing bifunctional beta-lactamase molecules with built-in target-recognizing module for prodrug therapy: identification of Enterobacter Cloacae P99 cephalosporinase loops suitable for randomization and phage-display selection.

    PubMed

    Shukla, Girja S; Krag, David N

    2009-01-01

    This study was focused on developing catalytically active beta-lactamase enzyme molecules that have target-recognizing sites built within their scaffold. Using phage-display approach, nine libraries were constructed by inserting the randomized linear or cysteine-constrained heptapeptides in the five different loops on the outer surface of P99 beta-lactamase molecule. The pIII signal peptide of Sec-pathway was employed for a periplasmic translocation of the beta-lactamase fusion protein, which we found more efficient than the DsbA signal peptide of SRP-pathway. The randomized heptapeptide loops replaced native amino acids between positions (34)Y-(37)K, (238)M-(246)A, (275)N-(280)A, (305)A-(311)S, or (329)I-(334)I of the P99 beta-lactamase molecules for generating the loop-1 to -5 libraries, respectively. The diversity of each loop library was judged by counting the primary and beta-lactamase-active clones. The linear peptide inserts in the loop-2 library showed the maximum number of the beta-lactamase-active clones, followed by the loop-5, loop-3, and loop-4. The insertion of the cysteine-constrained loops exhibited a dramatic loss of the enzyme-active beta-lactamase clones. The complexity of the loop-2 linear library, as determined by the frequency and diversity of amino acid distributions in the randomized region, appears consistent with the standards of other types of phage display library systems. The selection of the loop-2 linear library on streptavidin protein as a test target identified several beta-lactamase clones that specifically bound to streptavidin. In conclusion, this study identified the suitability of the loop-2 of P99 beta-lactamase for constructing a phage-display library of the beta-lactamase enzyme-active molecules that can be selected against a target. This is an enabling step in our long-term goal of developing bifunctional beta-lactamase molecules against cancer-specific targets for enzyme prodrug therapy of cancer.

  16. Dual-therapeutic reporter genes fusion for enhanced cancer gene therapy and imaging.

    PubMed

    Sekar, T V; Foygel, K; Willmann, J K; Paulmurugan, R

    2013-05-01

    Two of the successful gene-directed enzyme prodrug therapies include herpes simplex virus-thymidine kinase (HSV1-TK) enzyme-ganciclovir prodrug and the Escherichia coli nitroreductase (NTR) enzyme-CB1954 prodrug strategies; these enzyme-prodrug combinations produce activated cytotoxic metabolites of the prodrugs capable of tumor cell death by inhibiting DNA synthesis and killing quiescent cells, respectively. Both these strategies also affect significant bystander cell killing of neighboring tumor cells that do not express these enzymes. We have developed a dual-combination gene strategy, where we identified HSV1-TK and NTR fused in a particular orientation can effectively kill tumor cells when the tumor cells are treated with a fusion HSV1-TK-NTR gene- along with a prodrug combination of GCV and CB1954. In order to determine whether the dual-system demonstrate superior therapeutic efficacy than either HSV1-TK or NTR systems alone, we conducted both in vitro and in vivo tumor xenograft studies using triple negative SUM159 breast cancer cells, by evaluating the efficacy of cell death by apoptosis and necrosis upon treatment with the dual HSV1-TK genes-GCV-CB1954 prodrugs system, and compared the efficiency to HSV1-TK-GCV and NTR-CB1954. Our cell-based studies, tumor regression studies in xenograft mice, histological analyses of treated tumors and bystander studies indicate that the dual HSV1-TK-NTR-prodrug system is two times more efficient even with half the doses of both prodrugs than the respective single gene-prodrug system, as evidenced by enhanced apoptosis and necrosis of tumor cells in vitro in culture and xenograft of tumor tissues in animals.

  17. Crystal Structure of the Geobacillus stearothermophilus Carboxylesterase Est55 and Its Activation of Prodrug CPT-11

    PubMed Central

    Liu, Ping; Ewis, Hosam E.; Tai, Phang C.; Lu, Chung-Dar; Weber, Irene T.

    2007-01-01

    Several mammalian carboxylesterases were shown to activate the prodrug irinotecan (CPT-11) to produce SN-38, a topoisomerase inhibitor used in cancer therapy. However, the potential use of bacterial carboxylesterases, which have the advantage of high stability, has not been explored. We present the crystal structure of the carboxyesterase Est55 from Geobacillus stearothermophilus and evaluation of its enzyme activity on CPT-11. Crystal structures were determined at pH 6.2 and 6.8 and resolution of 2.0 and 1.58 Å, respectively. Est55 folds into three domains, a catalytic domain, an α/β domain and a regulatory domain. The structure is in an inactive form; the side chain of His409, one of the catalytic triad residues, is directed away from the other catalytic residues Ser194 and Glu310. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the binding of substrate, suggesting a regulatory role. However, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side chains were favorable, while polar serine was unfavorable for enzyme activity. Est55 was shown to hydrolyze CPT-11 into the active form SN-38. The mutant C408V provided a more stable enzyme for activation of CPT-11. Therefore, engineered thermostable Est55 is a candidate for use with irinotecan in enzyme-prodrug cancer therapy. PMID:17239398

  18. [Treatment of dyslipoproteinemia by systemic enzyme therapy in experimental glomerulonephritis].

    PubMed

    Mukhin, I V

    2002-01-01

    Patients with chronic glomerulonephritis (CG) develop disturbances of lipid blood spectrum leading to additional damage to renal structure. The existent methods of pathogenetic therapy have no effect on lipid imbalance. Recently, many autoimmune diseases have been treated with systemic enzyme therapy (SET). The authors studied SET effect in disturbed lipid metabolism in experimental glomerulonephritis. Experimental animals showed morphological and biochemical changes similar to those in CG of man. SET reduced renal tissue damage and symptoms of dyslipoproteinemia.

  19. Double suicide gene therapy using human neural stem cells against glioblastoma: double safety measures.

    PubMed

    Lee, Ji Yeoun; Lee, Do-Hun; Kim, Hyung A; Choi, Seung-Ah; Lee, Hong Jun; Park, Chul-Kee; Phi, Ji Hoon; Wang, Kyu-Chang; Kim, Seung U; Kim, Seung-Ki

    2014-01-01

    With recent advancements in stem cell-based gene therapy, concerns about safety have grown. Stem cell-based gene therapies may pose the risk of immunological problems and oncogenesis. We investigated the feasibility of treating glioblastomas with neural stem cells [(NSCs), HB1.F3 cells] expressing double prodrug enzymes [cytosine deaminase (CD) and tyrosine kinase (TK)] to eliminate the NSCs following treatment for safety purposes. First, the in vitro and in vivo therapeutic efficacies of NSCs engineered with double prodrug enzymes (HB1.F3-CD.TK cells) were compared to cells expressing a single prodrug enzyme (HB1.F3-CD). Second, the degree of safety achieved by NSC elimination was compared with an in vitro viability assay of the NSCs after treatment with the double prodrugs. We further compared the differences in in vivo proliferation of control, single prodrug enzyme and double prodrug enzyme expressing NSCs. HB1.F3-CD.TK cells showed a better or comparable treatment outcome than HB1.F3-CD cells in vitro and in vivo. For safety, HB1.F3-CD.TK cells showed the least viability in vitro after treatment with prodrugs compared to HB1.F3 and HB1.F3-CD cells. Additionally, the in vivo proliferation among the injected NSCs found in the tumor was the smallest for HB1.F3-CD.TK cells. Double-prodrug enzyme-directed gene therapy shows good therapeutic efficacy as well as efficient eradication of the NSCs to ensure safety for clinical applications of stem cell-based gene therapies.

  20. The phosphorylated prodrug FTY720 is a histone deacetylase inhibitor that reactivates ERα expression and enhances hormonal therapy for breast cancer.

    PubMed

    Hait, N C; Avni, D; Yamada, A; Nagahashi, M; Aoyagi, T; Aoki, H; Dumur, C I; Zelenko, Z; Gallagher, E J; Leroith, D; Milstien, S; Takabe, K; Spiegel, S

    2015-06-08

    Estrogen receptor-α (ERα)-negative breast cancer is clinically aggressive and does not respond to conventional hormonal therapies. Strategies that lead to re-expression of ERα could sensitize ERα-negative breast cancers to selective ER modulators. FTY720 (fingolimod, Gilenya), a sphingosine analog, is the Food and Drug Administration (FDA)-approved prodrug for treatment of multiple sclerosis that also has anticancer actions that are not yet well understood. We found that FTY720 is phosphorylated in breast cancer cells by nuclear sphingosine kinase 2 and accumulates there. Nuclear FTY720-P is a potent inhibitor of class I histone deacetylases (HDACs) that enhances histone acetylations and regulates expression of a restricted set of genes independently of its known effects on canonical signaling through sphingosine-1-phosphate receptors. High-fat diet (HFD) and obesity, which is now endemic, increase breast cancer risk and have been associated with worse prognosis. HFD accelerated the onset of tumors with more advanced lesions and increased triple-negative spontaneous breast tumors and HDAC activity in MMTV-PyMT transgenic mice. Oral administration of clinically relevant doses of FTY720 suppressed development, progression and aggressiveness of spontaneous breast tumors in these mice, reduced HDAC activity and strikingly reversed HFD-induced loss of estrogen and progesterone receptors in advanced carcinoma. In ERα-negative human and murine breast cancer cells, FTY720 reactivated expression of silenced ERα and sensitized them to tamoxifen. Moreover, treatment with FTY720 also re-expressed ERα and increased therapeutic sensitivity of ERα-negative syngeneic breast tumors to tamoxifen in vivo more potently than a known HDAC inhibitor. Our work suggests that a multipronged attack with FTY720 is a novel combination approach for effective treatment of both conventional hormonal therapy-resistant breast cancer and triple-negative breast cancer.

  1. The phosphorylated prodrug FTY720 is a histone deacetylase inhibitor that reactivates ERα expression and enhances hormonal therapy for breast cancer

    PubMed Central

    Hait, N C; Avni, D; Yamada, A; Nagahashi, M; Aoyagi, T; Aoki, H; Dumur, C I; Zelenko, Z; Gallagher, E J; Leroith, D; Milstien, S; Takabe, K; Spiegel, S

    2015-01-01

    Estrogen receptor-α (ERα)-negative breast cancer is clinically aggressive and does not respond to conventional hormonal therapies. Strategies that lead to re-expression of ERα could sensitize ERα-negative breast cancers to selective ER modulators. FTY720 (fingolimod, Gilenya), a sphingosine analog, is the Food and Drug Administration (FDA)-approved prodrug for treatment of multiple sclerosis that also has anticancer actions that are not yet well understood. We found that FTY720 is phosphorylated in breast cancer cells by nuclear sphingosine kinase 2 and accumulates there. Nuclear FTY720-P is a potent inhibitor of class I histone deacetylases (HDACs) that enhances histone acetylations and regulates expression of a restricted set of genes independently of its known effects on canonical signaling through sphingosine-1-phosphate receptors. High-fat diet (HFD) and obesity, which is now endemic, increase breast cancer risk and have been associated with worse prognosis. HFD accelerated the onset of tumors with more advanced lesions and increased triple-negative spontaneous breast tumors and HDAC activity in MMTV-PyMT transgenic mice. Oral administration of clinically relevant doses of FTY720 suppressed development, progression and aggressiveness of spontaneous breast tumors in these mice, reduced HDAC activity and strikingly reversed HFD-induced loss of estrogen and progesterone receptors in advanced carcinoma. In ERα-negative human and murine breast cancer cells, FTY720 reactivated expression of silenced ERα and sensitized them to tamoxifen. Moreover, treatment with FTY720 also re-expressed ERα and increased therapeutic sensitivity of ERα-negative syngeneic breast tumors to tamoxifen in vivo more potently than a known HDAC inhibitor. Our work suggests that a multipronged attack with FTY720 is a novel combination approach for effective treatment of both conventional hormonal therapy-resistant breast cancer and triple-negative breast cancer. PMID:26053034

  2. Enzyme replacement therapy for murine hypophosphatasia.

    PubMed

    Millán, José Luis; Narisawa, Sonoko; Lemire, Isabelle; Loisel, Thomas P; Boileau, Guy; Leonard, Pierre; Gramatikova, Svetlana; Terkeltaub, Robert; Camacho, Nancy Pleshko; McKee, Marc D; Crine, Philippe; Whyte, Michael P

    2008-06-01

    Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6-dependent seizures. There is no established medical treatment. Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one-step purification and a deca-aspartate sequence (D10) for targeting to mineralizing tissue (sALP-FcD10). TNALP-null mice (Akp2-/-), an excellent model for infantile HPP, were treated from birth using sALP-FcD10. Short-term and long-term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP-FcD10 for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP-FcD10 activity, calcium, PPi, and pyridoxal, as well as skeletal and dental manifestations using radiography, microCT, and histomorphometry. Akp2-/- mice receiving high-dose sALP-FcD10 grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PPi, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2-/- mice.

  3. Enzyme replacement and substrate reduction therapy for Gaucher disease.

    PubMed

    Shemesh, Elad; Deroma, Laura; Bembi, Bruno; Deegan, Patrick; Hollak, Carla; Weinreb, Neal J; Cox, Timothy M

    2015-03-27

    Gaucher disease, a rare disorder, is caused by inherited deficiency of the enzyme glucocerebrosidase. It is unique among the ultra-orphan disorders in that four treatments are currently approved by various regulatory authorities for use in routine clinical practice. Hitherto, because of the relatively few people affected worldwide, many of whom started therapy during a prolonged period when there were essentially no alternatives to imiglucerase, these treatments have not been systematically evaluated in studies such as randomized controlled trials now considered necessary to generate the highest level of clinical evidence. To summarize all available randomized controlled study data on the efficacy and safety of enzyme replacement therapies and substrate reduction therapy for treating Gaucher disease. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Inborn Errors of Metabolism Trials Register. Additional searches were conducted on ClinicalTrials.gov for any ongoing studies with potential interim results, and through PubMed. We also searched the reference lists of relevant articles and reviews.Date of last search: 07 August 2014. All randomized and quasi-randomized controlled studies (including open-label studies and cross-over studies) assessing enzyme replacement therapy or substrate reduction therapy, or both, in all types of Gaucher disease were included. Two authors independently assessed the risk of bias in the included studies, and extracted relevant data. Of the 488 studies retrieved by the electronic searches, eight met the inclusion criteria and were analysed (300 participants). Response parameters were restricted to haemoglobin concentration, platelet count, spleen and liver volume and serum biomarkers (chitotriosidase and CCL18). Only one publication reported a 'low risk of bias' score in all parameters assessed, and all studies included were randomized.Four studies reported the responses to enzyme replacement therapy of previously

  4. Activation of multiple chemotherapeutic prodrugs by the natural enzymolome of tumour-localised probiotic bacteria.

    PubMed

    Lehouritis, Panos; Stanton, Michael; McCarthy, Florence O; Jeavons, Matthieu; Tangney, Mark

    2016-01-28

    Some chemotherapeutic drugs (prodrugs) require activation by an enzyme for efficacy. We and others have demonstrated the ability of probiotic bacteria to grow specifically within solid tumours following systemic administration, and we hypothesised that the natural enzymatic activity of these tumour-localised bacteria may be suitable for activation of certain such chemotherapeutic drugs. Several wild-type probiotic bacteria; Escherichia coli Nissle, Bifidobacterium breve, Lactococcus lactis and Lactobacillus species, were screened against a panel of popular prodrugs. All strains were capable of activating at least one prodrug. E. coli Nissle 1917 was selected for further studies because of its ability to activate numerous prodrugs and its resistance to prodrug toxicity. HPLC data confirmed biochemical transformation of prodrugs to their toxic counterparts. Further analysis demonstrated that different enzymes can complement prodrug activation, while simultaneous activation of multiple prodrugs (CB1954, 5-FC, AQ4N and Fludarabine phosphate) by E. coli was confirmed, resulting in significant efficacy improvement. Experiments in mice harbouring murine tumours validated in vitro findings, with significant reduction in tumour growth and increase in survival of mice treated with probiotic bacteria and a combination of prodrugs. These findings demonstrate the ability of probiotic bacteria, without the requirement for genetic modification, to enable high-level activation of multiple prodrugs specifically at the site of action.

  5. Two absorption furosemide prodrugs.

    PubMed

    Mombrú, A W; Mariezcurrena, R A; Suescun, L; Pardo, H; Manta, E; Prandi, C

    1999-03-15

    The structures of two absorption furosemide prodrugs, hexanoyloxymethyl 4-chloro-N-furfuryl-5-sulfamoyl-anthranilate (C19H23CIN2O7S), (I), and benzoyloxymethyl 4-chloro-N-furfuryl-5-sulfamoylanthranilate (C20H17CIN2O7S), (II), are described in this paper and compared with furosemide and four other prodrugs. The molecular conformations of both compounds are similar to those of the other prodrugs; the packing and the crystal system are the primary differences. Compound (I) crystallizes in the trigonal space group R3 and compound (II) in the monoclinic space group P2(1)/n. The packing of both structures is stabilized by a three-dimensional hydrogen-bond network.

  6. Homology modeling of human γ-butyric acid transporters and the binding of pro-drugs 5-aminolevulinic acid and methyl aminolevulinic acid used in photodynamic therapy.

    PubMed

    Baglo, Yan; Gabrielsen, Mari; Sylte, Ingebrigt; Gederaas, Odrun A

    2013-01-01

    Photodynamic therapy (PDT) is a safe and effective method currently used in the treatment of skin cancer. In ALA-based PDT, 5-aminolevulinic acid (ALA), or ALA esters, are used as pro-drugs to induce the formation of the potent photosensitizer protoporphyrin IX (PpIX). Activation of PpIX by light causes the formation of reactive oxygen species (ROS) and toxic responses. Studies have indicated that ALA and its methyl ester (MAL) are taken up into the cells via γ-butyric acid (GABA) transporters (GATs). Uptake via GATs into peripheral sensory nerve endings may also account for one of the few adverse side effects of ALA-based PDT, namely pain. In the present study, homology models of the four human GAT subtypes were constructed using three x-ray crystal structures of the homologous leucine transporter (LeuT) as templates. Binding of the native substrate GABA and the possible substrates ALA and MAL was investigated by molecular docking of the ligands into the central putative substrate binding sites in the outward-occluded GAT models. Electrostatic potentials (ESPs) of the putative substrate translocation pathway of each subtype were calculated using the outward-open and inward-open homology models. Our results suggested that ALA is a substrate of all four GATs and that MAL is a substrate of GAT-2, GAT-3 and BGT-1. The ESP calculations indicated that differences likely exist in the entry pathway of the transporters (i.e. in outward-open conformations). Such differences may be exploited for development of inhibitors that selectively target specific GAT subtypes and the homology models may hence provide tools for design of therapeutic inhibitors that can be used to reduce ALA-induced pain.

  7. Homology Modeling of Human γ-Butyric Acid Transporters and the Binding of Pro-Drugs 5-Aminolevulinic Acid and Methyl Aminolevulinic Acid Used in Photodynamic Therapy

    PubMed Central

    Baglo, Yan; Gabrielsen, Mari; Sylte, Ingebrigt; Gederaas, Odrun A.

    2013-01-01

    Photodynamic therapy (PDT) is a safe and effective method currently used in the treatment of skin cancer. In ALA-based PDT, 5-aminolevulinic acid (ALA), or ALA esters, are used as pro-drugs to induce the formation of the potent photosensitizer protoporphyrin IX (PpIX). Activation of PpIX by light causes the formation of reactive oxygen species (ROS) and toxic responses. Studies have indicated that ALA and its methyl ester (MAL) are taken up into the cells via γ-butyric acid (GABA) transporters (GATs). Uptake via GATs into peripheral sensory nerve endings may also account for one of the few adverse side effects of ALA-based PDT, namely pain. In the present study, homology models of the four human GAT subtypes were constructed using three x-ray crystal structures of the homologous leucine transporter (LeuT) as templates. Binding of the native substrate GABA and the possible substrates ALA and MAL was investigated by molecular docking of the ligands into the central putative substrate binding sites in the outward-occluded GAT models. Electrostatic potentials (ESPs) of the putative substrate translocation pathway of each subtype were calculated using the outward-open and inward-open homology models. Our results suggested that ALA is a substrate of all four GATs and that MAL is a substrate of GAT-2, GAT-3 and BGT-1. The ESP calculations indicated that differences likely exist in the entry pathway of the transporters (i.e. in outward-open conformations). Such differences may be exploited for development of inhibitors that selectively target specific GAT subtypes and the homology models may hence provide tools for design of therapeutic inhibitors that can be used to reduce ALA-induced pain. PMID:23762315

  8. Cytochrome P450-activated prodrugs

    PubMed Central

    Ortiz de Montellano, Paul R

    2013-01-01

    A prodrug is a compound that has negligible, or lower, activity against a specified pharmacological target than one of its major metabolites. Prodrugs can be used to improve drug delivery or pharmacokinetics, to decrease toxicity, or to target the drug to specific cells or tissues. Ester and phosphate hydrolysis are widely used in prodrug design because of their simplicity, but such approaches are relatively ineffective for targeting drugs to specific sites. The activation of prodrugs by the cytochrome P450 system provides a highly versatile approach to prodrug design that is particularly adaptable for targeting drug activation to the liver, to tumors or to hypoxic tissues. PMID:23360144

  9. Prodrugs: design and clinical applications.

    PubMed

    Rautio, Jarkko; Kumpulainen, Hanna; Heimbach, Tycho; Oliyai, Reza; Oh, Dooman; Järvinen, Tomi; Savolainen, Jouko

    2008-03-01

    Prodrugs are bioreversible derivatives of drug molecules that undergo an enzymatic and/or chemical transformation in vivo to release the active parent drug, which can then exert the desired pharmacological effect. In both drug discovery and development, prodrugs have become an established tool for improving physicochemical, biopharmaceutical or pharmacokinetic properties of pharmacologically active agents. About 5-7% of drugs approved worldwide can be classified as prodrugs, and the implementation of a prodrug approach in the early stages of drug discovery is a growing trend. To illustrate the applicability of the prodrug strategy, this article describes the most common functional groups that are amenable to prodrug design, and highlights examples of prodrugs that are either launched or are undergoing human trials.

  10. 10-Boronic acid substituted camptothecin as prodrug of SN-38.

    PubMed

    Wang, Lei; Xie, Shao; Ma, Longjun; Chen, Yi; Lu, Wei

    2016-06-30

    Malignant tumor cells have been found to have high levels of reactive oxygen species such as hydrogen peroxide (H2O2), supporting the hypothesis that a prodrug could be activated by intracellular H2O2 and lead to a potential antitumor therapy. In this study, the 7-ethyl-10-boronic acid camptothecin (B1) was synthesized for the first time as prodrug of SN-38, by linking a cleavable aryl carbon-boron bond to the SN-38. Prodrug B1 selectively activated by H2O2, converted rapidly to the active form SN-38 under favorable oxidative conditions in cancer cells with elevated levels of H2O2. The cell survival assay showed that prodrug B1 was equally or more effective in inhibiting the growth of six different cancer cells, as compared to SN-38. Unexpectedly, prodrug B1 displayed even more potent Topo I inhibitory activity than SN-38, suggesting that it was not only a prodrug of SN-38 but also a typical Topo I inhibitor. Prodrug B1 also demonstrated a significant antitumor activity at 2.0 mg/kg in a xenograft model using human brain star glioblastoma cell lines U87MG.

  11. Development of a novel sulfonate ester-based prodrug strategy.

    PubMed

    Hanaya, Kengo; Yoshioka, Shohei; Ariyasu, Shinya; Aoki, Shin; Shoji, Mitsuru; Sugai, Takeshi

    2016-01-15

    A self-immolative γ-aminopropylsulfonate linker was investigated for use in the development of prodrugs that are reactive to various chemical or biological stimuli. To demonstrate their utility, a leucine-conjugated prodrug of 5-chloroquinolin-8-ol (5-Cl-8-HQ), which is a potent inhibitor against aminopeptidase from Aeromonas proteolytica (AAP), was synthesized. The sulfonate prodrug was considerably stable under physiological conditions, with only enzyme-mediated hydrolysis of leucine triggering the subsequent intramolecular cyclization to simultaneously release 5-Cl-8-HQ and form γ-sultam. It was also confirmed that this γ-aminopropylsulfonate linker was applicable for prodrugs of not only 8-HQ derivatives but also other drugs bearing a phenolic hydroxy group.

  12. Designer Bacteria as Intratumoural Enzyme Biofactories.

    PubMed

    Lehouritis, Panos; Hogan, Glenn; Tangney, Mark

    2017-09-12

    Bacterial-directed enzyme prodrug therapy (BDEPT) is an emerging form of treatment for cancer. It is a biphasic variant of gene therapy in which a bacterium, armed with an enzyme that can convert an inert prodrug into a cytotoxic compound, induces tumour cell death following tumour-specific prodrug activation. BDEPT combines the innate ability of bacteria to selectively proliferate in tumours, with the capacity of prodrugs to undergo contained, compartmentalised conversion into active metabolites in vivo. Although BDEPT has undergone clinical testing, it has received limited clinical exposure, and has yet to achieve regulatory approval. In this article, we review BDEPT from the system designer's perspective, and provide detailed commentary on how the designer should strategize its development de novo. We report on contemporary advancements in this field which aim to enhance BDEPT in terms of safety and efficacy. Finally, we discuss clinical and regulatory barriers facing BDEPT, and propose promising approaches through which these hurdles may best be tackled. Copyright © 2017. Published by Elsevier B.V.

  13. Enzyme-therapy approaches for the treatment of drug overdose and addiction

    PubMed Central

    Zheng, Fang; Zhan, Chang-Guo

    2015-01-01

    “Recent progress in the study of cocaine-metabolizing enzymes demonstrates that enzyme-therapy approaches using appropriately designed enzymes show promise for the treatment of drug overdose and addiction.” PMID:21428822

  14. Bioreductive prodrugs as cancer therapeutics: targeting tumor hypoxia.

    PubMed

    Guise, Christopher P; Mowday, Alexandra M; Ashoorzadeh, Amir; Yuan, Ran; Lin, Wan-Hua; Wu, Dong-Hai; Smaill, Jeff B; Patterson, Adam V; Ding, Ke

    2014-02-01

    Hypoxia, a state of low oxygen, is a common feature of solid tumors and is associated with disease progression as well as resistance to radiotherapy and certain chemotherapeutic drugs. Hypoxic regions in tumors, therefore, represent attractive targets for cancer therapy. To date, five distinct classes of bioreactive prodrugs have been developed to target hypoxic cells in solid tumors. These hypoxia-activated prodrugs, including nitro compounds, N-oxides, quinones, and metal complexes, generally share a common mechanism of activation whereby they are reduced by intracellular oxidoreductases in an oxygen-sensitive manner to form cytotoxins. Several examples including PR-104, TH-302, and EO9 are currently undergoing phase II and phase III clinical evaluation. In this review, we discuss the nature of tumor hypoxia as a therapeutic target, focusing on the development of bioreductive prodrugs. We also describe the current knowledge of how each prodrug class is activated and detail the clinical progress of leading examples.

  15. Design, synthesis, and preliminary pharmacological evaluation of new imidazolinones as L-DOPA prodrugs.

    PubMed

    Giorgioni, Gianfabio; Claudi, Francesco; Ruggieri, Sabrina; Ricciutelli, Massimo; Palmieri, Giovanni F; Di Stefano, Antonio; Sozio, Piera; Cerasa, Laura S; Chiavaroli, Annalisa; Ferrante, Claudio; Orlando, Giustino; Glennon, Richard A

    2010-03-01

    L-DOPA, the immediate biological precursor of dopamine, is still considered the drug of choice in the treatment of Parkinson's disease. However, therapy with L-DOPA is associated with a number of acute problems. With the aim to increase the bioavailability after oral administration, we designed a multi-protected L-DOPA prodrugs able to release the drug by both spontaneous chemical or enzyme catalyzed hydrolysis. The new compounds have been synthesized and preliminarily evaluated for their water solubility, log P, chemical stability, and enzymatic stability. The results indicate that the incorporation of the amino acidic moiety of L-DOPA into an imidazoline-4-one ring provides prodrugs sufficiently stable to potentially cross unchanged the acidic environment of the stomach, and to be absorbed from the intestine. They also might be able to release L-DOPA in human plasma after enzymatic hydrolysis. The ability of prodrugs 6a-b to increase basal levels of striatal DA, and influence brain neurochemistry associated with dopaminergic activity following oral administration, as well as the radical-scavenging activity against DPPH for compounds 6a-b and 15a are also reported.

  16. New Enzyme Prodrug and Methionine-Depletion Combination Therapy of Breast Cancer Designed for Effective Delivery to the Tumor

    DTIC Science & Technology

    2009-10-01

    methylselenol , α-ketobutyrate, and ammonia. Methylselenol has been shown to be cytotoxic to various cancer cells. To accomplish the specific aims...is planned to repeat this test for MDA-MB-231 cells with 750 µM L-methionine. 10 Figure 4: Effect of SeMet conversion to methylselenol ...to methylselenol on MCF-7 breast cancer cells. Cells were grown in medium adjusted to 2000 µM of L-methionine. Cell viability was assessed using

  17. New Enzyme Prodrug and Methionine-Depletion Combination Therapy of Breast Cancer Designed for Effective Delivery to the Tumor

    DTIC Science & Technology

    2010-10-01

    ketobutyrate, and ammonia. It also catalyzes the conversion of selenomethionine (SeMet) to toxic methylselenol , α-ketobutyrate, and ammonia... Methylselenol has been shown to be cytotoxic to various cancer cells. To accomplish the specific aims of this project, the L-methioninase-annexin V...and was relatively small and not statistically significant (p < 0.001). 9 Figure 4: Effect of SeMet conversion to methylselenol

  18. New Enzyme Prodrug and Methionine-Depletion Combination Therapy of Breast Cancer Designed for Effective Delivery to the Tumor

    DTIC Science & Technology

    2011-10-01

    It also catalyzes the conversion of selenomethionine (SeMet) to toxic methylselenol , α-ketobutyrate, and ammonia. Methylselenol has been shown to be...provide strong support for one of the basic ideas for the project, which is that the conversion of the SeMet to methylselenol at the surface of the...oxygen supply of the cancer cells. In addition, the breast cancer cells will be killed by toxic methylselenol being carried across the artery wall by

  19. The delivery and antinociceptive effects of morphine and its ester prodrugs from lipid emulsions.

    PubMed

    Wang, Jhi-Joung; Sung, K C; Yeh, Chih-Hui; Fang, Jia-You

    2008-04-02

    Long-acting analgesia is critical for patients suffering from long-acting pain. The purpose of this study was to develop lipid emulsions as parenteral drug delivery systems for morphine and its ester prodrugs. Morphine prodrugs with various alkyl chain lengths, including morphine propionate (MPR), morphine enanthate (MEN), and morphine decanoate (MDE), were synthesized. The prodrugs were stable against chemical hydrolysis in an aqueous solution, but were quickly hydrolyzed to the parent drug when exposed to esterase and plasma. Lipid emulsions were prepared using phosphatidylethanolamine (PE) as an emulsifier, while squalene was used as an inner oil phase. Drug release was found to be a function of the drug/prodrug lipophilicity, with a lower release rate for more-lipophilic drug/prodrugs. The inclusion of morphine and its prodrugs into lipid emulsions retarded their release. Lipid emulsions, which incorporated cholesterol, generally exhibited a drug/prodrug release comparable to that of emulsions without co-emulsifiers. Pluronic F68 (PF68) further slowed down the release of morphine and its prodrugs from the emulsions. The antinociceptive activity through the parenteral administration of these emulsions was examined using a cold ethanol tail-flick study. Compared with an aqueous solution, a prolonged analgesic duration was detected after application of the drug/prodrug emulsions. Incorporation of co-emulsifiers such as PF68 and cholesterol further increased the duration of action. The combination of prodrug strategy and lipid emulsions may be practically useful for improving analgesic therapy with morphine.

  20. Enzyme therapy for lysosomal acid lipase deficiency in the mouse.

    PubMed

    Du, H; Schiavi, S; Levine, M; Mishra, J; Heur, M; Grabowski, G A

    2001-08-01

    Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of the triglycerides (TG) and cholesteryl esters (CE) delivered to lysosomes. Its deficiency produces two human phenotypes, Wolman disease (WD) and cholesteryl ester storage disease (CESD). A targeted disruption of the LAL locus produced a null (lal( -/-)) mouse model that mimics human WD/CESD. The potential for enzyme therapy was tested using mannose terminated human LAL expressed in Pichia pastoris (phLAL), purified, and administered by tail vein injections to lal( -/-) mice. Mannose receptor (MR)-dependent uptake and lysosomal targeting of phLAL were evidenced ex vivo using competitive assays with MR-positive J774E cells, a murine monocyte/macrophage line, immunofluorescence and western blots. Following (bolus) IV injection, phLAL was detected in Kupffer cells, lung macrophages and intestinal macrophages in lal( -/-) mice. Two-month-old lal( -/-) mice received phLAL (1.5 U/dose) or saline injections once every 3 days for 30 days (10 doses). The treated lal( -/-) mice showed nearly complete resolution of hepatic yellow coloration; hepatic weight decreased by approximately 36% compared to PBS-treated lal( -/-) mice. Histologic analyses of numerous tissues from phLAL-treated mice showed reductions in macrophage lipid storage. TG and cholesterol levels decreased by approximately 50% in liver, 69% in spleen and 50% in small intestine. These studies provide feasibility for LAL enzyme therapy in human WD and CESD.

  1. Pancreatic enzyme replacement therapy for people with cystic fibrosis.

    PubMed

    Somaraju, Usha Rani; Solis-Moya, Arturo

    2014-10-13

    Most people with cystic fibrosis (80% to 90%) need pancreatic enzyme replacement therapy to prevent malnutrition. Enzyme preparations need to be taken whenever food is taken, and the dose needs to be adjusted according to the food consumed. A systematic review on the efficacy and safety of pancreatic enzyme replacement therapy is needed to guide clinical practice, as there is variability between centres with respect to assessment of pancreatic function, time of commencing treatment, dose and choice of supplements. To evaluate the efficacy and safety of pancreatic enzyme replacement therapy in children and adults with cystic fibrosis and to compare the efficacy and safety of different formulations of this therapy and their appropriateness in different age groups. Also, to compare the effects of pancreatic enzyme replacement therapy in cystic fibrosis according to different diagnostic subgroups (e.g. different ages at introduction of therapy and different categories of pancreatic function). We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches and handsearches of relevant journals and abstract books of conference proceedings. Most recent search: 14 August 2014.We also searched an ongoing trials website and the websites of the pharmaceutical companies who manufacture pancreatic enzyme replacements for any additional trials. Most recent search: 12 May 2014. Randomised and quasi-randomised controlled trials in people of any age, with cystic fibrosis and receiving pancreatic enzyme replacement therapy, at any dosage and in any formulation, for a period of not less than four weeks, compared to placebo or other pancreatic enzyme replacement therapy preparations. Two authors independently assessed trials and extracted outcome data. They also assessed the risk of bias of the trials included in the review. One parallel trial and 11 cross-over trials of children and

  2. Pancreatic enzyme replacement therapy for people with cystic fibrosis.

    PubMed

    Somaraju, Usha Rani; Solis-Moya, Arturo

    2016-11-23

    Most people with cystic fibrosis (80% to 90%) need pancreatic enzyme replacement therapy to prevent malnutrition. Enzyme preparations need to be taken whenever food is taken, and the dose needs to be adjusted according to the food consumed. A systematic review on the efficacy and safety of pancreatic enzyme replacement therapy is needed to guide clinical practice, as there is variability between centres with respect to assessment of pancreatic function, time of commencing treatment, dose and choice of supplements. This is an updated version of a published review. To evaluate the efficacy and safety of pancreatic enzyme replacement therapy in children and adults with cystic fibrosis and to compare the efficacy and safety of different formulations of this therapy and their appropriateness in different age groups. Also, to compare the effects of pancreatic enzyme replacement therapy in cystic fibrosis according to different diagnostic subgroups (e.g. different ages at introduction of therapy and different categories of pancreatic function). We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches and handsearches of relevant journals and abstract books of conference proceedings. Most recent search: 15 July 2016.We also searched an ongoing trials website and the websites of the pharmaceutical companies who manufacture pancreatic enzyme replacements for any additional trials. Most recent search: 22 July 2016. Randomised and quasi-randomised controlled trials in people of any age, with cystic fibrosis and receiving pancreatic enzyme replacement therapy, at any dosage and in any formulation, for a period of not less than four weeks, compared to placebo or other pancreatic enzyme replacement therapy preparations. Two authors independently assessed trials and extracted outcome data. They also assessed the risk of bias of the trials included in the review. One

  3. Expression of β-glucuronidase on the surface of bacteria enhances activation of glucuronide prodrugs.

    PubMed

    Cheng, C-M; Chen, F M; Lu, Y-L; Tzou, S-C; Wang, J-Y; Kao, C-H; Liao, K-W; Cheng, T-C; Chuang, C-H; Chen, B-M; Roffler, S; Cheng, T-L

    2013-05-01

    Extracellular activation of hydrophilic glucuronide prodrugs by β-glucuronidase (βG) was examined to increase the therapeutic efficacy of bacteria-directed enzyme prodrug therapy (BDEPT). βG was expressed on the surface of Escherichia coli by fusion to either the bacterial autotransporter protein Adhesin (membrane βG (mβG)/AIDA) or the lipoprotein (lpp) outermembrane protein A (mβG/lpp). Both mβG/AIDA and mβG/lpp were expressed on the bacterial surface, but only mβG/AIDA displayed enzymatic activity. The rate of substrate hydrolysis by mβG/AIDA-BL21cells was 2.6-fold greater than by pβG-BL21 cells, which express periplasmic βG. Human colon cancer HCT116 cells that were incubated with mβG/AIDA-BL21 bacteria were sensitive to a glucuronide prodrug (p-hydroxy aniline mustard β-D-glucuronide, HAMG) with an half maximal inhibitory concentration (IC50) value of 226.53±45.4 μM, similar to the IC50 value of the active drug (p-hydroxy aniline mustard, pHAM; 70.6±6.75 μM), indicating that mβG/AIDA on BL21 bacteria could rapidly and efficiently convert HAMG to an active anticancer agent. These results suggest that surface display of functional βG on bacteria can enhance the hydrolysis of glucuronide prodrugs and may increase the effectiveness of BDEPT.

  4. Hydrolysis-Sensitive Dithiolethione Prodrug Micelles.

    PubMed

    Hasegawa, Urara; Tateishi, Naoya; Uyama, Hiroshi; van der Vlies, André J

    2015-11-01

    Prodrug micelles carrying 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), a compound possessing chemopreventive properties, are prepared from amphiphilic block copolymers linking ADT-OH via an ester bond using glycine (PAM-PGlyADT) and isoleucine linkers (PAM-PIleADT). The release of ADT-OH from the PAM-PIleADT micelles is much slower than the PAM-PGlyADT micelles. The PAM-PGlyADT micelles show comparable toxicity with ADT-OH in different cancer cell lines, whereas the PAM-PIleADT micelles are not toxic up to 400 µM. This ADT-ester prodrug micelle approach enables to modulate the release rate of ADT-OH and thus might find application in cancer therapy and prevention. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Development of cytarabine prodrugs and delivery systems for leukemia treatment.

    PubMed

    Chhikara, Bhupender S; Parang, Keykavous

    2010-12-01

    Cytarabine is a polar nucleoside drug used for the treatment of myeloid leukemia and non-Hodgkin's lymphoma. The drug has a short plasma half-life, low stability and limited bioavailability. Overdosing of patients with continuous infusions may lead to side effects. Thus, various prodrug strategies and delivery systems have been explored extensively to enhance the half-life, stability and delivery of cytarabine. Among the recent cytarabine prodrugs, amino acid conjugate ValCytarabine and fatty acid derivative CP-4055 (in Phase III trials) have been investigated for the treatment of leukemia and solid tumors, respectively. Alternatively, delivery systems of cytarabine have emerged for the treatment of different cancers. The liposomal-cytarabine formulation (DepoCyt®, Pacira Pharmaceuticals Inc., New Jersey, USA) has been approved for the treatment of lymphomatous meningitis. Various prodrug strategies evaluated for cytarabine are discussed. Then, the review summarizes the drug delivery systems that have been used for more effective cancer therapy. This review provides in-depth discussion of the prodrug strategy and delivery systems of cytarabine derivatives for the treatment of cancer. The design of cytarabine prodrugs and delivery systems provides insights for designing the next generation of more effective anticancer agents with enhanced delivery and stability. Strategies on designing cytarabine prodrug and delivery formulations showed great promise in developing effective anticancer agents with better therapeutic profile. Similar studies with other anticancer nucleosides can be an alternative approach to gaining access to more effective anticancer agents.

  6. Stimuli-responsive PEGylated prodrugs for targeted doxorubicin delivery.

    PubMed

    Xu, Minghui; Qian, Junmin; Liu, Xuefeng; Liu, Ting; Wang, Hongjie

    2015-05-01

    In recent years, stimuli-sensitive prodrugs have been extensively studied for the rapid "burst" release of antitumor drugs to enhance chemotherapeutic efficiency. In this study, a novel stimuli-sensitive prodrug containing galactosamine as a targeting moiety, poly(ethylene glycol)-doxorubicin (PEG-DOX) conjugate, was developed for targeting HepG2 human liver cancer cells. To obtain the PEG-DOX conjugate, both galactosamine-decorated poly(ethylene glycol) aldehyde (Gal-PEG-CHO) and methoxy poly(ethylene glycol) aldehyde (mPEG-CHO) were firstly synthesized and functionalized with dithiodipropionate dihydrazide (TPH) through direct reductive amination via Schiff's base formation, and then DOX molecules were chemically conjugated to the hydrazide end groups of TPH-functionalized Gal-/m-PEG chains via pH-sensitive hydrazone linkages. The chemical structures of TPH-functionalized PEG and PEG-DOX prodrug were confirmed by (1)H NMR analysis. The PEG-DOX conjugate could self-assemble into spherical nanomicelles with a mean diameter of 140 nm, as indicated by transmission electron microscopy and dynamic light scattering. The drug loading content and loading efficiency in the prodrug nanomicelles were as high as 20 wt.% and 75 wt.%, respectively. In vitro drug release studies showed that DOX was released rapidly from the prodrug nanomicelles at the intracellular levels of pH and reducing agent. Cellular uptake and MTT experiments demonstrated that the galactosamine-decorated prodrug nanomicelles were more efficiently internalized into HepG2 cells via a receptor-mediated endocytosis process and exhibited a higher toxicity, compared with pristine prodrug nanomicelles. These results suggest that the novel Gal-PEG-DOX prodrug nanomicelles have tremendous potential for targeted liver cancer therapy.

  7. Smart Cu(II)-aptamer complexes based gold nanoplatform for tumor micro-environment triggered programmable intracellular prodrug release, photodynamic treatment and aggregation induced photothermal therapy of hepatocellular carcinoma

    PubMed Central

    Zhang, Da; Zheng, Aixian; Li, Juan; Wu, Ming; Wu, Lingjie; Wei, Zuwu; Liao, Naishun; Zhang, Xiaolong; Cai, Zhixiong; Yang, Huanghao; Liu, Gang; Liu, Xiaolong; Liu, Jingfeng

    2017-01-01

    This study describes smart Cu(II)-aptamer complexes based gold nanoplatform for tumor micro-environment triggered programmable prodrug release, in demand photodynamic therapy and aggregation induced photothermal ablation of hepatocellular carcinoma. The nanoplatform is consist of monodispersed gold nanoparticle (GNP) that is binding to HCC cell specific targeting aptamers (TLS11a) through Au-S bond; the aptamer is labeled with Ce6 at the 5'end and coordinated with Cu(II) through (GA)10 repeating bases to load AQ4N at the 3' end. In normal physiological conditions, the fluorescence and ROS generation ability of Ce6 are quenched by GNPs via RET; but in cancerous cells, the fluorescence and the ROS generation of Ce6 could be recovered by cleavage of Au-S bond through high level of intracellular GSH for real-time imaging and in demand PDT. Meanwhile, the prodrug AQ4N release could be triggered by acid-cleavage of coordination bonds, then accompanied by a release of Cu(II) that would induce the electrostatic aggregation of GNPs for photo-thermal ablation; furthermore, the significantly enhanced chemotherapy efficiency could be achieved by PDT produced hypoxia to convert AQ4N into AQ4. In summary, here described nanoplatform with tumor cell specific responsive properties and programmable PDT/PTT/chemotherapy functions, might be an interesting synergistic strategy for HCC treatment. PMID:28042325

  8. Strategies in the designing of prodrugs, taking into account the antiviral and anticancer compounds.

    PubMed

    Lesniewska-Kowiel, Monika A; Muszalska, Izabela

    2017-03-31

    Prodrugs are a wide group of substances of low or no pharmacological activity. The search for prodrugs is aimed at obtaining drugs characterized by better pharmacokinetic properties, pharmaceutical availability and selective activity of the active substance. Prodrug strategies involve chemical modifications and syntheses of new structures as well as the establishment of systems that deliver active substances for therapeutic aims that is prodrug-based treatments. The paper describes decisive factors in prodrug designing, such as enzymes participating in their activation, concepts of chemical modifications in the group of antiviral drugs and new anticancer treatments based on prodrugs (ADEPT, GDEPT, LEAPT). Prodrugs are seen as a possibility to design medicines which are selective for their therapeutic aim, for example a tumorous cell or a microorganism. Such an approach is possible thanks to the knowledge on: pathogenesis of diseases at molecular level, metabolism of healthy and affected cells as well as metabolism of microorganisms (bacteria, fungi, protozoa, etc.). Many drugs which have been used for years are still studied in relation to their metabolism and their molecular mechanism of operation, providing new knowledge on active substances. Many of them meet the criteria of being a prodrug. The paper indicates methods of discovering new structures or modifications of known structures and their synthesis as well as new therapeutic strategies using prodrugs, which are expected to be successful and to broaden the knowledge on what is happening to the drug in the body, in addition to providing a molecular explanation of xenobiotics activity.

  9. Stereoisomeric Prodrugs to Improve Corneal Absorption of Prednisolone: Synthesis and In Vitro Evaluation.

    PubMed

    Sheng, Ye; Yang, Xiaoyan; Wang, Zhiying; Mitra, Ashim K

    2016-06-01

    A series of stereoisomeric prodrugs have been designed to examine efficacy in generating higher corneal absorption relative to prednisolone. Prodrugs have been studied and identified with LC/MS/MS and NMR analyses. Prodrugs have been characterized for aqueous solubility, buffer stability, and cytotoxicity. Cellular uptake and permeability studies have been conducted across MDCK-MDR1 cells to determine prodrug affinity towards P-glycoprotein (P-gp) and peptide transporters. Enzyme-mediated degradation of prodrugs has been determined using Statens Seruminstitut rabbit cornea (SIRC) cell homogenates. Prodrugs exhibited higher aqueous solubility relative to prednisolone. Prodrugs circumvented P-gp-mediated cellular efflux and were recognized by peptide transporters. Prodrugs (DP, DDP) produced with D-isomers (D-valine) were significantly stable against both chemical and enzymatic hydrolyses. The order of degradation rate constants observed in chemical and enzymatic hydrolyses were in the same order, i.e., L-valine-L-valine-prednisolone (LLP) > L-valine-D-valine-prednisolone (LDP) > D-valine-L-valine-prednisolone (DLP) > D-valine-D-valine-prednisolone (DDP). Results obtained from this study clearly suggest that stereoisomeric prodrug approach is an effective strategy to overcome P-gp-mediated efflux and improve transcorneal permeability of prednisolone following topical administration.

  10. Amino Acid Prodrugs: An Approach to Improve the Absorption of HIV-1 Protease Inhibitor, Lopinavir

    PubMed Central

    Patel, Mitesh; Mandava, Nanda; Gokulgandhi, Mitan; Pal, Dhananjay; Mitra, Ashim K.

    2014-01-01

    Poor systemic concentrations of lopinavir (LPV) following oral administration occur due to high cellular efflux by P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs) and extensive metabolism by CYP3A4 enzymes. In this study, amino acid prodrugs of LPV were designed and investigated for their potential to circumvent efflux processes and first pass effects. Three amino acid prodrugs were synthesized by conjugating isoleucine, tryptophan and methionine to LPV. Prodrug formation was confirmed by the LCMS/MS and NMR technique. Interaction of LPV prodrugs with efflux proteins were carried out in P-gp (MDCK-MDR1) and MRP2 (MDCK-MRP2) transfected cells. Aqueous solubility studies demonstrated that prodrugs generate higher solubility relative to LPV. Prodrugs displayed higher stability under acidic conditions and degraded significantly with rise in pH. Uptake and transport data suggested that prodrugs carry significantly lower affinity towards P-gp and MRP2 relative to LPV. Moreover, prodrugs exhibited higher liver microsomal stability relative to LPV. Hence, amino acid prodrug modification might be a viable approach for enhancing LPV absorption across intestinal epithelial and brain endothelial cells which expresses high levels of P-gp and MRP2. PMID:24727459

  11. Prodrugs of aza nucleosides based on proton transfer reaction

    NASA Astrophysics Data System (ADS)

    Karaman, Rafik

    2010-12-01

    DFT calculation results for intramolecular proton transfer reactions in Kirby's enzyme models 1- 7 reveal that the reaction rate is quite responsive to geometric disposition, especially to distance between the two reactive centers, r GM, and the angle of attack, α (the hydrogen bonding angle). Hence, the study on the systems reported herein could provide a good basis for designing aza nucleoside prodrug systems that are less hydrophilic than their parental drugs and can be used, in different dosage forms, to release the parent drug in a controlled manner. For example, based on the calculated log EM, the cleavage process for prodrug 1ProD is predicted to be about 1010 times faster than that for prodrug 7ProD and about 104 times faster than prodrug 3ProD: rate 1ProD > rate 3ProD > rate 7ProD . Hence, the rate by which the prodrug releases the aza nucleoside drug can be determined according to the structural features of the linker (Kirby's enzyme model).

  12. Prodrug Strategies for Paclitaxel

    PubMed Central

    Meng, Ziyuan; Lv, Quanxia; Lu, Jun; Yao, Houzong; Lv, Xiaoqing; Jiang, Feng; Lu, Aiping; Zhang, Ge

    2016-01-01

    Paclitaxel is an anti-tumor agent with remarkable anti-tumor activity and wide clinical uses. However, it is also faced with various challenges especially for its poor water solubility and low selectivity for the target. To overcome these disadvantages of paclitaxel, approaches using small molecule modifications and macromolecule modifications have been developed by many research groups from all over the world. In this review, we discuss the different strategies especially prodrug strategies that are currently used to make paclitaxel more effective. PMID:27223283

  13. In vitro and in vivo evaluation of a sulfenamide prodrug of basic metformin.

    PubMed

    Huttunen, Kristiina M; Leppänen, Jukka; Vepsäläinen, Jouko; Sirviö, Jouni; Laine, Krista; Rautio, Jarkko

    2012-08-01

    In the present study, a previously described sulfenamide prodrug of a basic antidiabetic drug, metformin, was evaluated further. This sulfenamide prodrug was designed to improve the permeability and consequently the oral absorption and bioavailability (F) of the highly water-soluble metformin. Bioactivation of the prodrug was mediated by reduced glutathione, but it has been reported that sulfenamide prodrugs can also be bioactivated by other endogenous thiols like cysteine, and free thiol-containing proteins. Consistent with earlier findings for a sulfenamide prodrug of a weakly acid drug, linezolid, the permeability studies indicated that the metformin prodrug was also prematurely bioactivated on the apical surface of the Caco-2 cell monolayer. Nevertheless, the bioavailability of metformin was increased by approximately 25% after oral administration of the prodrug in rats, most probably because of better oral absorption. This indicates that the sulfenamide prodrug approach may be used to improve the moderate oral bioavailability of metformin, which may help to decrease the uncomfortable gastrointestinal adverse effects associated with metformin therapy as the daily doses of metformin can be reduced. Furthermore, the present study confirms that the applicability of the sulfenamide prodrug approach can be successfully extended from weak NH acids to very basic guanide-type drugs.

  14. Pharmacological Evaluation and Preliminary Pharmacokinetics Studies of a New Diclofenac Prodrug without Gastric Ulceration Effect

    PubMed Central

    dos Santos, Jean Leandro; Moreira, Vanessa; Campos, Michel Leandro; Chelucci, Rafael Consolin; Barbieri, Karina Pereira; de Castro Souto, Pollyana Cristina Maggio; Matsubara, Márcio Hideki; Teixeira, Catarina; Bosquesi, Priscila Longhin; Peccinini, Rosângela Gonçalves; Chin, Chung Man

    2012-01-01

    Long-term nonsteroidal anti-inflammatory drugs (NSAIDs) therapy has been associated with several adverse effects such as gastric ulceration and cardiovascular events. Among the molecular modifications strategies, the prodrug approach is a useful tool to discover new safe NSAIDs. The 1-(2,6-dichlorophenyl)indolin-2-one is a diclofenac prodrug which demonstrated relevant anti-inflammatory properties without gastro ulceration effect. In addition, the prodrug decreases PGE2 levels, COX-2 expression and cellular influx into peritoneal cavity induced by carrageenan treatment. Preliminary pharmacokinetic studies have shown in vivo bioconversion of prodrug to diclofenac. This prodrug is a new nonulcerogenic NSAID useful to treat inflammatory events by long-term therapy. PMID:23203127

  15. Cytomegalovirus protease targeted prodrug development.

    PubMed

    Sabit, Hairat; Dahan, Arik; Sun, Jing; Provoda, Chester J; Lee, Kyung-Dall; Hilfinger, John H; Amidon, Gordon L

    2013-04-01

    Human cytomegalovirus (HCMV) is a prevalent virus that infects up to 90% of the population. The goal of this research is to determine if small molecular prodrug substrates can be developed for a specific HCMV encoded protease and thus achieve site-specific activation. HCMV encodes a 256 amino acid serine protease that is responsible for capsid assembly, an essential process for herpes virus production. The esterase activity of the more stable HCMV A143T/A144T protease mutant was evaluated with model p-nitrophenol (ONp) esters, Boc-Xaa-ONp (Ala, Leu, Ile, Val, Gln, Phe at the Xaa position). We demonstrate that the A143T/A144T mutant has esterase activity toward specific small ester compounds, e.g., Boc-L-Ala-ONp. Mono amino acid and dipeptide prodrugs of ganciclovir (GCV) were also synthesized and evaluated for hydrolysis by the A143T/A144T protease mutant in solution. Hydrolysis of these prodrugs was also evaluated in Caco-2 cell homogenates, human liver microsomes (HLMs), and rat and human plasma. For the selectivity potential of the prodrugs, the hydrolysis ratio was evaluated as a percentage of prodrug hydrolyzed by the HCMV protease over the percentages of prodrug hydrolyses by Caco-2 cell homogenates, HLMs, and human/rat plasma. A dipeptide prodrug of ganciclovir, Ac-l-Gln-l-Ala-GCV, emerged as a potential selective prodrug candidate. The results of this research demonstrate that targeting prodrugs for activation by a specific protease encoded by the infectious HCMV pathogen may be achievable.

  16. Chemical and enzymatic stability of amino acid prodrugs containing methoxy, ethoxy and propylene glycol linkers.

    PubMed

    Gupta, Deepak; Gupta, Sheeba Varghese; Lee, Kyung-Dall; Amidon, Gordon L

    2009-01-01

    We evaluated the chemical and enzymatic stabilities of prodrugs containing methoxy, ethoxy and propylene glycol linkers in order to find a suitable linker for prodrugs of carboxylic acids with amino acids. l-Valine and l-phenylalanine prodrugs of model compounds (benzoic acid and phenyl acetic acid) containing methoxy, ethoxy and propylene glycol linkers were synthesized. The hydrolysis rate profile of each compound was studied at physiologically relevant pHs (1.2, 4, 6 and 7.4). Enzymatic hydrolysis of propylene glycol containing compounds was studied using Caco-2 homogenate as well as purified enzyme valacyclovirase. It was observed that the stability of the prodrugs increases with the linker length (propyl > ethyl > methyl). The model prodrugs were stable at acidic pH as compared to basic pH. It was observed that the prodrug with the aliphatic amino acid promoiety was more stable compared to its aromatic counterpart. The comparison between benzyl and the phenyl model compounds revealed that the amino acid side chain is significant in determining the stability of the prodrug whereas the benzyl or phenyl carboxylic acid had little or no effect on the stability. The enzymatic activation studies of propylene glycol linker prodrug in the presence of valacyclovirase and cell homogenate showed faster generation of the parent drug at pH 7.4. The half-life of prodrugs at pH 7.4 was more than 12 h, whereas in the presence of cell homogenate the half-lives were less than 1 h. Hydrolysis by Caco-2 homogenate generated the parent compound in two steps, where the prodrug was first converted to the intermediate, propylene glycol benzoate, which was then converted to the parent compound (benzoic acid). Enzymatic hydrolysis of propylene glycol containing prodrugs by valacyclovirase showed hydrolysis of the amino acid ester part to generate the propylene glycol ester of model compound (propylene glycol benzoate) as the major product. The amino acid prodrugs containing methoxy

  17. Enzyme-encapsulated silica nanoparticle for cancer chemotherapy

    NASA Astrophysics Data System (ADS)

    Chiu, Yi-Rong; Ho, Wei-Jen; Chao, Jiun-Shuan; Yuan, Chiun-Jye

    2012-03-01

    A novel horseradish peroxidase-encapsulated silica nanoparticle (SNP) was generated in this study under relatively mild conditions. The generated enzyme-encapsulated SNP were relatively uniform in size (average 70 ± 14.3 nm), monodispersed, and spherical, as characterized by transmission electron microscopy and scanning electron microscopy. The horseradish peroxidase encapsulated in silica nanoparticle exhibits biological properties, such as a pH-dependent activity profile and k m value, similar to that of free enzymes. Furthermore, enzyme-encapsulated SNP exhibited good operational stability for the repetitive usage with a relative standard deviation of 5.1 % ( n = 10) and a high stability for long term storage (>60 days) at 4 °C. The feasibility of using enzyme-encapsulated SNP in prodrug cancer therapy was also demonstrated by its capability to convert the prodrug indole-3-acetic acid into cytotoxic peroxyl radicals and trigger the death of tumor cells. These results indicate that the developed enzyme-encapsulated SNP has potential in the applications of prodrug cancer therapy.

  18. Synthesis of a novel legumain-cleavable colchicine prodrug with cell-specific toxicity.

    PubMed

    Smith, Robert Løvsletten; Åstrand, Ove Alexander Høgmoen; Nguyen, Luan Minh; Elvestrand, Tina; Hagelin, Gunnar; Solberg, Rigmor; Johansen, Harald Thidemann; Rongved, Pål

    2014-07-01

    Conventional chemotherapy has undesirable toxic side-effects to healthy tissues due to low cell selectivity of cytotoxic drugs. One approach to increase the specificity of a cytotoxic drug is to make a less toxic prodrug which becomes activated at the tumour site. The cysteine protease legumain have remarkable restricted substrate specificity and is the only known mammalian asparaginyl (Asn) endopeptidase. Over-expression of legumain is reported in cancers and unstable atherosclerotic plaques, and utilizing legumain is a promising approach to activate prodrugs. In this study we have synthesized the legumain-cleavable peptide sequence N-Boc-Ala-Ala-Asn-Val-OH. The peptide was subsequently conjugated to deacetyl colchicine during three steps to produce Suc-Ala-Ala-Asn-Val-colchicine (prodrug) with >90% chemical purity. Several cell lines with different expressions and activities of legumain were used to evaluate the general toxicity, specificity and efficacy of the microtubule inhibitor colchicine, valyl colchicine and the legumain-cleavable colchicine prodrug. The prodrug was more toxic to the colorectal cancer HCT116 cells (expressing both the 36kDa active and 56kDa proform of legumain) than SW620 cells (only expressing the 56kDa prolegumain) indicating a relationship between toxicity of the prodrug and activity of legumain in the cells. Also, in monoclonal legumain over-expressing HEK293 cells the prodrug toxicity was higher compared to native HEK293 cells. Furthermore, co-administration of the prodrug either with the potent legumain inhibitor cystatin E/M or the endocytosis inhibitor Dyngo-4a inhibited cell death, indicating that the prodrug toxicity was dependent on both asparaginyl endopeptidase activity and endocytosis. This colchicine prodrug adds to a legumain-activated prodrug strategy approach and could possibly be of use both in targeted anticancer and anti-inflammatory therapy.

  19. Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

    PubMed

    Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

    2017-06-16

    Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

  20. Antiviral prodrugs - the development of successful prodrug strategies for antiviral chemotherapy.

    PubMed

    De Clercq, Erik; Field, Hugh J

    2006-01-01

    Following the discovery of the first effective antiviral compound (idoxuridine) in 1959, nucleoside analogues, especially acyclovir (ACV) for the treatment of herpesvirus infections, have dominated antiviral therapy for several decades. However, ACV and similar acyclic nucleosides suffer from low aqueous solubility and low bioavailability following oral administration. Derivatives of acyclic nucleosides, typically esters, were developed to overcome this problem and valaciclovir, the valine ester of ACV, was among the first of a new series of compounds that were readily metabolized upon oral administration to produce the antiviral nucleoside in vivo, thus increasing the bioavailility by several fold. Concurrently, famciclovir was developed as an oral formulation of penciclovir. These antiviral 'prodrugs' thus established a principle that has led to many successful drugs including both nucleoside and nucleotide analogues for the control of several virus infections, notably those caused by herpes-, retro- and hepatitisviruses. This review will chart the origins and development of the most important of the antiviral prodrugs to date.

  1. β-Glucuronidase-responsive prodrugs for selective cancer chemotherapy: an update.

    PubMed

    Tranoy-Opalinski, Isabelle; Legigan, Thibaut; Barat, Romain; Clarhaut, Jonathan; Thomas, Mikaël; Renoux, Brigitte; Papot, Sébastien

    2014-03-03

    The design of novel antitumor agents allowing the destruction of malignant cells while sparing healthy tissues is one of the major challenges in medicinal chemistry. In this context, the use of non-toxic prodrugs programmed to be selectively activated by beta-glucuronidase present at high concentration in the microenvironment of most solid tumors has attracted considerable attention. This review summarizes the major progresses that have been realized in this field over the past ten years. This includes the new prodrugs that have been designed to target a wide variety of anticancer drugs, the prodrugs employed in the course of a combined therapy, the dendritic glucuronide prodrugs and the concept of β-glucuronidase-responsive albumin binding prodrugs. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  2. Development and validation of LC-MS/MS assay for the determination of the prodrug Midodrine and its active metabolite Desglymidodrine in plasma of ascitic patients: Application to individualized therapy and comparative pharmacokinetics.

    PubMed

    Ali, Ahmed A; Al-Ghobashy, Medhat A; Farid, Samar F; Kassem, Mohamed A

    2015-06-01

    Midodrine (MD) is a prodrug that is converted after oral administration to Desglymidodrine (DMD). In this study, an LC-MS/MS assay was developed and validated for investigation of the pharmacokinetics of MD and DMD in non azotemic patients with liver cirrhosis and tense ascites. Results were compared to those noted with healthy volunteers following the adminstration of a single oral dose of MD. Sample preparation was performed by liquid-liquid extraction using t-butyl methyl ether. HPLC separation was carried out using RP C18 column (4.6mm×50mm, 5μm). Isocratic elution was performed using methanol:0.2% formic acid (70:30, v/v) as the mobile phase, at a flow rate of 0.7mL/min. Tandem mass spectrometric detection was employed at positive electrospray ionization in MRM mode for the determination of MD and DMD. Analysis was carried out within 1.0min over a concentration range of 0.50-40.00ng/mL for the prodrug and its active metabolite. The assay was validated according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained. The applicability of the assay for the determination of the pharmacokinetic parameters of MD and DMD and personalized therapy was demonstrated in healthy volunteers and ascitic patients. Results revealed significant differences in pharmacokinetic parameters among the studied groups. Such differences were explained on the basis of the medical condition and co-adminstered medications exerting possible drug-drug interaction. Results confirmed the need for implementation of reliable analysis tools for therapeutic dose adjustment. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Development of macromolecular prodrug for rheumatoid arthritis☆

    PubMed Central

    Yuan, Fang; Quan, Ling-dong; Cui, Liao; Goldring, Steven R.; Wang, Dong

    2012-01-01

    Rheumatoid arthritis (RA) is a chronic autoimmune disease that is considered to be one of the major public health problems worldwide. The development of therapies that target tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and co-stimulatory pathways that regulate the immune system have revolutionized the care of patients with RA. Despite these advances, many patients continue to experience symptomatic and functional impairment. To address this issue, more recent therapies that have been developed are designed to target intracellular signaling pathways involved in immunoregulation. Though this approach has been encouraging, there have been major challenges with respect to off-target organ side effects and systemic toxicities related to the widespread distribution of these signaling pathways in multiple cell types and tissues. These limitations have led to an increasing interest in the development of strategies for the macromolecularization of anti-rheumatic drugs, which could target them to the inflamed joints. This approach enhances the efficacy of the therapeutic agent with respect to synovial inflammation, while markedly reducing non-target organ adverse side effects. In this manuscript, we provide a comprehensive overview of the rational design and optimization of macromolecular prodrugs for treatment of RA. The superior and the sustained efficacy of the prodrug may be partially attributed to their Extravasation through Leaky Vasculature and subsequent Inflammatory cell-mediated Sequestration (ELVIS) in the arthritic joints. This biologic process provides a plausible mechanism, by which macromolecular prodrugs preferentially target arthritic joints and illustrates the potential benefits of applying this therapeutic strategy to the treatment of other inflammatory diseases. PMID:22433784

  4. [Systemic enzyme therapy in the treatment of recurrent vulvovaginal candidiasis].

    PubMed

    Unzeitig, V; Dvořák, V; Hlaváčková, O; Malík, T; Nový, J; Stará, A; Skřivánek, A; Stěpán, J

    2013-04-01

    Phase I: A prospective evaluation of long-term systemic enzyme therapy (Wobenzym; WE) effects on the frequency of recurrent vulvovaginal candidiasis (RVVC) episodes. Phase II: A retrospective analysis of possible positive effects remaining in the next 3 years. Original work - pilot project consisting of prospective phase I and retrospective phase II trials. Composition of animal and plant proteo-lytic enzymes (WE) is a common component in the treatment of chronic or recurrent inflammatory diseases and has been also shown to have immunomodulatory effects. Project involving 7 gynecology practices has been started in 2005 - 2007 to evaluate the effectiveness of WE in the complex treatment of RVVC. The trial involved 62 women with at least 4 microscopically confirmed episodes of RVVC in the last 12 months (year 1; 4-9 episodes, mean 4.4 episodes per patient-year). From the beginning of the trial, participants took WE in the dose of 2× 8 tbl/day for 10 weeks and were monitored for 12 months (year +1). All infections of RVVC were treated according to usual practice of the particular gynecologist. The number of RVVC episodes during the year -1 was compared to the number of RVVC during the year +1. To evaluate possible long-term effects of the WE treatment, retrospective analysis of the data from 3 years following the phase I (year +2, +3, +4) was performed. Complete data for 54 women were collected (87.1% of the former group of patients). All data were processed with regular statistics methods. Mean number of RVVC in the year +1 has decreased from 4.4 to 0.5 per patient-year (i.e. by 88.5%; p < 0.001). All women experienced an improvement, 63% of them experienced no recurrence. The lower incidence of RVVC remained also for the phase II (year +2: 0.91; year +3: 0.57; year +4: 0.52 episodes of RVVC per patient-year). The difference, as compared to the mean incidence of RVVC before the treatment (year -1), remains significant (p < 0.001) although women, who became pregnant

  5. Angiotensin-Converting Enzyme Inhibitors' Influence on Antiplatelet Therapy of Clopidogrel in ACS.

    PubMed

    Yang, Shuo; Cui, Chanjuan; Zhang, Jie; Qiao, Rui

    2016-10-01

    Clopidogrel is a prodrug, the minority of which is converted to an active metabolite by hepatic cytochrome P450 (CYP2C19), however, most of it is metabolized to inactive substance by hepatic carboxylesterase1 (CES1). Meanwhile angiotensin-converting enzyme inhibitors (ACEIs) are mostly metabolized by CES1. We aimed to assess the impact of ACEIs on platelet inhibition by clopidogrel. We genotyped variants CES1, CYP2C19*2 and *3 in 502 patients with acute coronary syndrome (ACS) receiving clopidogrel therapy, and analyzed the effects of ACEIs on responsiveness to clopidogrel by the vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay and ADP-stimulated impedance whole blood platelet aggregation assay. It showed that the allele frequency of CES1 c.428A was 0% in these patients. 45.22% (227/502) of these patients were carriers of CYP2C19*2 or CYP2C9*3 loss-of-function alleles. Among them, 57.71% (131/227) of the patients with CYP2C19 variants received ACEIs therapy. In a total of 502 patients, there was no difference in the VASP-PRI or the impedance whole blood platelet aggregation assay between the ACEIs group and non-ACEIs group [56.26 ± 14.55% versus 57.76 ± 13.56%, p = 0.241; 0 (0 - 2) Ω vs. 0 (0 - 2) Ω, p = 0.856]. In the CYP2C19 variant patients, there was no difference in the VASP-PRI or the impedance whole blood platelet aggregation assay between ACEIs group and non-ACEIs group [57.24 ± 15.12% versus 58.07 ± 13.90%, p = 0.667; 0 (0 - 2) Ω versus 0 (0 - 2) Ω, p = 0.536]. In the subgroups of ACS patients (unstable angina, non-ST-segment elevation myocardial infarction, ST-segment elevation myocardial infarction), there was no difference in the VASP-PRI between the ACEIs group and non-ACEIs group [55.81 ± 15.24% versus 58.37 ± 13.31%, p = 0.103; 55.76 ± 15.20% versus 49.09 ± 15.22%, p = 0.098; 58.13 ± 11.48% versus 61.87 ± 10.34%, p = 0.221], and there was no difference in the impedance whole blood platelet aggregation assay between

  6. [The enzyme therapy within a complex treatment of hemophthalmos in patients with diabetes mellitus].

    PubMed

    Polunin, G S; Andzhelova, D V; Kiseleva, T N

    2004-01-01

    The efficiency of the enzyme therapy was studied in patients with hemophthalmos concurrent with diabetes mellitus of different types. Sixty-eight patients received parabulbar injections of the hemase and collelizin enzymes and another 25 patients underwent the routine therapy comprising injections of dicynone, emoxipin and heparinum. The method of ultrasound B-scanning was used to determine the area of hemorrhage spread into the vitreous body. A high efficiency of the enzyme therapy, as used within the complex treatment of hemophthalmos in patients with diabetes mellitus, was demonstrated. Ultrasound B-scanning is a highly informative technique in evaluating the enzyme-therapy efficiency. The enzyme therapy can be recommended as the most effective method for the treatment of hemophthalmos in patients with diabetes mellitus.

  7. Computer-assisted design for paracetamol masking bitter taste prodrugs.

    PubMed

    Hejaz, Hatem; Karaman, Rafik; Khamis, Mustafa

    2012-01-01

    It is believed that the bitter taste of paracetamol, a pain killer drug, is due to its hydroxyl group. Hence, it is expected that blocking the hydroxy group with a suitable linker could inhibit the interaction of paracetamol with its bitter taste receptor/s and hence masking its bitterness. Using DFT theoretical calculations we calculated proton transfers in ten different Kirby's enzyme models, 1-10. The calculation results revealed that the reaction rate is linearly correlated with the distance between the two reactive centers (r(GM)) and the angle of the hydrogen bonding (α) formed along the reaction pathway. Based on these results three novel tasteless paracetamol prodrugs were designed and the thermodynamic and kinetic parameters for their proton transfers were calculated. Based on the experimental t(1/2) (the time needed for the conversion of 50% of the reactants to products) and EM (effective molarity) values for processes 1-10 we have calculated the t(1/2) values for the conversion of the three prodrugs to the parental drug, paracetamol. The calculated t(1/2) values for ProD 1-3 were found to be 21.3 hours, 4.7 hours and 8 minutes, respectively. Thus, the rate by which the paracetamol prodrug undergoes cleavage to release paracetamol can be determined according to the nature of the linker of the prodrug (Kirby's enzyme model 1-10). Further, blocking the phenolic hydroxyl group by a linker moiety is believed to hinder the paracetamol bitterness.

  8. Improved peptide prodrugs of 5-ALA for PDT: rationalization of cellular accumulation and protoporphyrin IX production by direct determination of cellular prodrug uptake and prodrug metabolization.

    PubMed

    Giuntini, Francesca; Bourré, Ludovic; MacRobert, Alexander J; Wilson, Michael; Eggleston, Ian M

    2009-07-09

    Twenty-seven dipeptide derivatives of general structure Ac-Xaa-ALA-OR were synthesized as potential prodrugs for 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT). Xaa is an alpha-amino acid, chosen to provide a prodrug with appropriately tailored lipophilicity and water solubility. Although no simple correlation is observed between downstream production of protoporphyrin IX (PpIX) in PAM212 keratinocytes and HPLC-derived descriptors of compound lipophilicity, quantification of prodrug uptake reveals that most of the dipeptides are actually more efficiently accumulated than ALA in PAM212 and also A549 and Caco-2 cell lines. Subsequent ALA release is the limiting factor, which emphasizes the importance of decoupling prodrug uptake and intracellular metabolization when assessing the efficacy of ALA derivatives for PDT. In agreement with PpIX fluorescence studies, at a concentration of 0.1 mM, l-Phe derivatives 4m and 4o, and l-Leu, l-Met, and l-Glu derivatives 4f, 4k, and 4u, exhibit significantly enhanced photoxicity in PAM212 cells compared to ALA.

  9. Prodrugs--from serendipity to rational design.

    PubMed

    Huttunen, Kristiina M; Raunio, Hannu; Rautio, Jarkko

    2011-09-01

    The prodrug concept has been used to improve undesirable properties of drugs since the late 19th century, although it was only at the end of the 1950s that the actual term prodrug was introduced for the first time. Prodrugs are inactive, bioreversible derivatives of active drug molecules that must undergo an enzymatic and/or chemical transformation in vivo to release the active parent drug, which can then elicit its desired pharmacological effect in the body. In most cases, prodrugs are simple chemical derivatives that are only one or two chemical or enzymatic steps away from the active parent drug. However, some prodrugs lack an obvious carrier or promoiety but instead result from a molecular modification of the prodrug itself, which generates a new active compound. Numerous prodrugs designed to overcome formulation, delivery, and toxicity barriers to drug utilization have reached the market. In fact, approximately 20% of all small molecular drugs approved during the period 2000 to 2008 were prodrugs. Although the development of a prodrug can be very challenging, the prodrug approach represents a feasible way to improve the erratic properties of investigational drugs or drugs already on the market. This review introduces in depth the rationale behind the use of the prodrug approach from past to present, and also considers the possible problems that can arise from inadequate activation of prodrugs.

  10. Prodrugs for the treatment of neglected diseases.

    PubMed

    Chung, Man Chin; Ferreira, Elizabeth Igne; Santos, Jean Leandro; Giarolla, Jeanine; Rando, Daniela Gonçales; Almeida, Adélia Emília; Bosquesi, Priscila Longhin; Menegon, Renato Farina; Blau, Lorena

    2007-03-19

    Recently, World Health Organization (WHO) and Medicins San Frontieres (MSF) proposed a classification of diseases as global, neglected and extremely neglected. Global diseases, such as cancer, cardiovascular and mental (CNS) diseases represent the targets of the majority of the R&D efforts of pharmaceutical companies. Neglected diseases affect millions of people in the world yet existing drug therapy is limited and often inappropriate. Furthermore, extremely neglected diseases affect people living under miserable conditions who barely have access to the bare necessities for survival. Most of these diseases are excluded from the goals of the R&D programs in the pharmaceutical industry and therefore fall outside the pharmaceutical market. About 14 million people,mainly in developing countries, die each year from infectious diseases. From 1975 to 1999,1393 new drugs were approved yet only 1% were for the treatment of neglected diseases[3]. These numbers have not changed until now, so in those countries there is an urgent need for the design and synthesis of new drugs and in this area the prodrug approach is a very interesting field. It provides, among other effects, activity improvements and toxicity decreases for current and new drugs, improving market availability. It is worth noting that it is essential in drug design to save time and money, and prodrug approaches can be considered of high interest in this respect. The present review covers 20 years of research on the design of prodrugs for the treatment of neglected and extremely neglected diseases such as Chagas' disease (American trypanosomiasis), sleeping sickness (African trypanosomiasis), malaria, sickle cell disease, tuberculosis, leishmaniasis and schistosomiasis.

  11. Progress and problems with the use of suicide genes for targeted cancer therapy.

    PubMed

    Karjoo, Zahra; Chen, Xuguang; Hatefi, Arash

    2016-04-01

    Among various gene therapy methods for cancer, suicide gene therapy attracts a special attention because it allows selective conversion of non-toxic compounds into cytotoxic drugs inside cancer cells. As a result, therapeutic index can be increased significantly by introducing high concentrations of cytotoxic molecules to the tumor environment while minimizing impact on normal tissues. Despite significant success at the preclinical level, no cancer suicide gene therapy protocol has delivered the desirable clinical significance yet. This review gives a critical look at the six main enzyme/prodrug systems that are used in suicide gene therapy of cancer and familiarizes readers with the state-of-the-art research and practices in this field. For each enzyme/prodrug system, the mechanisms of action, protein engineering strategies to enhance enzyme stability/affinity and chemical modification techniques to increase prodrug kinetics and potency are discussed. In each category, major clinical trials that have been performed in the past decade with each enzyme/prodrug system are discussed to highlight the progress to date. Finally, shortcomings are underlined and areas that need improvement in order to produce clinical significance are delineated. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Pancreatic enzyme replacement therapy for pancreatic exocrine insufficiency in the 21st century

    PubMed Central

    Trang, Tony; Chan, Johanna; Graham, David Y

    2014-01-01

    Restitution of normal fat absorption in exocrine pancreatic insufficiency remains an elusive goal. Although many patients achieve satisfactory clinical results with enzyme therapy, few experience normalization of fat absorption, and many, if not most, will require individualized therapy. Increasing the quantity of lipase administered rarely eliminates steatorrhea but increases the cost of therapy. Enteric coated enzyme microbead formulations tend to separate from nutrients in the stomach precluding coordinated emptying of enzymes and nutrients. Unprotected enzymes mix well and empty with nutrients but are inactivated at pH 4 or below. We describe approaches for improving the results of enzyme therapy including changing to, or adding, a different product, adding non-enteric coated enzymes, (e.g., giving unprotected enzymes at the start of the meal and acid-protected formulations later), use of antisecretory drugs and/or antacids, and changing the timing of enzyme administration. Because considerable lipid is emptied in the first postprandial hour, it is prudent to start therapy with enteric coated microbead prior to the meal so that some enzymes are available during that first hour. Patients with hyperacidity may benefit from adjuvant antisecretory therapy to reduce the duodenal acid load and possibly also sodium bicarbonate to prevent duodenal acidity. Comparative studies of clinical effectiveness of different formulations as well as the characteristics of dispersion, emptying, and dissolution of enteric-coated microspheres of different diameter and density are needed; many such studies have been completed but not yet made public. We discuss the history of pancreatic enzyme therapy and describe current use of modern preparations, approaches to overcoming unsatisfactory clinical responses, as well as studies needed to be able to provide reliably effective therapy. PMID:25206255

  13. [Systemic enzyme therapy in the treatment of supracondylar fractures of the humerus in children].

    PubMed

    Gál, P; Tecl, F; Skotáková, J; Mach, V

    1998-12-01

    The authors present their experience with enzyme therapy--the preparation Wobenzym--in comprehensive treatment of supracondylar fractures of the humerus in children. On monitoring the condition of the extremity by Doppler ultrasound, where the flow through the radial artery was quantified they obtained better results in the group of patients treated by systemic enzyme therapy than in the control group. Systemic enzyme therapy is recommended as a suitable supplement in comprehensive treatment where the most important part is played by correct and early treatment along with precise monitoring of the extremity during the postoperative course.

  14. [Enzyme therapy in treatment of mastopathy. A randomized double-blind clinical study].

    PubMed

    Rammer, E; Friedrich, F

    1996-01-01

    In this randomized double-blind clinical study the efficacy of an enzyme preparation (Wobenzym) was compared with hormone therapy (Lynestrenol) in 29 women with mastopathy. There was a significantly greater decrease in number of hardenings of the mammary gland after 2 months of enzyme therapy than Lynestrenol therapy: improvement in the former group was 100%, in the latter group 78.6%. No significant difference was observed regarding the numbers of lumps, or number and size of cysts, sensitivity to touch, feeling of tension, spontaneous pain, and pain on pressure. The efficacy of both medicines is valued as good. Wobenzym therapy was tolerated very well. No side effects appeared at all. Enzyme therapy is an alternative, low-risk therapy for the management of mastopathy, which does not interfere with the already upset hormonal balance of the patients.

  15. Computationally-designed phenylephrine prodrugs - a model for enhancing bioavailability

    NASA Astrophysics Data System (ADS)

    Karaman, Rafik; Karaman, Donia; Zeiadeh, Isra'

    2013-11-01

    DFT calculations at B3LYP 6-31G (d,p) for intramolecular proton transfer in a number of Kirby's enzyme models demonstrated that the driving force for the proton transfer efficiency is the distance between the two reactive centres (rGM) and the attack angle (α); and the rate of the reaction is linearly correlated with rGM2 and sin (180°- α). Based on these results three phenylephrine prodrugs were designed to provide phenylephrine with higher bioavailability than their parent drug. Using the experimental t1/2 (the time needed for the conversion of 50% of the reactants to products) and EM (effective molarity) values for these processes the t1/2 values for the conversion of the three prodrugs to the parent drug, phenylephrine were calculated. The calculated t1/2 values for ProD 1 and ProD 2 were very high (145 days and several years, respectively) whereas that of ProD 3 was found to be about 35 hours. Therefore, the intra-conversion rates of the phenylephrine prodrugs to phenylephrine can be programmed according to the nature of the prodrug linker.

  16. Duocarmycin-based prodrugs for cancer prodrug monotherapy.

    PubMed

    Tietze, Lutz F; Schuster, Heiko J; Schmuck, Kianga; Schuberth, Ingrid; Alves, Frauke

    2008-06-15

    The synthesis and biological evaluation of novel prodrugs based on the cytotoxic antibiotic duocarmycin SA (1) for a selective treatment of cancer using a prodrug monotherapy (PMT) are described. Transformation of the phenol 8 with the glucuronic acid benzyl ester trichloroacetimidate 9b followed by reaction with DMAI x HCl (10) gives the glucuronide 11b, which is deprotected to afford the desired prodrug 4a containing a glucuronic acid moiety. In addition, the prodrug 4b with a glucuronic methyl ester unit is prepared. The cytotoxicity of the glucuronides is determined using a HTCFA-assay with IC(50) values of 610 nM for 4a and 3300 nM for 4b. In the presence of beta-glucuronidase, 4a expresses an IC(50) value of 0.9 nM and 4b of 2.1 nM resulting in QIC(50) values of about 700 for 4a and 1600 for 4b.

  17. TH-302, a hypoxia-activated prodrug with broad in vivo preclinical combination therapy efficacy: optimization of dosing regimens and schedules

    PubMed Central

    Liu, Qian; Sun, Jessica D.; Wang, Jingli; Ahluwalia, Dharmendra; Baker, Amanda F.; Cranmer, Lee D.; Ferraro, Damien; Wang, Yan; Duan, Jian-Xin; Ammons, W. Steve; Curd, John G.; Matteucci, Mark D.

    2014-01-01

    Purpose Subregional hypoxia is a common feature of tumors and is recognized as a limiting factor for the success of radiotherapy and chemotherapy. TH-302, a hypoxia-activated prodrug selectively targeting hypoxic regions of solid tumors, delivers a cytotoxic warhead to the tumor, while maintaining relatively low systemic toxicity. The antitumor activity, different dosing sequences, and dosing regimens of TH-302 in combination with commonly used conventional chemotherapeutics were investigated in human tumor xenograft models. Methods Seven chemotherapeutic drugs (docetaxel, cisplatin, pemetrexed, irinotecan, doxorubicin, gemcitabine, and temozolomide) were tested in combination with TH-302 in eleven human xenograft models, including non-small cell lung cancer (NSCLC), colon cancer, prostate cancer, fibrosarcoma, melanoma, and pancreatic cancer. Results The antitumor activity of docetaxel, cisplatin, pemetrexed, irinotecan, doxorubicin, gemcitabine, and temozolomide was increased when combined with TH-302 in nine out of eleven models tested. Administration of TH-302 2–8 h prior to the other chemotherapeutics yielded superior efficacy versus other sequences tested. Simultaneous administration of TH-302 and chemotherapeutics increased toxicity versus schedules with dosing separations. In a dosing optimization study, TH-302 administered daily at 50 mg/kg intraperitoneally for 5 days per week in the H460 NSCLC model showed the optimal response with minimal toxicity. Conclusions TH-302 enhances the activity of a wide range of conventional anti-neoplastic agents in a broad panel of in vivo xenograft models. These data highlight in vivo effects of schedule and order of drug administration in regimen efficacy and toxicity and have relevance to the design of human regimens incorporating TH-302. PMID:22382881

  18. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  19. [The pathogenetic basis for and clinical use of systemic enzyme therapy in traumatology and orthopedics].

    PubMed

    Neverov, V A; Klimov, A V

    1999-01-01

    The authors have generalized their experiences with using "Wobenzym" in 140 patients. Schemes of the effective systemic enzyme therapy are proposed for patients of the orthopedic-traumatological profile.

  20. Musings on genome medicine: enzyme-replacement therapy of the lysosomal storage diseases

    PubMed Central

    2009-01-01

    The lysosomal storage diseases, such as Gaucher's disease, mucopolysaccharidosis I, II and IV, Fabry's disease, and Pompe's disease, are rare inherited disorders whose symptoms result from enzyme deficiency causing lysosomal accumulation. Until effective gene-replacement therapy is developed, expensive, and at best incomplete, enzyme-replacement therapy is the only hope for sufferers of rare lysosomal storage diseases. Preventive strategies involving carrier detection should be a priority toward the successful management of these conditions. PMID:20017892

  1. GSH-Activated NIR Fluorescent Prodrug for Podophyllotoxin Delivery.

    PubMed

    Liu, Yajing; Zhu, Shaojia; Gu, Kaizhi; Guo, Zhiqian; Huang, Xiaoyu; Wang, Mingwei; Amin, Hesham M; Zhu, Weihong; Shi, Ping

    2017-09-06

    Theranostic prodrug therapy enables the targeted delivery of anticancer drugs with minimized adverse effects and real-time in situ monitoring of activation of the prodrugs. In this work, we report the synthesis and biological assessment of the near-infrared (NIR) prodrug DCM-S-PPT and its amphiphilic copolymer (mPEG-DSPE)-encapsulated nanoparticles. DCM-S-PPT is composed of podophyllotoxin (PPT) as the anticancer moiety and a dicyanomethylene-4H-pyran (DCM) derivative as the NIR fluorescent reporter, which are linked by a thiol-specific cleavable disulfide bond. In vitro experiments indicated that DCM-S-PPT has low cytotoxicity and that glutathione (GSH) can activate DCM-S-PPT resulting in PPT release and a concomitant significant enhancement in NIR fluorescence at 665 nm. After being intravenously injected into tumor-bearing nude mice, DCM-S-PPT exhibited excellent tumor-activated performance. Furthermore, we have demonstrated that mPEG-DSPE as a nanocarrier loaded with DCM-S-PPT (mPEG-DSPE/DCM-S-PPT) showed even greater tumor-targeting performance than DCM-S-PPT on account of the enhanced permeability and retention effect. Its tumor-targeting ability and specific drug release in tumors make DCM-S-PPT a promising prodrug that could provide a significant strategy for theranostic drug delivery systems.

  2. [Enzyme therapy in the treatment of lymphedema in the arm after breast carcinoma surgery].

    PubMed

    Adámek, J; Prausová, J; Wald, M

    1997-04-01

    The authors evaluate the contribution of proteolytic enzymes used in the treatment of the lymphatic oedema of the arm after mastectomy and radiotherapy for breast cancer. Proteolytic enzymes were successfully administered in monotherapy of lymphatic oedema as well as supportive therapy in other therapeutically ways.

  3. A biochemical and physicochemical comparison of two recombinant enzymes used for enzyme replacement therapies of hunter syndrome.

    PubMed

    Chung, Yo Kyung; Sohn, Young Bae; Sohn, Jong Mun; Lee, Jieun; Chang, Mi Sun; Kwun, Younghee; Kim, Chi Hwa; Lee, Jin Young; Yook, Yeon Joo; Ko, Ah-Ra; Jin, Dong-Kyu

    2014-05-01

    Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs). For enzyme replacement therapy (ERT) of Hunter syndrome, two recombinant enzymes, idursulfase (Elaprase(®), Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Hunterase(®), Green Cross Corporation, Yongin, Korea), are currently available in Korea. To compare the biochemical and physicochemical differences between idursulfase and idursulfase beta, we examined the formylglycine (FGly) content, specific enzyme activity, mannose-6-phosphate (M6P) content, sialic acid content, and in vitro cell uptake activity of normal human fibroblasts of these two enzymes.The FGly content, which determines the enzyme activity, of idursulfase beta was significantly higher than that of idursulfase (79.4 ± 0.9 vs. 68.1 ± 2.2 %, P < 0.001). In accordance with the FGly content, the specific enzyme activity of idursulfase beta was significantly higher than that of idursulfase (42.6 ± 1.1 vs. 27.8 ± 0.9 nmol/min/μg protein, P < 0.001). The levels of M6P and sialic acid were not significantly different (2.4 ± 0.1 vs 2.4 ± 0.3 mol/mol protein for M6P and 12.3 ± 0.7 vs. 12.4 ± 0.4 mol/mol protein for sialic acid). However, the cellular uptake activity of the normal human fibroblasts in vitro showed a significant difference (Kuptake, 5.09 ± 0.96 vs. 6.50 ± 1.28 nM protein, P = 0.017).In conclusion, idursulfase beta exhibited significantly higher specific enzyme activity than idursulfase, resulting from higher FGly content. These biochemical differences may be partly attributed to clinical efficacy. However, long-term clinical evaluations of Hunter syndrome patients treated with these two enzymes will be needed to demonstrate the clinical implications of significant difference of the enzyme activity and the FGly content.

  4. Crystallization and preliminary X-ray characterization of the Bacillus amyloliquefaciens YwrO enzyme

    SciTech Connect

    AbuKhader, Majed M.; Heap, John; De Matteis, Cristina I.; Doughty, Stephen W.; Minton, Nigel; Paoli, Max

    2007-09-01

    The novel bacterial nitroreductase YwrO from B. amyloliquefaciens has been crystallized in two different crystal forms. An initial molecular-replacement solution has been obtained using the mammalian NQO2 structure. CB1954 is an anticancer prodrug that is currently in clinical trials coupled with the Escherichia coli flavoenzyme nitroreductase (NTR) for use in directed-enzyme prodrug therapy (DEPT). The NTR enzyme is responsible for the conversion of the prodrug into a cytotoxic agent. The bifunctional alkylating agent produced by this bioactivation process leads to DNA damage and death of cancer cells. Recently, a novel flavoenzyme from Bacillus amyloliquefaciens, YwrO (Bam YwrO), was reported to be able to reduce CB1954 from its noncytotoxic form into its active form. The crystallization and preliminary X-ray diffraction analysis of two crystal forms of Bam YwrO are reported. The first crystal form is orthorhombic, with space group P22{sub 1}2{sub 1}, and diffracts X-rays to 2.18 Å resolution. The second crystal form is tetragonal, with space group P4{sub 1}, and diffracts X-rays to 3.4 Å. Determination of the Bam YwrO crystal structure will provide an understanding of the molecular recognition between this enzyme and the anticancer prodrug CB1954.

  5. [Basic studies on enzyme therapy of immune complex diseases].

    PubMed

    Steffen, C; Menzel, J

    1985-04-12

    Several in vitro investigations and animal experiments are described which may be used as experimental basis for the enzymatic treatment of rheumatoid arthritis and, possibly, also other immune complex diseases. Demonstration of absorption of unaltered orally-administered radiolabelled enzymes is shown in guinea pigs and rabbits. In vitro experiments with 4 types of soluble immune complexes which were incubated with gradually increasing amounts of enzymes showed dose-dependent cleavage of complexes. Antigen-induced experimental arthritis of rabbits, fed different amounts of a therapeutically used mixture of enzymes at different times, could be inhibited by this treatment, in dependence of dosage and time of feeding. With respect to the therapeutic applications of this study, the results favour the use of a high dosage repeated daily administration, since duration of effect seems limited.

  6. Dipeptidyl-peptidase IV (DPP IV/CD26)-activated prodrugs: a successful strategy for improving water solubility and oral bioavailability.

    PubMed

    Velázquez, Sonsoles; de Castro, Sonia; Diez-Torrubia, Alberto; Balzarini, Jan; Camarasa, María-José

    2015-01-01

    In the search of novel enzyme-based prodrug approaches to improve pharmacological properties of therapeutic drugs such as solubility and bioavailability, dipeptidyl-peptidase IV (DPP IV, also termed as CD26) enzyme activity provides a previously unexplored successful prodrug strategy. This review covers key aspects of the enzyme useful for the design of CD26-directed prodrugs. The proof-of-concept of this prodrug technology is provided for amine-containing agents by directly linking appropriate di- (or oligo)peptide moieties to a free amino group of a non-peptidic drug through an amide bond which is specifically hydrolized by DPP IV/CD26. Special emphasis is also made in discussing the design and synthesis of more elaborated tripartite prodrug systems, for further extension of the strategy to hydroxy-containing drugs. The application of this technology to improve water solubility and oral bioavailability of prominent examples of antiviral nucleosides is highlighted.

  7. Comparative enzyme-inducing effects of chlorpromazine and fluphenazine therapies in psychotic patients.

    PubMed

    Harman, A W; Frewin, D B; Priestly, B G

    1980-01-01

    Antipyrine elimination kinetics were measured in psychotic patients receiving either long-term chlorpromazine or fluphenazine decanoate therapy and in non-medicated control subjects. Patients receiving chlorpromazine metabolised antipyrine faster than the controls while, in patients receiving fluphenazine decanoate, there was not change. The results suggest that long-term chlorpromazine therapy induced the activity of drug-metabolising enzymes, whereas fluphenazine decanoate therapy had no effect.

  8. Trial watch – inhibiting PARP enzymes for anticancer therapy

    PubMed Central

    Sistigu, Antonella; Manic, Gwenola; Obrist, Florine; Vitale, Ilio

    2016-01-01

    ABSTRACT Poly(ADP-ribose) polymerases (PARPs) are a members of family of enzymes that catalyze poly(ADP-ribosyl)ation (PARylation) and/or mono(ADP-ribosyl)ation (MARylation), two post-translational protein modifications involved in crucial cellular processes including (but not limited to) the DNA damage response (DDR). PARP1, the most abundant family member, is a nuclear protein that is activated upon sensing distinct types of DNA damage and contributes to their resolution by PARylating multiple DDR players. Recent evidence suggests that, along with DDR, activated PARP1 mediates a series of prosurvival and proapoptotic processes aimed at preserving genomic stability. Despite this potential oncosuppressive role, upregulation and/or overactivation of PARP1 or other PARP enzymes has been reported in a variety of human neoplasms. Over the last few decades, several pharmacologic inhibitors of PARP1 and PARP2 have been assessed in preclinical and clinical studies showing potent antineoplastic activity, particularly against homologous recombination (HR)-deficient ovarian and breast cancers. In this Trial Watch, we describe the impact of PARP enzymes and PARylation in cancer, discuss the mechanism of cancer cell killing by PARP1 inactivation, and summarize the results of recent clinical studies aimed at evaluating the safety and therapeutic profile of PARP inhibitors in cancer patients. PMID:27308587

  9. Prodrugs of phosphonates and phosphates: crossing the membrane barrier.

    PubMed

    Wiemer, Andrew J; Wiemer, David F

    2015-01-01

    A substantial portion of metabolism involves transformation of phosphate esters, including pathways leading to nucleotides and oligonucleotides, carbohydrates, isoprenoids and steroids, and phosphorylated proteins. Because the natural substrates bear one or more negative charges, drugs that target these enzymes generally must be charged as well, but small charged molecules can have difficulty traversing the cell membrane by means other than endocytosis. The resulting dichotomy has stimulated a great deal of effort to develop effective prodrugs, compounds that carry little or no charge to enable them to transit biological membranes, but able to release the parent drug once inside the target cell. This chapter presents recent studies on advances in prodrug forms, along with representative examples of their application to marketed and developmental drugs.

  10. Prodrugs of phosphonates and phosphates: crossing the membrane barrier

    PubMed Central

    Wiemer, Andrew J.; Wiemer, David F.

    2016-01-01

    A substantial portion of metabolism involves transformation of phosphate esters, including pathways leading to nucleotides and oligonucleotides, carbohydrates, isoprenoids and steroids, and phosphorylated proteins. Because the natural substrates bear one or more negative charges, drugs that target these enzymes generally must be charged as well but small charged molecules can have difficulty traversing the cell membrane other than by endocytosis. The resulting dichotomy has stimulated abundant effort to develop effective prodrugs, compounds that carry little or no charge to enable them to transit biological membranes but then able to release the parent drug once inside the target cell. This chapter will present recent studies on advances in prodrug forms, along with representative examples of their application to marketed and developmental drugs. PMID:25391982

  11. A screen for and validation of prodrug antimicrobials.

    PubMed

    Fleck, Laura E; North, E Jeffrey; Lee, Richard E; Mulcahy, Lawrence R; Casadei, Gabriele; Lewis, Kim

    2014-01-01

    The rise of resistant pathogens and chronic infections tolerant to antibiotics presents an unmet need for novel antimicrobial compounds. Identifying broad-spectrum leads is challenging due to the effective penetration barrier of Gram-negative bacteria, formed by an outer membrane restricting amphipathic compounds, and multidrug resistance (MDR) pumps. In chronic infections, pathogens are shielded from the immune system by biofilms or host cells, and dormant persisters tolerant to antibiotics are responsible for recalcitrance to chemotherapy with conventional antibiotics. We reasoned that the dual need for broad-spectrum and sterilizing compounds could be met by developing prodrugs that are activated by bacterium-specific enzymes and that these generally reactive compounds could kill persisters and accumulate over time due to irreversible binding to targets. We report the development of a screen for prodrugs, based on identifying compounds that nonspecifically inhibit reduction of the viability dye alamarBlue, and then eliminate generally toxic compounds by testing for cytotoxicity. A large pilot of 55,000 compounds against Escherichia coli produced 20 hits, 3 of which were further examined. One compound, ADC111, is an analog of a known nitrofuran prodrug nitrofurantoin, and its activity depends on the presence of activating enzymes nitroreductases. ADC112 is an analog of another known antimicrobial tilbroquinol with unknown mechanism of action, and ADC113 does not belong to an approved class. All three compounds had a good spectrum and showed good to excellent activity against persister cells in biofilm and stationary cultures. These results suggest that screening for overlooked prodrugs may present a viable platform for antimicrobial discovery.

  12. Sulforaphane Preconditioning Sensitizes Human Colon Cancer Cells towards the Bioreductive Anticancer Prodrug PR-104A

    PubMed Central

    Erzinger, Melanie M.; Bovet, Cédric; Hecht, Katrin M.; Senger, Sabine; Winiker, Pascale; Sobotzki, Nadine; Cristea, Simona; Beerenwinkel, Niko; Shay, Jerry W.; Marra, Giancarlo; Wollscheid, Bernd; Sturla, Shana J.

    2016-01-01

    The chemoprotective properties of sulforaphane (SF), derived from cruciferous vegetables, are widely acknowledged to arise from its potent induction of xenobiotic-metabolizing and antioxidant enzymes. However, much less is known about the impact of SF on the efficacy of cancer therapy through the modulation of drug-metabolizing enzymes. To identify proteins modulated by a low concentration of SF, we treated HT29 colon cancer cells with 2.5 μM SF. Protein abundance changes were detected by stable isotope labeling of amino acids in cell culture. Among 18 proteins found to be significantly up-regulated, aldo-keto reductase 1C3 (AKR1C3), bioactivating the DNA cross-linking prodrug PR-104A, was further characterized. Preconditioning HT29 cells with SF reduced the EC50 of PR-104A 3.6-fold. The increase in PR-104A cytotoxicity was linked to AKR1C3 abundance and activity, both induced by SF in a dose-dependent manner. This effect was reproducible in a second colon cancer cell line, SW620, but not in other colon cancer cell lines where AKR1C3 abundance and activity were absent or barely detectable and could not be induced by SF. Interestingly, SF had no significant influence on PR-104A cytotoxicity in non-cancerous, immortalized human colonic epithelial cell lines expressing either low or high levels of AKR1C3. In conclusion, the enhanced response of PR-104A after preconditioning with SF was apparent only in cancer cells provided that AKR1C3 is expressed, while its expression in non-cancerous cells did not elicit such a response. Therefore, a subset of cancers may be susceptible to combined food-derived component and prodrug treatments with no harm to normal tissues. PMID:26950072

  13. Self-assemblies of pH-activatable PEGylated multiarm poly(lactic acid-co-glycolic acid)-doxorubicin prodrugs with improved long-term antitumor efficacies.

    PubMed

    Ding, Jianxun; Li, Di; Zhuang, Xiuli; Chen, Xuesi

    2013-10-01

    Two pH-activatable star-shaped prodrugs are synthesized through the condensation reaction between Y- or dumbbell-shaped poly(ethylene glycol)-poly(lactic acid-co-glycolic acid) (PEG-PLGA) copolymer and acid-sensitive cis-aconityl-doxorubicin. The prodrugs self-assemble into micelles with favorable hydrodynamic radii and relatively low critical micelle concentrations. In vitro DOX release from prodrug micelles is accelerated by the decrease of the PLGA content or at the late endosomal pH. The efficient cellular uptake and intracellular DOX release of the prodrug micelles are confirmed and the improved long-term anti-proliferative activities of prodrug micelles are revealed. These features suggest that the prodrugs provide a favorable approach to construct effective polymeric drug delivery systems for malignancy therapy.

  14. The Role of Mannosylated Enzyme and the Mannose Receptor in Enzyme Replacement Therapy

    PubMed Central

    Du, Hong; Levine, Mark; Ganesa, Chandrashekar; Witte, David P.; Cole, Edward S.; Grabowski, Gregory A.

    2005-01-01

    Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. LAL defects cause Wolman disease (WD) and CE storage disease (CESD). An LAL null (lal−/−) mouse model closely mimics human WD/CESD, with hepatocellular, Kupffer cell and other macrophage, and adrenal cortical storage of CEs and TGs. The effect on the cellular targeting of high-mannose and complex oligosaccharide–type oligosaccharide chains was tested with human LAL expressed in Pichia pastoris (phLAL) and CHO cells (chLAL), respectively. Only chLAL was internalized by cultured fibroblasts, whereas both chLAL and phLAL were taken up by macrophage mannose receptor (MMR)–positive J774E cells. After intraperitoneal injection into lal−/− mice, phLAL and chLAL distributed to macrophages and macrophage-derived cells of various organs. chLAL was also detected in hepatocytes. Ten injections of either enzyme over 30 d into 2- and 2.5-mo-old lal−/− mice produced normalization of hepatic color, decreased liver weight (50%–58%), and diminished hepatic cholesterol and TG storage. Lipid accumulations in macrophages were diminished with either enzyme. Only chLAL cleared lipids in hepatocytes. Mice double homozygous for the LAL and MMR deficiences (lal−/−;MMR−/−) showed phLAL uptake into Kupffer cells and hepatocytes, reversal of macrophage histopathology and lipid storage in all tissues, and clearance of hepatocytes. These results implicate MMR-independent and mannose 6-phosphate receptor–independent pathways in phLAL uptake and delivery to lysosomes in vivo. In addition, these studies show specific cellular targeting and physiologic effects of differentially oligosaccharide-modified human LALs mediated by MMR and that lysosomal targeting of mannose-terminated glycoproteins occurs and storage can be eliminated effectively without MMR. PMID:16380916

  15. Enzyme and combination therapy with cyclosporin A in the rat developing adjuvant arthritis.

    PubMed

    Rovenská, E; Svík, K; Stancíková, M; Rovenský, J

    1999-01-01

    Recent knowledge of the pathophysiology of rheumatoid arthritis and the mechanism of drug effects have enabled the use of new drugs and drug combinations in rheumatoid arthritis therapy. This study investigates the efficacy of both enzyme therapy and combined therapy with cyclosporin in rats with adjuvant arthritis. Rats with adjuvant-induced arthritis were administered either cyclosporin A (2.5 or 5.0 mg/kg/day per os), a mixture of enzymes (Phlogenzym (PHL); 45 mg/kg twice daily intrarectally), or a combination of 2.5 mg cyclosporin A and 90 mg PHL for a period of 40 days from the adjuvant application. Levels of serum albumin, changes in hind paw swelling and bone erosions were measured in rats as variables of inflammation and arthritis-associated destructive changes. Treatment with 5 mg of cyclosporin A, as well as with the combination therapy with cyclosporin A plus PHL, significantly inhibited both the inflammation and destructive arthritis-associated changes. However, 2.5 mg of cyclosporin A and PHL alone inhibited these disease markers, although to a lesser extent and at a later stage of arthritis development. The results show the inhibitory effect of enzyme therapy on rat adjuvant arthritis, as well as the efficacy of a low dose of cyclosporin A given in combination with enzyme therapy, which may be useful in the treatment of rheumatoid arthritis.

  16. Pronounced antitumor efficacy of doxorubicin when given as the prodrug DOX-GA3 in combination with a monoclonal antibody beta-glucuronidase conjugate.

    PubMed

    Houba, P H; Boven, E; van der Meulen-Muileman, I H; Leenders, R G; Scheeren, J W; Pinedo, H M; Haisma, H J

    2001-02-15

    A glucuronide doxorubicin prodrug N-[4-doxorubicin-N-carbonyl (oxymethyl) phenyl] O-beta-glucuronyl carbamate (DOX-GA3) has been developed to improve the antitumor effects of doxorubicin (DOX). The prodrug was originally designed to be activated into drug by human beta-glucuronidase (GUS) released from tumor cells in necrotic areas of tumor lesions. The aim of this study was to further improve the antitumor effects of DOX-GA3 by means of antibody-directed enzyme prodrug therapy (ADEPT). We thus investigated if the administration of an enzyme-immunoconjugate prepared from the pancarcinoma Ep-CAM specific monoclonal antibody (MAb) 323/A3 and beta-glucuronidase would result in improved antitumor effects because of additional enzyme localization in tumor tissue. In vitro, the prodrug DOX-GA3 was found to be 12-times less toxic than the parent drug DOX in a human ovarian cancer cell line. Immunospecific and complete activation of the prodrug took place when the cells were pretreated with 323/A3-beta-glucuronidase conjugate. In nude mice bearing s.c. human ovarian cancer xenografts (FMa) the maximum tolerated dose (MTD) of DOX-GA3 (500 mg/kg weekly x 2) was much higher when compared with that of DOX (8 mg/kg weekly x 2). In mice bearing well-established FMa xenografts, the standard treatment of DOX at the MTD (8 mg/kg weekly x 2) resulted in a tumor growth inhibition of 67%. Treatment with DOX-GA3 at a single dose of 500 mg/kg resulted in a better tumor growth inhibition of 87%. The combination of DOX-GA3 (500 mg/kg) with 323/A3-mGUS conjugate and anti-GUS MAb 105, to clear circulating conjugate, improved the antitumor effect even further to 98%. At the lower dose of 250 mg/kg DOX-GA3 tumor growth inhibition (34%) was not better than that of DOX. The combination, however, of DOX-GA3 at 250 mg/kg and 323/A3-mGUS conjugate plus MAb 105 again greatly improved the antitumor effect (growth inhibition of 93%). DOX given at 8 mg/kg weekly x 2 did not result in tumor regressions

  17. Intrathecal enzyme replacement therapy: Successful treatment of brain disease via the cerebrospinal fluid

    PubMed Central

    Dickson, Patricia; McEntee, Michael; Vogler, Carole; Le, Steven; Levy, Beth; Peinovich, Maryn; Hanson, Stephen; Passage, Merry; Kakkis, Emil

    2010-01-01

    Treatment of brain disease with recombinant proteins is difficult due to the blood-brain barrier. As an alternative to direct injections into the brain, we studied whether application of high concentrations of therapeutic enzymes via intrathecal (IT) injections could successfully drive uptake across the ependyma to treat brain disease. We studied IT enzyme replacement therapy with recombinant human iduronidase (rhIDU) in canine mucopolysaccharidosis I (MPS I, Hurler syndrome), a lysosomal storage disorder with brain and meningeal involvement. Monthly or quarterly IT treatment regimens with rhIDU achieved supranormal iduronidase enzyme levels in the brain, spinal cord, and spinal meninges. All regimens normalized total brain glycosaminoglycan (GAG) storage and reduced spinal meningeal GAG storage by 58–70%. The improvement in GAG storage levels persisted three months after the final IT dose. The successful use of enzyme therapy via the CSF represents a potentially useful approach for lysosomal storage disorders. PMID:17321776

  18. Cognitive Development in Infantile-Onset Pompe Disease Under Very Early Enzyme Replacement Therapy.

    PubMed

    Lai, Chih-Jou; Hsu, Ting-Rong; Yang, Chia-Feng; Chen, Shyi-Jou; Chuang, Ya-Chin; Niu, Dau-Ming

    2016-12-01

    Most patients with infantile-onset Pompe disease die in early infancy before beginning enzyme replacement therapy, which has made it difficult to evaluate the impact of Pompe disease on cognitive development. Patients with infantile-onset Pompe disease can survive with enzyme replacement therapy, and physicians can evaluate cognitive development in these patients. We established an effective newborn screening program with quick clinical diagnostic criteria. Cognitive and motor development were evaluated using the Bayley Scales of Infant and Toddler Development-Third Edition at 6, 12, and 24 months of age. The patients who were treated very early demonstrate normal cognitive development with no significant change in cognition during this period (P = .18 > .05). The cognitive development was positively correlated with motor development (r = 0.533, P = .011). The results indicated that very early enzyme replacement therapy could protect cognitive development in patients with infantile-onset Pompe disease up to 24 months of age.

  19. Activity of phosphatase-sensitive 5-aminolevulinic acid prodrugs in cancer cell lines.

    PubMed

    Herceg, Viktorija; Lange, Norbert; Allémann, Eric; Babič, Andrej

    2017-06-01

    5-aminolevulinc acid (5-ALA)-based photodynamic therapy (PDT) and photodiagnosis (PD) present many advantages over treatments with conventional photosensitizers (PS). It offers great tumor specificity, reduced photosensitivity reactions caused by PS accumulation in non-targeted tissues and also inherent PS metabolism into endogenous non-fluorescent heme. However, chemical instability, low bioavailability and poor pharmacokinetic profile limit systemic efficacy of 5-ALA. Here, we present a comprehensive in vitro evaluation of novel phosphatase-sensitive prodrugs of 5-ALA. These prodrugs are designed to be activated by ubiquitously expressed phosphatases with much improved chemical stability and reduced acute toxicity profile. PpIX kinetic measurements and flow cytometry show accumulation of PpIX upon incubation with phosphatase-sensitive prodrugs in PC3 human prostate cell cancer, MCF7 breast adenocarcinoma, U87MG glioblastoma, T24 bladder cancer and A549 lung carcinoma cells. They revealed a different fluorescence kinetics and dose-response curves for the different types of 5-ALA prodrugs. These experiments have allowed us to identify the most promising cancer cell types for phospho- 5-ALA prodrugs. Confocal fluorescence microscopy provided further evidence of fluorescent protoporphyrin IX accumulation and sub-cellular localisation. These findings, together with the low toxicity profile of phosphatase-sensitive prodrugs of 5-ALA and good response to PDT provide solid basis for future translational development in PC3, MCF7 and U87MG cancer types. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Sustained release of metformin via red blood cell accumulated sulfenamide prodrug.

    PubMed

    Peura, Lauri; Huttunen, Kristiina M

    2014-07-01

    Metformin is a first-line antidiabetic drug to treat type 2 diabetes. It is rapidly eliminated from plasma but also accumulated into red blood cells (RBCs) from which it is slowly released back into plasma. The aim of the study was to evaluate whether the amount of metformin in the RBCs could be increased by a sulfenamide prodrug approach, which could provide longer duration of metformin in systemic circulation. Pharmacokinetic properties of metformin and its cyclohexyl sulfenamide prodrug were evaluated in plasma and in whole blood after intravenous and oral administration in rats. Once the sulfenamide prodrug reached the bloodstream, it was rapidly and efficiently accumulated into the RBCs, where it was converted to metformin by free thiols. The RBC-whole blood ratio of metformin was increased approximately from 42% to 96% when metformin was administered intravenously as its sulfenamide prodrug, and the proportion of metformin in the RBCs was found to be concentration and time independent. Because metformin was slowly liberated into plasma, the prodrug showed a sustained-release pharmacokinetic profile and longer plasma half-life for metformin after oral administration. Therefore, this sulfenamide prodrug has great potential to improve metformin therapy as the daily doses could be reduced.

  1. Human β-glucuronidase: structure, function, and application in enzyme replacement therapy.

    PubMed

    Naz, Huma; Islam, Asimul; Waheed, Abdul; Sly, William S; Ahmad, Faizan; Hassan, Imtaiyaz

    2013-10-01

    Lysosomal storage diseases occur due to incomplete metabolic degradation of macromolecules by various hydrolytic enzymes in the lysosome. Despite structural differences, most of the lysosomal enzymes share many common features including a lysosomal targeting motif and phosphotransferase recognition sites. β-Glucuronidase (GUSB) is an important lysosomal enzyme involved in the degradation of glucuronate-containing glycosaminoglycan. The deficiency of GUSB causes mucopolysaccharidosis type VII (MPSVII), leading to lysosomal storage in the brain. GUSB is a well-studied protein for its expression, sequence, structure, and function. The purpose of this review is to summarize our current understanding of sequence, structure, function, and evolution of GUSB and its lysosomal enzyme targeting. Enzyme replacement therapy reported for this protein is also discussed.

  2. Application of homology modeling to generate CYP1A1 mutants with enhanced activation of the cancer chemotherapeutic prodrug dacarbazine.

    PubMed

    Lewis, Benjamin C; Mackenzie, Peter I; Miners, John O

    2011-11-01

    The chemotherapeutic prodrug dacarbazine (DTIC) has limited efficacy in human malignancies and exhibits numerous adverse effects that arise from systemic exposure to the cytotoxic metabolite. DTIC is activated by CYP1A1 and CYP1A2 catalyzed N-demethylation. However, structural features of these enzymes that confer DTIC N-demethylation have not been characterized. A validated homology model of CYP1A1 was employed to elucidate structure-activity relationships and to engineer CYP1A1 enzymes with altered DTIC activation. In silico docking demonstrated that DTIC orientates proximally to Ser122, Phe123, Asp313, Ala317, Ile386, Tyr259, and Leu496 of human CYP1A1. The site of metabolism is positioned 5.6 Å from the heme iron at an angle of 105.3°. Binding in the active site is stabilized by H-bonding between Tyr259 and the N(2) position of the imidazole ring. Twenty-seven CYP1A1 mutants were generated and expressed in Escherichia coli in yields ranging from 9 to 225 pmol P450/mg. DTIC N-demethylation by the E161K, E256K, and I458V mutants exhibited Michaelis-Menten kinetics, with decreases in K(m) (183-249 μM) that doubled the catalytic efficiency (p < 0.05) relative to wild-type CYP1A1 (K(m), 408 ± 43 μM; V(max), 28 ± 4 pmol · min(-1) · pmol of P450(-1)). The generation of enzymes with catalytically enhanced DTIC activation highlights the potential use of mutant CYP1A1 proteins in P450-based gene-directed enzyme prodrug therapy for the treatment of metastatic malignant melanoma.

  3. Computer-assisted design of pro-drugs for antimalarial atovaquone.

    PubMed

    Karaman, Rafik; Hallak, Hussein

    2010-10-01

    Density Functional Theory (DFT) and ab initio calculation results for the proton transfer reaction in Kirby's enzyme models 1-6 reveal that the reaction rate is largely dependent on the existence of a hydrogen bonding net in the reactants and the corresponding transition states. Further, the distance between the two reacting centers and the angle of the hydrogen bonding formed along the reaction path has profound effects on the rate. Hence, the study on the systems reported herein could provide a good basis for designing antimalarial (atovaquone) pro-drug systems that can be used to release the parent drug in a controlled manner. For example, based on the calculated log EM, the cleavage process for pro-drug 1Pro may be predicted to be about 10¹¹ times faster than that for a pro-drug 4Pro and about 10⁴ times faster than pro-drug 2Pro: rate (1Pro) > rate (2Pro > rate (4Pro). Thus, the rate by which the pro-drug releases the antimalarial drug can be determined according to the nature of the linker (Kirby's enzyme model 1-6).

  4. Prodrug approach: An overview of recent cases.

    PubMed

    Abet, Valentina; Filace, Fabiana; Recio, Javier; Alvarez-Builla, Julio; Burgos, Carolina

    2017-02-15

    In this review we highlight the most modern trends in the prodrug strategy. In drug research and development, the prodrug concept has found a number of useful applications. Selected examples of this approach are provided in this paper and they are classified according to the aim of their design.

  5. Improvement with ongoing Enzyme Replacement Therapy in advanced late-onset Pompe disease: a case study.

    PubMed

    Case, Laura E; Koeberl, Dwight D; Young, Sarah P; Bali, Deeksha; DeArmey, Stephanie M; Mackey, Joanne; Kishnani, Priya S

    2008-12-01

    Benefits of enzyme replacement therapy with Myozyme (alglucosidase alfa), anecdotally reported in late-onset Pompe disease, range from motor and pulmonary improvement in less severely affected patients, to stabilization with minimal improvement in those with advanced disease. We report a case of a 63-year-old patient with significant morbidity who made notable motor and pulmonary function gains after two years on therapy. Thus, improvements in those with advanced disease may be possible after long-term treatment.

  6. Production and purification of the multifunctional enzyme horseradish peroxidase

    PubMed Central

    Spadiut, Oliver; Herwig, Christoph

    2014-01-01

    The oxidoreductase horseradish peroxidase (HRP) is used in numerous industrial and medical applications. In this review, we briefly describe this well-studied enzyme and focus on its promising use in targeted cancer treatment. In combination with a plant hormone, HRP can be used in specific enzyme–prodrug therapies. Despite this outstanding application, HRP has not found its way as a biopharmaceutical into targeted cancer therapy yet. The reasons therefore lie in the present low-yield production and cumbersome purification of this enzyme from its natural source. However, surface glycosylation renders the recombinant production of HRP difficult. Here, we compare different production hosts for HRP and summarize currently used production and purification strategies for this enzyme. We further present our own strategy of glycoengineering this powerful enzyme to allow recombinant high-yield production in Pichia pastoris and subsequent simple downstream processing. PMID:24683473

  7. Immune tolerance improves the efficacy of enzyme replacement therapy in canine mucopolysaccharidosis I

    PubMed Central

    Dickson, Patricia; Peinovich, Maryn; McEntee, Michael; Lester, Thomas; Le, Steven; Krieger, Aimee; Manuel, Hayden; Jabagat, Catherine; Passage, Merry; Kakkis, Emil D.

    2008-01-01

    Mucopolysaccharidoses (MPSs) are lysosomal storage diseases caused by a deficit in the enzymes needed for glycosaminoglycan (GAG) degradation. Enzyme replacement therapy with recombinant human α-l-iduronidase successfully reduces lysosomal storage in canines and humans with iduronidase-deficient MPS I, but therapy usually also induces antibodies specific for the recombinant enzyme that could reduce its efficacy. To understand the potential impact of α-l-iduronidase–specific antibodies, we studied whether inducing antigen-specific immune tolerance to iduronidase could improve the effectiveness of recombinant iduronidase treatment in canines. A total of 24 canines with MPS I were either tolerized to iduronidase or left nontolerant. All canines received i.v. recombinant iduronidase at the FDA-approved human dose or a higher dose for 9–44 weeks. Nontolerized canines developed iduronidase-specific antibodies that proportionally reduced in vitro iduronidase uptake. Immune-tolerized canines achieved increased tissue enzyme levels at either dose in most nonreticular tissues and a greater reduction in tissue GAG levels, lysosomal pathology, and urinary GAG excretion. Tolerized MPS I dogs treated with the higher dose received some further benefit in the reduction of GAGs in tissues, urine, and the heart valve. Therefore, immune tolerance to iduronidase improved the efficacy of enzyme replacement therapy with recombinant iduronidase in canine MPS I and could potentially improve outcomes in patients with MPS I and other lysosomal storage diseases. PMID:18654665

  8. Oxidative stress in patients with mucopolysaccharidosis type II before and during enzyme replacement therapy.

    PubMed

    Filippon, Letícia; Vanzin, Camila S; Biancini, Giovana B; Pereira, Izabela N; Manfredini, Vanusa; Sitta, Angela; Peralba, Maria do Carmo R; Schwartz, Ida V D; Giugliani, Roberto; Vargas, Carmen R

    2011-06-01

    Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by deficiency of the enzyme iduronate-2-sulfatase, responsible for the degradation of glycosaminoglycans dermatan and heparan sulfate. Once the generation of free radicals is involved in the pathogenesis of many diseases, including some inborn errors of metabolism, the aim of this study was to evaluate blood oxidative stress parameters in MPS II patients, before and during 6 months of enzyme replacement therapy. We found significantly increased levels of malondialdehyde and carbonyl groups in plasma as well as erythrocyte catalase activity in patients before treatment compared to the control group. Plasma sulfhydryl group content and total antioxidant status were significantly reduced before treatment, while superoxide dismutase enzyme was not altered at this time when compared to controls. During enzyme replacement therapy, there was a significant reduction in levels of malondialdehyde when compared to pretreatment. Sulfhydryl groups were significantly increased until three months of treatment in MPS II patients in comparison to pretreatment. There were no significant alterations in plasma total antioxidant status and carbonyl groups as well as in catalase and superoxide dismutase activities during treatment in relation to pretreatment. The results indicate that MPS II patients are subject to lipid and protein oxidative damage and present reduction in non-enzymatic antioxidants, suggesting a possible involvement of free radicals in the pathophysiology of this disease. Also, the results may suggest that enzyme replacement therapy seems to protect against lipid peroxidation and protein damage in these patients.

  9. Prodrug behaviour of nicotinoylmorphine esters.

    PubMed

    Broekkamp, C L; Oosterloo, S K; Rijk, H W

    1988-06-01

    Morphine and its nicotinoyl esters, dinicotinoylmorphine (nicomorphine), 6-mononicotinoylmorphine (6-MNM) and 3-mononicotinoylmorphine (3-MNM) were tested in mice for central activity to obtain time-effect profiles of these compounds in rats. Two effects, analgesia with the hot plate test and locomotor stimulation in activity cages were measured and nicomorphine, 6-MNM and 3-MNM were found to have a faster onset of action compared with morphine. The effects of 3-MNM and morphine lasted longer than the effect of nicomorphine and 6-MNM. The prodrug behaviour of 3-MNM and nicomorphine for morphine and 6-MNM, respectively, is discussed.

  10. Modulation of porcine biotransformation enzymes by anthelmintic therapy with fenbendazole and flubendazole.

    PubMed

    Savlík, M; Fimanová, K; Szotáková, B; Lamka, J; Skálová, L

    2006-06-01

    Fenbendazole (FEN) and flubendazole (FLU) are benzimidazole anthelmintics often used in pig management for the control of nematodoses. The in vivo study presented here was designed to test the influence of FLU and FEN on cytochrome P4501A and other cytochrome P450 (CYP) isoforms, UDP-glucuronosyl transferase and several carbonyl reducing enzymes. The results indicated that FEN (in a single therapeutic dose as well as in repeated therapeutic doses) caused significant induction of pig CYP1A, while FLU did not show an inductive effect towards this isoform. Some of the other hepatic and intestinal biotransformation enzymes that were assayed were moderately influenced by FEN or FLU. Strong CYP1A induction following FEN therapy in pigs may negatively affect the efficacy and pharmacokinetics of FEN itself or other simultaneously or consecutively administered drugs. From the perspective of biotransformation enzyme modulation, FLU would appear to be a more convenient anthelmintic therapy of pigs than FEN.

  11. Application of a Microfluidic Reactor for Screening Cancer Prodrug Activation Using Silica-Immobilized Nitrobenzene Nitroreductase

    DTIC Science & Technology

    2006-01-01

    the corresponding electron- donating hydroxylamine is useful in a variety of biotechnological applications. Activation of prodrugs for cancer...withdrawing nitro group to the correspond- ing electron-donating hydroxylamine results in a very large electronic change, providing an effective enzyme... hydroxylamine derivative by bacterial nitroreductases.2-4 DEPT using Escherichia coli ni- troreductase and CB1954 has been demonstrated to be an

  12. Cell-permeable succinate prodrugs bypass mitochondrial complex I deficiency

    PubMed Central

    Ehinger, Johannes K.; Piel, Sarah; Ford, Rhonan; Karlsson, Michael; Sjövall, Fredrik; Frostner, Eleonor Åsander; Morota, Saori; Taylor, Robert W.; Turnbull, Doug M.; Cornell, Clive; Moss, Steven J.; Metzsch, Carsten; Hansson, Magnus J.; Fliri, Hans; Elmér, Eskil

    2016-01-01

    Mitochondrial complex I (CI) deficiency is the most prevalent defect in the respiratory chain in paediatric mitochondrial disease. This heterogeneous group of diseases includes serious or fatal neurological presentations such as Leigh syndrome and there are very limited evidence-based treatment options available. Here we describe that cell membrane-permeable prodrugs of the complex II substrate succinate increase ATP-linked mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres. Lactate accumulation in platelets due to rotenone-induced CI inhibition is reversed and rotenone-induced increase in lactate:pyruvate ratio in white blood cells is alleviated. Metabolomic analyses demonstrate delivery and metabolism of [13C]succinate. In Leigh syndrome patient fibroblasts, with a recessive NDUFS2 mutation, respiration and spare respiratory capacity are increased by prodrug administration. We conclude that prodrug-delivered succinate bypasses CI and supports electron transport, membrane potential and ATP production. This strategy offers a potential future therapy for metabolic decompensation due to mitochondrial CI dysfunction. PMID:27502960

  13. A 24-Year Enzyme Replacement Therapy in an Adenosine-deaminase-Deficient Patient.

    PubMed

    Tartibi, Hana M; Hershfield, Michael S; Bahna, Sami L

    2016-01-01

    Severe combined immunodeficiency (SCID) is a fatal childhood disease unless immune reconstitution is performed early in life, with either hematopoietic stem cell transplantation or gene therapy. One of its subtypes is caused by adenosine deaminase (ADA) enzyme deficiency, which leads to the accumulation of toxic metabolites that impair lymphocyte development and function. With the development of polyethylene glycol-conjugated adenosine deaminase (PEG-ADA) enzyme replacement therapy, many ADA-deficient children with SCID who could not receive a hematopoietic stem cell transplantation or gene therapy survived and had longer and healthier lives. We report a 24-year course of treatment in a patient who was diagnosed with ADA deficiency at 4 months of age. The patient was treated with PEG-ADA, which was the only therapy available for him. The patient's plasma ADA level was regularly monitored and the PEG-ADA dose adjusted accordingly. This treatment has resulted in near-normalization of lymphocyte counts, and his clinical course has been associated with only minor to moderate infections. Thus far, he has had no manifestations of autoimmune or lymphoproliferative disorders. This patient is among the longest to be maintained on PEG-ADA enzyme replacement therapy.

  14. [Enzyme therapy in comparison with immune complex determinations in chronic polyarthritis].

    PubMed

    Steffen, C; Smolen, J; Miehlke, K; Hörger, I; Menzel, J

    1985-01-01

    42 patients with definite or classic rheumatoid arthritis were treated with an enzyme mixture (Wobenzym) for 6 weeks. Circulating immune complexes were investigated with a Clq-solid phase RIA in weekly intervals and also rheumatoid factors were determined together with other routine laboratory tests. The condition of patients after therapy was recorded together with amount of circulating immune complexes during treatment. 26 (61.9%) patients improved, 13 (30.9%) patients remained unchanged, and 3 (17.1%) showed impairment. No side effects were observed. Patients who primarily showed no immune complexes or patients who had immune complexes which became negative during enzyme therapy, showed improvement more often than patients which had immune complexes and remained positive during the entire course of therapy.

  15. [Systemic enzyme therapy in the treatment of children with lower orbital wall fractures].

    PubMed

    Dubovskaia, L A; Guseva, M R; Gorbunova, E D; Gorbunov, A V; Kazinskaia, N V

    2006-01-01

    Wobenzim, a drug for systemic enzyme therapy, was used in 26 children with lower orbital wall fractures as monotherapy in early periods (on days 1 to 7) after brain injury to improve healing conditions and to minimize the formation of scar tissues in the fractural area. A control group comprised 24 patients who received systemic dehydration and vessel-strengthening therapy. Systemic enzyme therapy with Wobenzim could reduce the attenuation time of inflammatory manifestations of injury, prevent complications, such as strangulation of the musculus rectus inferior in the fractural area and development of strangulated orbital soft tissue hernia, and substantially decrease the number of patients having indications for surgical plastic repair of the orbital wall, as compared with the control group.

  16. Synthesis and biological evaluation of phosphate prodrugs of 4-phospho-D-erythronohydroxamic acid, an inhibitor of 6-phosphogluconate dehydrogenase.

    PubMed

    Ruda, Gian Filippo; Alibu, Vincent P; Mitsos, Christos; Bidet, Olivier; Kaiser, Marcel; Brun, Reto; Barrett, Michael P; Gilbert, Ian H

    2007-08-01

    We have previously reported the discovery of potent and selective inhibitors of 6-phosphogluconate dehydrogenase, the third enzyme of the phosphate pentose pathway, from Trypanosoma brucei, the causative organism of human African trypanosomiasis. These inhibitors were charged phosphate derivatives with restricted capacity to enter cells. Herein, we report the synthesis of five different classes of prodrugs: phosphoramidate; bis-S-acyl thioethyl esters (bis-SATE); bis-pivaloxymethyl (bis-POM); CycloSaligenyl; and phenyl, S-acyl thioethyl mixed phosphate esters (mix-SATE). Prodrugs were studied for stability and activity against the intact parasites. Most prodrugs caused inhibition of the growth of the parasites. The activity of the prodrugs against the parasites appeared to be related to their stability in aqueous buffer.

  17. Enzyme replacement therapy for murine mucopolysaccharidosis type VII leads to improvements in behavior and auditory function.

    PubMed Central

    O'Connor, L H; Erway, L C; Vogler, C A; Sly, W S; Nicholes, A; Grubb, J; Holmberg, S W; Levy, B; Sands, M S

    1998-01-01

    Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is one of a group of lysosomal storage diseases that share many clinical features, including mental retardation and hearing loss. Lysosomal storage in neurons of the brain and the associated behavioral abnormalities characteristic of a murine model of MPS VII have not been shown to be corrected by either bone marrow transplantation or gene therapy. However, intravenous injections of recombinant beta-glucuronidase initiated at birth reduce the pathological evidence of disease in MPS VII mice. In this study we present evidence that enzyme replacement initiated at birth improved the behavioral performance and reduced hearing loss in MPS VII mice. Enzyme-treated MPS VII mice performed similarly to normal mice and significantly better than mock- treated MPS VII mice in every phase of the Morris Water Maze test. In addition, the auditory function of treated MPS VII mice was dramatically improved, and was indistinguishable from normal mice. These data indicate that some of the learning, memory, and hearing deficits can be prevented in MPS VII mice if enzyme replacement therapy is initiated early in life. These data also provide functional correlates to the biochemical and histopathological improvements observed after enzyme replacement therapy. PMID:9525982

  18. Mutational and oxidative stress analysis in patients with mucopolysaccharidosis type I undergoing enzyme replacement therapy.

    PubMed

    Pereira, Vanessa Gonçalves; Martins, Ana Maria; Micheletti, Cecília; D'Almeida, Vânia

    2008-01-01

    Mucopolysaccharidosis type I (MPS I) patients present a wide range of clinical manifestations, which could be due to the high molecular heterogeneity of the IDUA gene and to pathological events besides the enzyme deficiency. The aim of this study was to identify the most common MPS I causing mutations and to evaluate some oxidative stress markers in Brazilian patients. 3 common mutations in the IDUA gene were searched in 11 MPS I patients by PCR-RFLP. Activities of antioxidant enzymes catalase and superoxide dismutase, and levels of total glutathione and thiobarbituric acid reactive substances were evaluated by spectrophotometric and colorimetric methods, during different periods of enzyme replacement therapy. The most common mutations were P533R and W402X, with allelic frequencies of 33.33% and 27.8% respectively. MPS I patients presented high levels of lipid peroxidation and enzyme replacement therapy led to an increase of catalase and a decrease of superoxide dismutase activities. P533R and W402X accounted for more than 60% of the alleles, but no genotype-phenotype correlation could be established. The alterations in antioxidant enzyme activities suggest that oxidative stress may be an important event among MPS I patients, which could contribute to the physiopathology of the disease.

  19. Amino acid deprivation using enzymes as a targeted therapy for cancer and viral infections.

    PubMed

    Fernandes, H S; Silva Teixeira, C S; Fernandes, P A; Ramos, M J; Cerqueira, N M F S A

    2017-03-01

    Amino acid depletion in the blood serum is currently being exploited and explored for therapies in tumors or viral infections that are auxotrophic for a certain amino acid or have a metabolic defect and cannot produce it. The success of these treatments is because normal cells remain unaltered since they are less demanding and/or can synthesize these compounds in sufficient amounts for their needs by other mechanisms. Areas covered: This review is focused on amino acid depriving enzymes and their formulations that have been successfully used in the treatment of several types of cancer and viral infections. Particular attention will be given to the enzymes L-asparaginase, L-arginase, L-arginine deiminase, and L-methionine-γ-lyase. Expert opinion: The immunogenicity and other toxic effects are perhaps the major limitations of these therapies, but they have been successfully decreased either through the expression of these enzymes from other organisms, recombination processes, pegylation of the selected enzymes or by specific mutations in the proteins. In 2006, FDA has already approved the use of L-asparaginase in the treatment of acute lymphoblastic leukemia. Other enzymes and in particular L-arginase, L-arginine deiminase, and L-methioninase have been showing promising results in vitro and in vivo studies.

  20. Design, Synthesis, and Evaluation of Novel Prodrugs of Transition State Inhibitors of Norovirus 3CL Protease.

    PubMed

    Galasiti Kankanamalage, Anushka C; Kim, Yunjeong; Rathnayake, Athri D; Alliston, Kevin R; Butler, Michelle M; Cardinale, Steven C; Bowlin, Terry L; Groutas, William C; Chang, Kyeong-Ok

    2017-07-27

    Ester and carbamate prodrugs of aldehyde bisulfite adduct inhibitors were synthesized in order to improve their pharmacokinetic and pharmacodynamic properties. The inhibitory activity of the compounds against norovirus 3C-like protease in enzyme and cell-based assays was determined. The ester and carbamate prodrugs displayed equivalent potency to those of the precursor aldehyde bisulfite adducts and precursor aldehydes. Furthermore, the rate of ester cleavage was found to be dependent on alkyl chain length. The generated prodrugs exhibited low cytotoxicity and satisfactory liver microsomes stability and plasma protein binding. The methodology described herein has wide applicability and can be extended to the bisulfite adducts of common warheads employed in the design of transition state inhibitors of serine and cysteine proteases of medical relevance.

  1. DNA damage in leukocytes from pretreatment mucopolysaccharidosis type II patients; protective effect of enzyme replacement therapy.

    PubMed

    Filippon, Letícia; Wayhs, Carlos A Y; Atik, Diana M; Manfredini, Vanusa; Herber, Silvani; Carvalho, Clarissa G; Schwartz, Ida V D; Giugliani, Roberto; Vargas, Carmen R

    2011-04-03

    Mucopolysaccharidosis type II (MPS II) is an X-linked recessive disease caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase, leading to progressive accumulation of glycosaminoglycans in nearly all cell types, tissues and organs. Enzyme replacement therapy reduces the storage of these substances in the lysosomes. Oxidative stress is related to the pathophysiology of many disorders, including inborn errors of metabolism. Oxidative damage to protein and lipid has been described in MPS types I and III. The aim of this study was to analyze DNA damage, as determined by the alkaline comet assay using silver staining, in peripheral leukocytes from MPS II patients before treatment and during the first six months of enzyme replacement therapy. We also correlated DNA damage with lipid and protein oxidative damages, analyzed by plasma malondialdehyde levels and carbonyl group content, respectively. We found a significant increase in lipid and protein damage in MPS II patients before treatment when compared to controls. Also, our results showed greater DNA damage in terms of damage index (DI) in pretreatment MPS II patients (DI=18.0 ± 2.4) when compared to controls (DI=66.0 ± 2.0). Enzyme replacement therapy led to a significant decrease in levels of malondialdehyde and DNA damage when compared to pretreatment, but did not reach control values. Significant positive correlations between DNA damage and malondialdehyde levels, as well as carbonyl group content, were observed. Our findings indicate that MPS II patients are subject to DNA damage and that enzyme replacement therapy is able to protect against this process.

  2. Synthesis and Evaluation as Prodrugs of Hydrophilic Carbamate Ester Analogues of Resveratrol.

    PubMed

    Azzolini, Michele; Mattarei, Andrea; La Spina, Martina; Marotta, Ester; Zoratti, Mario; Paradisi, Cristina; Biasutto, Lucia

    2015-09-08

    Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is an unfulfilled promise for health care: its exploitation is hindered by rapid conjugative metabolism in enterocytes and hepatocytes; low water solubility is a serious practical problem. To advantageously modify the physicochemical properties of the compound we have developed prodrugs in which all or part of the hydroxyl groups are linked via an N-monosubstituted carbamate ester bond to promoieties derived from glycerol or galactose, conferring higher water solubility. Kinetic studies of hydrolysis in aqueous solutions and in blood indicated that regeneration of resveratrol takes place in an appropriate time frame for delivery via oral administration. Despite their hydrophilicity some of the synthesized compounds were absorbed in the gastrointestinal tract of rats. In these cases the species found in blood after administration of a bolus consisted mainly of partially deprotected resveratrol derivatives and of the products of their glucuronidation, thus providing proof-of-principle evidence of behavior as prodrugs. The soluble compounds largely reached the lower intestinal tract. Upon administration of resveratrol, the major species found in this region was dihydroresveratrol, produced by enzymes of the intestinal flora. In experiments with a fully protected (trisubstituted) deoxygalactose containing prodrug, the major species were the prodrug itself and partially deprotected derivatives, along with small amounts of dihydroresveratrol. We conclude that the N-monosubstituted carbamate moiety is suitable for use in prodrugs of polyphenols.

  3. Integrase Inhibitor Prodrugs: Approaches to Enhancing the Anti-HIV Activity of β-Diketo Acids.

    PubMed

    Nair, Vasu; Okello, Maurice

    2015-07-13

    HIV integrase, encoded at the 3'-end of the HIV pol gene, is essential for HIV replication. This enzyme catalyzes the incorporation of HIV DNA into human DNA, which represents the point of "no-return" in HIV infection. Integrase is a significant target in anti-HIV drug discovery. This review article focuses largely on the design of integrase inhibitors that are β-diketo acids constructed on pyridinone scaffolds. Methodologies for synthesis of these compounds are discussed. Integrase inhibition data for the strand transfer (ST) step are compared with in vitro anti-HIV data. The review also examines the issue of the lack of correlation between the ST enzymology data and anti-HIV assay results. Because this disconnect appeared to be a problem associated with permeability, prodrugs of these inhibitors were designed and synthesized. Prodrugs dramatically improved the anti-HIV activity data. For example, for compound, 96, the anti-HIV activity (EC50) improved from 500 nM for this diketo acid to 9 nM for its prodrug 116. In addition, there was excellent correlation between the IC50 and IC90 ST enzymology data for 96 (6 nM and 97 nM, respectively) and the EC50 and EC90 anti-HIV data for its prodrug 116 (9 nM and 94 nM, respectively). Finally, it was confirmed that the prodrug 116 was rapidly hydrolyzed in cells to the active compound 96.

  4. Enzyme therapy for pompe disease with recombinant human alpha-glucosidase from rabbit milk.

    PubMed

    Van den Hout, J M; Reuser, A J; de Klerk, J B; Arts, W F; Smeitink, J A; Van der Ploeg, A T

    2001-04-01

    Pompe disease is a metabolic myopathy caused by deficiency of lysosomal acid alpha-glucosidase. In this report we review the first 36 weeks of a clinical study on the safety and efficacy of enzyme therapy aimed at correcting the deficiency. Four patients with infantile Pompe disease were enrolled. They received recombinant human alpha-glucosidase from transgenic rabbit milk. The product is generally well tolerated and reaches the primary target tissues. Normalization of alpha-glucosidase activity in skeletal muscle was obtained and degradation of PAS-positive material was seen in tissue sections. The clinical condition of all patients improved. The effect on heart was most significant, with an impressive reduction of the left ventricular mass index (LVMI). Motor function improved. The positive preliminary results stimulate continuation and extension of efforts towards the realization of enzyme therapy for Pompe disease.

  5. Exercise testing in late-onset glycogen storage disease type II patients undergoing enzyme replacement therapy.

    PubMed

    Marzorati, Mauro; Porcelli, Simone; Bellistri, Giuseppe; Morandi, Lucia; Grassi, Bruno

    2012-12-01

    Enzyme replacement therapy (ERT) has recently became available for patients with glycogen storage disease type II. Previous studies have demonstrated clinical efficacy of enzyme replacement therapy, however, data on physiological variables related to exercise tolerance are scarce. Four glycogen storage disease type II late-onset patients (45 ± 6 years) performed an incremental exercise on a cycle ergometer, up to voluntary exhaustion, before (BEFORE) and after 12 months of ERT (AFTER). Peak workload, oxygen uptake, heart rate, cardiac output (by impedance cardiography) and vastus lateralis oxygenation indices (by continuous-wave near-infrared spectroscopy, NIRS) were determined. Peak workload and oxygen uptake values significantly increased during ERT (54 ± 30 vs. 63 ± 31 watt, and 17.2 ± 4.4 vs. 19.7 ± 3.5 ml/kg/min, respectively, in BEFORE vs. AFTER). On the other hand, for both peak cardiac output (12.3 ± 5.3 vs. 14.8 ± 4.5L/min) and the NIRS-determined peak skeletal muscle fractional O(2) extraction, expressed as a percentage of the maximal values during a transient limb ischemia (30 ± 39% vs. 38 ± 28%), the observed increases were not statistically significant. Our findings suggest that in glycogen storage disease type II patients enzyme replacement therapy is associated with a mild improvement of exercise tolerance. The findings need to be validated during a longer follow-up on a larger group of patients.

  6. Biocatalytic approaches applied to the synthesis of nucleoside prodrugs.

    PubMed

    Iglesias, Luis E; Lewkowicz, Elizabeth S; Medici, Rosario; Bianchi, Paola; Iribarren, Adolfo M

    2015-01-01

    Nucleosides are valuable bioactive molecules, which display antiviral and antitumour activities. Diverse types of prodrugs are designed to enhance their therapeutic efficacy, however this strategy faces the troublesome selectivity issues of nucleoside chemistry. In this context, the aim of this review is to give an overview of the opportunities provided by biocatalytic procedures in the preparation of nucleoside prodrugs. The potential of biocatalysis in this research area will be presented through examples covering the different types of nucleoside prodrugs: nucleoside analogues as prodrugs, nucleoside lipophilic prodrugs and nucleoside hydrophilic prodrugs.

  7. Enhanced cytotoxicity with a novel system combining the paclitaxel-2'-ethylcarbonate prodrug and an HSV amplicon with an attenuated replication-competent virus, HF10 as a helper virus.

    PubMed

    Ishida, Daisuke; Nawa, Akihiro; Tanino, Tadatoshi; Goshima, Fumi; Luo, Chen Hong; Iwaki, Masahiro; Kajiyama, Hiroaki; Shibata, Kiyosumi; Yamamoto, Eiko; Ino, Kazuhiko; Tsurumi, Tatsuya; Nishiyama, Yukihiro; Kikkawa, Fumitaka

    2010-02-01

    We previously demonstrated that HF10, which is a natural, non-engineered HSV-1, has potent oncolytic activity in the treatment of solid malignant tumors in vitro and in vivo [H. Takakuwa, F. Goshima, N. Nozawa, T. Yoshikawa, H. Kimata, A. Nakao, et al., Oncolytic viral therapy using a spontaneously generated herpes simplex virus type 1 variant for disseminated peritoneal tumor in immunocompetent mice, Arch. Virol. 148 (2003) 813-825; S. Kohno, C. Lou, F. Goshima, Y. Nishiyama, T. Sata, Y. Ono, Herpes simplex virus type 1 mutant HF10 oncolytic viral therapy for bladder cancer, Urology 66 (2005) 1116-1121; D. Watanabe, F. Goshima, I. Mori, Y. Tamada, Y. Matsumoto, Y. Nishiyama, Oncolytic virotherapy for malignant melanoma with herpes simplex virus type 1 mutant HF10, J. Dermatol. Sci. 50 (2008) 185-196; A. Nawa, C. Luo, L. Zhang, Y. Ushijima, D. Ishida, M. Kamakura, et al., Non-engineered, naturally oncolytic herpes simplex virus HSV1 HF10: applications for cancer gene therapy, Curr. Gene. Ther. 8 (2008) 208-221]. Previous reports have also shown that a combination of HF10 and paclitaxel (TAX) was more efficacious than either regimen alone for some types of malignant tumors [S. Shimoyama, F. Goshima, O. Teshigahara, H. Kasuya, Y. Kodera, A. Nakao, et al., Enhanced efficacy of herpes simplex virus mutant HF10 combined with paclitaxel in peritoneal cancer dissemination models, Hepatogastroenterology 54 (2007) 1038-1042]. In this study, we investigated the efficacy of gene-directed enzyme prodrug therapy (GDEPT) using a novel system that combines the paclitaxel-2'-ethylcarbonate prodrug (TAX-2'-Et) and an HSV amplicon expressing rabbit-carboxylesterase (CES) with HF10 as a helper virus. This GDEPT system aims to produce high level of CES at the tumor site, resulting in efficient local conversion of the TAX-2'-Et prodrug into the active drug TAX [A. Nawa, T. Tanino, C. Lou, M. Iwaki, H. Kajiyama, K. Shibata, et al., Gene directed enzyme prodrug therapy for ovarian cancer

  8. Update on the Genetic Polymorphisms of Drug-Metabolizing Enzymes in Antiepileptic Drug Therapy

    PubMed Central

    Saruwatari, Junji; Ishitsu, Takateru; Nakagawa, Kazuko

    2010-01-01

    Genetic polymorphisms in the genes that encode drug-metabolizing enzymes are implicated in the inter-individual variability in the pharmacokinetics and pharmaco-dynamics of antiepileptic drugs (AEDs). However, the clinical impact of these polymorphisms on AED therapy still remains controversial. The defective alleles of cytochrome P450 (CYP) 2C9 and/or CYP2C19 could affect not only the pharmacokinetics, but also the pharmacodynamics of phenytoin therapy. CYP2C19 deficient genotypes were associated with the higher serum concentration of an active metabolite of clobazam, N-desmethylclobazam, and with the higher clinical efficacy of clobazam therapy than the other CYP2C19 genotypes. The defective alleles of CYP2C9 and/or CYP2C19 were also found to have clinically significant effects on the inter-individual variabilities in the population pharmacokinetics of phenobarbital, valproic acid and zonisamide. EPHX1 polymorphisms may be associated with the pharmacokinetics of carbamazepine and the risk of phenytoin-induced congenital malformations. Similarly, the UDP-glucuronosyltransferase 2B7 genotype may affect the pharmacokinetics of lamotrigine. Gluthatione S-transferase null genotypes are implicated in an increased risk of hepatotoxicity caused by carbamazepine and valproic acid. This article summarizes the state of research on the effects of mutations of drug-metabolizing enzymes on the pharmacokinetics and pharmacodynamics of AED therapies. Future directions for the dose-adjustment of AED are discussed. PMID:27713373

  9. β2 Agonists Enhance the Efficacy of Simultaneous Enzyme Replacement Therapy in Murine Pompe Disease

    PubMed Central

    Koeberl, Dwight D.; Li, Songtao; Dai, Jian; Thurberg, Beth L.; Bali, Deeksha; Kishnani, Priya S.

    2011-01-01

    Enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) has improved clinical outcomes in patients with Pompe disease; however, the response of skeletal muscle and the central nervous system to ERT has been attenuated. The poor response of skeletal muscle to ERT has been attributed to the low abundance of the cation-independent mannose-6-phosphate receptor (CI-MPR), which mediates receptor-mediated uptake of rhGAA. Hence the ability of adjunctive therapy with β2-agonists to increase CI-MPR expression in skeletal muscle was evaluated during ERT in murine Pompe disease with regard to reversal of neuromuscular involvement. Mice with Pompe disease were treated with weekly rhGAA injections (20 mg/kg) and a selective β2-agonist, either albuterol (30 mg/l in drinking water) or low-dose clenbuterol (6 mg/l in drinking water). Biochemical correction was enhanced by β2-agonist treatment in both muscle and the cerebellum, indicating that adjunctive therapy could enhance efficacy from ERT in Pompe disease with regard to neuromuscular involvement. Intriguingly, clenbuterol slightly reduced muscle glycogen content independent of CI-MPR expression, as demonstrated in CI-MPR knockout/GAA knockout mice that were otherwise resistant to ERT. Thus, adjunctive therapy with β2 agonists might improve the efficacy of ERT in Pompe disease and possibly other lysosomal storage disorders through enhancing receptor-mediated uptake of recombinant lysosomal enzymes. PMID:22154081

  10. β2 Agonists enhance the efficacy of simultaneous enzyme replacement therapy in murine Pompe disease.

    PubMed

    Koeberl, Dwight D; Li, Songtao; Dai, Jian; Thurberg, Beth L; Bali, Deeksha; Kishnani, Priya S

    2012-02-01

    Enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) has improved clinical outcomes in patients with Pompe disease; however, the response of skeletal muscle and the central nervous system to ERT has been attenuated. The poor response of skeletal muscle to ERT has been attributed to the low abundance of the cation-independent mannose-6-phosphate receptor (CI-MPR), which mediates receptor-mediated uptake of rhGAA. Hence the ability of adjunctive therapy with β2-agonists to increase CI-MPR expression in skeletal muscle was evaluated during ERT in murine Pompe disease with regard to reversal of neuromuscular involvement. Mice with Pompe disease were treated with weekly rhGAA injections (20 mg/kg) and a selective β2-agonist, either albuterol (30 mg/l in drinking water) or low-dose clenbuterol (6 mg/l in drinking water). Biochemical correction was enhanced by β2-agonist treatment in both muscle and the cerebellum, indicating that adjunctive therapy could enhance efficacy from ERT in Pompe disease with regard to neuromuscular involvement. Intriguingly, clenbuterol slightly reduced muscle glycogen content independent of CI-MPR expression, as demonstrated in CI-MPR knockout/GAA knockout mice that were otherwise resistant to ERT. Thus, adjunctive therapy with β2 agonists might improve the efficacy of ERT in Pompe disease and possibly other lysosomal storage disorders through enhancing receptor-mediated uptake of recombinant lysosomal enzymes.

  11. Anticancer Drug Released from Near IR-activated Prodrug Overcomes Spatiotemporal Limits of Singlet Oxygen

    PubMed Central

    Rajaputra, Pallavi; Bio, Moses; Nkepang, Gregory; Thapa, Pritam; Woo, Sukyung; You, Youngjae

    2016-01-01

    Photodynamic therapy (PDT) is a cancer treatment modality where photosensitizer (PS) is activated by visible and near IR light to produce singlet oxygen (1O2). However, 1O2 has a short lifetime (< 40 ns) and cannot diffuse (< 20 nm) beyond the cell diameter (e.g., ~ 1,800 nm). Thus, 1O2 damage is both spatially and temporally limited and does not produce bystander effect. In a heterogeneous tumor, cells escaping 1O2 damage can regrow after PDT treatment. To overcome these limitations, we developed a prodrug concept (PS-L-D) composed of a photosensitizer (PS), an anti-cancer drug (D), and an 1O2-cleavable linker (L). Upon illumination of the prodrug, 1O2 is generated, which damages the tumor and also releases anticancer drug. The locally released drug could cause spatially broader and temporally sustained damage, killing the surviving cancer cells after the PDT damage. In our previous report, we presented the superior activity of our prodrug of CA4 (combretastatin A-4), Pc-(L-CA4)2, compared to its non-cleavable analog, Pc-(NCL-CA4)2, that produced only PDT effects. Here, we provide clear evidence demonstrating that the released anticancer drug, CA4, indeed damages the surviving cancer cells over and beyond the spatial and temporal limits of 1O2. In the limited light illumination experiment, cells in the entire well were killed due to the effect of released anticancer drug, whereas only a partial damage was observed in the pseudo-prodrug treated wells. A time-dependent cell survival study showed more cell death in the prodrug-treated cells due to the sustained damage by the released CA4. Cell cycle analysis and microscopic imaging data demonstrated the typical damage patterns by CA4 in the prodrug treated cells. A time-dependent histological study showed that prodrug-treated tumors lacked mitotic bodies, and the prodrug caused broader and sustained tumor size reduction compared to those seen in the tumors treated with the pseudo-prodrug. This data consistently

  12. Anticancer drug released from near IR-activated prodrug overcomes spatiotemporal limits of singlet oxygen.

    PubMed

    Rajaputra, Pallavi; Bio, Moses; Nkepang, Gregory; Thapa, Pritam; Woo, Sukyung; You, Youngjae

    2016-04-01

    Photodynamic therapy (PDT) is a cancer treatment modality where photosensitizer (PS) is activated by visible and near IR light to produce singlet oxygen ((1)O2). However, (1)O2 has a short lifetime (<40 ns) and cannot diffuse (<20 nm) beyond the cell diameter (e.g., ∼ 1800 nm). Thus, (1)O2 damage is both spatially and temporally limited and does not produce bystander effect. In a heterogeneous tumor, cells escaping (1)O2 damage can regrow after PDT treatment. To overcome these limitations, we developed a prodrug concept (PS-L-D) composed of a photosensitizer (PS), an anti-cancer drug (D), and an (1)O2-cleavable linker (L). Upon illumination of the prodrug, (1)O2 is generated, which damages the tumor and also releases anticancer drug. The locally released drug could cause spatially broader and temporally sustained damage, killing the surviving cancer cells after the PDT damage. In our previous report, we presented the superior activity of our prodrug of CA4 (combretastatin A-4), Pc-(L-CA4)2, compared to its non-cleavable analog, Pc-(NCL-CA4)2, that produced only PDT effects. Here, we provide clear evidence demonstrating that the released anticancer drug, CA4, indeed damages the surviving cancer cells over and beyond the spatial and temporal limits of (1)O2. In the limited light illumination experiment, cells in the entire well were killed due to the effect of released anti-cancer drug, whereas only a partial damage was observed in the pseudo-prodrug treated wells. A time-dependent cell survival study showed more cell death in the prodrug-treated cells due to the sustained damage by the released CA4. Cell cycle analysis and microscopic imaging data demonstrated the typical damage patterns by CA4 in the prodrug treated cells. A time-dependent histological study showed that prodrug-treated tumors lacked mitotic bodies, and the prodrug caused broader and sustained tumor size reduction compared to those seen in the tumors treated with the pseudo-prodrug. This data

  13. Enzyme replacement in murine mucopolysaccharidosis type VII: neuronal and glial response to beta-glucuronidase requires early initiation of enzyme replacement therapy.

    PubMed

    Vogler, C; Levy, B; Galvin, N J; Thorpe, C; Sands, M S; Barker, J E; Baty, J; Birkenmeier, E H; Sly, W S

    1999-06-01

    We have previously shown that mucopolysaccharidosis type VII (MPS VII) mice receiving six weekly injections of recombinant beta-glucuronidase from birth had improved cognitive ability and reduced central nervous system lysosomal storage. However, a single beta-glucuronidase injection at 5 wk of age did not correct neuronal storage. We define the age at which central nervous system storage in MPS VII mice becomes resistant to beta-glucuronidase therapy and determine the effect of enzyme on other tissues by comparing the histology of mice begun on therapy at various times after birth. MPS VII mice received injections on the day of birth and then weekly for 5 wk with 16,000U/g beta-glucuronidase had reduced lysosomal storage in brain. The same therapy begun on d 14 of life or thereafter failed to correct neuronal storage, even when treatment was continued for six doses. Glial responsiveness or accessibility to enzyme also depended on early treatment. In contrast, leptomeningeal, osteoblast, and retinal pigment epithelial storage reduction depended on enzyme dose rather than age at initiation of therapy. Fixed tissue macrophage storage was reduced in all treated MPS VII mice, even those receiving a single dose. These observations indicate that fixed tissue macrophages in MPS VII mice remain sensitive to enzyme replacement therapy well into adulthood although neurons are responsive or accessible to enzyme therapy early in life. Because early initiation of enzyme replacement is important to achieve a central nervous system response, these studies emphasize the importance of newborn screening for lysosomal storage diseases so that early treatment can maximize the likelihood of a favorable therapeutic response.

  14. L-Methionase: A Therapeutic Enzyme to Treat Malignancies

    PubMed Central

    Sharma, Bhupender; Singh, Sukhdev; Kanwar, Shamsher S.

    2014-01-01

    Cancer is an increasing cause of mortality and morbidity throughout the world. L-methionase has potential application against many types of cancers. L-Methionase is an intracellular enzyme in bacterial species, an extracellular enzyme in fungi, and absent in mammals. L-Methionase producing bacterial strain(s) can be isolated by 5,5′-dithio-bis-(2-nitrobenzoic acid) as a screening dye. L-Methionine plays an important role in tumour cells. These cells become methionine dependent and eventually follow apoptosis due to methionine limitation in cancer cells. L-Methionine also plays an indispensable role in gene activation and inactivation due to hypermethylation and/or hypomethylation. Membrane transporters such as GLUT1 and ion channels like Na2+, Ca2+, K+, and Cl− become overexpressed. Further, the α-subunit of ATP synthase plays a role in cancer cells growth and development by providing them enhanced nutritional requirements. Currently, selenomethionine is also used as a prodrug in cancer therapy along with enzyme methionase that converts prodrug into active toxic chemical(s) that causes death of cancerous cells/tissue. More recently, fusion protein (FP) consisting of L-methionase linked to annexin-V has been used in cancer therapy. The fusion proteins have advantage that they have specificity only for cancer cells and do not harm the normal cells. PMID:25250324

  15. Design, synthesis, and evaluation of new cyclophosphamide-based anticancer prodrugs

    SciTech Connect

    Moon, Ki-Young.

    1993-01-01

    Cyclophosphamide (CP,1) is a prodrug that is activated by hepatic microsomal mixed-function oxidase (MFO) catalyzed C[sub 4]-hydroxylation. The resulting 4-hydroxycyclophosphamide (4-OH-CP) undergoes ring opening to aldophosphamide (Aldo), followed by generation of cytotoxic phosphoramide mustard (PDA,2) and acrolein by [beta]-elimination. The cytotoxic activity of CP is attributed to the aziridinium ion species derived from PDA that cross-links interstrand DNA. The aim of this research is to design, synthesize, and evaluate new cyclophosphamide-based alkylating agents to achieve improved therapeutic efficacy against neoplastic cells. Benzyl phosphoramide mustard (Benzyl PDA,4), 2.4-difluorobenzyl phosphoramide mustard (2,4-Difluorobenzyl PDA,5) and methyl phosphoramide mustard (Methyl PDA,6) were examined as lipophilic, chemically stable prodrugs of PDA (2). These phosphorodiamidic esters were designed to undergo biotransformation by hepatic microsomal enzymes to produce 2 without generation of acrolein and to be active against CP-resistant tumor cells. Several N-methyl-4-(alkylthio)cyclophosphamide derivatives were synthesized and examined as chemically stable, biooxidative prodrugs of 4-OH-CP, the activated species of CP. All of the prodrugs underwent N-demethylation in a time-dependent manner when incubated with rat hepatic microsomes, which resulted in formation of formaldehyde as well as alkylating species. Among the prodrugs, N-methyl-4-(diethyldithiocarbamoyl)cyclophosphamide (N-CH[sub 3]-4-DDTC-CP,15) showed exceptional in vitro cytotoxicity against 3T3 cells as well as against a panel of human tumor cell lines, with a particular sensitivity to leukemia and small cell lung cancer cell lines. Preliminary in vivo antitumor evaluation against L1210 leukemia in mice showed that all of the prodrugs were active.

  16. Hydrophilic Prodrug Approach for Reduced Pigment Binding and Enhanced Transscleral Retinal Delivery of Celecoxib

    PubMed Central

    Malik, Pradip; Kadam, Rajendra S.; Cheruvu, Narayan P.S.; Kompella, Uday B.

    2012-01-01

    Purpose Transscleral retinal delivery of celecoxib, an anti-inflammatory and anti-VEGF agent is restricted by its poor solubility and binding to the melanin pigment in choroid-RPE. The purpose of this study was to develop soluble prodrugs of celecoxib with reduced pigment binding and enhanced retinal delivery. Methods Three hydrophilic amide prodrugs of celecoxib were synthesized and characterized for solubility and lipophilicity. In vitro melanin binding to natural melanin (Sepia Officinalis) was estimated for all three prodrugs. In vitro transport studies across isolated bovine sclera and sclera-choroid-RPE (SCRPE) were performed. Prodrug with the highest permeability across SCRPE was characterized for metabolism and cytotoxicity and its in vivo transscleral delivery in pigmented rats. Results Celecoxib succinamidic acid (CSA), celecoxib maleamidic acid (CMA), and celecoxib acetamide (CAA) were synthesized and characterized. Aqueous solubilities of CSA, CMA, and CAA were 300-, 182-, and 76-fold higher, respectively, than celecoxib. Melanin binding affinity and capacity was significantly lower than celecoxib for all three prodrugs. Rank order for the % in vitro transport across bovine sclera and SCRPE was CSA > CMA ~ CAA ~ celecoxib, with the transport being 8-fold higher for CSA than celecoxib. CSA was further assessed for its metabolic stability and in vivo delivery. CSA showed optimum metabolic stability in all eye tissues with only 10–20 % conversion to parent celecoxib in 30 minutes. Metabolic enzymes responsible for bioconversion included amidases, esterase, and cytochrome P-450. In vivo delivery in pigmented BN rats showed that CSA had 4.7-, 1.4-, 3.3-, 6.0-, and 4.5- fold higher delivery to sclera, choroid-RPE, retina, vitreous, and lens than celecoxib. CSA has no cytotoxicity in ARPE-19 cells in the concentration range of 0.1 to 1000 μM. Conclusions Celecoxib succinamidic acid is a soluble prodrug of celecoxib with reduced melanin binding which

  17. Lipophilic Prodrugs of SN38: Synthesis and in Vitro Characterization toward Oral Chemotherapy.

    PubMed

    Bala, Vaskor; Rao, Shasha; Li, Peng; Wang, Shudong; Prestidge, Clive A

    2016-01-04

    SN38 (7-ethyl-10-hydroxy camptothecin) is a potent anticancer agent belonging to the camptothecin family; however, its oral delivery is extensively restricted by poor solubility in pharmaceutically acceptable excipients and low transmucosal permeability. Lipid-based carriers are well-known for their ability to improve oral absorption and bioavailability of lipid soluble and highly permeable compounds. Thus, this study has focused on improving solubility in lipid excipients, controlling stability, and enhancing transmucosal permeability of SN38 by specific chemical modification. To achieve these aims, a series of lipophilic prodrugs were designed and synthesized by esterification at the C10 and/or C20 positon(s) of SN38 with dietary fatty acids of diverse hydrocarbon chain lengths. The solubility of these novel prodrugs in long-chain triglycerides was increased up to 444-fold, and cytotoxicity was significantly reduced in comparison to SN38. The prodrugs were stable in simulated gastric fluids but exhibited different rates of hydrolysis (t1/2 < 5 min to t1/2 > 2 h) in simulated intestinal fluids (in the presence of enzymes) depending on the alkyl chain length and the position modified. A predictable reconversion of prodrugs to SN38 in plasma was also confirmed. On the basis of these studies, SN38-undecanoate (C20) was identified as the optimal prodrug. Finally, in vitro permeability and uptake studies in rat intestinal mucosal membrane using an Ussing chamber showed significant improvement in transepithelial drug transport and cellular uptake. Together, these results indicate that well designed lipophilic prodrugs have potential for the efficacious and safe oral delivery of SN38.

  18. Hydrophilic prodrug approach for reduced pigment binding and enhanced transscleral retinal delivery of celecoxib.

    PubMed

    Malik, Pradip; Kadam, Rajendra S; Cheruvu, Narayan P S; Kompella, Uday B

    2012-03-05

    Transscleral retinal delivery of celecoxib, an anti-inflammatory and anti-VEGF agent, is restricted by its poor solubility and binding to the melanin pigment in choroid-RPE. The purpose of this study was to develop soluble prodrugs of celecoxib with reduced pigment binding and enhanced retinal delivery. Three hydrophilic amide prodrugs of celecoxib, celecoxib succinamidic acid (CSA), celecoxib maleamidic acid (CMA), and celecoxib acetamide (CAA) were synthesized and characterized for solubility and lipophilicity. In vitro melanin binding to natural melanin (Sepia officinalis) was estimated for all three prodrugs. In vitro transport studies across isolated bovine sclera and sclera-choroid-RPE (SCRPE) were performed. Prodrug with the highest permeability across SCRPE was characterized for metabolism and cytotoxicity and its in vivo transscleral delivery in pigmented rats. Aqueous solubilities of CSA, CMA, and CAA were 300-, 182-, and 76-fold higher, respectively, than celecoxib. Melanin binding affinity and capacity were significantly lower than for celecoxib for all three prodrugs. Rank order for the % in vitro transport across bovine sclera and SCRPE was CSA > CMA ~ CAA ~ celecoxib, with the transport being 8-fold higher for CSA than celecoxib. CSA was further assessed for its metabolic stability and in vivo delivery. CSA showed optimum metabolic stability in all eye tissues with only 10-20% conversion to parent celecoxib in 30 min. Metabolic enzymes responsible for bioconversion included amidases, esterase, and cytochrome P-450. In vivo delivery in pigmented BN rats showed that CSA had 4.7-, 1.4-, 3.3-, 6.0-, and 4.5-fold higher delivery to sclera, choroid-RPE, retina, vitreous, and lens than celecoxib. CSA has no cytotoxicity in ARPE-19 cells in the concentration range of 0.1 to 1000 μM. Celecoxib succinamidic acid, a soluble prodrug of celecoxib with reduced melanin binding, enhances transscleral retinal delivery of celecoxib.

  19. Low-dose Gene Therapy Reduces the Frequency of Enzyme Replacement Therapy in a Mouse Model of Lysosomal Storage Disease

    PubMed Central

    Alliegro, Marialuisa; Ferla, Rita; Nusco, Edoardo; De Leonibus, Chiara; Settembre, Carmine; Auricchio, Alberto

    2016-01-01

    Enzyme replacement therapy (ERT) is the standard of care for several lysosomal storage diseases (LSDs). ERT, however, requires multiple and costly administrations and has limited efficacy. We recently showed that a single high dose administration of adeno-associated viral vector serotype 8 (AAV2/8) is at least as effective as weekly ERT in a mouse model of mucopolysaccharidosis type VI (MPS VI). However, systemic administration of high doses of AAV might result in both cell-mediated immune responses and insertional mutagenesis. Here we evaluated whether the combination of low doses of AAV2/8 with a less frequent (monthly) than canonical (weekly) ERT schedule may be as effective as the single treatments at high doses or frequent regimen. A greater reduction of both urinary glycosaminoglycans, considered a sensitive biomarker of therapeutic efficacy, and storage in the myocardium and heart valves was observed in mice receiving the combined than the single therapies. Importantly, these levels of correction were similar to those we obtained in a previous study following either high doses of AAV2/8 or weekly ERT. Our data show that low-dose gene therapy can be used as a means to rarify ERT administration, thus reducing both the risks and costs associated with either therapies. PMID:27658524

  20. NIR Fluorogenic Dye as a Modular Platform for Prodrug Assembly: Real-Time in vivo Monitoring of Drug Release.

    PubMed

    Redy-Keisar, Orit; Ferber, Shiran; Satchi-Fainaro, Ronit; Shabat, Doron

    2015-06-01

    The ability to monitor drug release in vivo provides essential pharmacological information. We developed a new modular approach for the preparation of theranostic prodrugs with a turn-ON near-infrared (NIR) fluorescence mode of action. The prodrugs release their chemotherapeutic cargo and an active cyanine fluorophore upon reaction with a specific analyte. The prodrug platform is based on the fluorogenic dye QCy7; upon removal of a triggering substrate, the dye fluoresces, and the free drug is released. The evaluated camptothecin prodrug was activated by endogenous hydrogen peroxide produced in tumor cells in vitro and in vivo. Drug release and in vitro cytotoxicity were correlated with the emitted fluorescence. The prodrug activation was effectively imaged in real time in mice bearing tumors. The modular design of the QCy7 fluorogenic platform should allow the preparation of numerous other prodrugs with various triggering substrates and chemotherapeutic agents. We anticipate that the development of real-time in vivo monitoring tools such as that described herein will pave the way for personalized therapy.

  1. Drug Delivery Nanoparticles with Locally Tunable Toxicity Made Entirely from a Light-Activatable Prodrug of Doxorubicin.

    PubMed

    Schutt, Carolyn; Ibsen, Stuart; Zahavy, Eran; Aryal, Santosh; Kuo, Stacey; Esener, Selin; Berns, Michael; Esener, Sadik

    2017-08-08

    A major challenge facing nanoparticle-based delivery of chemotherapy agents is the natural and unavoidable accumulation of these particles in healthy tissue resulting in local toxicity and dose-limiting side effects. To address this issue, we have designed and characterized a new prodrug nanoparticle with controllable toxicity allowing a locally-delivered light trigger to convert the payload of the particle from a low to a high toxicity state. The nanoparticles are created entirely from light-activatable prodrug molecules using a nanoprecipitation process. The prodrug is a conjugate of doxorubicin and photocleavable biotin (DOX-PCB). These DOX-PCB nanoparticles are 30 times less toxic to cells than doxorubicin, but can be activated to release pure therapeutic doxorubicin when exposed to 365 nm light. These nanoparticles have an average diameter of around 100 nm and achieve the maximum possible prodrug loading capacity since no support structure or coating is required to prevent loss of prodrug from the nanoparticle. These light activatable nanoparticles demonstrate tunable toxicity and can be used to facilitate future therapy development whereby light delivered specifically to the tumor tissue would locally convert the nanoparticles to doxorubicin while leaving nanoparticles accumulated in healthy tissue in the less toxic prodrug form.

  2. Enzyme replacement therapy in mucopolysaccharidosis type I: progress and emerging difficulties.

    PubMed

    Wraith, J E

    2001-04-01

    Mucopolysaccharidosis type I is due to a deficiency of the lysosomal enzyme alpha-L-iduronidase (EC 3.2.1.76) and is associated with a defect in the catabolism of the glycosaminoglycans heparan and dermatan sulphate. The clinical picture produced by this abnormal storage is diverse and ranges from a disorder that is fatal in the early months of life, due to cardiomyopathy, to a condition compatible with a normal life-span. It has become usual to describe the phenotypes associated with this spectrum of disorders after their eponymous names, Hurler (MPS IH, severe), Scheie (MPS IS, mild) and Hurler/Scheie (MPS IH/S, intermediate). Severely affected patients have progressive learning difficulties, facial and skeletal deformities, cardiac disease, corneal clouding, respiratory compromise and joint stiffness. Patients with MPS IH typically die in the first decade of life. MPS IH/S usually have normal intelligence and die in their twenties of cardiorespiratory disease. Patients with MPS IS may have joint stiffness, aortic valve disease and corneal clouding, but are often able to live a normal life-span. Enzyme replacement therapy has been developed as a potential therapy for some patients with MPS I. This process has been helped by the study of a naturally occurring canine model of the disease, which produces a phenotype similar to MPS IH/S in the human. This review details the progress that has been made in this area and also highlights some potential problems with the introduction of therapy.

  3. Enzyme augmentation therapy enhances the therapeutic efficacy of bone marrow transplantation in mucopolysaccharidosis type II mice.

    PubMed

    Akiyama, Kazumasa; Shimada, Yohta; Higuchi, Takashi; Ohtsu, Makoto; Nakauchi, Hiromitsu; Kobayashi, Hiroshi; Fukuda, Takahiro; Ida, Hiroyuki; Eto, Yoshikatsu; Crawford, Brett E; Brown, Jillian R; Ohashi, Toya

    2014-02-01

    Before the availability of an enzyme replacement therapy (ERT) for mucopolysaccharidosis type II (MPS II), patients were treated by bone marrow transplantation (BMT). However, the effectiveness of BMT for MPS II was equivocal, particularly at addressing the CNS manifestations. To study this further, we subjected a murine model of MPS II to BMT and evaluated the effect at correcting the biochemical and pathological aberrations in the viscera and CNS. Our results indicated that BMT reduced the accumulation of glycosaminoglycans (GAGs) in a variety of visceral organs, but not in the CNS. With the availability of an approved ERT for MPS II, we investigated and compared the relative merits of the two strategies either as a mono or combination therapy. We showed that the combination of BMT and ERT was additive at reducing tissue levels of GAGs in the heart, kidney and lung. Moreover, ERT conferred greater efficacy if the immunological response against the infused recombinant enzyme was low. Finally, we showed that pathologic GAGs might potentially represent a sensitive biomarker to monitor the therapeutic efficacy of therapies for MPS II. © 2013.

  4. Amino Acid Carbamates As Prodrugs Of Resveratrol.

    PubMed

    Mattarei, Andrea; Azzolini, Michele; La Spina, Martina; Zoratti, Mario; Paradisi, Cristina; Biasutto, Lucia

    2015-10-14

    Resveratrol (3, 5, 4'-trihydroxy-trans-stilbene), a plant polyphenol, has important drug-like properties, but its pharmacological exploitation in vivo is hindered by its rapid transformation via phase II conjugative metabolism. One approach to bypass this problem relies on prodrugs. We report here the synthesis, characterization, stability and in vivo pharmacokinetic behaviour of prodrugs of resveratrol in which the OH groups are engaged in an N-monosubstituted carbamate ester (-OC(O)NHR) linkage with a natural amino acid (Leu, Ile, Phe, Thr) to prevent conjugation and modulate the physicochemical properties of the molecule. We also report a convenient, high-yield protocol to obtain derivatives of this type. The new carbamate ester derivatives are stable at pH 1, while they undergo slow hydrolysis at physiological pH and hydrolyse with kinetics suitable for use in prodrugs in whole blood. After administration to rats by oral gavage the isoleucine-containing prodrug was significantly absorbed, and was present in the bloodstream as non-metabolized unaltered or partially deprotected species, demonstrating effective shielding from first-pass metabolism. We conclude that prodrugs based on the N-monosubstituted carbamate ester bond have the appropriate stability profile for the systemic delivery of phenolic compounds.

  5. Amino Acid Carbamates As Prodrugs Of Resveratrol

    PubMed Central

    Mattarei, Andrea; Azzolini, Michele; La Spina, Martina; Zoratti, Mario; Paradisi, Cristina; Biasutto, Lucia

    2015-01-01

    Resveratrol (3, 5, 4′-trihydroxy-trans-stilbene), a plant polyphenol, has important drug-like properties, but its pharmacological exploitation in vivo is hindered by its rapid transformation via phase II conjugative metabolism. One approach to bypass this problem relies on prodrugs. We report here the synthesis, characterization, stability and in vivo pharmacokinetic behaviour of prodrugs of resveratrol in which the OH groups are engaged in an N-monosubstituted carbamate ester (-OC(O)NHR) linkage with a natural amino acid (Leu, Ile, Phe, Thr) to prevent conjugation and modulate the physicochemical properties of the molecule. We also report a convenient, high-yield protocol to obtain derivatives of this type. The new carbamate ester derivatives are stable at pH 1, while they undergo slow hydrolysis at physiological pH and hydrolyse with kinetics suitable for use in prodrugs in whole blood. After administration to rats by oral gavage the isoleucine-containing prodrug was significantly absorbed, and was present in the bloodstream as non-metabolized unaltered or partially deprotected species, demonstrating effective shielding from first-pass metabolism. We conclude that prodrugs based on the N-monosubstituted carbamate ester bond have the appropriate stability profile for the systemic delivery of phenolic compounds. PMID:26463125

  6. Expression and purification of a human, soluble Arylsulfatase A for Metachromatic Leukodystrophy enzyme replacement therapy.

    PubMed

    Martino, Sabata; Consiglio, Antonella; Cavalieri, Cristina; Tiribuzi, Roberto; Costanzi, Egidia; Severini, Giovanni Maria; Emiliani, Carla; Bordignon, Claudio; Orlacchio, Aldo

    2005-05-25

    The production of active Arylsulfatase A is a key step in the development of enzyme replacement therapy for Metachromatic Leukodystrophy. To obtain large amounts of purified Arylsulfatase A for therapeutic use, we combined a retroviral expression system with a versatile and rapid purification protocol that can easily and reliably be adapted to high-throughput applications. The purification method consists of an initial ion-exchange DEAE-cellulose chromatography step followed by immuno-affinity purification using a polyclonal antibody against a 29-mer peptide of the Arylsulfatase A sequence. Immuno-adsorbed protein was eluted with a combination of acidic pH and an optimal concentration of the 29-mer peptide. This protocol reproducibly yielded approximately 100 microg of >99% pure human Arylsulfatase A, corresponding to 152 mU of enzyme activity, per liter of culture medium with properties similar to those of human non-recombinant protein.

  7. Delivery of a Protease-Activated Cytolytic Peptide Prodrug by Perfluorocarbon Nanoparticles.

    PubMed

    Jallouk, Andrew P; Palekar, Rohun U; Marsh, Jon N; Pan, Hua; Pham, Christine T N; Schlesinger, Paul H; Wickline, Samuel A

    2015-08-19

    Melittin is a cytolytic peptide derived from bee venom that inserts into lipid membranes and oligomerizes to form membrane pores. Although this peptide is an attractive candidate for treatment of cancers and infectious processes, its nonspecific cytotoxicity and hemolytic activity have limited its therapeutic applications. Several groups have reported the development of cytolytic peptide prodrugs that only exhibit cytotoxicity following activation by site-specific proteases. However, systemic administration of these constructs has proven difficult because of their poor pharmacokinetic properties. Here, we present a platform for the design of protease-activated melittin derivatives that may be used in conjunction with a perfluorocarbon nanoparticle delivery system. Although native melittin was substantially hemolytic (HD50: 1.9 μM) and cytotoxic (IC50: 2.4 μM), the prodrug exhibited 2 orders of magnitude less hemolytic activity (HD50: > 100 μM) and cytotoxicity (IC50: > 100 μM). Incubation with matrix metalloproteinase-9 (MMP-9) led to cleavage of the prodrug at the expected site and restoration of hemolytic activity (HD50: 3.4 μM) and cytotoxicity (IC50: 8.1 μM). Incubation of the prodrug with perfluorocarbon nanoparticles led to stable loading of 10,250 peptides per nanoparticle. Nanoparticle-bound prodrug was also cleaved and activated by MMP-9, albeit at a fourfold slower rate. Intravenous administration of prodrug-loaded nanoparticles in a mouse model of melanoma significantly decreased tumor growth rate (p = 0.01). Because MMPs and other proteases play a key role in cancer invasion and metastasis, this platform holds promise for the development of personalized cancer therapies directed toward a patient's individual protease expression profile.

  8. Anti-TNF-Alpha Therapy Enhances the Effects of Enzyme Replacement Therapy in Rats with Mucopolysaccharidosis Type VI

    PubMed Central

    Eliyahu, Efrat; Wolfson, Theodore; Ge, Yi; Jepsen, Karl J.; Schuchman, Edward H.; Simonaro, Calogera M.

    2011-01-01

    Background Although enzyme replacement therapy (ERT) is available for several lysosomal storage disorders, the benefit of this treatment to the skeletal system is very limited. Our previous work has shown the importance of the Toll-like receptor 4/TNF-alpha inflammatory pathway in the skeletal pathology of the mucopolysaccharidoses (MPS), and we therefore undertook a study to examine the additive benefit of combining anti-TNF-alpha therapy with ERT in a rat model of MPS type VI. Methodology/Principal Findings MPS VI rats were treated for 8 months with Naglazyme® (recombinant human N-acetyl-galactosamine-4-sulfatase), or by a combined protocol using Naglazyme® and the rat-specific anti-TNF-alpha drug, CNTO1081. Both protocols led to markedly reduced serum levels of TNF-alpha and RANKL, although only the combined treatment reduced TNF-alpha in the articular cartilage. Analysis of cultured articular chondrocytes showed that the combination therapy also restored collagen IIA1 expression, and reduced expression of the apoptotic marker, PARP. Motor activity and mobility were improved by ERT, and these were significantly enhanced by combination treatment. Tracheal deformities in the MPS VI animals were only improved by combination therapy, and there was a modest improvement in bone length. Ceramide levels in the trachea also were markedly reduced. MicroCT analysis did not demonstrate any significant positive effects on bone microarchitecture from either treatment, nor was there histological improvement in the bone growth plates. Conclusions/Significance The results demonstrate that combining ERT with anti-TNF- alpha therapy improved the treatment outcome and led to significant clinical benefit. They also further validate the usefulness of TNF-alpha, RANKL and other inflammatory molecules as biomarkers for the MPS disorders. Further evaluation of this combination approach in other MPS animal models and patients is warranted. PMID:21887218

  9. Exercise testing in late-onset glycogen storage disease type II patients undergoing enzyme replacement therapy

    PubMed Central

    Marzorati, Mauro; Porcelli, Simone; Bellistri, Giuseppe; Morandi, Lucia; Grassi, Bruno

    2012-01-01

    Enzyme replacement therapy (ERT) has recently became available for patients with glycogen storage disease type II. Previous studies have demonstrated clinical efficacy of enzyme replacement therapy, however, data on physiological variables related to exercise tolerance are scarce. Four glycogen storage disease type II late-onset patients (45 ± 6 years) performed an incremental exercise on a cycle ergometer, up to voluntary exhaustion, before (BEFORE) and after 12 months of ERT (AFTER). Peak workload, oxygen uptake, heart rate, cardiac output (by impedance cardiography) and vastus lateralis oxygenation indices (by continuous-wave near-infrared spectroscopy, NIRS) were determined. Peak workload and oxygen uptake values significantly increased during ERT (54 ± 30 vs. 63 ± 31 watt, and 17.2 ± 4.4 vs. 19.7 ± 3.5 ml/kg/min, respectively, in BEFORE vs. AFTER). On the other hand, for both peak cardiac output (12.3 ± 5.3 vs. 14.8 ± 4.5 L/min) and the NIRS-determined peak skeletal muscle fractional O2 extraction, expressed as a percentage of the maximal values during a transient limb ischemia (30 ± 39% vs. 38 ± 28%), the observed increases were not statistically significant. Our findings suggest that in glycogen storage disease type II patients enzyme replacement therapy is associated with a mild improvement of exercise tolerance. The findings need to be validated during a longer follow-up on a larger group of patients. PMID:23182645

  10. [Antioxidant enzymes in erythrocytes from hypertension patients receiving lisinopril monotherapy or combined lisinopril plus simvastatin therapy].

    PubMed

    Kosenko, E A; Suslikov, A V; Venediktova, N I; Kaminskiĭ, Iu G

    2011-01-01

    Statins and angiotensin-converting enzyme (ACE) inhibitors have beneficial impact on the serum cholesterol and blood pressure. It is supposed that statins and ACE inhibitors may modify the antioxidative status of erythrocytes. The study objective was to compare the effects of two treatments, lisinopril alone vs lisinopril plus simvastatin, on erythrocyte antioxidant enzyme activities. The study involved 32 patients with arterial hypertension, the initial serum total cholesterol, LDL-cholesterol and triglycerides within the normal range. Patients of two groups, each of 16 subjects, were treated with lisinopril (10 mg/day) or with lisinopril (10 mg/day) plus simvastatin (20 mg/day). Before and after 3 and 6 months of follow-up therapy, activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GLR) in purified erythrocytes were determined. In all patients, significantly higher catalase activity (by 79.3-106.5%, p < 0.0001) and significantly lower GPx activity (by 20.7-30.6%, p < 0.001) were observed after therapy as compared to the baselines. Just the same results were obtained in both groups (lisinopril and lisinopril + simvastatin), after both periods (3 and 6 month) of treatments. SOD activity was increased only in the lisinopril group and only after 6 months (p = 0.0345). No changes of GLR reductase activity were seen under all conditions indicated. Thus, the lisinopril monotherapy and combined lisinopril plus simvastatin therapy exhibit specific, pronounced and equipotent effects on antioxidant enzymes in human erythrocytes. Administration of lisinopril or lisinopril plus simvastatin may protect erythrocytes and other tissues from oxidative damage.

  11. [Radiation Anticarcinogenesis by Thiazolidine Pro-drug

    NASA Technical Reports Server (NTRS)

    Warters, Raymond L.; Roberts, Jeanette C.; Fain, Heidi

    1999-01-01

    The original goal of this work was to determine the capacity of selected aminothiols to modulate radiation induced cytotoxicity, mutagenesis and carcinogenesis in a human mammary epithelial cell line. The conclusions from this work are that WR-1065 is the "gold standard" for protection against radiation induced cytotoxicity, mutagenesis and carcinogenesis. While a potent radiation protector, WR-1065 is cytotoxic in vitro and in vivo. Our rationale for a study of the thiazolidine pro-drugs was that these compounds are neither toxic in vitro or in vivo. The results obtained during this funding period indicate that the thiazolidine pro-drugs are as potent as WR-1065 as protectors against radiation induced mutation induction, and thus presumably against radiation induced carcinogenesis. Our results indicate that the thiazolidine prodrugs are excellent candidates to test as non-toxic anticarcinogens for protecting astronauts from cancer induction during space travel.

  12. [Radiation Anticarcinogenesis by Thiazolidine Pro-drug

    NASA Technical Reports Server (NTRS)

    Warters, Raymond L.; Roberts, Jeanette C.; Fain, Heidi

    1999-01-01

    The original goal of this work was to determine the capacity of selected aminothiols to modulate radiation induced cytotoxicity, mutagenesis and carcinogenesis in a human mammary epithelial cell line. The conclusions from this work are that WR-1065 is the "gold standard" for protection against radiation induced cytotoxicity, mutagenesis and carcinogenesis. While a potent radiation protector, WR-1065 is cytotoxic in vitro and in vivo. Our rationale for a study of the thiazolidine pro-drugs was that these compounds are neither toxic in vitro or in vivo. The results obtained during this funding period indicate that the thiazolidine pro-drugs are as potent as WR-1065 as protectors against radiation induced mutation induction, and thus presumably against radiation induced carcinogenesis. Our results indicate that the thiazolidine prodrugs are excellent candidates to test as non-toxic anticarcinogens for protecting astronauts from cancer induction during space travel.

  13. Comparative efficacies of 2 cysteine prodrugs and a glutathione delivery agent in a colitis model

    PubMed Central

    OZ, HELIEH S.; CHEN, THERESA S.; NAGASAWA, HERBERT

    2007-01-01

    Oxidant-mediated injury plays an important role in the pathophysiology of inflammatory bowel disease (IBD). Recently, antioxidants were shown to modulate colitis in mice. In this study, the protective effects of L-cysteine and glutathione (GSH) prodrugs are further evaluated against progression of colitis in a murine model. ICR mice were fed compounds incorporated into chow as follows: Group (A) received chow supplemented with vehicle. Group (B) was provided 2-(RS)-n-propylthiazolidine-4(R)-carboxylic-acid (PTCA), a cysteine prodrug. Group (C) received D-ribose-L-cysteine (RibCys), another cysteine prodrug that releases L-cysteine. Group (D) was fed L-cysteine-glutathione mixed sulfide (CySSG), a ubiquitous GSH derivative present in mammalian cells. After 3 days, the animals were further provided with normal drinking water or water supplemented with dextran sodium sulfate (DSS). Mice administered DSS developed severe colitis and suffered weight loss. Colonic lesions significantly improved in animals treated with PTCA and RibCys and, to a lesser extent, with CySSG therapy. Hepatic GSH levels were depleted in colitis animals (control vs DSS, P < 0.001), and normalized with prodrug therapies (control vs treatments, P > 0.05). Protein expressions of serum amyloid A and inflammatory cytokines [interleukin (IL)-6, IL-12, tumor necrosis factor-alpha (TNF-α), osteopontin (OPN)] were significantly increased in colitis animals and improved with therapies. Immunohistochemistry and Western blot analyses showed significant upregulation of the macrophage-specific markers, COX-2 and CD68, which suggests macrophage activation and infiltration in the colonic lamina propria in colitis animals. These abnormalities were attenuated in prodrug-treated mice. In conclusion, these data strongly support the novel action of the PTCA against colitis, which further supports a possible therapeutic application for IBD patients. PMID:17656332

  14. Enzyme replacement therapy with idursulfase for mucopolysaccharidosis type II (Hunter syndrome).

    PubMed

    da Silva, Edina M K; Strufaldi, Maria Wany Louzada; Andriolo, Régis B; Silva, Laercio A

    2014-01-08

    Mucopolysaccharidosis II, also known as Hunter syndrome, is a rare, X-linked disease caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase, which catalyses a step in the catabolism of glycosaminoglycans. The glycosaminoglycans accumulate within tissues affecting multiple organs and physiologic systems. The clinical manifestations include neurologic involvement, severe airways obstruction, skeletal deformities and cardiomyopathy. The disease has a variable age of onset and variable rate of progression. In those with severe disease, death usually occurs in the second decade of life, whereas those patients with less severe disease may survive into adulthood. Enzyme replacement therapy with intravenous infusions of idursulfase has emerged as a new treatment for mucopolysaccharidosis type II. To evaluate the effectiveness and safety of enzyme replacement therapy with idursulfase compared to other interventions, placebo or no intervention, for treating mucopolysaccharidosis type II. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Trials Register (date of last search 22 July 2013).We also searched EMBASE, PubMed and the Literature Latino-Americana e do Caribe em Ciências da Saúde (LILACS) (date of last search 09 July 2013). Randomised and quasi-randomised controlled trials of enzyme replacement therapy with idursulfase compared to no intervention, placebo or other options (e.g. behavioral strategies, transplantation). Two authors independently screened the trials identified, appraised quality of papers and extracted data. One study (96 patients) met the inclusion criteria, although the primary outcome of this review - z score for height and weight, was not assessed in the study. This trial was considered to be of overall good quality. Following 53 weeks of treatment, patients in the weekly idursulfase 0.5 mg/kg group demonstrated a significant improvement rate compared with placebo for the primary outcome: distance walked in six

  15. Enzyme replacement therapy with idursulfase for mucopolysaccharidosis type II (Hunter syndrome).

    PubMed

    da Silva, Edina M K; Strufaldi, Maria Wany Louzada; Andriolo, Regis B; Silva, Laercio A

    2016-02-05

    Mucopolysaccharidosis II, also known as Hunter syndrome, is a rare, X-linked disease caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase, which catalyses a step in the catabolism of glycosaminoglycans. The glycosaminoglycans accumulate within tissues affecting multiple organs and physiologic systems. The clinical manifestations include neurologic involvement, severe airways obstruction, skeletal deformities and cardiomyopathy. The disease has a variable age of onset and variable rate of progression. In those with severe disease, death usually occurs in the second decade of life, whereas those individuals with less severe disease may survive into adulthood. Enzyme replacement therapy with intravenous infusions of idursulfase has emerged as a new treatment for mucopolysaccharidosis type II. This is an update of a previously published version of this review. To evaluate the effectiveness and safety of enzyme replacement therapy with idursulfase compared to other interventions, placebo or no intervention, for treating mucopolysaccharidosis type II. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Trials Register (date of last search 23 November 2015).We also searched Embase, PubMed and the Literature Latino-Americana e do Caribe em Ciências da Saúde (LILACS) (date of last search 28 November 2015). Randomised and quasi-randomised controlled trials of enzyme replacement therapy with idursulfase compared to no intervention, placebo or other options (e.g. behavioral strategies, transplantation). Two authors independently screened the trials identified, appraised quality of papers and extracted data. One study (96 male participants) met the inclusion criteria, although the primary outcome of this review - z score for height and weight, was not assessed in the study. This trial was considered to be of overall good quality. Following 53 weeks of treatment, participants in the weekly idursulfase 0.5 mg/kg group demonstrated a

  16. Enzyme replacement therapy with idursulfase for mucopolysaccharidosis type II (Hunter syndrome).

    PubMed

    da Silva, Edina Mk; Strufaldi, Maria Wany Louzada; Andriolo, Régis B; Silva, Laercio A

    2011-11-09

    Mucopolysaccharidosis II, also known as Hunter syndrome, is a rare, X-linked disease caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase, which catalyses a step in the catabolism of glycosaminoglycans. The glycosaminoglycans accumulate within tissues affecting multiple organs and physiologic systems. The clinical manifestations include neurologic involvement, severe airways obstruction, skeletal deformities and cardiomyopathy. The disease has a variable age of onset and variable rate of progression. In those with severe disease, death usually occurs in the second decade of life, whereas those patients with less severe disease may survive into adulthood. Enzyme replacement therapy with intravenous infusions of idursulfase has emerged as a new treatment for mucopolysaccharidosis type II. To evaluate the effectiveness and safety of enzyme replacement therapy with idursulfase compared to other interventions, placebo or no intervention, for treating mucopolysaccharidosis type II. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Trials Register (date of last search 01 September 2011).We also searched EMBASE, PubMed and the Literature Latino-Americana e do Caribe em Ciências da Saúde (LILACS) (date of last search October 2009). Randomised and quasi-randomised controlled trials of enzyme replacement therapy with idursulfase compared to no intervention, placebo or other options (e.g. behavioral strategies, transplantation). Two authors independently screened the trials identified, appraised quality of papers and extracted data. One study (96 patients) met the inclusion criteria, although the primary outcome of this review - z score for height and weight, was not assessed in the study. Following 53 weeks of treatment, patients in the weekly idursulfase 0.5 mg/kg group demonstrated a significant improvement rate compared with placebo for the primary outcome: distance walked in six minutes on the basis of the sum of ranks of change from

  17. Dipeptidyl peptidase IV-activated prodrugs of anti-varicella zoster virus bicyclic nucleoside analogues containing different self-cleavage spacer systems.

    PubMed

    Diez-Torrubia, Alberto; Cabrera, Silvia; De Meester, Ingrid; Camarasa, María-José; Balzarini, Jan; Velázquez, Sonsoles

    2012-09-01

    A new type of double prodrug of the antiviral family of bicyclic nucleoside analogues (BCNA) bearing cyclization self-cleavage spacers between the Val-Pro dipeptide sequence as well as the parent compound were synthesized and evaluated with regard to activation by the DPPIV/CD26 enzyme and for their stability in human and bovine serum. In buffer solution, carbamate and ester prodrugs were found to be chemically stable. Most prodrugs containing a dipeptidyl linker efficiently converted into the BCNA parent drug. In contrast, the Val-Pro alkyldiamino prodrugs converted predominantly into their alkyldiamino prodrug intermediates in the presence of CD26 and human serum. A marked increase in water solubility was observed for all prodrugs. In contrast to the parent compound, a tetrapeptide prodrug containing the Val-Val dipeptide as a self-cleavage spacer released substantial amounts of the BCNA parent drug at the basolateral side of Caco-2 cell cultures and exhibited 15- to 20-fold increased bioavailability in mice relative to the poorly bioavailable parent compound.

  18. Prodrugs of herpes simplex thymidine kinase inhibitors.

    PubMed

    Yanachkova, Milka; Xu, Wei-Chu; Dvoskin, Sofya; Dix, Edward J; Yanachkov, Ivan B; Focher, Federico; Savi, Lida; Sanchez, M Dulfary; Foster, Timothy P; Wright, George E

    2015-04-01

    Because guanine-based herpes simplex virus thymidine kinase inhibitors are not orally available, we synthesized various 6-deoxy prodrugs of these compounds and evaluated them with regard to solubility in water, oral bioavailability, and efficacy to prevent herpes simplex virus-1 reactivation from latency in a mouse model. Organic synthesis was used to prepare compounds, High Performance Liquid Chromatography (HPLC) to analyze hydrolytic conversion, Mass Spectrometry (MS) to measure oral bioavailability, and mouse latent infection and induced reactivation to evaluate the efficacy of a specific prodrug. Aqueous solubilities of prodrugs were improved, oxidation of prodrugs by animal cytosols occurred in vitro, and oral absorption of the optimal prodrug sacrovir™ (6-deoxy-mCF3PG) in the presence of the aqueous adjuvant Soluplus® and conversion to active compound N(2)-[3-(trifluoromethyl)pheny])guanine (mCF3PG) were accomplished in mice. Treatment of herpes simplex virus-1 latent mice with sacrovir™ in 1% Soluplus in drinking water significantly suppressed herpes simplex virus-1 reactivation and viral genomic replication. Ad libitum oral delivery of sacrovir™ was effective in suppressing herpes simplex virus-1 reactivation in ocularly infected latent mice as measured by the numbers of mice shedding infectious virus at the ocular surface, numbers of trigeminal ganglia positive for infectious virus, number of corneas that had detectable infectious virus, and herpes simplex virus-1 genome copy numbers in trigeminal ganglia following reactivation. These results demonstrate the statistically significant effect of the prodrug on suppressing herpes simplex virus-1 reactivation in vivo. © The Author(s) 2015.

  19. Lactic acid oligomers (OLAs) as prodrug moieties.

    PubMed

    Kruse, J; Lachmann, B; Lauer, R; Eppacher, S; Noe, C R

    2013-02-01

    In this paper we propose the use of lactic acid oligomers (OLAs) as prodrug moieties. Two synthetic approaches are presented, on the one hand a non selective oligomerisation of lactic acid and on the other hand a block synthesis to tetramers of lactic acid. Dimers of lactic acid were investigated with respect to their plasma stability and their adsorption to albumine. Ibuprofen was chosen as the first drug for OLAylation. The ester 19 of LA(1)-ibuprofen was evaluated with respect to the degradation to human plasma and the adsorption to albumine. All results indicate that lactic acid oligomers are promising prodrug moieties.

  20. [In vitro metabolism of fenbendazole prodrug].

    PubMed

    Wen, Ai-Dan; Duan, Li-Ping; Liu, Cong-Shan; Tao, Yi; Xue, Jian; Wu, Ning-Bo; Jiang, Bin; Zhang, Hao-Bing

    2013-02-01

    Synthesized fenbendazole prodrug N-methoxycarbonyl-N'-(2-nitro-4-phenylthiophenyl) thiourea (MPT) was analyzed in vitro in artificial gastric juice, intestinal juice and mouse liver homogenate model by using HPLC method, and metabolic curve was then generated. MPT was tested against Echinococcus granulosus protoscolices in vitro. The result showed that MPT could be metabolized in the three biological media, and to the active compound fenbendazole in liver homogenate, with a metabolic rate of 7.92%. Besides, the prodrug showed a weak activity against E. granulosus protoscolices with a mortality of 45.9%.

  1. Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing lipophilic gemcitabine prodrugs.

    PubMed

    Immordino, Maria Laura; Brusa, Paola; Rocco, Flavio; Arpicco, Silvia; Ceruti, Maurizio; Cattel, Luigi

    2004-12-10

    Gemcitabine is a known anticancer agent rapidly deaminated to the inactive metabolite 2',2'-difluorodeoxyuridine; it must therefore be administered at very high dose. Many different approaches have been tried to improve the metabolic stability; we synthesized a series of increasingly lipophilic prodrugs of gemcitabine by linking the 4-amino group with valeroyl, heptanoyl, lauroyl and stearoyl linear acyl derivatives. We studied their stability at storage, in plasma and with the lysosomal intracellular enzyme cathepsins. We studied incorporation of these lipophilic prodrugs in liposomes, where their encapsulation efficiency (EE) closely depends on the length of the saturated 4-(N)-acyl chain, the phospholipids chosen and the presence of cholesterol. A maximum EE of 98% was determined for 4-(N)-stearoyl-gemcitabine incorporated in DSPC/DSPG 9:1. This formulation was correlated with the highest stability in vitro and in vivo. Cytotoxicity of gemcitabine prodrugs, free or encapsulated in liposomes, was between two- and sevenfold that of free gemcitabine. Encapsulation of long-chain lipophilic prodrugs of gemcitabine in liposomes protected the drug from degradation in plasma, assuring a long plasma half-time and intracellular release of the free drug.

  2. Biomolecules damage and redox status abnormalities in Fabry patients before and during enzyme replacement therapy.

    PubMed

    Biancini, Giovana Brondani; Jacques, Carlos Eduardo; Hammerschmidt, Tatiane; de Souza, Heryk Motta; Donida, Bruna; Deon, Marion; Vairo, Filippo Pinto; Lourenço, Charles Marques; Giugliani, Roberto; Vargas, Carmen Regla

    2016-10-01

    Fabry disease (FD) is caused by deficient activity of the lysosomal enzyme α-galactosidase A. Its substrates, mainly globotriaosylceramide (Gb3), accumulate and seem to induce other pathophysiological findings of FD. Once enzyme replacement therapy (ERT) is not completely efficient on preventing disease progress in FD patients, elucidating the underlying mechanisms in FD pathophysiology is essential to the development of additional therapeutic strategies. We investigated 58 Fabry patients (23 male and 35 female) subdivided into two groups (at diagnosis and during long-term ERT) and compared them to healthy individuals. Fabry patients at diagnosis presented altered glutathione (GSH) metabolism (higher GSH levels, lower glutathione peroxidase - GPx - and normal glutathione reductase - GR - activities), higher lipid peroxidation levels (thiobarbituric acid reactive species - TBARS - and malondialdehyde - MDA), nitric oxide (NO(.)) equivalents and urinary Gb3. Fabry patients on ERT presented GSH metabolism similar to controls, although lipid peroxidation and urinary levels of NO(.) equivalents remained higher whereas Gb3 levels were lower than at diagnosis but still higher than controls. These data demonstrated that redox impairment occurs in Fabry patients before and after ERT, probably as a consequence of Gb3 accumulation, providing targets to future therapy approaches using antioxidants in combination with ERT in FD.

  3. Highly Versatile Polyelectrolyte Complexes for Improving the Enzyme Replacement Therapy of Lysosomal Storage Disorders.

    PubMed

    Giannotti, Marina I; Abasolo, Ibane; Oliva, Mireia; Andrade, Fernanda; García-Aranda, Natalia; Melgarejo, Marta; Pulido, Daniel; Corchero, José L; Fernández, Yolanda; Villaverde, Antonio; Royo, Miriam; García-Parajo, María F; Sanz, Fausto; Schwartz, Simó

    2016-10-05

    Lysosomal storage disorders are currently treated by enzyme replacement therapy (ERT) through the direct administration of the unprotected recombinant protein to the patients. Herein we present an ionically cross-linked polyelectrolyte complex (PEC) composed of trimethyl chitosan (TMC) and α-galactosidase A (GLA), the defective enzyme in Fabry disease, with the capability of directly targeting endothelial cells by incorporating peptide ligands containing the RGD sequence. We assessed the physicochemical properties, cytotoxicity, and hemocompatibility of RGD-targeted and untargeted PECs, the uptake by endothelial cells and the intracellular activity of PECs in cell culture models of Fabry disease. Moreover, we also explored the effect of different freeze-drying procedures in the overall activity of the PECs. Our results indicate that the use of integrin-binding RGD moiety within the PEC increases their uptake and the efficacy of the GLA enzyme, while the freeze-drying allows the activity of the therapeutic protein to remain intact. Overall, these results highlight the potential of TMC-based PECs as a highly versatile and feasible drug delivery system for improving the ERT of lysosomal storage disorders.

  4. Novel prodrugs with a spontaneous cleavable guanidine moiety.

    PubMed

    Hamada, Yoshio

    2016-04-01

    Water-soluble prodrug strategy is a practical alternative for improving the drug bioavailability of sparingly-soluble drugs with reduced drug efficacy. Many water-soluble prodrugs of sparingly-soluble drugs, such as the phosphate ester of a drug, have been reported. Recently, we described a novel water-soluble prodrug based on O-N intramolecular acyl migration. However, these prodrug approaches require a hydroxy group in the structure of their drugs, and other prodrug approaches are often restricted by the structure of the parent drugs. To develop prodrugs with no restriction in the structure, we focused on a decomposition reaction of arginine methyl ester. This reaction proceeds at room temperature under neutral conditions, and we applied this reaction to the prodrug strategy for drugs with an amino group. We designed and synthesized novel prodrugs of representative sparingly soluble drugs phenytoin and sulfathiazole. Phenytoin and sulfathiazole were obtained as stable salt that were converted to parent drugs under physiological conditions. Phenytoin prodrug 3 showed a short half-life (t1/2) of 13min, whereas sulfathiazole prodrug 7 had a moderate t1/2 of 40min. Prodrugs 3 and 7 appear to be suitable for use as an injectable formulation and orally administered drug, respectively.

  5. Enhanced response to enzyme replacement therapy in Pompe disease after the induction of immune tolerance.

    PubMed

    Sun, Baodong; Bird, Andrew; Young, Sarah P; Kishnani, Priya S; Chen, Y-T; Koeberl, Dwight D

    2007-11-01

    Pompe disease, which results from mutations in the gene encoding the glycogen-degrading lysosomal enzyme acid alpha -glucosidase (GAA) (also called "acid maltase"), causes death in early childhood related to glycogen accumulation in striated muscle and an accompanying infantile-onset cardiomyopathy. The efficacy of enzyme replacement therapy (ERT) with recombinant human GAA was demonstrated during clinical trials that prolonged subjects' overall survival, prolonged ventilator-free survival, and also improved cardiomyopathy, which led to broad-label approval by the U.S. Food and Drug Administration. Patients who lack any residual GAA expression and are deemed negative for cross-reacting immunologic material (CRIM) have a poor response to ERT. We previously showed that gene therapy with an adeno-associated virus (AAV) vector containing a liver-specific promoter elevated the GAA activity in plasma and prevented anti-GAA antibody formation in immunocompetent GAA-knockout mice for 18 wk, predicting that liver-specific expression of human GAA with the AAV vector would induce immune tolerance and enhance the efficacy of ERT. In this study, a very low number of AAV vector particles was administered before initiation of ERT, to prevent the antibody response in GAA-knockout mice. A robust antibody response was provoked in naive GAA-knockout mice by 6 wk after a challenge with human GAA and Freund's adjuvant; in contrast, administration of the AAV vector before the GAA challenge prevented the antibody response. Most compellingly, the antibody response was prevented by AAV vector administration during the 12 wk of ERT, and the efficacy of ERT was thereby enhanced. Thus, AAV vector-mediated gene therapy induced a tolerance to introduced GAA, and this strategy could enhance the efficacy of ERT in CRIM-negative patients with Pompe disease and in patients with other lysosomal storage diseases.

  6. Enhanced Response to Enzyme Replacement Therapy in Pompe Disease after the Induction of Immune Tolerance

    PubMed Central

    Sun, Baodong ; Bird, Andrew ; Young, Sarah P. ; Kishnani, Priya S. ; Chen, Y.-T. ; Koeberl, Dwight D. 

    2007-01-01

    Pompe disease, which results from mutations in the gene encoding the glycogen-degrading lysosomal enzyme acid α-glucosidase (GAA) (also called “acid maltase”), causes death in early childhood related to glycogen accumulation in striated muscle and an accompanying infantile-onset cardiomyopathy. The efficacy of enzyme replacement therapy (ERT) with recombinant human GAA was demonstrated during clinical trials that prolonged subjects’ overall survival, prolonged ventilator-free survival, and also improved cardiomyopathy, which led to broad-label approval by the U.S. Food and Drug Administration. Patients who lack any residual GAA expression and are deemed negative for cross-reacting immunologic material (CRIM) have a poor response to ERT. We previously showed that gene therapy with an adeno-associated virus (AAV) vector containing a liver-specific promoter elevated the GAA activity in plasma and prevented anti-GAA antibody formation in immunocompetent GAA-knockout mice for 18 wk, predicting that liver-specific expression of human GAA with the AAV vector would induce immune tolerance and enhance the efficacy of ERT. In this study, a very low number of AAV vector particles was administered before initiation of ERT, to prevent the antibody response in GAA-knockout mice. A robust antibody response was provoked in naive GAA-knockout mice by 6 wk after a challenge with human GAA and Freund’s adjuvant; in contrast, administration of the AAV vector before the GAA challenge prevented the antibody response. Most compellingly, the antibody response was prevented by AAV vector administration during the 12 wk of ERT, and the efficacy of ERT was thereby enhanced. Thus, AAV vector–mediated gene therapy induced a tolerance to introduced GAA, and this strategy could enhance the efficacy of ERT in CRIM-negative patients with Pompe disease and in patients with other lysosomal storage diseases. PMID:17924344

  7. Characterization of pre- and post-treatment pathology after enzyme replacement therapy for Pompe disease.

    PubMed

    Thurberg, Beth L; Lynch Maloney, Colleen; Vaccaro, Charles; Afonso, Kendra; Tsai, Anne Chun-Hui; Bossen, Edward; Kishnani, Priya S; O'Callaghan, Michael

    2006-12-01

    In Pompe disease, a genetic deficiency of lysosomal acid alpha-glucosidase, glycogen accumulates abnormally in the lysosomes of skeletal, cardiac and smooth muscle, and contributes to clinically progressive and debilitating muscle weakness. The present study involved 8 infantile-onset Pompe patients, treated weekly with 10 mg/kg of recombinant human acid alpha-glucosidase (rhGAA). Muscle biopsies were obtained at baseline, 12 and 52 weeks post-treatment to establish an indicator of efficacy. Several histologic strategies were employed to characterize changes in pre- and post-treatment samples, including high-resolution light microscopy and digital histomorphometry, electron microscopy, capillary density and fiber type analysis, and confocal microscopy for satellite cell activation analysis. Histomorphometric analysis was performed on muscle samples to assess glycogen depletion in response to enzyme replacement therapy (ERT). The extent of glycogen clearance varied widely among these patient samples, and correlated well with clinical outcome. Low glycogen levels, mild ultrastructural damage, a high proportion of type I fibers, and young age at baseline were all features associated with good histologic response. There was no correlation between capillary density and glycogen clearance, and activated satellite cell levels were shown to be higher in post-treatment biopsies with poor histologic responses. This histopathologic study of infantile Pompe disease provides detailed insight into the cellular progression of the disease and its response to therapy while highlighting a number of methodologies which may be employed to assess regression or progression of the associated pathology. As enzyme replacement therapy becomes more prevalent for the treatment of lysosomal storage diseases, such evaluation of post-treatment pathology will likely become a more common occurrence in the daily practice of pathologists.

  8. Gene therapy/bone marrow transplantation in ADA-deficient mice: roles of enzyme-replacement therapy and cytoreduction.

    PubMed

    Carbonaro, Denise A; Jin, Xiangyang; Wang, Xingchao; Yu, Xiao-Jin; Rozengurt, Nora; Kaufman, Michael L; Wang, Xiaoyan; Gjertson, David; Zhou, Yang; Blackburn, Michael R; Kohn, Donald B

    2012-11-01

    Gene therapy (GT) for adenosine deaminase-deficient severe combined immune deficiency (ADA-SCID) can provide significant long-term benefit when patients are given nonmyeloablative conditioning and ADA enzyme-replacement therapy (ERT) is withheld before autologous transplantation of γ-retroviral vector-transduced BM CD34+ cells. To determine the contributions of conditioning and discontinuation of ERT to the therapeutic effects, we analyzed these factors in Ada gene knockout mice (Ada(-/-)). Mice were transplanted with ADA-deficient marrow transduced with an ADA-expressing γ-retroviral vector without preconditioning or after 200 cGy or 900 cGy total-body irradiation and evaluated after 4 months. In all tissues analyzed, vector copy numbers (VCNs) were 100- to 1000-fold greater in mice receiving 900 cGy compared with 200 cGy (P < .05). In mice receiving 200 cGy, VCN was similar whether ERT was stopped or given for 1 or 4 months after GT. In unconditioned mice, there was decreased survival with and without ERT, and VCN was very low to undetectable. When recipients were conditioned with 200 cGy and received transduced lineage-depleted marrow, only recipients receiving ERT (1 or 4 months) had detectable vector sequences in thymocytes. In conclusion, cytoreduction is important for the engraftment of gene-transduced HSC, and short-term ERT after GT did not diminish the capacity of gene-corrected cells to engraft and persist.

  9. Prodrug approach to improve absorption of prednisolone

    PubMed Central

    Sheng, Ye; Yang, Xiaoyan; Pal, Dhananjay; Mitra, Ashim K.

    2015-01-01

    Amino acid and dipeptide prodrugs have been developed to examine their potential in enhancing aqueous solubility and permeability as well as to bypass P-glycoprotein (P-gp) mediated cellular efflux of prednisolone. Prodrugs have been synthesized and identified with LC/MS/MS and NMR. Prodrugs displayed significantly higher aqueous solubility relative to prednisolone. These compounds also exhibited higher stability under acidic conditions relative to basic medium. [14]-Erythromycin uptake remained unaltered in the presence of valine-valine-prednisolone (VVP) indicating lower affinity towards P-gp. Moreover, VVP generated significantly higher transepithelial permeability across MDCK-MDR1 cells compared to prednisolone. Importantly, [3H]-GlySar uptake diminished significantly in the presence of VVP indicating high affinity towards peptide transporters. Moreover, prednisolone was regenerated from VVP due to enzymatic hydrolysis in SIRC cell homogenate. Results obtained from these studies clearly suggest that peptide transporter targeted prodrugs is a viable strategy to improve aqueous solubility and overcome P-gp mediated cellular efflux of prednisolone. PMID:25888804

  10. Effects of the angiotensin converting enzyme inhibitor enalapril compared with diuretic therapy in elderly hypertensive patients.

    PubMed

    Verza, M; Cacciapuoti, F; Spiezia, R; D'Avino, M; Arpino, G; D'Errico, S; Sepe, J; Varricchio, M

    1988-11-01

    The aim of this study was to evaluate the usefulness of the angiotensin converting enzyme (ACE) inhibitor enalapril in a group of 30 patients (mean age 73.3 years) with moderate hypertension and normal haematological and chemical parameters (170 +/- 8.1 mmHg systolic and 104 +/- 5.8 mmHg diastolic blood pressure), who were receiving diuretic therapy with chlorthalidone (12.5 mg/day). This therapy caused a significant decrease in systolic and diastolic blood pressure (to 165 +/- 6.7 and 98 +/- 4.7 mmHg, respectively; P less than 0.001) but it also induced hypokalaemia (3.04 +/- 0.7 mmol/l; P less than 0.001) and multiple (greater than 10/h) and complex premature ventricular depolarizations (2nd, 3rd and 4th Lown grade). Enalapril treatment (5 mg/day for 5 days and 10 mg thereafter) was added to the diuretic therapy and after 2 months a further decrease in blood pressure was observed (to 158 +/- 5.6 mmHg systolic, P less than 0.001; 87.2 +/- 5.0 mmHg diastolic, P less than 0.001). Moreover, there was a significant reduction in the mean heart rate (from 79 to 72 beats/min, P less than 0.005) and an increase in serum potassium (to 4.19 +/- 0.2 mmol/l; P less than 0.001). In 80% of patients a 24-h dynamic electrocardiogram showed a significant reduction in both the number and complexity of premature ventricular depolarizations. Our findings suggest that ACE inhibitors can be useful in patients developing hypokalaemia during therapy. However, we are not yet able to explain the beneficial effects of enalapril in decreasing the frequency of premature ventricular depolarizations.

  11. Substrate Reduction Augments the Efficacy of Enzyme Therapy in a Mouse Model of Fabry Disease

    PubMed Central

    Marshall, John; Ashe, Karen M.; Bangari, Dinesh; McEachern, KerryAnne; Chuang, Wei-Lien; Pacheco, Joshua; Copeland, Diane P.; Desnick, Robert J.; Shayman, James A.; Scheule, Ronald K.; Cheng, Seng H.

    2010-01-01

    Fabry disease is an X-linked glycosphingolipid storage disorder caused by a deficiency in the activity of the lysosomal hydrolase α-galactosidase A (α-gal). This deficiency results in accumulation of the glycosphingolipid globotriaosylceramide (GL-3) in lysosomes. Endothelial cell storage of GL-3 frequently leads to kidney dysfunction, cardiac and cerebrovascular disease. The current treatment for Fabry disease is through infusions of recombinant α-gal (enzyme-replacement therapy; ERT). Although ERT can markedly reduce the lysosomal burden of GL-3 in endothelial cells, variability is seen in the clearance from several other cell types. This suggests that alternative and adjuvant therapies may be desirable. Use of glucosylceramide synthase inhibitors to abate the biosynthesis of glycosphingolipids (substrate reduction therapy, SRT) has been shown to be effective at reducing substrate levels in the related glycosphingolipidosis, Gaucher disease. Here, we show that such an inhibitor (eliglustat tartrate, Genz-112638) was effective at lowering GL-3 accumulation in a mouse model of Fabry disease. Relative efficacy of SRT and ERT at reducing GL-3 levels in Fabry mouse tissues differed with SRT being more effective in the kidney, and ERT more efficacious in the heart and liver. Combination therapy with ERT and SRT provided the most complete clearance of GL-3 from all the tissues. Furthermore, treatment normalized urine volume and uromodulin levels and significantly delayed the loss of a nociceptive response. The differential efficacies of SRT and ERT in the different tissues indicate that the combination approach is both additive and complementary suggesting the possibility of an improved therapeutic paradigm in the management of Fabry disease. PMID:21124789

  12. Pharmacogenetics, enzyme probes and therapeutic drug monitoring as potential tools for individualizing taxane therapy

    PubMed Central

    Krens, Stefanie D; McLeod, Howard L; Hertz, Daniel L

    2014-01-01

    The taxanes are a class of chemotherapeutic agents that are widely used in the treatment of various solid tumors. Although taxanes are highly effective in cancer treatment, their use is associated with serious complications attributable to large interindividual variability in pharmacokinetics and a narrow therapeutic window. Unpredictable toxicity occurrence necessitates close patient monitoring while on therapy and adverse effects frequently require decreasing, delaying or even discontinuing taxane treatment. Currently, taxane dosing is based primarily on body surface area, ignoring other factors that are known to dictate variability in pharmacokinetics or outcome. This article discusses three potential strategies for individualizing taxane treatment based on patient information that can be collected before or during care. The clinical implementation of pharmacogenetics, enzyme probes or therapeutic drug monitoring could enable clinicians to personalize taxane treatment to enhance efficacy and/or limit toxicity. PMID:23556452

  13. Impact of enzyme replacement therapy and hematopoietic stem cell transplantation in patients with Morquio A syndrome

    PubMed Central

    Tomatsu, Shunji; Sawamoto, Kazuki; Alméciga-Díaz, Carlos J; Shimada, Tsutomu; Bober, Michael B; Chinen, Yasutsugu; Yabe, Hiromasa; Montaño, Adriana M; Giugliani, Roberto; Kubaski, Francyne; Yasuda, Eriko; Rodríguez-López, Alexander; Espejo-Mojica, Angela J; Sánchez, Oscar F; Mason, Robert W; Barrera, Luis A; Mackenzie, William G; Orii, Tadao

    2015-01-01

    Patients with mucopolysaccharidosis IVA (MPS IVA) can present with systemic skeletal dysplasia, leading to a need for multiple orthopedic surgical procedures, and often become wheelchair bound in their teenage years. Studies on patients with MPS IVA treated by enzyme replacement therapy (ERT) showed a sharp reduction on urinary keratan sulfate, but only modest improvement based on a 6-minute walk test and no significant improvement on a 3-minute climb-up test and lung function test compared with the placebo group, at least in the short-term. Surgical remnants from ERT-treated patients did not show reduction of storage materials in chondrocytes. The impact of ERT on bone lesions in patients with MPS IVA remains limited. ERT seems to be enhanced in a mouse model of MPS IVA by a novel form of the enzyme tagged with a bone-targeting moiety. The tagged enzyme remained in the circulation much longer than untagged native enzyme and was delivered to and retained in bone. Three-month-old MPS IVA mice treated with 23 weekly infusions of tagged enzyme showed marked clearance of the storage materials in bone, bone marrow, and heart valves. When treatment was initiated at birth, reduction of storage materials in tissues was even greater. These findings indicate that specific targeting of the enzyme to bone at an early stage may improve efficacy of ERT for MPS IVA. Recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21 (DE3) (erGALNS) and in the methylotrophic yeast Pichia pastoris (prGALNS) has been produced as an alternative to the conventional production in Chinese hamster ovary cells. Recombinant GALNS produced in microorganisms may help to reduce the high cost of ERT and the introduction of modifications to enhance targeting. Although only a limited number of patients with MPS IVA have been treated with hematopoietic stem cell transplantation (HSCT), beneficial effects have been reported. A wheelchair-bound patient with a severe form of MPS

  14. Impact of enzyme replacement therapy and hematopoietic stem cell transplantation in patients with Morquio A syndrome.

    PubMed

    Tomatsu, Shunji; Sawamoto, Kazuki; Alméciga-Díaz, Carlos J; Shimada, Tsutomu; Bober, Michael B; Chinen, Yasutsugu; Yabe, Hiromasa; Montaño, Adriana M; Giugliani, Roberto; Kubaski, Francyne; Yasuda, Eriko; Rodríguez-López, Alexander; Espejo-Mojica, Angela J; Sánchez, Oscar F; Mason, Robert W; Barrera, Luis A; Mackenzie, William G; Orii, Tadao

    2015-01-01

    Patients with mucopolysaccharidosis IVA (MPS IVA) can present with systemic skeletal dysplasia, leading to a need for multiple orthopedic surgical procedures, and often become wheelchair bound in their teenage years. Studies on patients with MPS IVA treated by enzyme replacement therapy (ERT) showed a sharp reduction on urinary keratan sulfate, but only modest improvement based on a 6-minute walk test and no significant improvement on a 3-minute climb-up test and lung function test compared with the placebo group, at least in the short-term. Surgical remnants from ERT-treated patients did not show reduction of storage materials in chondrocytes. The impact of ERT on bone lesions in patients with MPS IVA remains limited. ERT seems to be enhanced in a mouse model of MPS IVA by a novel form of the enzyme tagged with a bone-targeting moiety. The tagged enzyme remained in the circulation much longer than untagged native enzyme and was delivered to and retained in bone. Three-month-old MPS IVA mice treated with 23 weekly infusions of tagged enzyme showed marked clearance of the storage materials in bone, bone marrow, and heart valves. When treatment was initiated at birth, reduction of storage materials in tissues was even greater. These findings indicate that specific targeting of the enzyme to bone at an early stage may improve efficacy of ERT for MPS IVA. Recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21 (DE3) (erGALNS) and in the methylotrophic yeast Pichia pastoris (prGALNS) has been produced as an alternative to the conventional production in Chinese hamster ovary cells. Recombinant GALNS produced in microorganisms may help to reduce the high cost of ERT and the introduction of modifications to enhance targeting. Although only a limited number of patients with MPS IVA have been treated with hematopoietic stem cell transplantation (HSCT), beneficial effects have been reported. A wheelchair-bound patient with a severe form of MPS

  15. Fabry disease: characterisation of the plasma proteome pre- and post-enzyme replacement therapy.

    PubMed

    Heo, Sun Hee; Kang, Eungu; Kim, Yoon-Myung; Go, Heounjeong; Kim, Kyung Yong; Jung, Jae Yong; Kang, Minji; Kim, Gu-Hwan; Kim, Jae-Min; Choi, In-Hee; Choi, Jin-Ho; Jung, Sung-Chul; Desnick, Robert J; Yoo, Han-Wook; Lee, Beom Hee

    2017-08-23

    Fabry disease is characterised by the progressive accumulation of globotriaosylceramide (Gb3) and related glycosphingolipids in vascular endothelial cells. Enzyme replacement therapy (ERT) clears this accumulation. We analysed plasma proteome profiles before and after ERT to characterise its molecular pathology. Two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation-time of flight tandem mass spectrometry (MALDI-TOF MS) and tandem mass spectrometry (MS/MS) were done using plasma samples before and after ERT in eight patients with classical Fabry disease RESULTS: After short-term ERT (4-12 months), the levels of 15 plasma proteins involved in inflammation, oxidative and ischaemic injury, or complement activation were reduced significantly. Among them, β-actin (ACTB), inactivated complement C3b (iC3b), and C4B were elevated significantly in pre-ERT Fabry disease plasma compared with control plasma. After longer-term ERT (46-96 months), iC3b levels gradually decreased, whereas the levels of other proteins varied. The gradual reduction of iC3b was comparable to that of Gb3 levels. In addition, iC3b increased significantly in pre-ERT Fabry disease mouse plasma, and C3 deposits were notable in renal tissues of pre-enzyme replacement therapy patients. These results indicated that C3-mediated complement activation might be altered in Fabry disease and ERT might promote its stabilisation. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  16. Cardiomyopathy and response to enzyme replacement therapy in a male mouse model for Fabry disease.

    PubMed

    Nguyen Dinh Cat, Aurelie; Escoubet, Brigitte; Agrapart, Vincent; Griol-Charhbili, Violaine; Schoeb, Trenton; Feng, Wenguang; Jaimes, Edgar; Warnock, David G; Jaisser, Frederic

    2012-01-01

    Fabry disease is an X-linked disorder of glycosphingolipid metabolism that results in progressive accumulation of neutral glycosphingolipids, (predominately globotriaosylceramide; GL-3) in lysosomes, as well as other cellular compartments and the extracellular space. Our aim was to characterize the cardiac phenotype of male knock-out mice that are deficient in alpha-galactosidase A activity, as a model for Fabry disease and test the efficacy of Enzyme Replacement Therapy with agalsidase-beta. Male mice (3-4 months of age) were characterized with awake blood pressure and heart rate measurements, cardiac echocardiography and electrocardiography measurements under light anesthesia, histological studies and molecular studies with real-time polymerase chain reaction. The Fabry knock-out mouse has bradycardia and lower blood pressure than control wild type (CB7BL/6J) mice. In Fabry knock-out mice, the cardiomyopathy associated mild hypertrophy at echography with normal systolic LV function and mild diastolic dysfunction. Premature atrial contractions were more frequent in without conduction defect. Heart weight normalized to tibial length was increased in Fabry knock-out mice. Ascending aorta dilatation was observed. Molecular studies were consistent with early stages of cardiac remodeling. A single dose of agalsidase-beta (3 mg/kg) did not affect the LV hypertrophy, function or heart rate, but did improve the mRNA signals of early cardiac remodeling. In conclusion, the alpha-galactosidase A deficient mice at 3 to 4 months of age have cardiac and vascular alterations similar to that described in early clinical stage of Fabry disease in children and adolescents. Enzyme replacement therapy affects cardiac molecular remodeling after a single dose.

  17. Cardiomyopathy and Response to Enzyme Replacement Therapy in a Male Mouse Model for Fabry Disease

    PubMed Central

    Nguyen Dinh Cat, Aurelie; Escoubet, Brigitte; Agrapart, Vincent; Griol-Charhbili, Violaine; Schoeb, Trenton; Feng, Wenguang; Jaimes, Edgar; Warnock, David G.; Jaisser, Frederic

    2012-01-01

    Fabry disease is an X-linked disorder of glycosphingolipid metabolism that results in progressive accumulation of neutral glycosphingolipids, (predominately globotriaosylceramide; GL-3) in lysosomes, as well as other cellular compartments and the extracellular space. Our aim was to characterize the cardiac phenotype of male knock-out mice that are deficient in alpha-galactosidase A activity, as a model for Fabry disease and test the efficacy of Enzyme Replacement Therapy with agalsidase-beta. Male mice (3–4 months of age) were characterized with awake blood pressure and heart rate measurements, cardiac echocardiography and electrocardiography measurements under light anesthesia, histological studies and molecular studies with real-time polymerase chain reaction. The Fabry knock-out mouse has bradycardia and lower blood pressure than control wild type (CB7BL/6J) mice. In Fabry knock-out mice, the cardiomyopathy associated mild hypertrophy at echography with normal systolic LV function and mild diastolic dysfunction. Premature atrial contractions were more frequent in without conduction defect. Heart weight normalized to tibial length was increased in Fabry knock-out mice. Ascending aorta dilatation was observed. Molecular studies were consistent with early stages of cardiac remodeling. A single dose of agalsidase-beta (3 mg/kg) did not affect the LV hypertrophy, function or heart rate, but did improve the mRNA signals of early cardiac remodeling. In conclusion, the alpha-galactosidase A deficient mice at 3 to 4 months of age have cardiac and vascular alterations similar to that described in early clinical stage of Fabry disease in children and adolescents. Enzyme replacement therapy affects cardiac molecular remodeling after a single dose. PMID:22574107

  18. Nutritional therapy improves growth and protein status of children with a urea cycle enzyme defect.

    PubMed

    Acosta, Phyllis B; Yannicelli, Steven; Ryan, Alan S; Arnold, Georgianne; Marriage, Barbara J; Plewinska, Magda; Bernstein, Laurie; Fox, Joyce; Lewis, Vyonne; Miller, Marvin; Velazquez, Antonio

    2005-12-01

    Poor growth has been described in patients with urea cycle enzyme defects treated with protein-restricted diets, while protein status is seldom reported. To assess the effects of nutritional therapy with a medical food on growth and protein status of patients with a urea cycle enzyme defect. A 6-mo multicenter outpatient study was conducted with infants and toddlers managed by nutrition therapy with Cyclinex-1 Amino Acid-Modified Medical Food with Iron (Ross Products Division, Abbott Laboratories, Columbus, OH). Main outcome variables were anthropometrics and plasma amino acids (selected), albumin, and transthyretin concentrations. Seventeen patients completed the study. Mean (+/-SE) baseline age was 11.30+/-3.20 months (median 4.40 months; range 0.22-38.84 months). Length and weight z-scores increased significantly during the 6-month study. Head circumference increased, but not significantly. Three patients were stunted and two were wasted (-2.0 z-score) at baseline while at study end, only one patient was both stunted and wasted. The majority of patients increased in length, head circumference, and weight z-scores during study. Mean (+/-SE) plasma albumin concentration increased from 34+/-2g/L at baseline to 38+/-1g/L at study end. Plasma transthyretin increased from a mean (+/-SE) of 177+/-13 mg/L at baseline to 231+/-15 mg/L at study end. No correlation was found between plasma NH(3) concentrations and medical food intake. Plasma NH(3) concentration was positively correlated with the percentage of Food and Agriculture Organization/World Health Organization/United Nations recommended protein ingested. Intakes of adequate protein and energy for age result in anabolism and linear growth without increasing plasma NH(3) concentrations. Medical food intakes did not correlate with plasma NH(3) concentrations.

  19. Bacterial Toxins for Oncoleaking Suicidal Cancer Gene Therapy.

    PubMed

    Pahle, Jessica; Walther, Wolfgang

    For suicide gene therapy, initially prodrug-converting enzymes (gene-directed enzyme-producing therapy, GDEPT) were employed to intracellularly metabolize non-toxic prodrugs into toxic compounds, leading to the effective suicidal killing of the transfected tumor cells. In this regard, the suicide gene therapy has demonstrated its potential for efficient tumor eradication. Numerous suicide genes of viral or bacterial origin were isolated, characterized, and extensively tested in vitro and in vivo, demonstrating their therapeutic potential even in clinical trials to treat cancers of different entities. Apart from this, growing efforts are made to generate more targeted and more effective suicide gene systems for cancer gene therapy. In this regard, bacterial toxins are an alternative to the classical GDEPT strategy, which add to the broad spectrum of different suicide approaches. In this context, lytic bacterial toxins, such as streptolysin O (SLO) or the claudin-targeted Clostridium perfringens enterotoxin (CPE) represent attractive new types of suicide oncoleaking genes. They permit as pore-forming proteins rapid and also selective toxicity toward a broad range of cancers. In this chapter, we describe the generation and use of SLO as well as of CPE-based gene therapies for the effective tumor cell eradication as promising, novel suicide gene approach particularly for treatment of therapy refractory tumors.

  20. Enzyme replacement therapy in newborn mucopolysaccharidosis IVA mice: early treatment rescues bone lesions?

    PubMed

    Tomatsu, Shunji; Montaño, Adriana M; Oikawa, Hirotaka; Dung, Vu Chi; Hashimoto, Amiko; Oguma, Toshihiro; Gutiérrez, Monica L; Takahashi, Tatsuo; Shimada, Tsutomu; Orii, Tadao; Sly, William S

    2015-02-01

    We treated mucopolysaccharidosis IVA (MPS IVA) mice to assess the effects of long-term enzyme replacement therapy (ERT) initiated at birth, since adult mice treated by ERT showed little improvement in bone pathology [1]. To conduct ERT in newborn mice, we used recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in a CHO cell line. First, to observe the tissue distribution pattern, a dose of 250units/g body weight was administered intravenously in MPS IVA mice at day 2 or 3. The infused enzyme was primarily recovered in the liver and spleen, with detectable activity in the bone and brain. Second, newborn ERT was conducted after a tissue distribution study. The first injection of newborn ERT was performed intravenously, the second to fourth weekly injections were intraperitoneal, and the remaining injections from 5th to 14th weeks were intravenous into the tail vein. MPS IVA mice treated with GALNS showed clearance of lysosomal storage in the liver and spleen, and sinus lining cells in bone marrow. The column structure of the growth plate was organized better than that in adult mice treated with ERT; however, hyaline and fibrous cartilage cells in the femur, spine, ligaments, discs, synovium, and periosteum still had storage materials to some extent. Heart valves were refractory to the treatment. Levels of serum keratan sulfate were kept normal in newborn ERT mice. In conclusion, the enzyme, which enters the cartilage before the cartilage cell layer becomes mature, prevents disorganization of column structure. Early treatment from birth leads to partial remission of bone pathology in MPS IVA mice. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Enzyme replacement therapy in newborn mucopolysaccharidosis IVA mice: early treatment rescues bone lesions?

    PubMed Central

    Tomatsu, Shunji; Montaño, Adriana M.; Oikawa, Hirotaka; Dung, Vu Chi; Hashimoto, Amiko; Oguma, Toshihiro; Takahashi, Tatsuo; Shimada, Tsutomu; Orii, Tadao; Sly, William S.

    2014-01-01

    We treated mucopolysaccharidosis IVA (MPS IVA) mice to assess the effects of long-term enzyme replacement therapy (ERT) initiated at birth, since adult mice treated by ERT showed little improvement in bone pathology (1). To conduct ERT in newborn mice, we used recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in a CHO cell line. First, to observe the tissue distribution pattern, a dose of 250 units/g body weight was administered intravenously in MPS IVA mice at day 2 or 3. The infused enzyme was primarily recovered in liver and spleen, with detectable activity in bone and brain. Second, newborn ERT was conducted after tissue distribution study. The first injection of newborn ERT was performed intravenously, the second to fourth weekly injections were intraperitoneal, and the remaining injections from 5th to 14th week were intravenous into the tail vein. MPS IVA mice treated with GALNS showed clearance of lysosomal storage in liver, spleen, and sinus lining cells in bone marrow. The column structure of the growth plate was organized better than adult mice treated with ERT; however, hyaline and fibrous cartilage cells in femur, spine, ligaments, discs, synovium, and periosteum still had storage materials to some extent. Heart valves were refractory to the treatment. Levels of serum keratan sulfate were kept normal in newborn ERT mice. In conclusion, the enzyme, which enters the cartilage before the cartilage cell layer becomes mature, prevents disorganization of column structure. Early treatment from birth leads to partial remission of bone pathology in MPS IVA mouse. PMID:24953405

  2. Development of nanoparticle-bound arylsulfatase B for enzyme replacement therapy of mucopolysaccharidosis VI.

    PubMed

    Mühlstein, A; Gelperina, S; Kreuter, J

    2013-07-01

    Lysosomal storage disorders like mucopolysaccharidosis (MPS) VI are rare diseases with a lack of well-suited treatments. Even though an enzyme replacement therapy (ERT) of recombinant arylsulfatase B (ASB) is available for MPS VI, the administration cannot positively affect the neurologic manifestations such as spinal cord compression. Since nanoparticles (NP) have shown to be effective drug carriers, the feasibility of arylsulfatase B adsorption onto poly(butyl cyanoacrylate) (PBCA) nanoparticles was investigated in this study. In order to advance the ERT of ASB, the adsorption of the latter on the surface of PBCA NP as well as in vitro release in serum was investigated. With alteration of parameters like temperature, incubation time, pH, and enzyme amount, the adsorption process revealed to be stable with a maximum capacity of 67 microg/mg NP at a pH of 6.3. In vitro release experiments demonstrated that the adsorption is stable for at least 60 minutes in human blood serum, indicating that the ASB-loaded PBCA nanoparticles represent a promising candidate for ERT of MPS VI.

  3. [Neuropathic Gaucher disease treated with long enzyme replacement therapy. Two clinical cases].

    PubMed

    Correa, Cecilia

    2013-01-01

    Gaucher disease (GD) is the most common of all inherited lipid storage diseases. It is an autosomal recessive disorder portraying catabolism and cerebroside deposit in the lysosomes, which is due to a lack of glucocerebrosidase enzyme. Though GD shows a panethnic pattern of presentation, it particularly affects the Ashkenazi Jewish population. Several mutations have been defined among GD patients, and some genotypes related to neurologic affection have been described (L444P--most common mutation for neuropathic GD--188S, V394L and G377S). Lipid material storage or deposit exerts multiorganic affection. Enzyme replacement therapy (ERT) has demonstrable efficacy in reversing organic damage related to GD, though its capability to stop neurologic affection is currently under controversy and particular research. This paper portrays two GD cases of Mexican children treated with ERT at general zone hospitals of the Instituto Mexicano del Seguro Social in recent years, both of them depicting characteristic type 3 GD mutations, and comparing their clinical evolution with and without neurological features.

  4. Engineering and Characterization of an Enzyme Replacement Therapy for Classical Homocystinuria.

    PubMed

    Majtan, Tomas; Park, Insun; Carrillo, Richard S; Bublil, Erez M; Kraus, Jan P

    2017-06-12

    Homocystinuria due to loss of cystathionine beta-synthase (CBS) causes accumulation of homocysteine and depletion of cysteine. Current treatments are suboptimal, and thus the development of an enzyme replacement therapy based on PEGylated human truncated CBS (PEG-CBS) has been initiated. Attenuation of potency was observed, which necessitated a screen of several PEG-CBS conjugates for their efficacy to correct and maintain the plasma metabolite profile of murine homocystinuria after repeated administrations interrupted with washouts. We found that CBS coupling with maleimide PEG inconsistently modified the enzyme. In contrast, the PEG-CBS conjugate with 20 kDa N-hydroxysuccinimide-PEG showed very little loss of potency likely due to a reproducible PEGylation resulting in species modified with five PEGs per subunit on average. We developed assays suitable for monitoring the extent of CBS PEGylation and demonstrated a sustainable partial normalization of homocystinuria upon continuous PEG-CBS administration via osmotic pumps. Taken together, we identified the PEG-CBS conjugate suitable for manufacturing and clinical development.

  5. Stepwise-activable multifunctional peptide-guided prodrug micelles for cancerous cells intracellular drug release

    NASA Astrophysics Data System (ADS)

    Zhang, Jing; Li, Mengfei; Yuan, Zhefan; Wu, Dan; Chen, Jia-da; Feng, Jie

    2016-10-01

    A novel type of stepwise-activable multifunctional peptide-guided prodrug micelles (MPPM) was fabricated for cancerous cells intracellular drug release. Deca-lysine sequence (K10), a type of cell-penetrating peptide, was synthesized and terminated with azido-glycine. Then a new kind of molecule, alkyne modified doxorubicin (DOX) connecting through disulfide bond (DOX-SS-alkyne), was synthesized. After coupling via Cu-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reaction, reduction-sensitive peptide-guided prodrug was obtained. Due to the amphiphilic property of the prodrug, it can assemble to form micelles. To prevent the nanocarriers from unspecific cellular uptake, the prodrug micelles were subsequently modified with 2,3-dimethyl maleic anhydride to obtain MPPM with a negatively charged outer shell. In vitro studies showed that MPPM could be shielded from cells under psychological environment. However, when arriving at mild acidic tumor site, the cell-penetrating capacity of MPPM would be activated by charge reversal of the micelles via hydrolysis of acid-labile β-carboxylic amides and regeneration of K10, which enabled efficient internalization of MPPM by tumor cells as well as following glutathione- and protease-induced drug release inside the cancerous cells. Furthermore, since the guide peptide sequences can be accurately designed and synthesized, it can be easily changed for various functions, such as targeting peptide, apoptotic peptide, even aptamers, only need to be terminated with azido-glycine. This method can be used as a template for reduction-sensitive peptide-guided prodrug for cancer therapy.

  6. Reduced glucosylceramide in the mouse model of Fabry disease: correction by successful enzyme replacement therapy.

    PubMed

    Quinta, Rui; Rodrigues, Daniel; Assunção, Marisa; Macedo, Maria Fatima; Azevedo, Olga; Cunha, Damião; Oliveira, Pedro; Sá Miranda, Maria Clara

    2014-02-15

    Fabry disease is an X-linked lysosomal storage disease (LSD) caused by deficient activity of α-Galactosidase A (α-Gal A). As a result, glycosphingolipids, mainly globotriaosylceramide (Gb3), progressively accumulate in body fluids and tissues. Studies aiming at the identification of secondary lipid alterations in Fabry disease may be potentially useful for the monitorization of the response to enzyme replacement therapy (ERT) and development of future therapies. The focus of this study was to evaluate if α-Gal A deficiency has an effect on two key groups of molecules of sphingolipids metabolism: glucosylceramides (GlucCers) and ceramides (Cers). Studies performed in a mouse model of Fabry disease showed reduced level of GlucCer and normal level of Cer in plasma, liver, spleen, kidney and heart. Moreover, analysis of GlucCer isoforms in Fabry knockout mice showed that GlucCer isoforms are unequally reduced in different tissues of these animals. ERT had a specific effect on the liver's GlucCer levels of Fabry knockout mice, increasing hepatic GlucCer to the levels observed in wild type mice. In contrast to Fabry knockout mice, plasma of Fabry patients had normal GlucCer and Cer but an increased GlucCer/Cer ratio. This alteration showed a positive correlation with plasma globotriaosylsphingosine (lyso-Gb3) concentration. In conclusion, this work reveals novel secondary lipid imbalances caused by α-Gal A deficiency.

  7. Dialysis and transplantation in Fabry disease: indications for enzyme replacement therapy.

    PubMed

    Mignani, Renzo; Feriozzi, Sandro; Schaefer, Roland M; Breunig, Frank; Oliveira, João Paulo; Ruggenenti, Piero; Sunder-Plassmann, Gere

    2010-02-01

    ESRD is a major cause of morbidity and premature mortality in Fabry disease, particularly in classically affected males. The decline of renal function in Fabry nephropathy is adversely affected by male gender, advanced chronic kidney disease (CKD), and severe proteinuria. The diagnosis of Fabry nephropathy may be missed if not specifically addressed in progressive CKD and patients have been first identified in screening programs of dialysis patients. Fabry patients have worse 3-year survival rates on dialysis as compared with nondiabetic controls. The 5-year survival rate of transplanted Fabry patients is also lower than that of controls. However, because Fabry nephropathy does not recur in the allograft and transplanted Fabry patients appear to have better overall outcomes than those maintained on dialysis, kidney transplantation should be recommended as a first choice in renal replacement therapy (RRT) for Fabry disease. Appropriately designed and powered studies are not available to answer the question whether enzyme replacement therapy (ERT) influences outcomes, the course of cardiomyopathy, events, or survival in Fabry patients on RRT. The authors are not aware of compelling indications for ERT in RRT patients because progression of cardiomyopathy was documented during ERT. Whether the excess mortality risk of Fabry patients on RRT can be prevented by ERT is unknown. Despite observational reports of symptomatic improvement, the available evidence supporting ERT for such patients is not compelling enough. To clarify this issue, studies are needed to test the effectiveness of agalsidases in preventing cardiac and cerebrovascular complications in Fabry patients with ESRD.

  8. Successful immune tolerance induction to enzyme replacement therapy in CRIM-negative infantile Pompe disease

    PubMed Central

    Messinger, Yoav H.; Mendelsohn, Nancy J.; Rhead, William; Dimmock, David; Hershkovitz, Eli; Champion, Michael; Jones, Simon A.; Olson, Rebecca; White, Amy; Wells, Cara; Bali, Deeksha; Case, Laura E.; Young, Sarah P.; Rosenberg, Amy S.; Kishnani, Priya S.

    2013-01-01

    Purpose Infantile Pompe disease resulting from a deficiency of lysosomal acid α-glucosidase (GAA) requires enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA). Cross-reactive immunologic material negative (CRIM-negative) Pompe patients develop high-titer antibody to the rhGAA and do poorly. We describe successful tolerance induction in CRIM-negative patients. Methods Two CRIM-negative patients with preexisting anti-GAA antibodies were treated therapeutically with rituximab, methotrexate, and gammaglobulins. Two additional CRIM-negative patients were treated prophylactically with a short course of rituximab and methotrexate, in parallel with initiating rhGAA. Results In both patients treated therapeutically, anti-rhGAA was eliminated after 3 and 19 months. All four patients are immune tolerant to rhGAA, off immune therapy, showing B-cell recovery while continuing to receive ERT at ages 36 and 56 months (therapeutic) and 18 and 35 months (prophylactic). All patients show clinical response to ERT, in stark contrast to the rapid deterioration of their nontolerized CRIM-negative counterparts. Conclusion The combination of rituximab with methotrexate ± intravenous gammaglobulins (IVIG) is an option for tolerance induction of CRIM-negative Pompe to ERT when instituted in the naïve setting or following antibody development. It should be considered in other conditions in which antibody response to the therapeutic protein elicits robust antibody response that interferes with product efficacy. PMID:22237443

  9. Targeted delivery of a model immunomodulator to the lymphatic system: comparison of alkyl ester versus triglyceride mimetic lipid prodrug strategies.

    PubMed

    Han, Sifei; Quach, Tim; Hu, Luojuan; Wahab, Anisa; Charman, William N; Stella, Valentino J; Trevaskis, Natalie L; Simpson, Jamie S; Porter, Christopher J H

    2014-03-10

    A lipophilic prodrug approach has been used to promote the delivery of a model immunomodulator, mycophenolic acid (MPA), to the lymphatic system after oral administration. Lymphatic transport was employed to facilitate enhanced drug uptake into lymphocytes, as recent studies demonstrate that targeted drug delivery to lymph resident lymphocytes may enhance immunomodulatory effects. Two classes of lymph-directing prodrugs were synthesised. Alkyl chain derivatives (octyl mycophenolate, MPA-C8E; octadecyl mycophenolate, MPA-C18E; and octadecyl mycophenolamide, MPA-C18AM), to promote passive partitioning into lipids in lymphatic transport pathways, and a triglyceride mimetic prodrug (1,3-dipalmitoyl-2-mycophenoloyl glycerol, 2-MPA-TG) to facilitate metabolic integration into triglyceride deacylation-reacylation pathways. Lymphatic transport, lymphocyte uptake and plasma pharmacokinetics were assessed in mesenteric lymph and carotid artery cannulated rats following intraduodenal infusion of lipid-based formulations containing MPA or MPA prodrugs. Patterns of prodrug hydrolysis in rat digestive fluid, and cellular re-esterification in vivo, were evaluated to examine the mechanisms responsible for lymphatic transport. Poor enzyme stability and low absorption appeared to limit lymphatic transport of the alkyl derivatives, although two of the three alkyl chain prodrugs - MPA-C18AM (6-fold) and MPA-C18E (13-fold) still increased lymphatic drug transport when compared to MPA. In contrast, 2-MPA-TG markedly increased lymphatic drug transport (80-fold) and drug concentrations in lymphocytes (103-fold), and this was achieved via biochemical incorporation into triglyceride deacylation-reacylation pathways. The prodrug was hydrolysed rapidly to 2-mycophenoloyl glycerol (2-MPA-MG) in the presence of rat digestive fluid, and 2-MPA-MG was subsequently re-esterified in the enterocyte with oleic acid (most likely originating from the co-administered formulation) prior to accessing the

  10. Facile Fabrication of Tumor Redox-Sensitive Nanoassemblies of Small-Molecule Oleate Prodrug as Potent Chemotherapeutic Nanomedicine.

    PubMed

    Luo, Cong; Sun, Jin; Sun, Bingjun; Liu, Dan; Miao, Lei; Goodwin, Tyler Jay; Huang, Leaf; He, Zhonggui

    2016-12-01

    The conjugate of paclitaxel (PTX) and docosahexaenoic acid has entered into clinical trials. However, the most recent clinical outcomes fell short of expectations, due to the extremely slow drug release from the hydrophobic conjugates. Herein, a novel prodrug-based nanoplatform self-assembled by the disulfide bond linked conjugates of PTX and oleic acid for rapid and differential release of PTX in tumor cells is reported. This redox-responsive prodrug-nanosystem demonstrates multiple therapeutic advantages, including one-step facile fabrication, high drug-loading efficiency (56%, w/w), on-demand drug release responding to redox stimuli, as well as favorable cellular uptake and biodistribution. These advantages result in significantly enhanced antitumor efficacy in vivo, with the tumor almost completely disappearing in mice. Such a uniquely engineered prodrug-nanosystem has great potential to be used as potent chemotherapeutic nanomedicine in clinical cancer therapy.

  11. Enzyme replacement therapy for mucopolysaccharidosis VI: long-term cardiac effects of galsulfase (Naglazyme®) therapy.

    PubMed

    Braunlin, E; Rosenfeld, H; Kampmann, C; Johnson, J; Beck, M; Giugliani, R; Guffon, N; Ketteridge, D; Sá Miranda, C M; Scarpa, M; Schwartz, I V; Leão Teles, E; Wraith, J E; Barrios, P; Dias da Silva, E; Kurio, G; Richardson, M; Gildengorin, G; Hopwood, J J; Imperiale, M; Schatz, A; Decker, C; Harmatz, P

    2013-03-01

    Characteristic cardiac valve abnormalities and left ventricular hypertrophy are present in untreated patients with mucopolysaccharidosis type VI (MPS VI). Cardiac ultrasound was performed to investigate these findings in subjects during long-term enzyme replacement therapy (ERT) with recombinant human arylsulfatase B (rhASB, rhN-acetylgalactosamine 4-sulfatase, galsulfase, Naglazyme®). Studies were conducted in 54 subjects before ERT was begun and at specific intervals for up to 96 weeks of weekly infusions of rhASB at 1 mg/kg during phase 1/2, phase 2, and phase 3 trials of rhASB. At baseline, mitral and aortic valve obstruction was present and was significantly greater in those ≥12 years of age. Mild mitral and trace aortic regurgitation were present, the former being significantly greater in those <12 years. Left ventricular hypertrophy, with averaged z-scores ranging from 1.6-1.9 SD greater than normal, was present for ages both <12 and ≥12 years. After 96 weeks of ERT, ventricular septal hypertrophy regressed in those <12 years. For those ≥12 years, septal hypertrophy was unchanged, and aortic regurgitation increased statistically but not physiologically. Obstructive gradients across mitral and aortic valves remained unchanged. The results suggest that long-term ERT is effective in reducing intraventricular septal hypertrophy and preventing progression of cardiac valve abnormalities when administered to those <12 years of age.

  12. Where do the immunostimulatory effects of oral proteolytic enzymes ('systemic enzyme therapy') come from? Microbial proteolysis as a possible starting point.

    PubMed

    Biziulevicius, Gediminas A

    2006-01-01

    Enteric-coated proteolytic enzyme preparations like Wobenzym and Phlogenzym are widely used for the so-called 'systemic enzyme therapy' both in humans and animals. Numerous publications reveal that oral proteolytic enzymes are able to stimulate directly the activity of immune competent cells as well as to increase efficiency of some of their products. But origins of the immunostimulatory effects of oral proteolytic enzymes are still unclear. The hypothesis described here suggests that it may be proteolysis of intestinal microorganisms that makes the immune competent cells to work in the immunostimulatory manner. The hypothesis was largely formed by several scientific observations: First, microbial lysis products (lipopolysaccharides, muropeptides and other peptidoglycan fragments, beta-glucans, etc.) are well known for their immunostimulatory action. Second, a normal human being hosts a mass of intestinal microorganisms equivalent to about 1 kg. The biomass (mainly due to naturally occurring autolysis) continuously supplies the host's organism with immunostimulatory microbial cell components. Third, the immunostimulatory effects resulting from the oral application of exogenously acting antimicrobial (lytic) enzyme preparations, such as lysozyme and lysosubtilin, are likely to be a result of the action of microbial lysis products. Fourth, cell walls of most microorganisms contain a considerable amount of proteins/peptides, a possible target for exogenous proteolytic enzymes. In fact, several authors have already shown that a number of proteases possess an ability to lyse the microbial cells in vitro. Fifth, the pretreatment of microbial cells (at least of some species) in vitro with proteolytic enzymes makes them more sensitive to the lytic action of lysozyme and, otherwise, pretreatment with lysozyme makes them more susceptible to proteolytic degradation. Sixth, exogenous proteases, when in the intestines, may participate in final steps of food-protein digestion

  13. Enzyme replacement therapy for Mucopolysaccharidosis Type I among patients followed within the MPS Brazil Network.

    PubMed

    Dornelles, Alícia Dorneles; de Camargo Pinto, Louise Lapagesse; de Paula, Ana Carolina; Steiner, Carlos Eduardo; Lourenço, Charles Marques; Kim, Chong Ae; Horovitz, Dafne Dain Gandelman; Ribeiro, Erlane Marques; Valadares, Eugênia Ribeiro; Goulart, Isabela; Neves de Souza, Isabel C; da Costa Neri, João Ivanildo; Santana-da-Silva, Luiz Carlos; Silva, Luiz Roberto; Ribeiro, Márcia; de Oliveira Sobrinho, Ruy Pires; Giugliani, Roberto; Schwartz, Ida Vanessa Doederlein

    2014-03-01

    Mucopolysaccharidosis type I (MPS I) is a rare lysosomal disorder caused by deficiency of alpha-L-iduronidase. Few clinical trials have assessed the effect of enzyme replacement therapy (ERT) for this condition. We conducted an exploratory, open-label, non-randomized, multicenter cohort study of patients with MPS I. Data were collected from questionnaires completed by attending physicians at the time of diagnosis (T1; n = 34) and at a median time of 2.5 years later (T2; n = 24/34). The 24 patients for whom data were available at T2 were allocated into groups: A, no ERT (9 patients; median age at T1 = 36 months; 6 with severe phenotype); B, on ERT (15 patients; median age at T1 = 33 months; 4 with severe phenotype). For all variables in which there was no between-group difference at baseline, a delta of ≥ ± 20% was considered clinically relevant. The following clinically relevant differences were identified in group B in T2: lower rates of mortality and reported hospitalization for respiratory infection; lower frequency of hepatosplenomegaly; increased reported rates of obstructive sleep apnea syndrome and hearing loss; and stabilization of gibbus deformity. These changes could be due to the effect of ERT or of other therapies which have also been found more frequently in group B. Our findings suggest MPS I patients on ERT also receive a better overall care. ERT may have a positive effect on respiratory morbidity and overall mortality in patients with MPS I. Additional studies focusing on these outcomes and on other therapies should be performed.

  14. Enzyme replacement therapy for Mucopolysaccharidosis Type I among patients followed within the MPS Brazil Network

    PubMed Central

    Dornelles, Alícia Dorneles; de Camargo Pinto, Louise Lapagesse; de Paula, Ana Carolina; Steiner, Carlos Eduardo; Lourenço, Charles Marques; Kim, Chong Ae; Horovitz, Dafne Dain Gandelman; Ribeiro, Erlane Marques; Valadares, Eugênia Ribeiro; Goulart, Isabela; Neves de Souza, Isabel C.; da Costa Neri, João Ivanildo; Santana-da-Silva, Luiz Carlos; Silva, Luiz Roberto; Ribeiro, Márcia; de Oliveira Sobrinho, Ruy Pires; Giugliani, Roberto; Schwartz, Ida Vanessa Doederlein

    2014-01-01

    Mucopolysaccharidosis type I (MPS I) is a rare lysosomal disorder caused by deficiency of alpha-L-iduronidase. Few clinical trials have assessed the effect of enzyme replacement therapy (ERT) for this condition. We conducted an exploratory, open-label, non-randomized, multicenter cohort study of patients with MPS I. Data were collected from questionnaires completed by attending physicians at the time of diagnosis (T1; n = 34) and at a median time of 2.5 years later (T2; n = 24/34). The 24 patients for whom data were available at T2 were allocated into groups: A, no ERT (9 patients; median age at T1 = 36 months; 6 with severe phenotype); B, on ERT (15 patients; median age at T1 = 33 months; 4 with severe phenotype). For all variables in which there was no between-group difference at baseline, a delta of ≥ ± 20% was considered clinically relevant. The following clinically relevant differences were identified in group B in T2: lower rates of mortality and reported hospitalization for respiratory infection; lower frequency of hepatosplenomegaly; increased reported rates of obstructive sleep apnea syndrome and hearing loss; and stabilization of gibbus deformity. These changes could be due to the effect of ERT or of other therapies which have also been found more frequently in group B. Our findings suggest MPS I patients on ERT also receive a better overall care. ERT may have a positive effect on respiratory morbidity and overall mortality in patients with MPS I. Additional studies focusing on these outcomes and on other therapies should be performed. PMID:24688287

  15. Hepatotoxicity and liver enzyme alteration in patients with immunobullous diseases receiving immunosuppressive therapy.

    PubMed

    Shahshahani, Mostafa M; Azizahari, Sahar; Soori, Tahere; Manavi, Saeed; Balighi, Kamran; Daneshpazhooh, Maryam; Davatchi, Cheida S; Esmaili, Nasife

    2011-12-01

    To avoid complications of high dose corticosteroid, pemphigus patients are usually co-treated with other immunosuppressive agents. Liver enzyme abnormality occurs commonly during treatment and occasionally causes discontinuation of drugs. To assess the rate of therapy-induced hepatotoxicity in patients with immunobullous diseases, we conducted a study of 250 pemphigus patients under immunosuppressive therapy prospectively. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) plasma levels were recorded before the start of treatment and every week under treatment (up to 3 weeks). Hepatotoxicity was defined as the rise in the ALT plasma levels to greater than twice the upper normal limit. Approximately 81% of patients received prednisolone and azathioprine. Approximately 12% received only prednisolone. Hepatotoxicity occurred in 2.9% (n = 8) of patients after 1 week, in 7.8% (n = 20) after 2 weeks and in 11.5% (n = 29) after 3 weeks. No patient had jaundice or other clinical manifestations of hepatitis. The mean values of ALT and AST before the start of treatment were 20.7 ± 13.7 and 17.6 ± 10.8 U/L, respectively that grew to 47.5 ± 28.5 and 26.8 ± 14.5 U/L, 3 weeks after the initiation of treatment. Distribution of changes was not significantly different among groups of age, sex, immunosuppressive drugs and isoniazid consumption. Under usual treatment of pemphigus, hepatotoxicity occurs in 10% of patients during the first 3 weeks of therapy that does not seem to be associated with azathioprine or mycophenolate mofetil exclusively. High doses of prednisolone may play a role.

  16. Adjunctive albuterol enhances the response to enzyme replacement therapy in late-onset Pompe disease.

    PubMed

    Koeberl, Dwight D; Austin, Stephanie; Case, Laura E; Smith, Edward C; Buckley, Anne F; Young, Sarah P; Bali, Deeksha; Kishnani, Priya S

    2014-05-01

    Effective dosages for enzyme replacement therapy (ERT) in Pompe disease are much higher than for other lysosomal storage disorders, which has been attributed to low cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle. We have previously demonstrated the benefit of increased CI-MPR-mediated uptake of recombinant human acid-α-glucosidase during ERT in mice with Pompe disease following addition of albuterol therapy. Currently we have completed a pilot study of albuterol in patients with late-onset Pompe disease already on ERT for >2 yr, who were not improving further. The 6-min walk test (6MWT) distance increased in all 7 subjects at wk 6 (30±13 m; P=0.002), wk 12 (34±14 m; P=0.004), and wk 24 (42±37 m; P=0.02), in comparison with baseline. Grip strength was improved significantly for both hands at wk 12. Furthermore, individual subjects reported benefits; e.g., a female patient could stand up from sitting on the floor much more easily (time for supine to standing position decreased from 30 to 11 s), and a male patient could readily swing his legs out of his van seat (hip abduction increased from 1 to 2+ on manual muscle testing). Finally, analysis of the quadriceps biopsies suggested increased CI-MPR at wk 12 (P=0.08), compared with baseline. With the exception of 1 patient who succumbed to respiratory complications of Pompe disease in the first week, only mild adverse events have been reported, including tremor, transient difficulty falling asleep, and mild urinary retention (requiring early morning voiding). Therefore, this pilot study revealed initial safety and efficacy in an open label study of adjunctive albuterol therapy in patients with late-onset Pompe disease who had been stable on ERT with no improvements noted over the previous several years.

  17. Photoactivatable Caged Prodrugs of VEGFR-2 Kinase Inhibitors.

    PubMed

    Pinchuk, Boris; Horbert, Rebecca; Döbber, Alexander; Kuhl, Lydia; Peifer, Christian

    2016-04-29

    In this study, we report on the design, synthesis, photokinetic properties and in vitro evaluation of photoactivatable caged prodrugs for the receptor tyrosine kinase VEGFR-2. Highly potent VEGFR-2 inhibitors 1 and 3 were caged by introduction of a photoremovable protecting group (PPG) to yield the caged prodrugs 4 and 5. As expected, enzymatic and cellular proliferation assays showed dramatically diminished efficacy of caged prodrugs in vitro. Upon ultraviolet (UV) irradiation of the prodrugs original inhibitory activity was completely restored and even distinctly reinforced, as was the case for the prodrug 4. The presented results are a further evidence for caging technique being an interesting approach in the protein kinase field. It could enable spatial and temporal control for the inhibition of VEGFR-2. The described photoactivatable prodrugs might be highly useful as biological probes for studying the VEGFR-2 signal transduction.

  18. Prodrugs - an efficient way to breach delivery and targeting barriers.

    PubMed

    Huttunen, Kristiina M; Rautio, Jarkko

    2011-01-01

    The study of prodrugs that are chemically modified bioreversible derivatives of active drug compounds to alter their undesired properties has been expanded widely during the last decades. Despite the commercial success the prodrugs have afforded, the concept is still quite unknown among many scientist. Furthermore, many scientists regard prodrugs as a pure interest of academic research groups and not as a feasible solution to improve the delivery or targeting properties of new chemical entities, drug candidates failed in clinical trials, or drugs withdrawn from the market. Although there are still unmet needs that require addressing, prodrugs should be seen as fine-tuning tools for the successful drug research and development. This review represents the potential of prodrugs to improve the drug delivery by enhanced aqueous solubility or permeability as well as describes several targeted prodrug strategies.

  19. Synthesis and characterization of novel dipeptide ester prodrugs of acyclovir

    NASA Astrophysics Data System (ADS)

    Nashed, Yasser E.; Mitra, Ashim K.

    2003-07-01

    Four dipeptide (Gly-Gly, Gly-Val, Val-Val, Val-Gly) ester prodrugs of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir, ACV) were synthesized. LC/MS was used to characterize the new prodrugs. Both 1H NMR and 13C NMR spectra of the four prodrugs of ACV were measured and assigned based on spectral comparison with compounds of similar structures.

  20. Clinical and metabolic correction of pompe disease by enzyme therapy in acid maltase-deficient quail.

    PubMed Central

    Kikuchi, T; Yang, H W; Pennybacker, M; Ichihara, N; Mizutani, M; Van Hove, J L; Chen, Y T

    1998-01-01

    Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase (GAA), a glycogen degrading lysosomal enzyme. GAA-deficient (AMD) Japanese quails exhibit progressive myopathy and cannot lift their wings, fly, or right themselves from the supine position (flip test). Six 4-wk-old acid maltase-deficient quails, with the clinical symptoms listed, were intravenously injected with 14 or 4.2 mg/kg of precursor form of recombinant human GAA or buffer alone every 2-3 d for 18 d (seven injections). On day 18, both high dose-treated birds (14 mg/kg) scored positive flip tests and flapped their wings, and one bird flew up more than 100 cm. GAA activity increased in most of the tissues examined. In heart and liver, glycogen levels dropped to normal and histopathology was normal. In pectoralis muscle, morphology was essentially normal, except for increased glycogen granules. In sharp contrast, sham-treated quail muscle had markedly increased glycogen granules, multi-vesicular autophagosomes, and inter- and intrafascicular fatty infiltrations. Low dose-treated birds (4.2 mg/kg) improved less biochemically and histopathologically than high dose birds, indicating a dose-dependent response. Additional experiment with intermediate doses and extended treatment (four birds, 5.7-9 mg/kg for 45 d) halted the progression of the disease. Our data is the first to show that an exogenous protein can target to muscle and produce muscle improvement. These data also suggest enzyme replacement with recombinant human GAA is a promising therapy for human Pompe disease. PMID:9466978

  1. Clinical and metabolic correction of pompe disease by enzyme therapy in acid maltase-deficient quail.

    PubMed

    Kikuchi, T; Yang, H W; Pennybacker, M; Ichihara, N; Mizutani, M; Van Hove, J L; Chen, Y T

    1998-02-15

    Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase (GAA), a glycogen degrading lysosomal enzyme. GAA-deficient (AMD) Japanese quails exhibit progressive myopathy and cannot lift their wings, fly, or right themselves from the supine position (flip test). Six 4-wk-old acid maltase-deficient quails, with the clinical symptoms listed, were intravenously injected with 14 or 4.2 mg/kg of precursor form of recombinant human GAA or buffer alone every 2-3 d for 18 d (seven injections). On day 18, both high dose-treated birds (14 mg/kg) scored positive flip tests and flapped their wings, and one bird flew up more than 100 cm. GAA activity increased in most of the tissues examined. In heart and liver, glycogen levels dropped to normal and histopathology was normal. In pectoralis muscle, morphology was essentially normal, except for increased glycogen granules. In sharp contrast, sham-treated quail muscle had markedly increased glycogen granules, multi-vesicular autophagosomes, and inter- and intrafascicular fatty infiltrations. Low dose-treated birds (4.2 mg/kg) improved less biochemically and histopathologically than high dose birds, indicating a dose-dependent response. Additional experiment with intermediate doses and extended treatment (four birds, 5.7-9 mg/kg for 45 d) halted the progression of the disease. Our data is the first to show that an exogenous protein can target to muscle and produce muscle improvement. These data also suggest enzyme replacement with recombinant human GAA is a promising therapy for human Pompe disease.

  2. Antibody-mediated enzyme replacement therapy targeting both lysosomal and cytoplasmic glycogen in Pompe disease.

    PubMed

    Yi, Haiqing; Sun, Tao; Armstrong, Dustin; Borneman, Scott; Yang, Chunyu; Austin, Stephanie; Kishnani, Priya S; Sun, Baodong

    2017-02-02

    Pompe disease is characterized by accumulation of both lysosomal and cytoplasmic glycogen primarily in skeletal and cardiac muscles. Mannose-6-phosphate receptor-mediated enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) targets the enzyme to lysosomes and thus is unable to digest cytoplasmic glycogen. Studies have shown that anti-DNA antibody 3E10 penetrates living cells and delivers "cargo" proteins to the cytosol or nucleus via equilibrative nucleoside transporter ENT2. We speculate that 3E10-mediated ERT with GAA will target both lysosomal and cytoplasmic glycogen in Pompe disease. A fusion protein (FabGAA) containing a humanized Fab fragment derived from the murine 3E10 antibody and the 110 kDa human GAA precursor was constructed and produced in CHO cells. Immunostaining with an anti-Fab antibody revealed that the Fab signals did not co-localize with the lysosomal marker LAMP2 in cultured L6 myoblasts or Pompe patient fibroblasts after incubation with FabGAA. Western blot with an anti-GAA antibody showed presence of the 150 kDa full-length FabGAA in the cell lysates, in addition to the 95- and 76 kDa processed forms of GAA that were also seen in the rhGAA-treated cells. Blocking of mannose-6-phosphate receptor with mannose-6-phosphate markedly reduced the 95- and the 76 kDa forms but not the 150 kDa form. In GAA-KO mice, FabGAA achieved similar treatment efficacy as rhGAA at an equal molar dose in reducing tissue glycogen contents. Our data suggest that FabGAA retains the ability of rhGAA to treat lysosomal glycogen accumulation and has the beneficial potential over rhGAA to reduce cytoplasmic glycogen storage in Pompe disease.

  3. An open-label clinical trial of agalsidase alfa enzyme replacement therapy in children with Fabry disease who are naïve to enzyme replacement therapy

    PubMed Central

    Goker-Alpan, Ozlem; Longo, Nicola; McDonald, Marie; Shankar, Suma P; Schiffmann, Raphael; Chang, Peter; Shen, Yinghua; Pano, Arian

    2016-01-01

    Background Following a drug manufacturing process change, safety/efficacy of agalsidase alfa were evaluated in enzyme replacement therapy (ERT)-naïve children with Fabry disease. Methods In an open-label, multicenter, Phase II study (HGT-REP-084; Shire), 14 children aged ≥7 years received 0.2 mg/kg agalsidase alfa every other week for 55 weeks. Primary endpoints: safety, changes in autonomic function (2-hour Holter monitoring). Secondary endpoints: estimated glomerular filtration rate, left ventricular mass index (LVMI), midwall fractional shortening, pharmacodynamic parameters, and patient-reported quality-of-life. Results Among five boys (median 10.2 [range 6.7, 14.4] years) and nine girls (14.8 [10.1, 15.9] years), eight patients experienced infusion-related adverse events (vomiting, n=4; nausea, n=3; dyspnea, n=3; chest discomfort, n=2; chills, n=2; dizziness, n=2; headache, n=2). One of these had several hypersensitivity episodes. However, no patient discontinued for safety reasons and no serious adverse events occurred. One boy developed immunoglobulin G (IgG) and neutralizing antidrug antibodies. Overall, no deterioration in cardiac function was observed in seven patients with low/abnormal SDNN (standard deviation of all filtered RR intervals; <100 ms) and no left ventricular hypertrophy: mean (SD) baseline SDNN, 81.6 (20.9) ms; mean (95% confidence interval [CI]) change from baseline to week 55, 17.4 (2.9, 31.9) ms. Changes in SDNN correlated with changes in LVMI (r=−0.975). No change occurred in secondary efficacy endpoints: mean (95% CI) change from baseline at week 55 in LVMI, 0.16 (−3.3, 3.7) g/m2.7; midwall fractional shortening, −0.62% (−2.7%, 1.5%); estimated glomerular filtration rate, 0.15 (−11.4, 11.7) mL/min/1.73 m2; urine protein, −1.8 (−6.0, 2.4) mg/dL; urine microalbumin, 0.6 (−0.5, 1.7) mg/dL; plasma globotriaosylceramide (Gb3), −5.71 (−10.8, −0.6) nmol/mL; urinary Gb3, −1,403.3 (−3,714.0, 907.4) nmol/g creatinine

  4. Can chronic maternal drug therapy alter the nursing infant's hepatic drug metabolizing enzyme pattern?

    PubMed

    Toddywalla, V S; Patel, S B; Betrabet, S S; Kulkarni, R D; Kombo, I; Saxena, B N

    1995-10-01

    This study was carried out to investigate whether minute quantities of maternal drugs ingested over an extended period of time by a breast-feeding infant can alter the activity pattern of the infant's hepatic drug metabolizing enzyme (HDME). The HDME activity patterns of 12 breast-fed infants whose mothers were not on drug therapy were compared with those of 11 infants whose mothers had been taking 30 micrograms levo-norgesterel daily for 90 to 195 days (oral contraceptives group) and of 10 infants whose mothers had been taking ethambutol and isoniazid daily since pregnancy (tuberculosis group). As 6 beta hydroxycortisol in urine is considered to be a good and acceptable reflector of HDME activity, it was estimated from the infants' urine using enzyme-linked immunosorbent assay (ELISA) technique. A comparison of the patterns between 90 days of age and 195 days of age of the infants in the control group and the two study groups indicated an increase from 36.6 ng/mL to 58.4 ng/mL at 195 days in the control group. An initial decrease from 36.6 ng/mL to 26.2 ng/mL was noted with commencement of maternal levo-norgesterel therapy, followed by a slow and steady rise to 47.8 ng/mL at 195 days of age, with a shift in the peak from 120 to 135 days of infants age in the oral contraceptive group. A suppressed pattern with decreased levels of 6 beta hydroxycortisol ranging from 19.3 ng/mL to 26.5 ng/mL at 195 days was found in the tuberculosis group. The data were analyzed by two-way analysis of variance (ANOVA) coupled with Duncan's Multiple range test. Both treatment group showed significant differences from the control group at the 0.050 level. The HDME plays an important role in determining the final outcome of any drug in humans, as it controls the metabolism of drugs. Hence, alterations in its activity caused by the transfer of maternal drugs over a prolonged period of time could pose a serious problem to nurslings when they require drugs for their own benefit.

  5. Enhanced efficacy of enzyme replacement therapy in Pompe disease through mannose-6-phosphate receptor expression in skeletal muscle.

    PubMed

    Koeberl, Dwight D; Luo, Xiaoyan; Sun, Baodong; McVie-Wylie, Alison; Dai, Jian; Li, Songtao; Banugaria, Suhrad G; Chen, Y-T; Bali, Deeksha S

    2011-06-01

    Enzyme replacement therapy (ERT) with acid α-glucosidase has become available for Pompe disease; however, the response of skeletal muscle, as opposed to the heart, has been attenuated. The poor response of skeletal muscle has been attributed to the low abundance of the cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle compared to heart. To further understand the role of CI-MPR in Pompe disease, muscle-specific CI-MPR conditional knockout (KO) mice were crossed with GAA-KO (Pompe disease) mice. We evaluated the impact of CI-MPR-mediated uptake of GAA by evaluating ERT in CI-MPR-KO/GAA-KO (double KO) mice. The essential role of CI-MPR was emphasized by the lack of efficacy of ERT as demonstrated by markedly reduced biochemical correction of GAA deficiency and of glycogen accumulations in double KO mice, in comparison with the administration of the same therapeutic doses in GAA-KO mice. Clenbuterol, a selective β(2)-agonist, enhanced the CI-MPR expression in skeletal tissue and also increased efficacy from GAA therapy, thereby confirming the key role of CI-MPR with regard to enzyme replacement therapy in Pompe disease. Biochemical correction improved in both muscle and non-muscle tissues, indicating that therapy could be similarly enhanced in other lysosomal storage disorders. In summary, enhanced CI-MPR expression might improve the efficacy of enzyme replacement therapy in Pompe disease through enhancing receptor-mediated uptake of GAA.

  6. Enhanced Efficacy of Enzyme Replacement Therapy in Pompe Disease Through Mannose-6-Phosphate Receptor Expression in Skeletal Muscle

    PubMed Central

    Koeberl, Dwight D.; Luo, Xiaoyan; Sun, Baodong; McVie-Wylie, Alison; Dai, Jian; Li, Songtao; Banugaria, Suhrad G.; Chen, Y-T; Bali, Deeksha S.

    2011-01-01

    Enzyme replacement therapy (ERT) with acid α-glucosidase has become available for Pompe disease; however, the response of skeletal muscle, as opposed to the heart, has been attenuated. The poor response of skeletal muscle has been attributed to the low abundance of the cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle compared to heart. To further understand the role of CI-MPR in Pompe disease, muscle-specific CI-MPR conditional knockout (KO) mice were crossed with GAA-KO (Pompe disease) mice. We evaluated the impact of CI-MPR-mediated uptake of GAA by evaluating ERT in CI-MPR-KO/GAA-KO (double KO) mice. The essential role of CI-MPR was emphasized by the lack of efficacy of ERT as demonstrated by markedly reduced biochemical correction of GAA deficiency and of glycogen accumulations in double KO mice, in comparison with administration of the same therapeutic doses in GAA-KO mice. Clenbuterol, a selective β2-agonist, enhanced CI-MPR expression in skeletal tissue and also increased efficacy from GAA therapy, thereby confirming the key role of CI-MPR with regard to enzyme replacement therapy in Pompe disease. Biochemical correction improved in both muscle and non-muscle tissues, indicating that therapy could be similarly enhanced in other lysosomal storage disorders. In summary, enhanced CI-MPR expression might improve the efficacy of enzyme replacement therapy in Pompe disease through enhancing receptor-mediated uptake of GAA. PMID:21397538

  7. Serum-Mediated Inhibition of Enzyme Replacement Therapy in Fabry Disease.

    PubMed

    Lenders, Malte; Stypmann, Jörg; Duning, Thomas; Schmitz, Boris; Brand, Stefan-Martin; Brand, Eva

    2016-01-01

    Fabry disease (FD) is a progressive multisystemic disorder, treatable with recombinant enzyme replacement therapy (agalsidase). However, recent studies suggest an endogenous inhibition of agalsidase in patients with FD, as reported for other lysosomal storage diseases. To assess the clinical consequences of serum-mediated agalsidase inhibition in affected patients, we determined the agalsidase inhibition status of 168 patients (68 male) with FD and compared outcomes of inhibition-positive patients with those of inhibition-negative patients. The assessment included clinical events during time on agalsidase, determination of renal and cardiac function, and evaluation of FD-related symptoms. The frequency of serum-mediated agalsidase inhibition was 40% in agalsidase-treated males. Inhibition did not depend on the compound initially used (agalsidase-α or -β). Agalsidase inhibition was associated with higher lyso-globotriaosylceramide levels and worse disease severity scores in patients. Compared with agalsidase inhibition-negative men, agalsidase inhibition-positive men showed greater left ventricular mass (P=0.02) and substantially lower renal function (difference in eGFR of about -30 ml/min per 1.73 m(2); P=0.04), which was confirmed by a longitudinal 5-year retrospective analysis. Additionally, affected patients presented more often with FD-typical symptoms, such as diarrhea, fatigue, and neuropathic pain, among others. Therefore, patients with poor clinical outcome on agalsidase should be tested for agalsidase inhibition. Future studies are warranted to determine if affected patients with FD benefit from acute reduction of anti-agalsidase antibodies or long-term immune modulation therapies to suppress agalsidase inhibition and to identify mechanisms that minimize antibody generation against agalsidase.

  8. Far-Red Light Activatable, Multifunctional Prodrug for Fluorescence Optical Imaging and Combinational Treatment

    PubMed Central

    2015-01-01

    We recently developed “photo-unclick chemistry”, a novel chemical tool involving the cleavage of aminoacrylate by singlet oxygen, and demonstrated its application to visible light-activatable prodrugs. In this study, we prepared an advanced multifunctional prodrug, Pc-(L-CA4)2, composed of the fluorescent photosensitizer phthalocyanine (Pc), an SO-labile aminoacrylate linker (L), and a cytotoxic drug combretastatin A-4 (CA4). Pc-(L-CA4)2 had reduced dark toxicity compared with CA4. However, once illuminated, it showed improved toxicity similar to CA4 and displayed bystander effects in vitro. We monitored the time-dependent distribution of Pc-(L-CA4)2 using optical imaging with live mice. We also effectively ablated tumors by the illumination with far-red light to the mice, presumably through the combined effects of photodynamic therapy (PDT) and released chemotherapy drug, without any sign of acute systemic toxicity. PMID:24694092

  9. Genetically engineered pre-microRNA-34a prodrug suppresses orthotopic osteosarcoma xenograft tumor growth via the induction of apoptosis and cell cycle arrest

    PubMed Central

    Zhao, Yong; Tu, Mei-Juan; Wang, Wei-Peng; Qiu, Jing-Xin; Yu, Ai-Xi; Yu, Ai-Ming

    2016-01-01

    Osteosarcoma (OS) is the most common primary malignant bone tumor in children, and microRNA-34a (miR-34a) replacement therapy represents a new treatment strategy. This study was to define the effectiveness and safety profiles of a novel bioengineered miR-34a prodrug in orthotopic OS xenograft tumor mouse model. Highly purified pre-miR-34a prodrug significantly inhibited the proliferation of human 143B and MG-63 cells in a dose dependent manner and to much greater degrees than controls, which was attributed to induction of apoptosis and G2 cell cycle arrest. Inhibition of OS cell growth and invasion were associated with release of high levels of mature miR-34a from pre-miR-34a prodrug and consequently reduction of protein levels of many miR-34a target genes including SIRT1, BCL2, c-MET, and CDK6. Furthermore, intravenous administration of in vivo-jetPEI formulated miR-34a prodrug significantly reduced OS tumor growth in orthotopic xenograft mouse models. In addition, mouse blood chemistry profiles indicated that therapeutic doses of bioengineered miR-34a prodrug were well tolerated in these animals. The results demonstrated that bioengineered miR-34a prodrug was effective to control OS tumor growth which involved the induction of apoptosis and cell cycle arrest, supporting the development of bioengineered RNAs as a novel class of large molecule therapeutic agents. PMID:27216562

  10. Enzyme replacement therapy from birth in a feline model of mucopolysaccharidosis type VI.

    PubMed Central

    Crawley, A C; Niedzielski, K H; Isaac, E L; Davey, R C; Byers, S; Hopwood, J J

    1997-01-01

    We report evidence of a dose responsive effect of enzyme replacement therapy in mucopolysaccharidosis type VI cats from birth, at the clinical, biochemical, and histopathological level. Cats treated with weekly, intravenous recombinant human N-acetylgalactosamine-4-sulfatase at 1 and 5 mg/kg, were heavier, more flexible, had greatly reduced or no spinal cord compression, and had almost normal urinary glycosaminoglycan levels. There was near normalization or complete reversal of lysosomal storage in heart valve, aorta, skin, dura, liver, and brain perivascular cells. No reduction in lysosomal vacuolation was observed in cartilage or cornea; however, articular cartilage was thinner and external ear pinnae were larger in some treated cats. Degenerative joint changes were not obviously delayed in treated cats. Skeletal pathology was reduced, with more normalized bone dimensions and with more uniform bone density and trabecular pattern clearly visible on radiographs by 5 to 6 mo; however, differences between 1 and 5 mg/kg dose rates were not clearly distinguishable. At a dose of 0.2 mg/kg, disease was not significantly altered in the majority of parameters examined. Lysosomal storage was present in all tissues examined in the midterm mucopolysaccharidosis type VI fetus and increased rapidly in extent and severity from birth. PMID:9045867

  11. Continuous infusion of enzyme replacement therapy is inferior to weekly infusions in MPS I dogs

    PubMed Central

    Passage, M.B.; Krieger, A.W.; Peinovich, M.C.; Lester, T.; Le, S.Q.; Dickson, P.I.; Kakkis, E.D.

    2010-01-01

    Summary Intravenous enzyme replacement therapy with recombinant human α-l-iduronidase (rhIDU) is used weekly to treat mucopolysaccharidosis (MPS) I. We tested continuous administration of rhIDU at two dosing levels (0.58 mg/kg/week and 2 mg/kg/week) in MPS I dogs, and compared the efficacy of continuous to the clinically-used 0.58 mg/kg weekly three-hour infusion. Peak plasma concentrations of rhIDU were much higher in weekly-treated dogs (mean 256 units/ml) than steady-state concentrations in dogs treated with continuous infusion (mean 1.97 units/ml at 0.58 mg/kg/week; 10.1 units/ml at 2 mg/kg/week). Dogs receiving continuous IV rhIDU, even at a higher (2 mg/kg/week) dose, had consistently lower iduronidase levels in tissues than dogs receiving a weekly (0.58 mg/kg/week) dose. GAG storage was also less improved by continuous intravenous infusion. Adverse events were similar in all dosing groups. We found that continuous administration of 2 mg/kg/week rhIDU to MPS I dogs was insufficient to achieve GAG storage reduction comparable to 0.58 mg/kg weekly dosing. PMID:19562502

  12. Bortezomib-induced enzyme-targeted radiation therapy in herpesvirus-associated tumors.

    PubMed

    Fu, De-Xue; Tanhehco, Yvette; Chen, Jianmeng; Foss, Catherine A; Fox, James J; Chong, Ja-Mun; Hobbs, Robert F; Fukayama, Masashi; Sgouros, George; Kowalski, Jeanne; Pomper, Martin G; Ambinder, Richard F

    2008-10-01

    We investigated the possibility of using a pharmacologic agent to modulate viral gene expression to target radiotherapy to tumor tissue. In a mouse xenograft model, we had previously shown targeting of [(125)I]2'-fluoro-2'-deoxy-beta-D-5-iodouracil-arabinofuranoside ([(125)I]FIAU) to tumors engineered to express the Epstein-Barr virus thymidine kinase (EBV-TK). Here we extend those results to targeting of a therapeutic radiopharmaceutical [(131)I]FIAU to slow or stop tumor growth or to achieve tumor regression. These outcomes were achieved in xenografts with tumors that constitutively expressed the EBV-TK. With naturally infected EBV tumor cell lines (Burkitt's lymphoma and gastric carcinoma), activation of viral gene expression by pretreatment with bortezomib was required. Marked changes in tumor growth could also be achieved in naturally infected Kaposi's sarcoma herpesvirus tumors after pretreatment with bortezomib. Bortezomib-induced enzyme-targeted radiation therapy illustrates the possibility of pharmacologically modulating tumor gene expression to result in targeted radiotherapy.

  13. Pancreatic enzyme replacement therapy in cystic fibrosis: dose, variability and coefficient of fat absorption.

    PubMed

    Calvo-Lerma, Joaquim; Martínez-Barona, Sandra; Masip, Etna; Fornés, Victoria; Ribes-Koninckx, Carmen

    2017-10-01

    Pancreatic enzyme replacement therapy (PERT) remains a backbone in the nutritional treatment of cystic fibrosis. Currently, there is a lack of an evidence-based tool that allows dose adjustment. To date, no studies have found an association between PERT dose and fat absorption. Therefore, the aim of the study was to assess the influence of both the PERT dose and the variability in this dose on the coefficient of fat absorption (CFA). This is a retrospective longitudinal study of 16 pediatric patients (192 food records) with three consecutive visits to the hospital over a twelve-month period. Dietary fat intake and PERT were assessed via a four-day food record and fat content in stools was determined by means of a three-day stool sample collection. A beta regression model was built to explain the association between the CFA and the interaction between the PERT dose (lipase units [LU]/g dietary fat) and the variability in the PERT dose (standard deviation [SD]). The coefficient of fat absorption increased with the PERT dose when the variability in the dose was low. In contrast, even at the highest PERT dose values, the CFA decreased when the variability was high. The confidence interval suggested an association, although the analysis was not statistically significant. The variability in the PERT dose adjustment should be taken into consideration when performing studies on PERT efficiency. A clinical goal should be the maintenance of a constant PERT dose rather than trying to obtain an optimal value.

  14. The Immune Response to Intrathecal Enzyme Replacement Therapy in Mucopolysaccharidosis I Patients

    PubMed Central

    Vera, Moin; Le, Steven; Kan, Shih-hsin; Garban, Hermes; Naylor, David; Mlikotic, Anton; Kaitila, Ilkka; Harmatz, Paul; Chen, Agnes; Dickson, Patricia

    2013-01-01

    BACKGROUND Intrathecal (IT) enzyme replacement therapy with recombinant human α-l-iduronidase (rhIDU) has been studied to treat glycosaminoglycan storage in the central nervous system of mucopolysaccharidosis (MPS) I dogs and is currently being studied in MPS I patients. METHODS We studied the immune response to IT rhIDU in MPS I subjects with spinal cord compression who had been previously treated with intravenous rhIDU. We measured concentrations of specific antibodies and cytokines in serum and cerebrospinal fluid collected prior to monthly IT rhIDU infusions and compared serologic findings to clinical adverse event reports to establish temporal correlations with clinical symptoms. RESULTS Five MPS I subjects participating in IT rhIDU trials were studied. One subject with symptomatic spinal cord compression had evidence of an inflammatory response with cerebrospinal fluid leukocytosis, elevated IL-5 and elevated IgG. This subject also complained of lower back pain and buttock parasthesias temporally correlated with serologic abnormalities. Clinical symptoms were managed with oral medication and serologic abnormalities resolved though this subject withdrew from the trial to have spinal decompressive surgery. CONCLUSION IT rhIDU was generally well tolerated in the subjects studied though one subject had moderate to severe clinical symptoms and serologic abnormalities consistent with an immune response. PMID:24002329

  15. Enzyme replacement therapy for treating mucopolysaccharidosis type IVA (Morquio A syndrome): effect and limitations

    PubMed Central

    Tomatsu, Shunji; Sawamoto, Kazuki; Shimada, Tsutomu; Bober, Michael B.; Kubaski, Francyne; Yasuda, Eriko; Mason, Robert W.; Khan, Shaukat; Alméciga-Díaz, Carlos J.; Barrera, Luis A.; Mackenzie, William G.

    2015-01-01

    Introduction Following a Phase III, randomized, double-blind, placebo (PBO)-controlled, multinational study in subjects with mucopolysaccharidosis IVA (MPS IVA), enzyme replacement therapy (ERT) of elosulfase alfa has been approved in several countries. The study was designed to evaluate safety and efficacy of elosulfase alfa in patients with MPS IVA aged 5 years and older. Areas covered Outcomes of clinical trials for MPS IVA have been described. Subjects received either 2.0 mg/kg/week, 2.0 mg/kg/every other week, or PBO, for 24 weeks. The primary endpoint was the change from baseline 6-min walk test (6MWT) distance compared to PBO. The 6MWT results improved in patients receiving 2 mg/kg weekly compared to PBO. The every other week regimen resulted in walk distances comparable to PBO. There was no change from baseline in the 3 Min Stair Climb Test in both treatment groups. Following completion of the initial study, patients, who continued to receive elosulfase alfa 2 mg/kg weekly (QW) for another 48 weeks (for a total of up to 72-week exposure), did not show additional improvement on 6MWT. Expert opinion We suggest that ERT is a therapeutic option for MPS IVA, providing a modest effect and the majority of the effects are seen in the soft tissues. PMID:26973801

  16. Enzyme replacement therapy for treating mucopolysaccharidosis type IVA (Morquio A syndrome): effect and limitations.

    PubMed

    Tomatsu, Shunji; Sawamoto, Kazuki; Shimada, Tsutomu; Bober, Michael B; Kubaski, Francyne; Yasuda, Eriko; Mason, Robert W; Khan, Shaukat; Alméciga-Díaz, Carlos J; Barrera, Luis A; Mackenzie, William G; Orii, Tadao

    2015-11-01

    Following a Phase III, randomized, double-blind, placebo (PBO)-controlled, multinational study in subjects with mucopolysaccharidosis IVA (MPS IVA), enzyme replacement therapy (ERT) of elosulfase alfa has been approved in several countries. The study was designed to evaluate safety and efficacy of elosulfase alfa in patients with MPS IVA aged 5 years and older. Outcomes of clinical trials for MPS IVA have been described. Subjects received either 2.0 mg/kg/week, 2.0 mg/kg/every other week, or PBO, for 24 weeks. The primary endpoint was the change from baseline 6-min walk test (6MWT) distance compared to PBO. The 6MWT results improved in patients receiving 2 mg/kg weekly compared to PBO. The every other week regimen resulted in walk distances comparable to PBO. There was no change from baseline in the 3 Min Stair Climb Test in both treatment groups. Following completion of the initial study, patients, who continued to receive elosulfase alfa 2 mg/kg weekly (QW) for another 48 weeks (for a total of up to 72-week exposure), did not show additional improvement on 6MWT. We suggest that ERT is a therapeutic option for MPS IVA, providing a modest effect and the majority of the effects are seen in the soft tissues.

  17. A beta-blocker, propranolol, decreases the efficacy from enzyme replacement therapy in Pompe disease

    PubMed Central

    Han, Sang-oh; Pope, Rand; Li, Songtao; Kishnani, Priya S.; Steet, Richard; Koeberl, Dwight D.

    2015-01-01

    Enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) fails to completely reverse muscle weakness in Pompe disease. β2-agonists enhanced ERT by increasing receptor-mediated uptake of rhGAA in skeletal muscles. Purpose To test the hypothesis that a β-blocker might reduce the efficacy of ERT, because the action of β-blockers opposes those of β2-agonists. Methods Mice with Pompe disease were treated with propranolol (a β-blocker) or clenbuterol in combination with ERT, or with ERT alone. Results Propranolol-treated mice had decreased weight gain (p<0.01), in comparison with clenbuterol-treated mice. Left ventricular mass was decreased (and comparable to wild-type) in ERT only and clenbuterol-treated groups of mice, and unchanged in propranolol-treated mice. GAA activity increased following either clenbuterol or propranolol in skeletal muscles. However, muscle glycogen was reduced only in clenbuterol-treated mice, not in propranolol-treated mice. Cell-based experiments confirmed that propranolol reduces uptake of rhGAA into Pompe fibroblasts and also demonstrated that the drug induces intracellular accumulation of glycoproteins at higher doses. Conclusion Propranolol, a commonly prescribed β-blocker, increased left ventricular mass and decreased glycogen clearance in skeletal muscle following ERT. β-blockers might therefore decrease the efficacy from ERT in patients with Pompe disease. PMID:26454691

  18. Angiotensin-converting enzyme inhibitors in the therapy of renal diseases.

    PubMed

    Lefebvre, H P; Toutain, P L

    2004-10-01

    Renal diseases, especially chronic renal failure (CRF), are common in canine and feline medicine. The renin-angiotensin-aldosterone system (RAAS) plays a pivotal role in these conditions in the development of renal lesions and the progression of kidney dysfunction. Angiotensin-converting enzyme inhibitors (ACEI) are currently considered as the most efficient agents in therapeutic strategies. The benefit of an ACEI treatment can be explained by at least three mechanisms: ACEI limit systemic and glomerular capillary hypertension, have an antiproteinuric effect, and retard the development of glomerulosclerosis and tubulointerstitial lesions. These effects have been studied in dogs and cats, and there is now some evidence to support the recommendation of ACEI therapy in dogs and cats with CRF. Nevertheless the prescription of ACEI in such patients should take into account the potential influence of renal impairment on ACEI disposition, and adverse effects on the renal function itself (especially hypotension and acute reductions in glomerular filtration rate). The risk of drug interaction with diuretics, nonsteroidal anti-inflammatory drugs and anesthetics, should not be overestimated. Furthermore, hypotension may occur in patients on a low sodium diet.

  19. A beta-blocker, propranolol, decreases the efficacy from enzyme replacement therapy in Pompe disease.

    PubMed

    Han, Sang-Oh; Pope, Rand; Li, Songtao; Kishnani, Priya S; Steet, Richard; Koeberl, Dwight D

    2016-02-01

    Enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) fails to completely reverse muscle weakness in Pompe disease. β2-agonists enhanced ERT by increasing receptor-mediated uptake of rhGAA in skeletal muscles. To test the hypothesis that a β-blocker might reduce the efficacy of ERT, because the action of β-blockers opposes those of β2-agonists. Mice with Pompe disease were treated with propranolol (a β-blocker) or clenbuterol in combination with ERT, or with ERT alone. Propranolol-treated mice had decreased weight gain (p<0.01), in comparison with clenbuterol-treated mice. Left ventricular mass was decreased (and comparable to wild-type) in ERT only and clenbuterol-treated groups of mice, and unchanged in propranolol-treated mice. GAA activity increased following either clenbuterol or propranolol in skeletal muscles. However, muscle glycogen was reduced only in clenbuterol-treated mice, not in propranolol-treated mice. Cell-based experiments confirmed that propranolol reduces uptake of rhGAA into Pompe fibroblasts and also demonstrated that the drug induces intracellular accumulation of glycoproteins at higher doses. Propranolol, a commonly prescribed β-blocker, reduced weight, increased left ventricular mass and decreased glycogen clearance in skeletal muscle following ERT. β-Blockers might therefore decrease the efficacy from ERT in patients with Pompe disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Deleterious effects of interruption followed by reintroduction of enzyme replacement therapy on a lysosomal storage disorder.

    PubMed

    Schneider, Ana Paula; Matte, Ursula; Pasqualim, Gabriela; Tavares, Angela Maria Vicente; Mayer, Fabiana Quoos; Martinelli, Barbara; Ribas, Graziela; Vargas, Carmen Regla; Giugliani, Roberto; Baldo, Guilherme

    2016-10-01

    Temporary interruption of enzyme replacement therapy (ERT) in patients with different lysosomal storage disorders may happen for different reasons (adverse reactions, issues with reimbursement, logistic difficulties, and so forth), and the impact of the interruption is still uncertain. In the present work, we studied the effects of the interruption of intravenous ERT (Laronidase, Genzyme) followed by its reintroduction in mice with the prototypical lysosomal storage disorder mucopolysaccharidosis type I, comparing to mice receiving continuous treatment, untreated mucopolysaccharidosis type I mice, and normal mice. In the animals which treatment was temporarily interrupted, we observed clear benefits of treatment in several organs (liver, lung, heart, kidney, and testis) after reintroduction, but a worsening in the thickness of the aortic wall was detected. Furthermore, these mice had just partial improvements in behavioral tests, suggesting some deterioration in the brain function. Despite worsening is some disease aspects, urinary glycosaminoglycans levels did not increase during interruption, which indicates that this biomarker commonly used to monitor treatment in patients should not be used alone to assess treatment efficacy. The deterioration observed was not caused by the development of serum antienzyme antibodies. All together our results suggest that temporary ERT interruption leads to deterioration of function in some organs and should be avoided whenever possible. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Progress in Enzyme Replacement Therapy in Glycogen Storage Disease Type II

    PubMed Central

    Semplicini, Claudio; Tonin, Paola; Filosto, Massimiliano; Pegoraro, Elena; Sorarù, Gianni; Fanin, Marina

    2009-01-01

    Glycogen storage disease type II (GSDII) is an autosomal recessive lysosomal disorder caused by mutations in the gene encoding alpha-glucosidase (GAA). The disease can be clinically classified into three types: a severe infantile form, a juvenile and an adultonset form. Cases with juvenile or adult onset GSDII mimic limb-girdle muscular dystrophy or polymyositis and are often characterized by respiratory involvement. GSDII patients are diagnosed by biochemical assay and by molecular characterization of the GAA gene. Ascertaining a natural history of patients with heterogeneous late-onset GSDII is useful for evaluating their progressive functional disability. A significant decline is observed over the years in skeletal and respiratory muscle function. Enzyme replacement therapy (ERT) has provided encouraging results in the infantile form. It is not yet known if ERT is effective in late-onset GSDII. We examined a series of 11 patients before and after ERT evaluating muscle strength by MRC, timed and graded functional tests, 6-minute walk test (6MWT), respiratory function by spirometric parameters and quality of life. We observed a partial improvement during a prolonged follow-up from 3 to 18 months. The use of different clinical parameters in the proposed protocol seems crucial to determine the efficacy of ERT, since not all late-onset patients respond similarly to ERT. PMID:21179524

  2. In vivo and in situ tracking cancer chemotherapy by highly photostable NIR fluorescent theranostic prodrug.

    PubMed

    Wu, Xumeng; Sun, Xuanrong; Guo, Zhiqian; Tang, Jianbin; Shen, Youqing; James, Tony D; Tian, He; Zhu, Weihong

    2014-03-05

    In vivo monitoring of the biodistribution and activation of prodrugs is urgently required. Near infrared (NIR) fluorescence-active fluorophores with excellent photostability are preferable for tracking drug release in vivo. Herein, we describe a NIR prodrug DCM-S-CPT and its polyethylene glycol-polylactic acid (PEG-PLA) loaded nanoparticles as a potent cancer therapy. We have conjugated a dicyanomethylene-4H-pyran derivative as the NIR fluorophore with camptothecin (CPT) as the anticancer drug using a disulfide linker. In vitro experiments verify that the high intracellular glutathione (GSH) concentrations in tumor cells cause cleavage of the disulfide linker, resulting in concomitantly the active drug CPT release and significant NIR fluorescence turn-on with large Stokes shift (200 nm). The NIR fluorescence of DCM-S-CPT at 665 nm with fast response to GSH can act as a direct off-on signal reporter for the GSH-activatable prodrug. Particularly, DCM-S-CPT possesses much better photostability than ICG, which is highly desirable for in situ fluorescence-tracking of cancer chemotherapy. DCM-S-CPT has been successfully utilized for in vivo and in situ tracking of drug release and cancer therapeutic efficacy in living animals by NIR fluorescence. DCM-S-CPT exhibits excellent tumor-activatable performance when intravenously injected into tumor-bearing nude mice, as well as specific cancer therapy with few side effects. DCM-S-CPT loaded in PEG-PLA nanoparticles shows even higher antitumor activity than free CPT, and is also retained longer in the plasma. The tumor-targeting ability and the specific drug release in tumors make DCM-S-CPT as a promising prodrug, providing significant advances toward deeper understanding and exploration of theranostic drug-delivery systems.

  3. Long-term and high-dose trials of enzyme replacement therapy in the canine model of mucopolysaccharidosis I.

    PubMed

    Kakkis, E D; McEntee, M F; Schmidtchen, A; Neufeld, E F; Ward, D A; Gompf, R E; Kania, S; Bedolla, C; Chien, S L; Shull, R M

    1996-08-01

    Enzyme replacement is a potential therapy for mucopolysaccharidosis I (MPS I), a lysosomal storage disorder caused by alpha-L-iduronidase deficiency. Previous work showed improvement in the tissues of MPS I dogs treated intravenously for 3 months with recombinant human alpha-L-iduronidase (25,000 units or approximately 0.1 mg/kg/week). We have now treated an MPS I-affected dog for 13 months to assess the clinical effects of enzyme replacement. The treated dog gained more weight, was more active, and had less joint stiffness than the untreated littermate. Biochemical and histologic studies demonstrated uptake of alpha-L-iduronidase and decreased lysosomal storage in the liver, kidney, spleen, lymph nodes, synovium, adrenals, and lungs. The brain had detectable enzyme activity and decreased glycosaminoglycan storage although histologic improvement was not evident. Cartilage and heart valve did not show any detectable improvement. A fivefold higher dose (approximately 0.5 mg/kg) administered five times over 10 days to two other dogs resulted in higher tissue enzyme activity and similarly decreased glycosaminoglycan storage and excretion. Antibodies to human alpha-L-iduronidase were induced in all treated dogs and may be associated with immune complex deposition and proteinuria. Recombinant canine alpha-L-iduronidase also induced antibody formation to a similar degree. The results support the conclusion that enzyme replacement is a promising therapy for MPS I though immunologic complications may occur.

  4. Lipophilic pyrazinoic acid amide and ester prodrugs stability, activation and activity against M. tuberculosis.

    PubMed

    Simões, Marta Filipa; Valente, Emília; Gómez, M José Rodríguez; Anes, Elsa; Constantino, Luís

    2009-06-28

    Pyrazinamide (PZA) is active against M. tuberculosis and is a first line agent for the treatment of human tuberculosis. PZA is itself a prodrug that requires activation by a pyrazinamidase to form its active metabolite pyrazinoic acid (POA). Since the specificity of cleavage is dependent on a single bacterial enzyme, resistance to PZA is often found in tuberculosis patients. Esters of POA have been proposed in the past as alternatives to PZA however the most promising compounds were rapidly degraded in the presence of serum. In order to obtain compounds that could survive during the transport phase, we synthesized lipophilic ester and amide POA derivatives, studied their activity against M. tuberculosis, their stability in plasma and rat liver homogenate and also their activation by a mycobacterial homogenate. The new lipophilic ester prodrugs were found to be active in concentrations 10-fold lower than those needed for PZA to kill sensitive M. tuberculosis and also have a suitable stability in the presence of plasma. Amides of POA although more stable in plasma have lower activity. The reason can probably be found in the rate of activation of both types of prodrugs; while esters are easily activated by mycobacterial esterases, amides are resistant to activation and are not transformed into POA at a suitable rate.

  5. Submicron lipid emulsion as a drug delivery system for nalbuphine and its prodrugs.

    PubMed

    Wang, Jhi-Joung; Sung, K C; Hu, Oliver Yoa-Pu; Yeh, Chih-Hui; Fang, Jia-You

    2006-10-10

    This study investigates the submicron lipid emulsion as a potential parenteral drug delivery system for nalbuphine and its ester prodrugs. Submicron emulsions were prepared using egg phospholipid as the main emulsifier, various co-emulsifiers were also incorporated, including Brij 30, Brij 98, and stearylamine. Squalene as the oil phase formed stable emulsions with small particles. Drug release was affected by incorporating various co-emulsifiers and drugs with various lipophilicity. The loading of nalbuphine into lipid emulsions resulted in the slower and sustained release of nalbuphine. Lipid emulsions containing Brij 98 could further enhance the release of prodrugs as compared to the aqueous solution (control) especially for nalbuphine enanthate (NAE). Hemolysis caused by the interaction between erythrocytes and lipid emulsions was investigated. Brij 30 and Brij 98 could shield the hemolytic activity of phospholipids in the oil/water interface, decreasing the acute toxicological potential of the emulsions. The in vivo analgesic activity of various emulsions was examined by a cold ethanol tail-flick test. The analgesic duration and potency were significantly increased by incorporating nalbuphine and NAE into Brij 98-containing emulsions. There was no need for nalbuphine benzoate (NAB) to show a controlled delivery manner by encapsulating into emulsions, since NAB itself could prolong the analgesic duration of nalbuphine due to the slow enzyme degradation. The in vivo analgesic activity correlated well to the profiles of in vivo pharmacokinetic profiles. The study demonstrates the feasibility of using submicron lipid emulsion as the parenteral drug delivery system for nalbuphine and its prodrugs.

  6. Dipeptide Prodrug Approach to Evade Efflux Pumps and CYP3A4 Metabolism of Lopinavir

    PubMed Central

    Patel, Mitesh; Sheng, Ye; Mandava, Nanda K.; Pal, Dhananjay; Mitra, Ashim K.

    2014-01-01

    Oral absorption of lopinavir (LPV) is limited due to P-glycoprotein (P-gp) and multidrug resistance-associated protein2 (MRP2) mediated efflux by intestinal epithelial cells. Moreover, LPV is extensively metabolized by CYP3A4 enzymes. In the present study, dipeptide prodrug approach was employed to circumvent efflux pumps (P-gp and MRP2) and CYP3A4 mediated metabolism of LPV. Valine-isoleucine-LPV (Val-Ile-LPV) was synthesized and identified by LCMS and NMR techniques. The extent of LPV and Val-Ile-LPV interactions with P-gp and MRP2 was studied by uptake and transport studies across MDCK-MDR1 and MDCK-MRP2 cells. To determine the metabolic stability, time and concentration dependent degradation study was performed in liver microsomes. Val-Ile-LPV exhibited significantly higher aqueous solubility relative to LPV. This prodrug generated higher stability under acidic pH. Val-Ile-LPV demonstrated significantly lower affinity towards P-gp and MRP2 relative to LPV. Transepithelial transport of Val-Ile-LPV was significantly higher in the absorptive direction (apical to basolateral) relative to LPV. Importantly, Val-Ile-LPV was recognized as an excellent substrate by peptide transporter. Moreover, Val-Ile-LPV displayed significantly higher metabolic stability relative to LPV. Results obtained from this study suggested that dipeptide prodrug approach is a viable option to elevate systemic levels of LPV following oral administration PMID:25261710

  7. Prodrug delivery of novel PTP1B inhibitors to enhance insulin signalling.

    PubMed

    Erbe, D V; Klaman, L D; Wilson, D P; Wan, Z-K; Kirincich, S J; Will, S; Xu, X; Kung, L; Wang, S; Tam, S; Lee, J; Tobin, J F

    2009-06-01

    A growing percentage of the population is resistant to two key hormones - insulin and leptin - as a result of increased obesity, often leading to significant health consequences such as type 2 diabetes. Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of signalling by both of these hormones, so that inhibitors of this enzyme may provide promise for correcting endocrine abnormalities in both diabetes and obesity. As with other tyrosine phosphatases, identification of viable drug candidates targeting PTP1B has been elusive because of the nature of its active site. Beginning with novel phosphotyrosine mimetics, we have designed some of the most potent PTP1B inhibitors. However, their highly acidic structures limit intrinsic permeability and pharmacokinetics. Ester prodrugs of these inhibitors improve their drug-like properties with the goal of delivering these nanomolar inhibitors to the cytoplasm of cells within target tissues. In addition to identifying prodrugs that is able to deliver active drugs into cells to inhibit PTP1B and increase insulin signalling, these compounds were further modified to gain a variety of cleavage properties for targeting activity in vivo. One such prodrug candidate improved insulin sensitivity in ob/ob mice, with lowered fasting blood glucose levels seen in the context of lowered fasting insulin levels following 4 days of intraperitoneal dosing. The results presented in this study highlight the potential for design of orally active drug candidates targeting PTP1B, while also delineating the considerable challenges remaining.

  8. Synthesis and analysis of activity of a potential anti-melanoma prodrug with a hydrazine linker.

    PubMed

    Frąckowiak-Wojtasek, Bożena; Gąsowska-Bajger, Beata; Mazurek, Magdalena; Raniszewska, Agnieszka; Logghe, Marieke; Smolarczyk, Ryszard; Cichoń, Tomasz; Szala, Stanisław; Wojtasek, Hubert

    2014-01-01

    A potential anti-melanoma prodrug containing a phenolic activator, a hydrazine linker, and a nitrogen mustard effector - (N-{4-[bis-(2-chloroethyl)amino]benzoyl}-N'-(4-hydroxybenzyl)hydrazine) has been synthesized in seven steps. Spectrophotometric measurements of its oxidation by tyrosinase showed a rapid increase of absorbance at 337 nm. HPLC analysis demonstrated that two major products were formed. However, during the reaction one of the products was converted into the other. The stable product with a maximum of absorption at 337 nm was isolated and identified as 5,6-dihydroxy-1H-indazol-1-yl 4-[bis-(2-chloroethyl)amino]benzoate. It was formed by a cyclization of the enzymatically generated o-quinone. This reaction was unexpected, since the acylated hydrazine nitrogen atom should not be sufficiently nucleophilic to attack the o-quinone ring. This cyclization prevented the effector release from the enzyme-activated prodrug. As a result, the prodrug showed only limited specificity for B16-F10 murine melanoma cells compared to reference cell lines. When applied in solid tumors in mice it showed slightly higher activity than the parent mustard drug (4-[bis-(2-chloroethyl)amino]benzoic cid), but significantly lower activity than melphalan, a commercial mustard drug with a structure resembling tyrosine, occasionally used in the treatment of melanoma.

  9. Modeling of Pharmacokinetics of Cocaine in Human Reveals the Feasibility for Development of Enzyme Therapies for Drugs of Abuse

    PubMed Central

    Zheng, Fang; Zhan, Chang-Guo

    2012-01-01

    A promising strategy for drug abuse treatment is to accelerate the drug metabolism by administration of a drug-metabolizing enzyme. The question is how effectively an enzyme can actually prevent the drug from entering brain and producing physiological effects. In the present study, we have developed a pharmacokinetic model through a combined use of in vitro kinetic parameters and positron emission tomography data in human to examine the effects of a cocaine-metabolizing enzyme in plasma on the time course of cocaine in plasma and brain of human. Without an exogenous enzyme, cocaine half-lives in both brain and plasma are almost linearly dependent on the initial cocaine concentration in plasma. The threshold concentration of cocaine in brain required to produce physiological effects has been estimated to be 0.22±0.07 µM, and the threshold area under the cocaine concentration versus time curve (AUC) value in brain (denoted by AUC2∞) required to produce physiological effects has been estimated to be 7.9±2.7 µM·min. It has been demonstrated that administration of a cocaine hydrolase/esterase (CocH/CocE) can considerably decrease the cocaine half-lives in both brain and plasma, the peak cocaine concentration in brain, and the AUC2∞. The estimated maximum cocaine plasma concentration which a given concentration of drug-metabolizing enzyme can effectively prevent from entering brain and producing physiological effects can be used to guide future preclinical/clinical studies on cocaine-metabolizing enzymes. Understanding of drug-metabolizing enzymes is key to the science of pharmacokinetics. The general insights into the effects of a drug-metabolizing enzyme on drug kinetics in human should be valuable also in future development of enzyme therapies for other drugs of abuse. PMID:22844238

  10. Anti-BlyS antibody reduces the immune reaction against enzyme and enhances the efficacy of enzyme replacement therapy in Fabry disease model mice.

    PubMed

    Sato, Yohei; Ida, Hiroyuki; Ohashi, Toya

    2017-02-02

    Formation of antibodies against a therapeutic enzyme is an important complication during enzyme replacement therapy (ERT) for lysosomal storage diseases. Fabry disease (FD) is caused by a deficiency of alpha-galactosidase (GLA), which results in the accumulation of globotriaosylceramide (GL-3). We have shown immune tolerance induction (ITI) during ERT in FD model mice by using an anti-B lymphocyte stimulator (anti-BlyS) antibody (belimumab). A single dose of the anti-BlyS antibody temporarily lowered the percentage of B cells and IgG antibody titer against recombinant human GLA. Administration of a low maintenance dose of the anti-BlyS antibody suppressed the B cell population and immunotolerance was induced in 20% of mice, but antibody formation could not be prevented. We then increased the maintenance dose of the anti-BlyS antibody and immunotolerance was induced in 50% of mice. Therapeutic enzyme distribution and clearance of GL-3 were also enhanced by a high maintenance dose of the anti-BlyS antibody.

  11. Respiratory muscle training with enzyme replacement therapy improves muscle strength in late - onset Pompe disease.

    PubMed

    Mitja, Jevnikar; Metka, Kodric; Fabiana, Cantarutti; Cifaldi, Rossella; Longo, Cinzia; Rossana, Della Porta; Bruno, Bembi; Marco, Confalonieri

    2015-12-01

    Pompe disease is an autosomal recessive metabolic disorder caused by the deficiency of the lysosomal enzyme acid α-glucosidase. This deficiency leads to glycogen accumulation in the lysosomes of muscle tissue causing progressive muscular weakness particularly of the respiratory system. Enzyme replacement therapy (ERT) has demonstrated efficacy in slowing down disease progression in infants. Despite the large number of studies describing the effects of physical training in juvenile and adult late onset Pompe disease (LOPD). There are very few reports that analyze the benefits of respiratory muscle rehabilitation or training. The effectiveness of respiratory muscle training was investigated using a specific appliance with adjustable resistance (Threshold). The primary endpoint was effect on respiratory muscular strength by measurements of MIP and MEP. Eight late-onset Pompe patients (aged 13 to 58 years; 4 female, 4 male) with respiratory muscle deficiency on functional respiratory tests were studied. All patients received ERT at the dosage of 20 mg/kg/every 2 weeks and underwent training with Threshold at specified pressures for 24 months. A significant increase in MIP was observed during the follow-up of 24 month: 39.6 cm H2O (+ 25.0%) at month 3; 39.5 cm H2O (+ 24.9%) at month 6; 39.1 cm H2O (+ 23.7%) at month 9; 37.3 cm H2O (+ 18.2%) at month 12; and 37.3 cm H2O (+ 17.8%) at month 24. Median MEP values also showed a significant increase during the first 9 months: 29.8 cm H2O, (+ 14.3%) at month 3; 31.0 cm H2O (+ 18.6) at month 6; and 29.5 cm H2O (+ 12.9) at month 9. MEP was then shown to be decreased at months 12 and 24; median MEP was 27.2 cm H2O (+ 4.3%) at 12 months and 26.6 cm H2O (+ 1.9%) at 24 months. The FVC remain stable throughout the study. An increase in respiratory muscular strength was demonstrated with Threshold training when used in combination with ERT.

  12. Enzyme replacement therapy for Fabry disease: a systematic review of available evidence.

    PubMed

    Schaefer, Roland M; Tylki-Szymańska, Anna; Hilz, Max J

    2009-11-12

    Fabry disease is a progressive and life-threatening glycolipid storage disorder affecting both males and females. The primary driver of the disease is the accumulation of glycolipids (globotriaosylceramide [GL-3]) in a variety of cell types, including vascular endothelial cells, a range of renal cell types, cardiomyocytes and neurons, which is caused by deficient activity of the lysosomal enzyme, alpha-galactosidase. The disease typically presents during childhood or adolescence. First manifestations reflect involvement of small nerve fibres of the peripheral and autonomic nervous systems. With age, severe complications involving the kidneys, heart and brain cause considerable morbidity and premature death. Outside the US, enzyme replacement therapy (ERT) with agalsidase alfa 0.2 mg/kg every other week (EOW) and agalsidase beta 1.0 mg/kg EOW is available for the treatment of patients with Fabry disease, while agalsidase beta 1.0 mg/kg EOW is the only approved drug in the US. To analyse the evidence for ERT, a systematic review of the literature was performed to identify prospectively designed randomized, controlled trials (RCTs) and open-label studies on the efficacy of agalsidase alfa and agalsidase beta. MEDLINE and EMBASE databases were searched; inclusion criteria for the systematic review were prospectively designed clinical studies evaluating ERT with quantifiable endpoints: double-blind and open-label studies were eligible. Exclusion criteria were review articles, case reports, case studies, letters to the editor and articles based on registry data (Fabry Outcome Survey or Fabry Registry). In addition, any studies with a retrospective design or data based on post hoc analyses were excluded. The evidence was reviewed with respect to the clinical benefits of ERT at the level of the end organ. A total of 9 RCTs and 23 open-label studies were identified for inclusion. The efficacy of ERT in Fabry disease has been measured against a variety of endpoints, the

  13. Design, Synthesis, and Evaluation of Prodrugs of Ertapenem

    PubMed Central

    2013-01-01

    Carbapenems are intravenous lifesaving hospital antibiotics. Once patients leave the hospital, they are sent home with antibiotics other than carbapenems since they cannot be administered orally due to lack of oral absorption primarily because of very highly polarity. A prodrug approach is a bona fide strategy to improve oral absorption of compounds. Design and synthesis, in vitro and in vivo evaluation of diversified prodrugs of ertapenem, one of the only once daily dosed carbapenems is described. Many of the prodrugs prepared for evaluation are rapidly hydrolyzed in rat plasma. Only bis-(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (medoxomil) ester prodrug was rapidly hydrolyzed in most of the plasmas including rat, human, dog, and monkey. Although the rate of conversion of ertapenem diethyl ester prodrug (6) was slow in in vitro plasma hydrolysis, it showed the best in vivo pharmacokinetic profile in dog by an intraduodenal dosing giving >31% total oral absorption. PMID:24900737

  14. Evaluation of Antimalarial Activity and Toxicity of a New Primaquine Prodrug

    PubMed Central

    Davanço, Marcelo Gomes; Aguiar, Anna Caroline Campos; dos Santos, Leandro Alves; Padilha, Elias Carvalho; Campos, Michel Leandro; de Andrade, Cleverton Roberto; da Fonseca, Luiz Marcos; dos Santos, Jean Leandro; Chin, Chung Man; Krettli, Antoniana Ursine; Peccinini, Rosangela Gonçalves

    2014-01-01

    Plasmodium vivax is the most prevalent of the five species causing malaria in humans. The current available treatment for P. vivax malaria is limited and unsatisfactory due to at least two drawbacks: the undesirable side effects of primaquine (PQ) and drug resistance to chloroquine. Phenylalanine-alanine-PQ (Phe-Ala-PQ) is a PQ prodrug with a more favorable pharmacokinetic profile compared to PQ. The toxicity of this prodrug was evaluated in in vitro assays using a human hepatoma cell line (HepG2), a monkey kidney cell line (BGM), and human red blood cells deficient in the enzyme glucose-6-phosphate-dehydrogenase (G6PD). In addition, in vivo toxicity assays were performed with rats that received multiple doses of Phe-Ala-PQ to evaluate biochemical, hematological, and histopathological parameters. The activity was assessed by the inhibition of the sporogonic cycle using a chicken malaria parasite. Phe-Ala-PQ blocked malaria transmission in Aedes mosquitoes. When compared with PQ, it was less cytotoxic to BGM and HepG2 cells and caused less hemolysis of G6PD-deficient red blood cells at similar concentrations. The prodrug caused less alteration in the biochemical parameters than did PQ. Histopathological analysis of the liver and kidney did show differences between the control and Phe-Ala-PQ-treated groups, but they were not statistically significant. Taken together, the results highlight the prodrug as a novel lead compound candidate for the treatment of P. vivax malaria and as a blocker of malaria transmission. PMID:25133630

  15. Evaluation of antimalarial activity and toxicity of a new primaquine prodrug.

    PubMed

    Davanço, Marcelo Gomes; Aguiar, Anna Caroline Campos; Dos Santos, Leandro Alves; Padilha, Elias Carvalho; Campos, Michel Leandro; de Andrade, Cleverton Roberto; da Fonseca, Luiz Marcos; Dos Santos, Jean Leandro; Chin, Chung Man; Krettli, Antoniana Ursine; Peccinini, Rosangela Gonçalves

    2014-01-01

    Plasmodium vivax is the most prevalent of the five species causing malaria in humans. The current available treatment for P. vivax malaria is limited and unsatisfactory due to at least two drawbacks: the undesirable side effects of primaquine (PQ) and drug resistance to chloroquine. Phenylalanine-alanine-PQ (Phe-Ala-PQ) is a PQ prodrug with a more favorable pharmacokinetic profile compared to PQ. The toxicity of this prodrug was evaluated in in vitro assays using a human hepatoma cell line (HepG2), a monkey kidney cell line (BGM), and human red blood cells deficient in the enzyme glucose-6-phosphate-dehydrogenase (G6PD). In addition, in vivo toxicity assays were performed with rats that received multiple doses of Phe-Ala-PQ to evaluate biochemical, hematological, and histopathological parameters. The activity was assessed by the inhibition of the sporogonic cycle using a chicken malaria parasite. Phe-Ala-PQ blocked malaria transmission in Aedes mosquitoes. When compared with PQ, it was less cytotoxic to BGM and HepG2 cells and caused less hemolysis of G6PD-deficient red blood cells at similar concentrations. The prodrug caused less alteration in the biochemical parameters than did PQ. Histopathological analysis of the liver and kidney did show differences between the control and Phe-Ala-PQ-treated groups, but they were not statistically significant. Taken together, the results highlight the prodrug as a novel lead compound candidate for the treatment of P. vivax malaria and as a blocker of malaria transmission.

  16. Dual delivery systems based on polyamine analog BENSpm as prodrug and gene delivery vectors

    NASA Astrophysics Data System (ADS)

    Zhu, Yu

    Combination drug and gene therapy shows promise in cancer treatment. However, the success of such strategy requires careful selection of the therapeutic agents, as well as development of efficient delivery vectors. BENSpm (N 1, N11-bisethylnorspermine), a polyamine analogue targeting the intracellular polyamine pathway, draws our special attention because of the following reasons: (1) polyamine pathway is frequently dysregulated in cancer; (2) BENSpm exhibits multiple functions to interfere with the polyamine pathway, such as to up-regulate polyamine metabolism enzymes and down-regulate polyamine biosynthesis enzymes. Therefore BENSpm depletes all natural polyamines and leads to apoptosis and cell growth inhibition in a wide range of cancers; (3) preclinical studies proved that BENSpm can act synergistically with various chemotherapy agents, making it a promising candidate in combination therapy; (4) multiple positive charges in BENSpm enable it as a suitable building block for cationic polymers, which can be further applied to gene delivery. In this dissertation, our goal was to design dual-function delivery vector based on BENSpm that can function as a gene delivery vector and, after intracellular degradation, as an active anticancer agent targeting dysregulated polyamine metabolism. We first demonstrated strong synergism between BENSpm and a potential therapeutic gene product TRAIL. Strong synergism was obtained in both estrogen-dependent MCF-7 breast cancer cells and triple-negative MDA-MB-231 breast cancer cells. Significant dose reduction of TRAIL in combination with BENSpm in MDA-MB-231 cells, together with the fact that BENSpm rendered MCF-7 cells more sensitive to TRAIL treatment verified our rationale of designing BENSpm-based delivery platform. This was expected to be beneficial for overcoming drug resistance in chemotherapy, as well as boosting the therapeutic effect of therapeutic genes. We first designed a lipid-based BENSpm dual vector (Lipo

  17. Partial resolution of bone lesions. A child with severe combined immunodeficiency disease and adenosine deaminase deficiency after enzyme-replacement therapy

    SciTech Connect

    Yulish, B.S.; Stern, R.C.; Polmar, S.H.

    1980-01-01

    A child with severe combined immunodeficiency disease and adenosine deaminase deficiency, with characteristic bone dysplasia, was treated with transfusions of frozen irradiated RBCs as a means of enzyme replacement. This therapy resulted in restoration of immunologic competence and partial resolution of the bone lesions. Although the natural history of these lesions without therapy is not known, enzyme-replacement therapy may have played a role in the resolution of this patient's bone lesions.

  18. COMPARING THE ENZYME REPLACEMENT THERAPY COST IN POST PANCREATECTOMY PATIENTS DUE TO PANCREATIC TUMOR AND CHRONIC PANCREATITIS.

    PubMed

    Fragoso, Anna Victoria; Pedroso, Martha Regina; Herman, Paulo; Montagnini, André Luis

    2016-01-01

    Among late postoperative complications of pancreatectomy are the exocrine and endocrine pancreatic insufficiencies. The presence of exocrine pancreatic insufficiency imposes, as standard treatment, pancreatic enzyme replacement. Patients with chronic pancreatitis, with intractable pain or any complications with surgical treatment, are likely to present exocrine pancreatic insufficiency or have this condition worsened requiring adequate dose of pancreatic enzymes. The aim of this study is to compare the required dose of pancreatic enzyme and the enzyme replacement cost in post pancreatectomy patients with and without chronic pancreatitis. Observational cross-sectional study. In the first half of 2015 patients treated at the clinic of the Department of Gastrointestinal Surgery at Hospital das Clínicas, Universidade de São Paulo, Brazil, who underwent pancreatectomy for at least 6 months and in use of enzyme replacement therapy were included in this series. The study was approved by the Research Ethics Committee. The patients were divided into two groups according to the presence or absence of chronic pancreatitis prior to pancreatic surgery. For this study, P<0.05 was considered statistically significant. The annual cost of the treatment was R$ 2150.5 ± 729.39; R$ 2118.18 ± 731.02 in patients without pancreatitis and R$ 2217.74 ± 736.30 in patients with pancreatitis. There was no statistically significant difference in the cost of treatment of enzyme replacement post pancreatectomy in patients with or without chronic pancreatitis prior to surgical indication.

  19. Development of Targeted, Enzyme-Activated Nano-Conjugates for Hepatic Cancer Therapy

    NASA Astrophysics Data System (ADS)

    Kuruvilla, Sibu Philip

    Hepatocellular carcinoma (HCC) is the 5th most commonly-occurring cancer worldwide and the 2nd highest cause for cancer-related deaths globally. The current treatment strategy is the direct injection of a chemotherapeutic agent (e.g. doxorubicin; DOX) into the hepatic artery, through a process called hepatic arterial infusion (HAI). Unfortunately, HAI is severely hindered by limited therapeutic efficacy against the tumor and high systemic toxicity to surrounding organs (e.g. cardiotoxicity). This thesis focuses on the development of a targeted, nanoparticle-based drug delivery system aimed to improve the clinical treatment of HCC. In particular, we employ generation 5 (G5) poly(amido amine) (PAMAM) dendrimers targeted to hepatic cancer cells via N-acetylgalactosamine (NAcGal) ligands attached to the surface through a poly(ethylene glycol) (PEG) brush. DOX is attached to the G5 surface through two different enzyme-sensitive linkages, L3 or L4, to achieve controllable release of the drug inside hepatic cancer cells. The combination of NAcGal-PEG targeting branches with either L3- or L4-DOX linkages led to the development of P1 and P2 particles, respectively. In Part 1, we discuss the development of these particles and measure their ability to target and kill hepatic cancer cells in vitro. In Part 2, we investigate the antitumor activity of P1 and P2 particles in tumor-bearing mice in comparison to the free drug, and we measure the cardiac function of mice undergoing treatment to assess differences in DOX-induced cardiotoxicity. Finally, in Part 3, we explore multi-valent targeting of G5 dendrimers in pursuit of further improving their specificity to hepatic cancer cells. Ultimately, this thesis provides insight into the utility of nanoparticle-based drug delivery systems that can potentially be translated to the clinic to improve cancer therapy.

  20. The right ventricle in Fabry disease: natural history and impact of enzyme replacement therapy.

    PubMed

    Niemann, Markus; Breunig, Frank; Beer, Meinrad; Herrmann, Sebastian; Strotmann, Jörg; Hu, Kai; Emmert, Andrea; Voelker, Wolfram; Ertl, Georg; Wanner, Christoph; Weidemann, Frank

    2010-12-01

    Storage of globotriaosylceramides is present in the left and right ventricles of patients with Fabry disease. Improvement of left ventricular morphology and function during enzyme replacement therapy (ERT) has previously been reported. To analyse the effects of long term ERT on right ventricular morphology and function. This was a prospective follow-up of 75 genetically confirmed consecutive Fabry patients for 3.1±1.8 years. According to treatment guidelines the natural history was followed in 18 patients, whereas 57 patients received ERT. Standard echocardiography, strain rate imaging for regional deformation of the right and left ventricle, and magnetic resonance tomography with late enhancement (LE) imaging for the detection of fibrosis were all performed at yearly intervals. Right ventricular hypertrophy was evident in 53 patients (71%) at baseline. A significant positive correlation was found between left and right ventricular wall thickness (r=0.76; p<0.0001). LE was detected in half of the patients (n=38) in the left ventricle at baseline while no patient ever had LE of the right ventricle. Patients with LE in the left ventricle presented with the lowest right ventricular deformation properties. In contrast to the left ventricle, there was no change in right ventricular wall thickness (baseline 6.9±1.6 mm vs follow-up 6.7±1.5 mm; p=0.44) or systolic strain rate (2.2±0.7/s vs 2.1±0.8/s; p=0.31) during follow-up with ERT. The degree of right ventricular involvement in Fabry disease is related to the left ventricular cardiomyopathy stage. ERT seems to have no direct impact on right ventricular morphology and function.

  1. Effectiveness of enzyme replacement therapy in Fabry disease: Long term experience in Argentina.

    PubMed

    Cabrera, Gustavo; Politei, Juan; Antongiovani, Norberto; Amartino, Hernán

    2017-06-01

    Evidence regarding long term effectiveness of enzyme replacement therapy (ERT) in Fabry disease (FD) is needed. The aim of this study was to analyze in a cohort of FD patients in Argentina, the long term effectiveness of ERT on renal, cardiac and cerebrovascular parameters. Patients with genetically proven FD were included from GADYTEF (Argentinean group for the treatment of FD) between 2001 and 2014. Renal, cardiac, and cerebral outcomes were prospectively studied in patients treated with ERT. Additionally, the occurrence of major cardiac complications, stroke, end-stage renal disease and death was analyzed during follow up. During the follow-up 8 major complications occurred in 5 patients (n = 2 deaths, n = 4 cases of end stage renal disease and n = 1 atrial fibrillation), 4 of them males and only 1 female who suffered an atrial fibrillation. Sudden death or stroke did not occur. Four (40%) of 10 males with baseline left ventricular hypertrophy (LVH) reduced left ventricular mass index (LVMI) from 163.1 ± 64.7 to 123.4 ± 49.8 g/m(2), 2 stabilized LVMI and 4 increased LVMI from157.9 ± 32.3 to 261.6 ± 48.6 g/m(2). Estimated glomerular filtration was stable in 30 patients (17 males and 13 females). We observed a few major complications during the follow up. Future studies are necessary to show the effectiveness of ERT in affected patients.

  2. Pregnancy is associated with elevation of liver enzymes in HIV-positive women on antiretroviral therapy.

    PubMed

    Huntington, Susie; Thorne, Claire; Newell, Marie-Louise; Anderson, Jane; Taylor, Graham P; Pillay, Deenan; Hill, Teresa; Tookey, Pat A; Sabin, Caroline

    2015-04-24

    The objective of this study is to assess whether pregnancy is associated with an increased risk of liver enzyme elevation (LEE) and severe LEE in HIV-positive women on antiretroviral therapy (ART). Two observational studies: the UK Collaborative HIV Cohort (UK CHIC) study and the UK and Ireland National Study of HIV in Pregnancy and Childhood (NSHPC). Combined data from UK CHIC and NSHPC were used to identify factors associated with LEE (grade 1-4) and severe LEE (grade 3-4). Women starting ART in 2000-2012 were included irrespective of pregnancy status. Cox proportional hazards were used to assess fixed and time-dependent covariates including pregnancy status, CD4 cell count, drug regimen and hepatitis B virus/hepatitis C virus (HBV/HCV) coinfection. One-quarter (25.7%, 982/3815) of women were pregnant during follow-up, 14.2% (n = 541) when starting ART. The rate of LEE was 14.5/100 person-years in and 6.0/100 person-years outside of pregnancy. The rate of severe LEE was 3.9/100 person-years in and 0.6/100 person-years outside of pregnancy. The risk of LEE and severe LEE was increased during pregnancy [LEE: adjusted hazard ratio (aHR) 1.66 (1.31-2.09); severe LEE: aHR 3.57 (2.30-5.54)], including in secondary analyses excluding 541 women pregnant when starting ART. Other factors associated with LEE and severe LEE included lower CD4 cell count (<250 cells/μl), HBV/HCV coinfection and calendar year. Although few women developed severe LEE, this study provides further evidence that pregnancy is associated with an increased risk of LEE and severe LEE, reinforcing the need for regular monitoring of liver biomarkers during pregnancy.

  3. A phase I/II clinical trial of enzyme replacement therapy in mucopolysaccharidosis II (Hunter syndrome).

    PubMed

    Muenzer, Joseph; Gucsavas-Calikoglu, Muge; McCandless, Shawn E; Schuetz, Thomas J; Kimura, Alan

    2007-03-01

    To evaluate the safety and explore the efficacy of idursulfase (recombinant human iduronate-2-sulfatase) treatment for mucopolysaccharidosis II (MPS II). Twelve patients were enrolled into a randomized, double-blind, placebo-controlled trial for 24 weeks followed by an open-label extension study. Three groups of 4 patients were enrolled sequentially, with 3 patients in each group receiving idursulfase and 1 patient receiving placebo. The first group received idursulfase at 0.15 mg/kg infused every other week with the 2nd and 3rd groups receiving 0.5 and 1.5 mg/kg, respectively. After 24 weeks the placebo-treated patients were changed to idursulfase at the dose of their group. The primary endpoint was a change from baseline in urinary excretion of glycosaminoglycans. Results were pooled for analysis by ANOVA and compared to baseline. Urinary glycosaminoglycans were reduced within 2 weeks of initiating idursulfase and were decreased 49% after 48 weeks of treatment (P<0.0001). Both liver and spleen volume were decreased at 24 weeks (P<0.01) and 48 weeks (P<0.001). The 6-minute walk test distance increased an average of 48 meters after 48 weeks (P=0.013). Six patients in the higher dose groups developed IgG antibodies that did not influence the clinical effects of idursulfase. This study describes the first experience with enzyme replacement therapy for the treatment of patients with MPS II. Idursulfase was generally well tolerated and was associated with reductions in urine glycosaminoglycans levels and organ size, as well as an increased 6-minute walk test distance.

  4. Taliglucerase alfa: an enzyme replacement therapy using plant cell expression technology.

    PubMed

    Grabowski, Gregory A; Golembo, Myriam; Shaaltiel, Yoseph

    2014-05-01

    Gaucher disease (GD) is a rare, genetic lysosomal storage disorder caused by functional defects of acid β-glucosidase that results in multiple organ dysfunction. Glycosylation of recombinant acid human β-glucosidase and exposure of terminal mannose residues are critical to the success of enzyme replacement therapy (ERT) for the treatment of visceral and hematologic manifestations in GD. Three commercially available ERT products for treatment of GD type 1 (GD1) include imiglucerase, velaglucerase alfa, and taliglucerase alfa. Imiglucerase and velaglucerase alfa are produced in different mammalian cell systems and require production glycosylation modifications to expose terminal α-mannose residues, which are needed for mannose receptor-mediated uptake by target macrophages. Such modifications add to production costs. Taliglucerase alfa is a plant cell-expressed acid β-glucosidase approved in the United States and other countries for ERT in adults with GD1. A plant-based expression system, using carrot root cell cultures, was developed for production of taliglucerase alfa and does not require additional processing for postproduction glycosidic modifications. Clinical trials have demonstrated that taliglucerase alfa is efficacious, with a well-established safety profile in adult, ERT-naïve patients with symptomatic GD1, and for such patients previously treated with imiglucerase. These included significant improvements in organomegaly and hematologic parameters as early as 6months, and maintenance of achieved therapeutic values in previously treated patients. Ongoing clinical trials will further characterize the long-term efficacy and safety of taliglucerase alfa in more diverse patient populations, and may help to guide clinical decisions for achieving optimal outcomes for patients with GD1.

  5. Pregnancy is associated with elevation of liver enzymes in HIV-positive women on antiretroviral therapy

    PubMed Central

    HUNTINGTON, Susie; THORNE, Claire; NEWELL, Marie-Louise; ANDERSON, Jane; TAYLOR, Graham P; PILLAY, Deenan; HILL, Teresa; TOOKEY, Pat A; SABIN, Caroline

    2015-01-01

    Objective To assess whether pregnancy is associated with an increased risk of liver enzyme elevation (LEE) and severe LEE in HIV-positive women on antiretroviral therapy (ART). Design Two observational studies; the UK Collaborative HIV Cohort (UK CHIC) study and the UK and Ireland National Study of HIV in Pregnancy and Childhood (NSHPC). Methods Combined data from UK CHIC and NSHPC were used to identify factors associated with LEE (grade 1-4) and severe LEE (grade 3-4). Women starting ART in 2000-2012 were included irrespective of pregnancy status. Cox proportional hazards were used to assess fixed and time-dependent covariates including pregnancy status, CD4 count, drug regimen and hepatitis B/C (HBV/HCV) co-infection. Results One-quarter (25.7%, 982/3815) of women were pregnant during follow-up; 14.2% (n=541) when starting ART. The rate of LEE was 14.5/100 person-years (PY) in and 6.0/100 PY outside of pregnancy. The rate of severe LEE was 3.9/100 PY in and 0.6/100 PY outside of pregnancy. The risk of LEE and severe LEE was increased during pregnancy (LEE: aHR 1.66 [1.31-2.09]; severe LEE: aHR 3.57 [2.30-5.54]), including in secondary analyses excluding 541 women pregnant when starting ART. Other factors associated with LEE and severe LEE included lower CD4 count (<250 cells/mm3), HBV/HCV co-infection and calendar year. Conclusions Although few women developed severe LEE, this study provides further evidence that pregnancy is associated with increased risk of LEE and severe LEE, reinforcing the need for regular monitoring of liver biomarkers during pregnancy. PMID:25710412

  6. Light-emitting diode therapy reduces persistent inflammatory pain: Role of interleukin 10 and antioxidant enzymes.

    PubMed

    Martins, D F; Turnes, B L; Cidral-Filho, F J; Bobinski, F; Rosas, R F; Danielski, L G; Petronilho, F; Santos, A R S

    2016-06-02

    During the last decades, the use of light-emitting diode therapy (LEDT) has increased significantly for the treatment of wound healing, analgesia and inflammatory processes. Nevertheless, scientific data on the mechanisms responsible for the therapeutic effect of LEDT are still insufficient. Thus, this study investigated the analgesic, anti-inflammatory and anti-oxidative effect of LEDT in the model of chronic inflammatory hyperalgesia. Mice injected with Complete Freund's Adjuvant (CFA) underwent behavioral, i.e. mechanical and hot hyperalgesia; determination of cytokine levels (tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL-10), oxidative stress markers (protein carbonyls and thiobarbituric acid reactive species (TBARS)) and antioxidant enzymes (catalase (CAT) and superoxide dismutase (SOD)). Additionally, mice were pretreated with either naloxone or fucoidin and mechanical hyperalgesia was assessed. LEDT inhibited mechanical and thermal hyperalgesia induced by CFA injection. LEDT did not reduce paw edema, neither influenced the levels of TNF-α and IL1-β; although it increased the levels of IL-10. LEDT significantly prevented TBARS increase in both acute and chronic phases post-CFA injection; whereas protein carbonyl levels were reduced only in the acute phase. LEDT induced an increase in both SOD and CAT activity, with effects observable in the acute but not in the chronic. And finally, pre-administration of naloxone or fucoidin prevented LEDT analgesic effect. These data contribute to the understanding of the neurobiological mechanisms involved in the therapeutic effect of LEDT as well as provides additional support for its use in the treatment of painful conditions of inflammatory etiology. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  7. Expression of Carboxylesterase Isozymes and Their Role in the Behavior of a Fexofenadine Prodrug in Rat Skin.

    PubMed

    Imai, Teruko; Ariyoshi, Satomi; Ohura, Kayoko; Sawada, Takashi; Nakada, Yuichiro

    2016-02-01

    The expression of carboxylesterase (CES) and the transdermal movement of an ester prodrug were studied in rat skin. Ethyl-fexofenadine (ethyl-FXD) was used as a model lipophilic prodrug that is slowly hydrolyzed to its parent drug, FXD (MW 502). Among the CES1 and CES2 isozymes, Hydrolase A is predominant in rat skin and this enzyme was involved in 65% of the cutaneous hydrolysis of ethyl-FXD. The similarity of the permeation behavior of ethyl-FXD in full thickness and stripped skin indicated that the stratum corneum was not a barrier to penetration. However, only FXD was observed in receptor fluid, not ethyl-FXD, presumably because of the high degree of binding of ethyl-FXD in viable skin. The rate of hydrolysis of ethyl-FXD was much faster than steady-state flux, such that the influx rate was the rate-limiting process for transdermal permeation. Although Hydrolase A levels gradually increased in skin taken from rats aged from 8 to 90 weeks, variations in the expression levels of the esterase hardly affected the conversion of prodrug. The present data suggest that the slow hydrolysis of the prodrug of an active ingredient in viable skin followed by slow diffusion of active drug may provide a useful approach to topical application.

  8. The use of capecitabine in the combined-modality therapy for rectal cancer.

    PubMed

    Liauw, Stanley L; Minsky, Bruce D

    2008-03-01

    Locally advanced rectal adenocarcinoma is treated by combined-modality therapy, which consists of surgery, chemotherapy, and radiation therapy. A series of randomized trials established a preferred treatment sequence of preoperative radiation therapy and 5-fluorouracil(5-FU)-based chemotherapy, total mesorectal excision, and adjuvant 5-FU-based chemotherapy for patients with stage II/III disease. Capecitabine is an oral prodrug of 5-FU that has potential advantages compared with intravenous 5-FU, including ease of administration and potentially increased therapeutic effect. Capecitabine is converted by a 3-step enzymatic process; the last step involves the enzyme thymidine phosphorylase, which is overexpressed in tumor tissues and is stimulated by concurrent radiation therapy. Over the past 5 years, several phase I/II trials of capecitabine-based therapy were reported. This review discusses the evolution of combined-modality therapy for rectal cancer with specific attention given to the use of capecitabine in conjunction with radiation therapy.

  9. Retrospective study of long-term outcomes of enzyme replacement therapy in Fabry disease: Analysis of prognostic factors

    PubMed Central

    Biegstraaten, Marieke; Hughes, Derralynn A.; Mehta, Atul; Elliott, Perry M.; Oder, Daniel; Watkinson, Oliver T.; Vaz, Frédéric M.; van Kuilenburg, André B. P.; Wanner, Christoph; Hollak, Carla E. M.

    2017-01-01

    Despite enzyme replacement therapy, disease progression is observed in patients with Fabry disease. Identification of factors that predict disease progression is needed to refine guidelines on initiation and cessation of enzyme replacement therapy. To study the association of potential biochemical and clinical prognostic factors with the disease course (clinical events, progression of cardiac and renal disease) we retrospectively evaluated 293 treated patients from three international centers of excellence. As expected, age, sex and phenotype were important predictors of event rate. Clinical events before enzyme replacement therapy, cardiac mass and eGFR at baseline predicted an increased event rate. eGFR was the most important predictor: hazard ratios increased from 2 at eGFR <90 ml/min/1.73m2 to 4 at eGFR <30, compared to patients with an eGFR >90. In addition, men with classical disease and a baseline eGFR <60 ml/min/1.73m2 had a faster yearly decline (-2.0 ml/min/1.73m2) than those with a baseline eGFR of >60. Proteinuria was a further independent risk factor for decline in eGFR. Increased cardiac mass at baseline was associated with the most robust decrease in cardiac mass during treatment, while presence of cardiac fibrosis predicted a stronger increase in cardiac mass (3.36 gram/m2/year). Of other cardiovascular risk factors, hypertension significantly predicted the risk for clinical events. In conclusion, besides increasing age, male sex and classical phenotype, faster disease progression while on enzyme replacement therapy is predicted by renal function, proteinuria and to a lesser extent cardiac fibrosis and hypertension. PMID:28763515

  10. Photo-triggered fluorescent theranostic prodrugs as DNA alkylating agents for mechlorethamine release and spatiotemporal monitoring.

    PubMed

    Cao, Yanting; Pan, Rong; Xuan, Weimin; Wei, Yongyi; Liu, Kejian; Zhou, Jiahong; Wang, Wei

    2015-06-28

    We describe a new theranostic strategy for selective delivery and spatiotemporal monitoring of mechlorethamine, a DNA alkylating agent. A photo-responsive prodrug is designed and composed of a photolabile o-nitrophenylethyl group, a DNA alkylating mechlorethamine drug and a coumarin fluorophore. Masking of the "N" in mechlorethamine in a positively charged state in the prodrug renders it inactive, non-toxic, selective and non-fluorescent. Indeed, the stable prodrug shows negligible cytotoxicity towards normal cells with and without UV activation and is completely non-fluorescent. However, upon photo-irradiation, the active mechlorethamine is released and induces efficient DNA cross-links, accompanied by a strong fluorescence enhancement (152 fold). Furthermore, DNA cross-linking activity from the release can be transformed into anticancer activity observed in in vitro studies of tumor cells. Importantly, the drug release progress and the movement can be conveniently monitored by fluorescence spectroscopy. The mechanistic study proves that the DNA cross-linking activity is mainly due to the release of DNA alkylating mechlorethamine. Altogether, the studies show the power of the theranostic strategy for efficient therapy in cancer treatment.

  11. A Prodrug-doped Cellular Trojan Horse for the Potential Treatment of Prostate Cancer

    PubMed Central

    Levy, Oren; Brennen, W. Nathaniel; Han, Edward; Rosen, David Marc; Musabeyezu, Juliet; Safaee, Helia; Ranganath, Sudhir; Ngai, Jessica; Heinelt, Martina; Milton, Yuka; Wang, Hao; Bhagchandani, Sachin H.; Joshi, Nitin; Bhowmick, Neil; Denmeade, Samuel R.; Isaacs, John T.; Karp, Jeffrey M.

    2016-01-01

    Despite considerable advances in prostate cancer research, there is a major need for a systemic delivery platform that efficiently targets anti-cancer drugs to sites of disseminated prostate cancer while minimizing host toxicity. In this proof-of-principle study, human mesenchymal stem cells (MSCs) were loaded with poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) that encapsulate the macromolecule G114, a thapsigargin-based prostate specific antigen (PSA)-activated prodrug . G114-particles (~950nm in size) were internalized by MSCs, followed by the release of G114 as an intact prodrug from loaded cells. Moreover, G114 released from G114 MP-loaded MSCs selectively induced death of the PSA-secreting PCa cell line, LNCaP. Finally, G114 MP-loaded MSCs inhibited tumor growth when used in proof-of-concept co-inoculation studies with CWR22 PCa xenografts, suggesting that cell-based delivery of G114 did not compromise the potency of this pro-drug in-vitro or in-vivo. This study demonstrates a potentially promising approach to assemble a cell-based drug delivery platform, which inhibits cancer growth in-vivo without the need of genetic engineering. We envision that upon achieving efficient homing of systemically infused MSCs to cancer sites, this MSC-based platform may be developed into an effective, systemic ‘Trojan Horse’ therapy for targeted delivery of therapeutic agents to sites of metastatic PCa. PMID:27019026

  12. A prodrug-doped cellular Trojan Horse for the potential treatment of prostate cancer.

    PubMed

    Levy, Oren; Brennen, W Nathaniel; Han, Edward; Rosen, David Marc; Musabeyezu, Juliet; Safaee, Helia; Ranganath, Sudhir; Ngai, Jessica; Heinelt, Martina; Milton, Yuka; Wang, Hao; Bhagchandani, Sachin H; Joshi, Nitin; Bhowmick, Neil; Denmeade, Samuel R; Isaacs, John T; Karp, Jeffrey M

    2016-06-01

    Despite considerable advances in prostate cancer research, there is a major need for a systemic delivery platform that efficiently targets anti-cancer drugs to sites of disseminated prostate cancer while minimizing host toxicity. In this proof-of-principle study, human mesenchymal stem cells (MSCs) were loaded with poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) that encapsulate the macromolecule G114, a thapsigargin-based prostate specific antigen (PSA)-activated prodrug. G114-particles (∼950 nm in size) were internalized by MSCs, followed by the release of G114 as an intact prodrug from loaded cells. Moreover, G114 released from G114 MP-loaded MSCs selectively induced death of the PSA-secreting PCa cell line, LNCaP. Finally, G114 MP-loaded MSCs inhibited tumor growth when used in proof-of-concept co-inoculation studies with CWR22 PCa xenografts, suggesting that cell-based delivery of G114 did not compromise the potency of this pro-drug in-vitro or in-vivo. This study demonstrates a potentially promising approach to assemble a cell-based drug delivery platform, which inhibits cancer growth in-vivo without the need of genetic engineering. We envision that upon achieving efficient homing of systemically infused MSCs to cancer sites, this MSC-based platform may be developed into an effective, systemic 'Trojan Horse' therapy for targeted delivery of therapeutic agents to sites of metastatic PCa. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Studies on synthesis, stability, release and pharmacodynamic profile of a novel diacerein-thymol prodrug.

    PubMed

    Dhaneshwar, Suneela; Patel, Vriha; Patil, Dipmala; Meena, Gourav

    2013-01-01

    Involvement of oxidative stress, leading to chondrocyte senescence and cartilage ageing has been implicated in the pathogenesis of osteoarthritis (OA). New efforts to prevent the development and progression of OA include strategies and interventions aimed at reducing oxidative damage in articular cartilage using antioxidants as adjuncts to conservative therapy. Diacerein is an anthraquinone derivative with a marked disease modifying effect on OA owing to IL-1 β inhibition. In the present work an attempt was made at design and development of a co-drug of diacerein with antioxidant thymol. Structural elucidation was carried out by spectral analysis. When release kinetics of prodrug was studied in phosphate buffer (pH 7.4) and small intestinal homogenates of rats, 91% and 94% diacerein was available respectively at the end of 4.5 h. Chemical linkage of thymol with diacerein improved its lipophilicity and hence bioavailability. Screening of prodrug in Freud's adjuvant-induced arthritis and ulcerogenic potential by Rainsford's cold stress model exhibited significant reduction in paw volume, joint diameter and ulcer index with superior anti-inflammatory/anti-arthritic activities than the standards. Results of histopathology of tibio-tarsal joint indicated that animals treated with diacerein exhibited moderate synovitis while thymol and physical mixture-treated animals showed mild synovitis. Interestingly in prodrug-treated animals synovitis was not observed. The results of this study underline the promising potential of co-drug of diacerein and thymol in the management of OA.

  14. ELQ-300 prodrugs for enhanced delivery and single-dose cure of malaria.

    PubMed

    Miley, Galen P; Pou, Sovitj; Winter, Rolf; Nilsen, Aaron; Li, Yuexin; Kelly, Jane X; Stickles, Allison M; Mather, Michael W; Forquer, Isaac P; Pershing, April M; White, Karen; Shackleford, David; Saunders, Jessica; Chen, Gong; Ting, Li-Min; Kim, Kami; Zakharov, Lev N; Donini, Cristina; Burrows, Jeremy N; Vaidya, Akhil B; Charman, Susan A; Riscoe, Michael K

    2015-09-01

    ELQ-300 is a preclinical candidate that targets the liver and blood stages of Plasmodium falciparum, as well as the forms that are crucial to transmission of disease: gametocytes, zygotes, and ookinetes. A significant obstacle to the clinical development of ELQ-300 is related to its physicochemical properties. Its relatively poor aqueous solubility and high crystallinity limit absorption to the degree that only low blood concentrations can be achieved following oral dosing. While these low blood concentrations are sufficient for therapy, the levels are too low to establish an acceptable safety margin required by regulatory agencies for clinical development. One way to address the challenging physicochemical properties of ELQ-300 is through the development of prodrugs. Here, we profile ELQ-337, a bioreversible O-linked carbonate ester prodrug of the parent molecule. At the molar equivalent dose of 3 mg/kg of body weight, the delivery of ELQ-300 from ELQ-337 is enhanced by 3- to 4-fold, reaching a maximum concentration of drug in serum (C max) of 5.9 μM by 6 h after oral administration, and unlike ELQ-300 at any dose, ELQ-337 provides single-dose cures of patent malaria infections in mice at low-single-digit milligram per kilogram doses. Our findings show that the prodrug strategy represents a viable approach to overcome the physicochemical limitations of ELQ-300 to deliver the active drug to the bloodstream at concentrations sufficient for safety and toxicology studies, as well as achieving single-dose cures.

  15. A Distinct Urinary Biomarker Pattern Characteristic of Female Fabry Patients That Mirrors Response to Enzyme Replacement Therapy

    PubMed Central

    Kistler, Andreas D.; Siwy, Justyna; Breunig, Frank; Jeevaratnam, Praveen; Scherl, Alexander; Mullen, William; Warnock, David G.; Wanner, Christoph; Hughes, Derralynn A.; Mischak, Harald; Wüthrich, Rudolf P.; Serra, Andreas L.

    2011-01-01

    Female patients affected by Fabry disease, an X-linked lysosomal storage disorder, exhibit a wide spectrum of symptoms, which renders diagnosis, and treatment decisions challenging. No diagnostic test, other than sequencing of the alpha-galactosidase A gene, is available and no biomarker has been proven useful to screen for the disease, predict disease course and monitor response to enzyme replacement therapy. Here, we used urine proteomic analysis based on capillary electrophoresis coupled to mass spectrometry and identified a biomarker profile in adult female Fabry patients. Urine samples were taken from 35 treatment-naïve female Fabry patients and were compared to 89 age-matched healthy controls. We found a diagnostic biomarker pattern that exhibited 88.2% sensitivity and 97.8% specificity when tested in an independent validation cohort consisting of 17 treatment-naïve Fabry patients and 45 controls. The model remained highly specific when applied to additional control patients with a variety of other renal, metabolic and cardiovascular diseases. Several of the 64 identified diagnostic biomarkers showed correlations with measures of disease severity. Notably, most biomarkers responded to enzyme replacement therapy, and 8 of 11 treated patients scored negative for Fabry disease in the diagnostic model. In conclusion, we defined a urinary biomarker model that seems to be of diagnostic use for Fabry disease in female patients and may be used to monitor response to enzyme replacement therapy. PMID:21698285

  16. Methylselenol prodrug enhances MDV3100 efficacy for treatment of castration-resistant prostate cancer.

    PubMed

    Zhan, Yang; Cao, Bo; Qi, Yanfeng; Liu, Shuang; Zhang, Qi; Zhou, Weidong; Xu, Duo; Lu, Hua; Sartor, Oliver; Kong, Wei; Zhang, Haitao; Dong, Yan

    2013-11-01

    The next-generation antiandrogen MDV3100 prolongs overall survival of patients with metastatic castration-resistant prostate cancer (CRPC). However, patient responses are variable, and survival benefit remains relatively small. Developing effective modality to improve MDV3100 efficacy is urgently needed. Recent evidence suggests that constitutively active androgen receptor splice variants (AR-Vs) drive resistance to MDV3100. In our study, we show that methylselenol prodrug downregulates the expression and activity of both the full-length AR (AR-FL) and AR-Vs. The downregulation is independent of androgen and could be attributable to repressed transcription of the AR gene. Cotreatment with methylselenol prodrug and MDV3100 suppresses AR signaling more dramatically than either agent alone, and synergistically inhibits the growth of CRPC cells in vitro. The combinatorial efficacy is observed in not only AR-V-expressing cells but also cells expressing predominantly AR-FL, likely owing to the ability of the two drugs to block the AR signaling cascade at distinct steps. Ectopic expression of AR-FL or AR-V7 attenuates the combinatorial efficacy, indicating that downregulating AR-FL and AR-V7 is importantly involved in mediating the combinatorial efficacy. Significantly, methylselenol prodrug also downregulates AR-FL and AR-Vs in vivo and substantially improves the antitumor efficacy of MDV3100. These findings support a potential combination therapy for improving MDV3100 efficacy, and provide a rationale for evaluating the clinical application of combining methylselenol prodrug with MDV3100 for the treatment of CRPC. © 2013 UICC.

  17. Methylselenol prodrug enhances MDV3100 efficacy for treatment of castration-resistant prostate cancer

    PubMed Central

    Zhan, Yang; Cao, Bo; Qi, Yanfeng; Liu, Shuang; Zhang, Qi; Zhou, Weidong; Xu, Duo; Lu, Hua; Sartor, Oliver; Kong, Wei; Zhang, Haitao; Dong, Yan

    2013-01-01

    The next-generation antiandrogen MDV3100 prolongs overall survival of patients with metastatic castration-resistant prostate cancer (CRPC). However, patient responses are variable, and survival benefit remains relatively small. Developing effective modality to improve MDV3100 efficacy is urgently needed. Recent evidence suggests that constitutively active androgen receptor splice variants (AR-Vs) drive resistance to MDV3100. In our study, we show that methylselenol prodrug downregulates the expression and activity of both the full-length AR (AR-FL) and AR-Vs. The downregulation is independent of androgen and could be attributable to repressed transcription of the AR gene. Cotreatment with methylselenol prodrug and MDV3100 suppresses AR signaling more dramatically than either agent alone, and synergistically inhibits the growth of CRPC cells in vitro. The combinatorial efficacy is observed in not only AR-V-expressing cells but also cells expressing predominantly AR-FL, likely owing to the ability of the two drugs to block the AR signaling cascade at distinct steps. Ectopic expression of AR-FL or AR-V7 attenuates the combinatorial efficacy, indicating that downregulating AR-FL and AR-V7 is importantly involved in mediating the combinatorial efficacy. Significantly, methylselenol prodrug also downregulates AR-FL and AR-Vs in vivo and substantially improves the antitumor efficacy of MDV3100. These findings support a potential combination therapy for improving MDV3100 efficacy, and provide a rationale for evaluating the clinical application of combining methylselenol prodrug with MDV3100 for the treatment of CRPC. PMID:23575870

  18. Evaluation of miglustat as maintenance therapy after enzyme therapy in adults with stable type 1 Gaucher disease: a prospective, open-label non-inferiority study

    PubMed Central

    2012-01-01

    Background Previous studies have provided equivocal data on the use of miglustat as maintenance therapy in Gaucher disease type 1. We report findings from a clinical trial evaluating the effects of miglustat treatment in patients with stable type 1 Gaucher disease after enzyme therapy. Methods Adult type 1 Gaucher disease patients stabilized during at least 3 years of previous enzyme therapy were included in this 2-year, prospective, open-label non-inferiority study. The primary endpoint was percent change from baseline in liver volume. Secondary endpoints included changes in spleen volume, hemoglobin concentration and platelet count. Results Forty-two patients were enrolled (mean±SD age, 45.1±12.7 years; previous enzyme therapy duration 9.5±4.0 years). Median (range) exposure to miglustat 100 mg t.i.d. was 658 (3–765) days. Twenty-one patients discontinued treatment prematurely; 13 due to adverse events, principally gastrointestinal. The upper 95% confidence limit of mean percent change in liver volume from baseline to end of treatment was below the non-inferiority margin of 10% (–1.1%; 95%CI −6.0, 3.9%). Mean (95%CI) changes in spleen volume, hemoglobin concentration and platelet count were 102 (24,180) mL, –0.95 (−1.38, –0.53) g/dL and −44.1 (–57.6, –30.7) ×109/L, respectively. Conclusions The primary efficacy endpoint was met; overall there was no change in liver volume during 24 months of miglustat therapy. Several patients showed a gradual deterioration in some disease manifestations, suggesting that miglustat could maintain clinical stability, but not in all patients. Miglustat demonstrated a predictable profile of safety and tolerability that was consistent with that reported in previous clinical trials and experience in clinical practice. Trial registration Clinicaltrials.gov identifier NCT00319046 PMID:23270487

  19. Enhancement of Drug Delivery: Enzyme-replacement Therapy for Murine Morquio A Syndrome

    PubMed Central

    Tomatsu, Shunji; Montaño, Adriana M; Dung, Vu Chi; Ohashi, Amiko; Oikawa, Hirotaka; Oguma, Toshihiro; Orii, Tadao; Barrera, Luis; Sly, William S

    2010-01-01

    Mucopolysaccharidosis IVA (MPS IVA, Morquio A disease) is an inherited lysosomal storage disorder that features skeletal chondrodysplasia caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Human GALNS was bioengineered with the N-terminus extended by the hexaglutamate sequence (E6) to improve targeting to bone (E6-GALNS). We initially assessed blood clearance and tissue distribution. Next, to assess the effectiveness of storage clearance and reversal of pathological phenotype, a dose of 250 U/g of enzyme was given weekly to Morquio A mice (adults: 12 or 24 weeks, newborn: 8 weeks). Sulfatase modifier factor 1 (SUMF1) was co-transfected to activate the enzyme fully. The E6-GALNS tagged enzyme had markedly prolonged clearance from circulation, giving over 20 times exposure time in blood, compared to untagged enzyme. The tagged enzyme was retained longer in bone, with residual enzyme activity demonstrable at 48 hours after infusion. The pathological findings in adult mice treated with tagged enzyme showed substantial clearance of the storage materials in bone, bone marrow, and heart valves, especially after 24 weekly infusions. Mice treated from the newborn period showed marked reduction of storage materials in tissues investigated. These findings indicate the feasibility of using tagged enzyme to enhance delivery and pathological effectiveness in Morquio A mice. PMID:20332769

  20. Enhancement of drug delivery: enzyme-replacement therapy for murine Morquio A syndrome.

    PubMed

    Tomatsu, Shunji; Montaño, Adriana M; Dung, Vu Chi; Ohashi, Amiko; Oikawa, Hirotaka; Oguma, Toshihiro; Orii, Tadao; Barrera, Luis; Sly, William S

    2010-06-01

    Mucopolysaccharidosis IVA (MPS IVA, Morquio A disease) is an inherited lysosomal storage disorder that features skeletal chondrodysplasia caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Human GALNS was bioengineered with the N-terminus extended by the hexaglutamate sequence (E6) to improve targeting to bone (E6-GALNS). We initially assessed blood clearance and tissue distribution. Next, to assess the effectiveness of storage clearance and reversal of pathological phenotype, a dose of 250 U/g of enzyme was given weekly to Morquio A mice (adults: 12 or 24 weeks, newborn: 8 weeks). Sulfatase modifier factor 1 (SUMF1) was co-transfected to activate the enzyme fully. The E6-GALNS tagged enzyme had markedly prolonged clearance from circulation, giving over 20 times exposure time in blood, compared to untagged enzyme. The tagged enzyme was retained longer in bone, with residual enzyme activity demonstrable at 48 hours after infusion. The pathological findings in adult mice treated with tagged enzyme showed substantial clearance of the storage materials in bone, bone marrow, and heart valves, especially after 24 weekly infusions. Mice treated from the newborn period showed marked reduction of storage materials in tissues investigated. These findings indicate the feasibility of using tagged enzyme to enhance delivery and pathological effectiveness in Morquio A mice.

  1. Enhanced intracellular stability of dextran-horse radish peroxidase conjugate: an approach to enzyme replacement therapy.

    PubMed

    Mumtaz, S; Bachhawat, B K

    1992-09-15

    Horse radish peroxidase (HRP), a mannose-containing glycoprotein was covalently modified by conjugation with dextran. The rapid uptake of HRP by the liver is markedly inhibited by mannan. The uptake of dextran-HRP conjugate by the liver, though lower compared to that of the free enzyme, is also partially inhibited by mannan. Liposomes were therefore used as carriers for delivering the free and the modified HRP to the liver. The dextran-HRP conjugate showed greater stability intracellularly as compared to the free enzyme. The enhanced stability of enzymes upon their extensive glycosylation with nondegradable sugar polymers would be of importance in extending the catalytic life of therapeutically active enzymes and thereby improve their therapeutic potential for the treatment of certain enzyme deficiency disorders.

  2. Use of a Modified α-N-Acetylgalactosaminidase in the Development of Enzyme Replacement Therapy for Fabry Disease

    PubMed Central

    Tajima, Youichi; Kawashima, Ikuo; Tsukimura, Takahiro; Sugawara, Kanako; Kuroda, Mayuko; Suzuki, Toshihiro; Togawa, Tadayasu; Chiba, Yasunori; Jigami, Yoshifumi; Ohno, Kazuki; Fukushige, Tomoko; Kanekura, Takuro; Itoh, Kohji; Ohashi, Toya; Sakuraba, Hitoshi

    2009-01-01

    A modified α-N-acetylgalactosaminidase (NAGA) with α-galactosidase A (GLA)-like substrate specificity was designed on the basis of structural studies and was produced in Chinese hamster ovary cells. The enzyme acquired the ability to catalyze the degradation of 4-methylumbelliferyl-α-D-galactopyranoside. It retained the original NAGA's stability in plasma and N-glycans containing many mannose 6-phosphate (M6P) residues, which are advantageous for uptake by cells via M6P receptors. There was no immunological cross-reactivity between the modified NAGA and GLA, and the modified NAGA did not react to serum from a patient with Fabry disease recurrently treated with a recombinant GLA. The enzyme cleaved globotriaosylceramide (Gb3) accumulated in cultured fibroblasts from a patient with Fabry disease. Furthermore, like recombinant GLA proteins presently used for enzyme replacement therapy (ERT) for Fabry disease, the enzyme intravenously injected into Fabry model mice prevented Gb3 storage in the liver, kidneys, and heart and improved the pathological changes in these organs. Because this modified NAGA is hardly expected to cause an allergic reaction in Fabry disease patients, it is highly promising as a new and safe enzyme for ERT for Fabry disease. PMID:19853240

  3. Beta-lactamase targeted enzyme activatable photosensitizers for antimicrobial PDT

    NASA Astrophysics Data System (ADS)

    Zheng, Xiang; Verma, Sarika; Sallum, Ulysses W.; Hasan, Tayyaba

    2009-06-01

    Photodynamic therapy (PDT) as a treatment modality for infectious disease has shown promise. However, most of the antimicrobial photosensitizers (PS) non-preferentially accumulate in both bacteria and host tissues, causing host tissue phototoxicity during treatment. We have developed a new antimicrobial PDT strategy which exploits beta-lactam resistance mechanism, one of the major drug-resistance bacteria evolved, to achieve enhanced target specificity with limited host damage. Our strategy comprises a prodrug construct with a PS and a quencher linked by beta-lactam ring, resulting in a diminished phototoxicity. This construct, beta-lactamase enzyme-activated-photosensitizer (beta-LEAP), can only be activated in the presence of both light and bacteria, and remains inactive elsewhere such as mammalian tissue. Beta-LEAP construct had shown specific cleavage by purified beta-lactamase and by beta-lactamase over-expressing methicillin resistant Staphylococcus aureus (MRSA). Specific photodynamic toxicity was observed towards MRSA, while dark and light toxicity were equivalent to reference strains. The prodrug design, synthesis and photophysical properties will be discussed.

  4. [The Russian consensus on the diagnosis and treatment of chronic pancreatitis: Enzyme replacement therapy].

    PubMed

    Khatkov, I E; Maev, I V; Bordin, D S; Kucheryavyi, Yu A; Abdulkhakov, S R; Alekseenko, S A; Alieva, E I; Alikhanov, R B; Bakulin, I G; Baranovsky, A Yu; Beloborodova, E V; Belousova, E A; Buriev, I M; Bystrovskaya, E V; Vertyankin, S V; Vinokurova, L V; Galperin, E I; Gorelov, A V; Grinevich, V B; Danilov, M V; Darvin, V V; Dubtsova, E A; Dyuzheva, T G; Egorov, V I; Efanov, M G; Zakharova, N V; Zagainov, V E; Ivashkin, V T; Izrailov, R E; Korochanskaya, N V; Kornienko, E A; Korobka, V L; Kokhanenko, N Yu; Livzan, M A; Loranskaya, I D; Nikolskaya, K A; Osipenko, M F; Okhlobystin, A V; Pasechnikov, V D; Plotnikova, E Yu; Polyakova, S I; Sablin, O A; Simanenkov, V I; Ursova, N I; Tsvirkun, V V; Tsukanov, V V; Shabunin, A V

    2017-01-01

    Russian National Research Medical University, Ministry of Health of Russia, Moscow; 26A.M. Nikiforov All-Russian Center of Emergency and Radiation Medicine, Russian Ministry for Civil Defense, Emergencies and Elimination of Consequences of Natural Disasters, Saint Petersburg; 27Research Institute for Medical Problems of the North, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk; 28S.P. Botkin City Clinical Hospital, Moscow Healthcare Department, Moscow; 29Tver State Medical University, Ministry of Health of Russia, Tver The Russian consensus on the diagnosis and treatment of chronic pancreatitis has been prepared on the initiative of the Russian Pancreatology Club to clarify and consolidate the opinions of Russian specialists (gastroenterologists, surgeons, and pediatricians) on the most significant problems of diagnosis and treatment of chronic pancreatitis. This article continues a series of publications explaining the most significant interdisciplinary consensus statements and deals with enzyme replacement therapy.

  5. Cost-effectiveness of enzyme replacement therapy for type 1 Gaucher disease

    PubMed Central

    2014-01-01

    Objective To evaluate the cost-effectiveness of enzyme replacement therapy (ERT) compared to standard medical care without ERT in the Dutch cohort of patients with type 1 Gaucher disease (GD I). Design Cost-effectiveness analysis was performed using a life-time state-transition model of the disease’s natural course. Transition probabilities, effectiveness data and costs were derived from retrospective data and prospective follow-up of the Dutch study cohort. Setting The tertiary referral center for Gaucher disease in the Netherlands. Participants The Dutch cohort of patients with GD I. Intervention ERT versus standard medical care without ERT in symptomatic patients. Main outcome measures Years free of end organ damage (YFEOD) (splenectomy, bone complication, malignancy, multiple complications), quality adjusted life years (QALY), and costs. Results Over an 85 year lifetime, an untreated GD I patient will generate 48.9 YFEOD and 55.86 QALYs. Starting ERT in a symptomatic patient increases the YFEOD by 12.8 years, while the number of QALYs gained increases by 6.27. The average yearly ERT medication costs range between €124,000 and €258,000 per patient. The lifetime costs of ERT starting in the symptomatic stage are €5,716,473 against €171,780 without ERT, a difference of €5,544,693. Consequently, the extra costs per additional YFEOD or per additional QALY are €434,416 and €884,994 respectively. After discounting effects by 1.5% and costs by 4% and under a reasonable scenario of ERT unit cost reduction by 25%, these incremental cost-effectiveness ratios could decrease to €149,857 and €324,812 respectively. Discussion ERT is a highly potential drug for GD I with substantial health gains. The conservatively estimated incremental cost-effectiveness ratios are substantially lower than for Pompe and Fabry disease. We suggest that the high effectiveness has contributed importantly to acceptance of reimbursement of ERT for GD I. The present study may

  6. Membrane-permeable Triphosphate Prodrugs of Nucleoside Analogues.

    PubMed

    Gollnest, Tristan; Dinis de Oliveira, Thiago; Rath, Anna; Hauber, Ilona; Schols, Dominique; Balzarini, Jan; Meier, Chris

    2016-04-18

    The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent TriPPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Discovery of olmesartan hexetil: a new potential prodrug of olmesartan.

    PubMed

    El-Gamal, Mohammed I; Anbar, Hanan S; Chung, Hye Jin; Kim, Hyun-Il; Cho, Young-Jin; Lee, Bong Sang; Lee, Sun Ahe; Moon, Ji Yun; Lee, Dong Jin; Kwon, Dow; Choi, Won-Jai; Jeon, Hong-Ryeol; Oh, Chang-Hyun

    2013-03-01

    Synthesis of a new ester prodrug of olmesartan, olmesartan hexetil (1), is described. It is in vitro stabilities and in vivo pharmacokinetics (PK) were evaluated. It showed high stability in simulated gastric juice, and was rapidly hydrolyzed to olmesartan in rat liver microsomes and rat plasma in vitro. C(max) and AUC(last) for olmesartan were significantly increased in case of hexetil prodrug, compared with olmesartan medoxomil. Olmesartan hexetil is proposed to be an efficient prodrug of olmesartan with markedly increased oral bioavailability. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. First enzymatically activated Taxotere prodrugs designed for ADEPT and PMT.

    PubMed

    Bouvier, Emmanuel; Thirot, Sylvie; Schmidt, Frédéric; Monneret, Claude

    2004-03-01

    Described here are the syntheses and preliminary biological evaluations of the first two enzymatically activated prodrugs of docetaxel (Taxotere) reported to date. These prodrugs were designed as potential candidates for selective chemotherapy in ADEPT or PMT. They are constituted of a glucuronic acid moiety, a double spacer and the cytotoxic drug, differing only by the spacer substitution. The prodrugs were stable in a buffer, and the in vitro studies showed good detoxification and hydrolysis kinetics. As docetaxel was efficiently released in both cases, these compounds are very valuable candidates for further biological evaluations.

  9. Synthesis and biophysical characterization of chlorambucil anticancer ether lipid prodrugs.

    PubMed

    Pedersen, Palle J; Christensen, Mikkel S; Ruysschaert, Tristan; Linderoth, Lars; Andresen, Thomas L; Melander, Fredrik; Mouritsen, Ole G; Madsen, Robert; Clausen, Mads H

    2009-05-28

    The synthesis and biophysical characterization of four prodrug ether phospholipid conjugates are described. The lipids are prepared from the anticancer drug chlorambucil and have C16 and C18 ether chains with phosphatidylcholine or phosphatidylglycerol headgroups. All four prodrugs have the ability to form unilamellar liposomes (86-125 nm) and are hydrolyzed by phospholipase A(2), resulting in chlorambucil release. Liposomal formulations of prodrug lipids displayed cytotoxicity toward HT-29, MT-3, and ES-2 cancer cell lines in the presence of phospholipase A(2), with IC(50) values in the 8-36 microM range.

  10. Immune response to enzyme replacement therapy: 4-sulfatase epitope reactivity of plasma antibodies from MPS VI cats.

    PubMed

    Turner, C T; Hopwood, J J; Bond, C S; Brooks, D A

    1999-07-01

    The mucopolysaccharidoses (MPS) are a group of multiple pathology disorders which are part of a larger group of genetic diseases known as lysosomal storage disorders. Enzyme replacement therapy (ERT) has been developed as a therapy for MPS patients. However, immune responses to ERT have been reported in MPS animal models and in human Gaucher patients. Antibodies can have adverse effects during ERT, which include hypersensitivity/anaphylactic reactions, enzyme inactivation, and enzyme degradation. This study aimed to characterize the immune response to ERT in a feline model of MPS VI, by defining the epitope reactivity of cat plasma antibody against human recombinant N-acetylgalactosamine 4-sulfatase (4-sulfatase) replacement protein. For MPS VI cat plasma, antibody reactivity was observed prior to ERT, with distinct regions of 4-sulfatase linear sequence displaying low affinity antibody reactivity. There was an increase in antibody titer to 4-sulfatase for MPS VI cats post-ERT, with the majority of the immune response detected to linear sequence epitopes. One cat displayed a high titer and high affinity epitope reactivity following prolonged exposure (>/=9 months) to the replacement protein. MPS VI cats on shorter term ERT (3 months) showed high titers to 4-sulfatase and similar patterns of epitope reactivity, but lower affinity antibody reactivity, when compared to the latter cat. This study reports the linear amino acid sequence reactivity and nature of the immune response produced to 4-sulfatase before and after ERT. The monitoring of antibody production during replacement therapy is an important consideration for patient management, as high titer antibodies can affect the efficacy of therapy. Copyright 1999 Academic Press.

  11. Ocular drug metabolism of the bioactivating antioxidant N-acetylcarnosine for vision in ophthalmic prodrug and codrug design and delivery.

    PubMed

    Babizhayev, Mark A

    2008-10-01

    The basic idea in this study relates to the interesting research problem to employ with the knowledgeable pharmacy staff N-acetylcarnosine (NAC) in the developed suitable compounded prodrug ophthalmic preparations, which are currently used for the treatment of cataract and have antioxidant effect, in order to provide the molecular support to one of the most popular beliefs of the growing market for the treatment of senile cataract in patients and animals with efficacious NAC drug formulations worldwide patented by the author. This work presents the progress in ocular NAC prodrug and codrug design and delivery in light of revealed ocular metabolic activities. There is a considerable interest in the ophthalmic codrug design including NAC prodrug based on the strategies to improve ophthalmic drug delivery of the active peptide principal L-carnosine through the sustained intraocular metabolic activation of a dipeptide while making it resistant to enzymatic hydrolysis. Novel approaches to ocular NAC drug delivery, developed by Innovative Vision Products, Inc. (IVP), aim at enhancing the drug bioavailability by ensuring a prolonged retention of the medication in the eye, and/or by facilitating transcorneal penetration. IVP team studied the effects of lubricant eye drops designed as 1% NAC prodrug of L-carnosine containing a mucoadhesive cellulose-based and corneal absorption promoters in a drug delivery system. The predicted responses of the corneal and conjunctival penetrations to the synergistic promoters are useful in controlling the extent and pathway of the ocular and systemic absorptions of instilled NAC prodrug in designed ophthalmic formulations thereof. Utility of peptidase enzyme inhibitors in the codrug formulation to modulate the transport and metabolism of NAC prodrug appears to be a promising strategy for enhancing dipeptide drug transport across the cornea. The developed and officially CE mark registered by IVP NAC prodrug and codrug lubricating eye drop

  12. Long-term persistence to mono and combination therapies with angiotensin converting enzymes and angiotensin II receptor blockers in Australia.

    PubMed

    Gadzhanova, Svetla; Roughead, Elizabeth E; Bartlett, Louise E

    2016-06-01

    The purpose of this study was to compare the impact of initial antihypertensive therapy including angiotensin converting enzyme inhibitors (ACE) or angiotensin II receptor blockers (ARB) on long-term persistence to therapy. A retrospective cohort study using prescription claims data from the Australian Pharmaceutical Benefit Scheme (PBS). Kaplan-Meier analysis of prescription refills and cox proportional hazard models were used to compare the time on therapy (persistence) in people newly initiated to monotherapy or combination therapy including ACE or ARB, between April 2007 and March 2008. Differences in persistence to initial drug class or any antihypertensive therapy were reported at 4-year follow-up. About 119,500 persons initiated ACE or ARB: 47 % initiated ACE monotherapy; 32 % ARB monotherapy; 13 % ACE combinations; and 8 % ARB combinations. Persistence (% on treatment at 4 years) to index therapy was lower in people starting ACE and ARB combinations compared to ACE or ARB monotherapies: ACE combination (12 %) versus ACE monotherapy (25 %) and ARB combinations (22 %) versus ARB monotherapy (35 %). Persistence was higher in those initiating fixed dose combinations (FDC) versus separate pill combinations of ACEs (19 vs. 10 %) and ARBs (25 vs. 14 %). Persistence at 4 years to any antihypertensive therapy was similar between initiators to ACE or ARB monotherapy (60 and 61 %, p = 0.08), ACE or ARB combinations (56 %, p = 0.99), and was slightly higher for separate pill combinations (57-59 %) versus FDC (55 %). Choice of initial antihypertensive may have little impact on long-term persistence to therapy.

  13. Combination enzyme therapy for gastric digestion of dietary gluten in patients with celiac sprue.

    PubMed

    Gass, Jonathan; Bethune, Michael T; Siegel, Matthew; Spencer, Andrew; Khosla, Chaitan

    2007-08-01

    Celiac sprue is a multifactorial disease characterized by an inflammatory response to ingested gluten in the small intestine. Proteolytically resistant, proline- and glutamine-rich gluten peptides from wheat, rye, and barley persist in the intestinal lumen and elicit an immune response in genetically susceptible persons. We investigated a new combination enzyme product, consisting of a glutamine-specific endoprotease (EP-B2 from barley) and a prolyl endopeptidase (SC PEP from Sphingomonas capsulata), for its ability to digest gluten under gastric conditions. The ability of this combination enzyme to digest and detoxify whole-wheat bread gluten was investigated. In vitro and in vivo (rat) experimental systems were developed to simulate human gastric digestion, and the resulting material was analyzed by high-performance liquid chromatography, enzyme-linked immunoabsorbent assay, and patient-derived T-cell proliferation assays. The analysis revealed that EP-B2 extensively proteolyzes complex gluten proteins in bread, whereas SC PEP rapidly detoxifies the residual oligopeptide products of EP-B2 digestion. In vitro dose variation data suggests that an approximate 1:1 weight ratio of the 2 enzymes should maximize their synergistic potential. The efficacy of this 2-enzyme glutenase was verified in a rat model of gastric gluten digestion. By combining 2 enzymes with gastric activity and complementary substrate specificity, it should be possible to increase the safe threshold of ingested gluten, thereby ameliorating the burden of a highly restricted diet for patients with celiac sprue.

  14. Synergism of Selective Tumor Vascular Thrombosis and Protease Activated Prodrug

    DTIC Science & Technology

    2008-05-01

    Such a cytotoxin can be aldesleukin, 5- aminolevulinic acid, bleomycin sulfate, camptothecin, carboplatin, carmustine, cisplatin, cladribine, lyophilized... aminolevulinic acid, protoporphyrin IX, taxol or paclitaxel. In one embodiment, the prodrug is activated by asparaginyl proteases (e.g., legumain) and

  15. Switch from agalsidase beta to agalsidase alfa in the enzyme replacement therapy of patients with Fabry disease in Latin America.

    PubMed

    Ripeau, Diego; Amartino, Hernán; Cedrolla, Martín; Urtiaga, Luis; Urdaneta, Bella; Cano, Marilis; Valdez, Rita; Antongiovanni, Norberto; Masllorens, Francisca

    2017-01-01

    There are currently two available enzyme replacement therapies for Fabry disease and little information regarding efficacy and safety of switching therapies. Between 2009 and 2012 there was a worldwide shortage of agalsidase beta and patients on that enzyme were switched to agalsidase alfa. This retrospective observational study assessed a 2-year period of efficacy and safety in a population of Fabry patients, in Argentina (30 patients) and Venezuela (3 patients), who switched therapies from algasidase beta to agalsidase alfa. Thirty-three patients completed 24-months follow-up after the switch (age 32.4 ± 2.0, range 10.0-55.9 years; male: female 23:10). Measures of renal function such as estimated glomerular filtration rate remained almost unchanged in 31 patients without end stage renal disease over the 2 years after switching and urine protein excretion continued stable. Cardiac functional parameters: left ventricular mass index, interventricular septum, left ventricular posterior wall showed no significant change from baseline in the 33 patients. Quality of life, pain and disease severity scores were mostly unchanged after 24-months and agalsidase alfa was generally well tolerated. Our findings showed there is no significant change in the efficacy measured through the renal or cardiac function, quality of life, pain, disease severity scoring and safety for at least 2 years after switching from agalsidase beta to agalsidase alfa.

  16. Mucopolysaccharidosis: cardiologic features and effects of enzyme-replacement therapy in 24 children with MPS I, II and VI.

    PubMed

    Brands, Marion M M G; Frohn-Mulder, Ingrid M; Hagemans, Marloes L C; Hop, Wim C J; Oussoren, Esmee; Helbing, Wim A; van der Ploeg, Ans T

    2013-03-01

    We determined the cardiologic features of children with MPS I, II and VI, and evaluated the effect of enzyme-replacement therapy (ERT) on cardiac disease. Twenty-four children aged 1-18 years with MPS I, II or VI were prospectively evaluated with echocardiogram and electrocardiogram from the start of enzyme-replacement therapy up to 6 years of treatment. At start of therapy, 66% had abnormal cardiac geometric features. Left-ventricular mass index (LVMI) was increased in half of the patients, due mainly to concentric hypertrophy in MPS I and II and to eccentric hypertrophy in MPS VI. Regurgitation was most severe in a subgroup of young MPS VI patients (<5 years) at the mitral valve. At baseline, all patients had abnormal valves. The ECG showed no clear rhythm or conduction abnormalities; neither, in most patients, did it reflect the hypertrophy. After ERT, the LVMI Z-score normalized in 70% of the patients who had a Z-score > 2. LVMI Z-scores decreased significantly in patients with MPS I and MPS II (p = 0.04 and p = 0.032). Despite ERT, valve regurgitation increased in 60% of the patients. We conclude that all our MPS patients have cardiac abnormalities. The most severe cardiac disease was observed in a subgroup of young MPS VI patients. While ERT had an effect on LVMI and IVSd, it apparently had little or none on valve regurgitation.

  17. Click synthesis of a polyamidoamine dendrimer-based camptothecin prodrug

    PubMed Central

    Zolotarskaya, Olga Yu.; Xu, Leyuan; Valerie, Kristoffer; Yang, Hu

    2015-01-01

    In the present work we report on the click synthesis of a new camptothecin (CPT) prodrug based on anionic polyamidoamine (PAMAM) dendrimer intended for cancer therapy. We applied ‘click’ chemistry to improve polymer-drug coupling reaction efficiency. Specifically, CPT was functionalized with a spacer, 1-azido-3,6,9,12,15-pentaoxaoctadecan-18-oic acid (APO), via EDC/DMAP coupling reaction. In parallel, propargylamine (PPA) and methoxypoly(ethylene glycol) amine were conjugated to PAMAM dendrimer G4.5 in sequence using an effective coupling agent 4-(4,6-dimethoxy-(1,3,5)triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM). CPT-APO was then coupled to PEGylated PAMAM dendrimer G4.5-PPA via a click reaction using copper bromide/2,2’-bipyridine/ dimethyl sulfoxide (catalyst/ligand/solvent). Human glioma cells were exposed to the CPT-conjugate to determine toxicity and cell cycle effects using WST-1 assay and flow cytometry. The CPT-conjugate displayed a dose-dependent toxicity with an IC50 of 5 μM, a 185-fold increase relative to free CPT, presumably as a result of slow release. As expected, conjugated CPT resulted in G2/M arrest and cell death while the dendrimer itself had little to no toxicity. Altogether, highly efficient click chemistry allows for the synthesis of multifunctional dendrimers for sustained drug delivery. PMID:26640689

  18. Long-term enzyme replacement therapy in beta-glucuronidase--deficient mice by allogeneic bone marrow transplantation

    SciTech Connect

    Yatziv, S.; Weiss, L.; Morecki, S.; Fuks, Z.; Slavin, S.

    1982-06-01

    Enzyme replacement therapy was successfully accomplished in beta-Glu-deficient C3H/HeJ mice after transplantation of BM cells obtained from normal BALB/c donors. Marrow recipients were prepared for transplantation by fractionated TLI. Enzyme activity increased from 20.5 +/- 7.0 nmol/mg of protein per hour to 180 +/- 30.2 in the liver (p less than 0.001) and from 8.2 +/- 2.0 to 17.5 +/- 5.0 nmol/ml/hr in the plasma (p less than 0.05) at 50 days after marrow infusion. Normal enzyme activity was maintained in treated mice for at least 100 days after marrow transplantation, as documented by repeated liver biopsies and examination of plasma samples. The marrow donors and the recipients were fully histoincompatible. Both immunologic rejection of the marrow allograft and GVHD were prevented by the prior conditioning of the recipients with TLI, resulting in bilateral transplantation tolerance of host vs. graft and graft vs. host. The data suggest that allogeneic BM transplantation may provide a possible therapeutic approach for certain enzyme deficiency syndromes.

  19. [Influence of complex therapy on the activity of glutathione-dependent enzymes of saliva in patients with parodontitis].

    PubMed

    Gavriliuk, L A; Shevchenko, N V; Vartichan, A I; Lysyĭ, L T; Kepnataru, K F; Godorozha, P D

    2008-01-01

    The activities of antioxidative enzymes (glutathione reductase, glutathione S-transferase) and content of reduced glutathione (GSH), thiocyanate (SCN) and protein were determined in saliva of patients with parodontitis treated with traditional and complex therapy, which additionally included the antihomotoxic preparations Traumeel S ointment, Coenzyme compositum or Lymphomyosot. Inflammation process led to the metabolic disturbances and imbalance of the antioxidative defense system in the patients with parodontitis. The results suggest that complex therapy with the antihomotoxic preparations restored imbalance of the antioxidative defense and was more effective than the traditional therapy alone in the patients with parodontitis. Analysis of interrelation between salivary parameters in patients with parodontitis indicated positive correlation before and after the complex therapy (as an exception there was lack of correlation between content of protein and tiocyanate in the saliva of patients before the beginning of the therapeutic course). So these results reflect activityof pathological process and antioxidant defense imbalance in saliva of patients with parodontitis and may be a basis for recommendation of employment of the complex antihomotoxic therapy as the initial stage of pathological process.

  20. Ocular Sustained Release Nanoparticles Containing Stereoisomeric Dipeptide Prodrugs of Acyclovir

    PubMed Central

    Jwala, Jwala; Boddu, Sai H.S.; Shah, Sujay; Sirimulla, Suman; Pal, Dhananjay

    2011-01-01

    Abstract Purpose The objective of this study was to develop and characterize polymeric nanoparticles of appropriate stereoisomeric dipeptide prodrugs of acyclovir (L-valine-L-valine-ACV, L-valine-D-valine-ACV, D-valine-L-valine-ACV, and D-valine-D-valine-ACV) for the treatment of ocular herpes keratitis. Methods Stereoisomeric dipeptide prodrugs of acyclovir (ACV) were screened for bioreversion in various ocular tissues, cell proliferation, and uptake across the rabbit primary corneal epithelial cell line. Docking studies were carried out to examine the affinity of prodrugs to the peptide transporter protein. Prodrugs with optimum characteristics were selected for the preparation of nanoparticles using various grades of poly (lactic-co-glycolic acid) (PLGA). Nanoparticles were characterized for the entrapment efficiency, surface morphology, size distribution, and in vitro release. Further, the effect of thermosensitive gels on the release of prodrugs from nanoparticles was also studied. Results L-valine-L-valine-ACV and L-valine-D-valine-ACV were considered to be optimum in terms of enzymatic stability, uptake, and cytotoxicity. Docking results indicated that L-valine in the terminal position increases the affinity of the prodrugs to the peptide transporter protein. Entrapment efficiency values of L-valine-L-valine-ACV and L-valine-D-valine-ACV were found to be optimal with PLGA 75:25 and PLGA 65:35 polymers, respectively. In vitro release of prodrugs from nanoparticles exhibited a biphasic release behavior with initial burst phase followed by sustained release. Dispersion of nanoparticles in thermosensitive gels completely eliminated the burst release phase. Conclusion Novel nanoparticulate systems of dipeptide prodrugs of ACV suspended in thermosensitive gels may provide sustained delivery after topical administration. PMID:21500985

  1. The Prodrug Approach: A Successful Tool for Improving Drug Solubility.

    PubMed

    Jornada, Daniela Hartmann; dos Santos Fernandes, Guilherme Felipe; Chiba, Diego Eidy; de Melo, Thais Regina Ferreira; dos Santos, Jean Leandro; Chung, Man Chin

    2015-12-29

    Prodrug design is a widely known molecular modification strategy that aims to optimize the physicochemical and pharmacological properties of drugs to improve their solubility and pharmacokinetic features and decrease their toxicity. A lack of solubility is one of the main obstacles to drug development. This review aims to describe recent advances in the improvement of solubility via the prodrug approach. The main chemical carriers and examples of successful strategies will be discussed, highlighting the advances of this field in the last ten years.

  2. Ocular sustained release nanoparticles containing stereoisomeric dipeptide prodrugs of acyclovir.

    PubMed

    Jwala, Jwala; Boddu, Sai H S; Shah, Sujay; Sirimulla, Suman; Pal, Dhananjay; Mitra, Ashim K

    2011-04-01

    The objective of this study was to develop and characterize polymeric nanoparticles of appropriate stereoisomeric dipeptide prodrugs of acyclovir (L-valine-L-valine-ACV, L-valine-D-valine-ACV, D-valine-L-valine-ACV, and D-valine-D-valine-ACV) for the treatment of ocular herpes keratitis. Stereoisomeric dipeptide prodrugs of acyclovir (ACV) were screened for bioreversion in various ocular tissues, cell proliferation, and uptake across the rabbit primary corneal epithelial cell line. Docking studies were carried out to examine the affinity of prodrugs to the peptide transporter protein. Prodrugs with optimum characteristics were selected for the preparation of nanoparticles using various grades of poly (lactic-co-glycolic acid) (PLGA). Nanoparticles were characterized for the entrapment efficiency, surface morphology, size distribution, and in vitro release. Further, the effect of thermosensitive gels on the release of prodrugs from nanoparticles was also studied. L-valine-L-valine-ACV and L-valine-D-valine-ACV were considered to be optimum in terms of enzymatic stability, uptake, and cytotoxicity. Docking results indicated that L-valine in the terminal position increases the affinity of the prodrugs to the peptide transporter protein. Entrapment efficiency values of L-valine-L-valine-ACV and L-valine-D-valine-ACV were found to be optimal with PLGA 75:25 and PLGA 65:35 polymers, respectively. In vitro release of prodrugs from nanoparticles exhibited a biphasic release behavior with initial burst phase followed by sustained release. Dispersion of nanoparticles in thermosensitive gels completely eliminated the burst release phase. Novel nanoparticulate systems of dipeptide prodrugs of ACV suspended in thermosensitive gels may provide sustained delivery after topical administration.

  3. Personalized Cancer Therapy Considering Cytochrome P450 Variability.

    PubMed

    Preissner, Saskia; Simmaco, Maurizio; Gentile, Giovanna; Preissner, Robert

    2015-01-01

    The individual variability of pharmacokinetics is underestimated and few systematic studies exist in this field. In most cases, this leads to unwanted side effects or toxicity. In polychemotherapy, prodrugs (like ifosfamide), which have to be activated by cytochrome P450 enzymes (CYPs), play an important role. If patients are poor metabolizers for these drugs, the therapy will be ineffective. Furthermore, CYPs and transporters can be (over)expressed in target tissues, which is also not examined and considered in clinical routine. Here, we present a body map showing relevant enzymes in some organs and tissues. Finally, a typical case of a Caucasian chemotherapy patient with breast cancer is presented and discussed regarding a personalized cancer therapy considering the single nucleotide polymorphisms found via genotyping.

  4. Engineering reduced-immunogenicity enzymes for amino acid depletion therapy in cancer.

    PubMed

    Cantor, Jason R; Panayiotou, Vasiliki; Agnello, Giulia; Georgiou, George; Stone, Everett M

    2012-01-01

    Cancer has become the leading cause of death in the developed world and has remained one of the most difficult diseases to treat. One of the difficulties in treating cancer is that conventional chemotherapies often have unacceptable toxicities toward normal cells at the doses required to kill tumor cells. Thus, the demand for new and improved tumor specific therapeutics for the treatment of cancer remains high. Alterations to cellular metabolism constitute a nearly universal feature of many types of cancer cells. In particular, many tumors exhibit deficiencies in one or more amino acid synthesis or salvage pathways forcing a reliance on the extracellular pool of these amino acids to satisfy protein biosynthesis demands. Therefore, one treatment modality that satisfies the objective of developing cancer cell-selective therapeutics is the systemic depletion of that tumor-essential amino acid, which can result in tumor apoptosis with minimal side effects to normal cells. While this strategy was initially suggested over 50 years ago, it has been recently experiencing a renaissance owing to advances in protein engineering technology, and more sophisticated approaches to studying the metabolic differences between tumorigenic and normal cells. Dietary restriction is typically not sufficient to achieve a therapeutically relevant level of amino acid depletion for cancer treatment. Therefore, intravenous administration of enzymes is used to mediate the degradation of such amino acids for therapeutic purposes. Unfortunately, the human genome does not encode enzymes with the requisite catalytic or pharmacological properties necessary for therapeutic purposes. The use of heterologous enzymes has been explored extensively both in animal studies and in clinical trials. However, heterologous enzymes are immunogenic and elicit adverse responses ranging from anaphylactic shock to antibody-mediated enzyme inactivation, and therefore have had limited utility. The one notable exception

  5. Formulating a new basis for the treatment against botulinum neurotoxin intoxication: 3,4-diaminopyridine prodrug design and characterization

    PubMed Central

    Zakhari, Joseph S.; Kinoyama, Isao; Hixon, Mark S.; Di Mola, Antonia; Globisch, Daniel; Janda, Kim D.

    2011-01-01

    Botulism is a disease characterized by neuromuscular paralysis and is produced from botulinum neurotoxins (BoNTs) found within the Gram positive bacterium Clostridium botulinum. This bacteria produces the most deadliest toxin known, with lethal doses as low as 1 ng/kg. Due to the relative ease of production and transport, the use of these agents as potential bioterrorist weapons has become of utmost concern. No small molecule therapies against BoNT intoxication have been approved to date. However, 3,4-diaminopyridine, (3,4-DAP), a potent reversible inhibitor of voltage-gated potassium channels, is an effective cholinergic agonist used in the treatment of neuromuscular degenerative disorders that require cholinergic enhancement. 3,4-DAP has also been shown to facilitate recovery of neuromuscular action potential post botulinum intoxication by blocking K+ channels. Unfortunately, 3,4-DAP displays toxicity largely due to blood-brain-barrier (BBB) penetration. As a dual-action prodrug approach to cholinergic enhancement we have designed carbamate and amide conjugates of 3,4-DAP. The carbamate prodrug is intended to be a slowly reversible inhibitor of acetylcholinesterase (AChE) along the lines of the stigmines thereby allowing increased persistence of released acetylcholine within the synaptic cleft. As a secondary activity, cleavage of the carbamate prodrug by AChE will afford the localized release of 3,4-DAP, which in turn, will enhance the pre-synaptic release of additional acetylcholine. Being a competitive inhibitor with respect to acetylcholine, the activity of the prodrug will be greatest at the synaptic junctions most depleted of acetylcholine. Here we report upon the synthesis and biochemical characterization of three new classes of prodrugs intended to limit previously reported stability and toxicity issues. Of the prodrugs examined, compound 32, demonstrated the most clinically relevant half-life of 2.76 h, while selectively inhibiting AChE over

  6. In vitro characterization of the bioconversion of pomaglumetad methionil, a novel metabotropic glutamate 2/3 receptor agonist peptide prodrug.

    PubMed

    Moulton, Richard D; Ruterbories, Kenneth J; Bedwell, David W; Mohutsky, Michael A

    2015-05-01

    To characterize the hydrolysis of the peptide prodrug pomaglumetad methionil (LY2140023; (1R,4S,5S,6S)-4-(L-methionylamino)-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide), to the active drug LY404039 [(1R,4S,5S,6S)-4-amino-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide], a series of in vitro studies were performed in various matrices, including human intestinal, liver, kidney homogenate, and human plasma. The studies were performed to determine the tissue(s) and enzyme(s) responsible for the conversion of the prodrug to the active molecule. This could enable an assessment of the risk for drug interactions, an evaluation of pharmacogenomic implications, as well as the development of a Physiologically Based Pharmacokinetic (PBPK) model for formation of the active drug. Of the matrices examined, hydrolysis of pomaglumetad methionil was observed in intestinal and kidney homogenate preparations and plasma, but not in liver homogenate. Clearance values calculated after applying standard scaling factors suggest the intestine and kidney as primary sites of hydrolysis. Studies with peptidase inhibitors were performed in an attempt to identify the enzyme(s) catalyzing the conversion. Near complete inhibition of LY404039 formation was observed in intestinal and kidney homogenate and human plasma with the selective dehydropeptidase1 (DPEP1) inhibitor cilastatin. Human recombinant DPEP1 was expressed and shown to catalyze the hydrolysis, which was completely inhibited by cilastatin. These studies demonstrate pomaglumetad methionil can be converted to LY404039 via one or multiple enzymes completely inhibited by cilastatin, likely DPEP1, in plasma, the intestine, and the kidney, with the plasma and kidney involved in the clearance of the circulating prodrug. These experiments define a strategy for the characterization of enzymes responsible for the metabolism of other peptide-like compounds. Copyright © 2015 by The American Society for Pharmacology

  7. Enzyme replacement therapy (ERT) procedure for mucopolysaccharidosis type II (MPS II) by intraventricular administration (IVA) in murine MPS II.

    PubMed

    Higuchi, Takashi; Shimizu, Hiromi; Fukuda, Takahiro; Kawagoe, Shiho; Matsumoto, Juri; Shimada, Yohta; Kobayashi, Hiroshi; Ida, Hiroyuki; Ohashi, Toya; Morimoto, Hideto; Hirato, Tohru; Nishino, Katsuya; Eto, Yoshikatsu

    2012-09-01

    Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS) and is characterized by the accumulation of glycosaminoglycans (GAGs). MPS II has been treated by hematopoietic stem cell therapy (HSCT)/enzyme replacement therapy (ERT), but its effectiveness in the central nervous system (CNS) is limited because of poor enzyme uptake across the blood-brain barrier (BBB). To increase the efficacy of ERT in the brain, we tested an intraventricular ERT procedure consisting of repeated administrations of IDS (20 μg/mouse/3 weeks) in IDS-knockout, MPS II model mice. The IDS enzyme activity and the accumulation of total GAGs were measured in mouse brains. The IDS activity was significantly increased, and the accumulation of total GAGs was decreased in the MPS II mouse brains treated with multiple administrations of IDS via intraventricular ERT. Additionally, a high level of IDS enzyme activity was appreciated in other MPS II mouse tissues, such as the liver, spleen, testis and others. A Y-maze was used to test learning and memory after repeated intraventricular ERT with IDS. The IDS-treated mouse groups recovered the capacity for short-term memory and activity. Although large and small vacuoles were found at the margin of the cerebellar Purkinje cells in the disease-control mice, these vacuoles disappeared upon treated with IDS. Loss of vacuoles was also observed in other tissues (liver, kidney and testis). These results demonstrate the possible efficacy of an ERT procedure with intraventricular administration of IDS for the treatment of MPS II. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  8. Structure-activity relationship study of human liver microsomes-catalyzed hydrolysis rate of ester prodrugs of MENT by comparative molecular field analysis (CoMFA).

    PubMed

    Bursi, Roberta; Grootenhuis, Arijan; van der Louw, Jaap; Verhagen, Jos; de Gooyer, Marcel; Jacobs, Peter; Leysen, Dirk

    2003-03-01

    A series of MENT esters (3-71) was designed, prepared and tested to study the structure-activity relationship (SAR) of the hydrolysis rate with human liver microsomes of these prodrugs. Compounds were obtained covering a wide range of metabolic stability. The results are useful for the proper selection of prodrugs for different pharmaceutical formulations to deliver the potent and prostate-sparing androgen MENT. The MENT esters can especially be administered for male hormone replacement therapy and male contraception. Comparative molecular field analysis (CoMFA) was applied to a dataset of 28 esters, for which ED50 values could be obtained. The CoMFA model where the electrostatic and H-bond molecular fields were combined turned out to be most predictive. Despite the limited size of the dataset, CoMFA can help to rationalize the SAR of the ester hydrolysis rate of ester prodrugs of MENT.

  9. Home infusion program with enzyme replacement therapy for Fabry disease: The experience of a large Italian collaborative group.

    PubMed

    Concolino, D; Amico, L; Cappellini, M D; Cassinerio, E; Conti, M; Donati, M A; Falvo, F; Fiumara, A; Maccarone, M; Manna, R; Matucci, A; Musumeci, M B; Nicoletti, A; Nisticò, R; Papadia, F; Parini, R; Peluso, D; Pensabene, L; Pisani, A; Pistone, G; Rigoldi, M; Romani, I; Tenuta, M; Torti, G; Veroux, M; Zachara, E

    2017-09-01

    Fabry disease (FD) [OMIM 301500] is an X-linked lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A, resulting in progressive multisystem accumulation of globotriaosylceramide (Gb3). Although the introduction of Enzyme Replacement Therapy (ERT) resulted in a variety of clinical benefits, life-long intravenous (IV) treatment with ERT with an every other week schedule, may interfere with daily life activities and impact on QoL. We report here a multicentric, observational, longitudinal data analysis on a large cohort of 85 Italian FD patients (45 males, 40 females) from 11 out of 20 Italian regions, who received a cumulative number of 4269 home infusions of agalsidase alfa. For the whole cohort, the average duration of home therapy was 1 year and 11 months (range 3 months-4 years and 6 months), and during this period, compliance to treatment (number of infusions performed vs scheduled) reached 100%. The EQ-5 VAS scale was administered to patients to evaluate the self-reported QoL, 58% of patients showing an increase of EQ-5 VAS score at follow up compared to baseline (home treatment start) or remaining stable. A mild increase of average disease severity, measured through Mainz Severity Score Index (MSSI), was found during hospital treatment (p < 0,007), while it remained stable between the first home therapy infusion and last follow up. Interestingly, 4 out of 7 (57%) patients, showing an improvement in FD-related clinical status after starting home therapy, had previously a sub-optimal compliance to treatment during the period of hospital treatment management. Only 4 adverse non serious reactions (0,093%) were reported totally in 2 patients during home treatment. We conclude that home infusions in eligible patients with FD are safe, contribute to improve treatment compliance and therapeutic clinical outcomes, and may have a positive impact on self-perceived QoL.

  10. Experiments to optimize enzyme substitution therapy in pancreatic duct-ligated pigs.

    PubMed

    Kammlott, E; Karthoff, J; Stemme, K; Gregory, P; Kamphues, J

    2005-01-01

    Ligation of the pancreatic duct in pigs leads to severe maldigestion and malabsorption of crude nutrients. Supplementation with 24 capsules of Creon (Solvay Pharmaceuticals GmbH, Hannover, Germany) per meal led to an increased digestibility of crude nutrients. With regard to optimization of the treatment of EPI no essential improvements can be achieved by adding omeprazol or lecithin to the diet. In pancreatic duct-ligated pigs the isolated addition of omeprazol led to an increase of the pre-caecal digestibility of crude fat and organic matter. With additional enzyme substitution, the application of omeprazol did not result in an improved fat digestibility. Isolated addition of lecithin to the diet resulted in a reduced total digestibility of crude fat. Offering the diet twice a day and using a higher frequency of enzyme applications (four or six instead of only two applications) had no effects on the digestibilty of crude fat or organic matter. According to the observations in pancreatic duct-ligated pigs, the addition of missing enzymes to the diet led to the best treatment results in EPI. Administration of omeprazol or a higher feeding frequency as well as the application of enzymes in small proportion of the whole meal or dosages given consecutively over the day showed no advantages. Furthermore, the present study suggests that the addition of lecithin cannot be recommended in EPI, when given diets with butter as the predominant fat source as in human dietetics.

  11. Large amino acid transporter 1 (LAT1) prodrugs of valproic acid: new prodrug design ideas for central nervous system delivery.

    PubMed

    Peura, Lauri; Malmioja, Kalle; Laine, Krista; Leppänen, Jukka; Gynther, Mikko; Isotalo, Antti; Rautio, Jarkko

    2011-10-03

    Central nervous system (CNS) drug delivery is a major challenge in drug development because the blood-brain barrier (BBB) efficiently restricts the entry of drug molecules into the CNS at sufficient amounts. The brain uptake of poorly penetrating drugs could be improved by utilizing the transporters at the BBB with a prodrug approach. In this study, we designed four phenylalanine derivatives of valproic acid and studied their ability to utilize a large amino acid transporter 1 (LAT1) in CNS delivery with an aim to show that the meta-substituted phenylalanine prodrugs bind to LAT1 with a higher affinity compared with the affinity of the para-substituted derivatives. All of the prodrugs crossed the BBB carrier mediatedly via LAT1 in in situ rat brain perfusion. For the first time, we introduced a novel meta-substituted phenylalanine analogue promoiety which improved the LAT1 affinity 10-fold and more importantly the rat brain uptake of the prodrug 2-fold compared with those of the para-substituted derivatives. Therefore, we have characterized a new prodrug design idea for CNS drug delivery utilizing a transporter-mediated prodrug approach.

  12. HUMAN SKIN PERMEATION OF 3-O-ALKYL CARBAMATE PRODRUGS OF NALTREXONE

    PubMed Central

    Vaddi, Haranath K.; Banks, Stan L.; Chen, Jianhong; Hammell, Dana C.; Crooks, Peter A.; Stinchcomb, Audra L.

    2009-01-01

    N-Monoalkyl and N,N-dialkyl carbamate prodrugs of naltrexone (NTX), an opioid antagonist, were synthesized and their in vitro permeation across human skin was determined. Relevant physicochemical properties were also determined. Most prodrugs exhibited lower melting points, lower aqueous solubilities, and higher oil solubilities than NTX. The flux values from N-monoalkyl carbamate prodrugs were significantly higher than those from NTX and N,N-dialkyl carbamates. The melting points of N-monoalkyl carbamate prodrugs were quite low compared to the N,N-dialkyl carbamate prodrugs and NTX. Heats of fusion for the N,N-dialkyl carbamate prodrugs were higher than that for NTX. N-Monoalkyl carbamate prodrugs had higher stratum corneum/vehicle partition coefficients than their N,N-dialkyl counterparts. Higher percent prodrug bioconversion to NTX in skin appeared to be related to increased skin flux. N,N-Dialkyl carbamate prodrugs were more stable in buffer and in plasma than N-monoalkyl carbamate prodrugs. In conclusion, N-monoalkyl carbamate prodrugs of NTX improved the systemic delivery of NTX across human skin in vitro. N,N-Dialkyl substitution in the prodrug moiety decreased skin permeation and plasma hydrolysis to the parent drug. The cross-sectional area of the carbamate head group was the major determinant of flux of the N-monoalkyl and N,N-dialkyl carbamate prodrugs of NTX. PMID:18972573

  13. Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder--murine Pompe disease.

    PubMed

    Raben, Nina; Schreiner, Cynthia; Baum, Rebecca; Takikita, Shoichi; Xu, Sengen; Xie, Tao; Myerowitz, Rachel; Komatsu, Masaaki; Van der Meulen, Jack H; Nagaraju, Kanneboyina; Ralston, Evelyn; Plotz, Paul H

    2010-11-01

    Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alphaglucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50–60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy.

  14. Mucopolysaccharidosis type II (Hunter syndrome): a clinical review and recommendations for treatment in the era of enzyme replacement therapy

    PubMed Central

    Scarpa, Maurizio; Beck, Michael; Bodamer, Olaf A.; De Meirleir, Linda; Guffon, Nathalie; Meldgaard Lund, Allan; Malm, Gunilla; Van der Ploeg, Ans T.; Zeman, Jiri

    2007-01-01

    Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a rare X-linked recessive disease caused by deficiency of the lysosomal enzyme iduronate-2-sulphatase, leading to progressive accumulation of glycosaminoglycans in nearly all cell types, tissues and organs. Clinical manifestations include severe airway obstruction, skeletal deformities, cardiomyopathy and, in most patients, neurological decline. Death usually occurs in the second decade of life, although some patients with less severe disease have survived into their fifth or sixth decade. Until recently, there has been no effective therapy for MPS II, and care has been palliative. Enzyme replacement therapy (ERT) with recombinant human iduronate-2-sulphatase (idursulfase), however, has now been introduced. Weekly intravenous infusions of idursulfase have been shown to improve many of the signs and symptoms and overall wellbeing in patients with MPS II. This paper provides an overview of the clinical manifestations, diagnosis and symptomatic management of patients with MPS II and provides recommendations for the use of ERT. The issue of treating very young patients and those with CNS involvement is also discussed. ERT with idursulfase has the potential to benefit many patients with MPS II, especially if started early in the course of the disease. PMID:18038146

  15. Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder—murine Pompe disease

    PubMed Central

    Takikita, Shoichi; Xu, Sengen; Xie, Tao; Myerowitz, Rachel; Komatsu, Masaaki; Van Der Meulen, Jack H; Nagaraju, Kanneboyina; Ralston, Evelyn; Plotz, Paul H

    2010-01-01

    Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alpha-glucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50–60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy. PMID:20861693

  16. Variable cytochrome P450 2D6 expression and metabolism of codeine and other opioid prodrugs: implications for the Australian anaesthetist.

    PubMed

    Wilcox, R A; Owen, H

    2000-12-01

    Codeine is a popular opioid prodrug dependent on the activity of the specific cytochrome P450 enzyme 2D6 (CYP2D6). This enzyme catalyses the production of the potent analgesic metabolite morphine, but genetic studies have demonstrated that individuals from different ethnic groups exhibit considerable variability in the functional capacities of their expressed CYP2D6 enzymes, and pharmacological studies have shown many commonly prescribed drugs can reduce the action of CYP2D6 enzymes. These findings have significant clinical implications for the rational prescription of effective analgesia, especially in a multicultural country like Australia.

  17. Lipase-catalyzed synthesis of (S)-naproxen ester prodrug by transesterification in organic solvents.

    PubMed

    Tsai, S W; Tsai, C S; Chang, C S

    1999-06-01

    A lipase-catalyzed enantioselective transesterification process was developed for the synthesis of (S)-naproxen 2-N-morpholinoethyl ester prodrug from racemic 2,2,2-trifluoroethyl naproxen ester in organic solvents. By selecting isooctane and 37 degrees C as the best solvent and temperature, the apparent fits of the initial conversion rates for transesterification and hydrolysis side reaction suggest a ping-pong Bi-Bi enzymatic mechanism with the alcohol as a competitive enzyme inhibitor. Improvements in the initial conversion rate and the productivity for the desired (S)-ester product were obtained after comparing with the result of an enantioselective esterification process. Studies of water content in isooctane and alcohol containing various N,N-dialkylamino groups on the enzyme activity and enantioselectivity, as well as the recovery of (S)-ester product by using extraction, were also reported.

  18. Mucopolysaccharidoses in northern Brazil: Targeted mutation screening and urinary glycosaminoglycan excretion in patients undergoing enzyme replacement therapy

    PubMed Central

    Viana, Gustavo M.; de Lima, Nathália O.; Cavaleiro, Rosely; Alves, Erik; Souza, Isabel C.N.; Feio, Raimunda; Leistner-Segal, Sandra; Schwartz, Ida; Giugliani, Roberto; da Silva, Luiz C. Santana

    2011-01-01

    Mucopolysaccharidoses (MPS) are rare lysosomal disorders caused by the deficiency of specific lysosomal enzymes responsible for glycosaminoglycan (GAG) degradation. Enzyme Replacement Therapy (ERT) has been shown to reduce accumulation and urinary excretion of GAG, and to improve some of the patients’ clinical signs. We studied biochemical and molecular characteristics of nine MPS patients (two MPS I, four MPS II and three MPS VI) undergoing ERT in northern Brazil. The responsiveness of ERT was evaluated through urinary GAG excretion measurements. Patients were screened for eight common MPS mutations, using PCR, restriction enzyme tests and direct sequencing. Two MPS I patients had the previously reported mutation p.P533R. In the MPS II patients, mutation analysis identified the mutation p.R468W, and in the MPS VI patients, polymorphisms p.V358M and p.V376M were also found. After 48 weeks of ERT, biochemical analysis showed a significantly decreased total urinary GAG excretion in patients with MPS I (p < 0.01) and MPS VI (p < 0.01). Our findings demonstrate the effect of ERT on urinary GAG excretion and suggest the adoption of a screening strategy for genotyping MPS patients living far from the main reference centers. PMID:21931511

  19. Effects of chronic swimming training and oestrogen therapy on coronary vascular reactivity and expression of antioxidant enzymes in ovariectomized rats.

    PubMed

    Claudio, Erick R G; Endlich, Patrick W; Santos, Roger L; Moysés, Margareth R; Bissoli, Nazaré S; Gouvêa, Sônia A; Silva, Josiane F; Lemos, Virginia S; Abreu, Glaucia R

    2014-01-01

    The aim of this study was to evaluate the effects of swimming training (SW) and oestrogen replacement therapy (ERT) on coronary vascular reactivity and the expression of antioxidant enzymes in ovariectomized rats. Animals were randomly assigned to one of five groups: sham (SH), ovariectomized (OVX), ovariectomized with E2 (OE2), ovariectomized with exercise (OSW), and ovariectomized with E2 plus exercise (OE2+SW). The SW protocol (5×/week, 60 min/day) and/or ERT were conducted for 8 weeks; the vasodilator response to bradykinin was analysed (Langendorff Method), and the expression of antioxidant enzymes (SOD-1 and 2, catalase) and eNOS and iNOS were evaluated by Western blotting. SW and ERT improved the vasodilator response to the highest dose of bradykinin (1000 ng). However, in the OSW group, this response was improved at 100, 300 and 1000 ng when compared to OVX (p<0,05). The SOD-1 expression was increased in all treated/trained groups compared to the OVX group (p<0,05), and catalase expression increased in the OSW group only. In the trained group, eNOS increased vs. OE2, and iNOS decreased vs. SHAM (p<0,05). SW may represent an alternative to ERT by improving coronary vasodilation, most likely by increasing antioxidant enzyme and eNOS expression and augmenting NO bioavailability.

  20. Review of aerobic glycolysis and its key enzymes – new targets for lung cancer therapy

    PubMed Central

    Li, Xue-bing; Gu, Jun-dong; Zhou, Qing-hua

    2015-01-01

    Most tumor cells show different metabolic pathways than normal cells. Even under the conditions of sufficient oxygen, they produce energy by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, which is known as aerobic glycolysis or the Warburg effect. Lung cancer is a malignant tumor with one of the highest incidence and mortality rates in the world at present. However, the exact mechanisms underlying lung cancer development remain unclear. The three key enzymes of glycolysis are hexokinase, phosphofructokinase, and pyruvate kinase. Lactate dehydrogenase catalyzes the transfer of pyruvate to lactate. All four enzymes have been reported to be overexpressed in tumors, including lung cancer, and can be regulated by many oncoproteins to promote tumor proliferation, migration, and metastasis with dependence or independence of glycolysis. The discovery of aerobic glycolysis in the 1920s has provided new means and potential therapeutic targets for lung cancer. PMID:26273330

  1. A new extracorporeal reactor-dialyzer for enzyme therapy using immobilized L-asparaginase.

    PubMed

    Jackson, J A; Halvorson, H R; Furlong, J W; Lucast, K D; Shore, J D

    1979-05-01

    Depletion of circulating L-asparagine has proved beneficial in the treatment of some acute lymphoyctic leukemias. To avoid the immunologic sequelae of administering L-asparaginase parenterally, we have covalently attached the enzyme to the outside of the fibers of a conventional hollow fiber hemodialyzer. This provides ready access of the substrate to the enzyme, while simultaneously isolating the foreign protein from the immune system. Such reactor-dialyzers perform well, both in vitro and in vivo. Circulating L-asparagine in the healthy dog is reduced from about 50 micrometer to less than 2 micrometer within 30 min of connecting the reactor-dialyzer and the reduction persists for at least 4 hr after cessation of treatment.

  2. Targeted anticancer prodrug with mesoporous silica nanoparticles as vehicles

    NASA Astrophysics Data System (ADS)

    Fan, Jianquan; Fang, Gang; Wang, Xiaodan; Zeng, Fang; Xiang, Yufei; Wu, Shuizhu

    2011-11-01

    A targeted anticancer prodrug system was fabricated with 180 nm mesoporous silica nanoparticles (MSNs) as carriers. The anticancer drug doxorubicin (DOX) was conjugated to the particles through an acid-sensitive carboxylic hydrazone linker which is cleavable under acidic conditions. Moreover, folic acid (FA) was covalently conjugated to the particle surface as the targeting ligand for folate receptors (FRs) overexpressed in some cancer cells. The in vitro release profiles of DOX from the MSN-based prodrug systems showed a strong dependence on the environmental pH values. The fluorescent dye FITC was incorporated in the MSNs so as to trace the cellular uptake on a fluorescence microscope. Cellular uptakes by HeLa, A549 and L929 cell lines were tested for FA-conjugated MSNs and plain MSNs respectively, and a much more efficient uptake by FR-positive cancer cells (HeLa) can be achieved by conjugation of folic acid onto the particles because of the folate-receptor-mediated endocytosis. The cytotoxicities for the FA-conjugated MSN prodrug, the plain MSN prodrug and free DOX against three cell lines were determined, and the result indicates that the FA-conjugated MSN prodrug exhibits higher cytotoxicity to FR-positive cells, and reduced cytotoxicity to FR-negative cells. Thus, with 180 nm MSNs as the carriers for the prodrug system, good drug loading, selective targeting and sustained release of drug molecules within targeted cancer cells can be realized. This study may provide useful insights for designing and improving the applicability of MSNs in targeted anticancer prodrug systems.

  3. Metabolic Activation of the Anti-Hepatitis C Virus Nucleotide Prodrug PSI-352938

    PubMed Central

    Niu, Congrong; Tolstykh, Tatiana; Bao, Haiying; Park, Yeojin; Babusis, Darius; Lam, Angela M.; Bansal, Shalini; Du, Jinfa; Chang, Wonsuk; Reddy, P. Ganapati; Zhang, Hai-Ren; Woolley, Joseph; Wang, Li-Quan; Chao, Piyun B.; Ray, Adrian S.; Otto, Michael J.; Sofia, Michael J.

    2012-01-01

    PSI-352938 is a novel cyclic phosphate prodrug of β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methylguanosine-5′-monophosphate with potent anti-HCV activity. In order to inhibit the NS5B RNA-dependent RNA polymerase, PSI-352938 must be metabolized to the active triphosphate form, PSI-352666. During in vitro incubations with PSI-352938, significantly larger amounts of PSI-352666 were formed in primary hepatocytes than in clone A hepatitis C virus (HCV) replicon cells. Metabolism and biochemical assays were performed to define the molecular mechanism of PSI-352938 activation. The first step, removal of the isopropyl group on the 3′,5′-cyclic phosphate moiety, was found to be cytochrome P450 (CYP) 3A4 dependent, with other CYP isoforms unable to catalyze the reaction. The second step, opening of the cyclic phosphate ring, was catalyzed by phosphodiesterases (PDEs) 2A1, 5A, 9A, and 11A4, all known to be expressed in the liver. The role of these enzymes in the activation of PSI-352938 was confirmed in primary human hepatocytes, where prodrug activation was reduced by inhibitors of CYP3A4 and PDEs. The third step, removal of the O6-ethyl group on the nucleobase, was shown to be catalyzed by adenosine deaminase-like protein 1. The resulting monophosphate was consecutively phosphorylated to the diphosphate and to the triphosphate PSI-352666 by guanylate kinase 1 and nucleoside diphosphate kinase, respectively. In addition, formation of nucleoside metabolites was observed in primary hepatocytes, and ecto-5′-nucleotidase was able to dephosphorylate the monophosphate metabolites. Since CYP3A4 is highly expressed in the liver, the CYP3A4-dependent metabolism of PSI-352938 makes it an effective liver-targeted prodrug, in part accounting for the potent antiviral activity observed clinically. PMID:22526308

  4. Enzyme replacement therapy started at birth improves outcome in difficult-to-treat organs in mucopolysaccharidosis I mice.

    PubMed

    Baldo, Guilherme; Mayer, Fabiana Q; Martinelli, Bárbara Z; de Carvalho, Talita G; Meyer, Fabiola S; de Oliveira, Patrícia G; Meurer, Luise; Tavares, Angela; Matte, Ursula; Giugliani, Roberto

    2013-05-01

    Since we previously observed that in patients with mucopolysaccharidosis (MPS) the storage of undegraded glycosaminoglycans (GAG) occurs from birth, in the present study we aimed to compare normal, untreated MPS I mice (knockout for alpha-l-iduronidase-IDUA), and MPS I mice treated with enzyme replacement therapy (ERT, Laronidase, 1.2mg/kg every 2 weeks) started from birth (ERT-neo) or from 2 months of age (ERT-ad). All mice were sacrificed at 6 months. Both treatments were equally effective in normalizing GAG levels in the viscera but had no detectable effect on the joint. Heart function was also improved with both treatments. On the other hand, mice treated from birth presented better outcomes in the difficult-to-treat aortas and heart valves. Surprisingly, both groups had improvements in behavior tests, and normalization of GAG levels in the brain and IDUA injection resulted in detectable levels of enzyme in the brain tissue 1h after administration. ERT-ad mice developed significantly more anti-IDUA-IgG antibodies, and mice that didn't develop antibodies had better performances in behavior tests, indicating that development of antibodies may reduce enzyme bioavailability. Our results suggest that ERT started from birth leads to better outcomes in the aorta and heart valves, as well as a reduction in antibody levels. Some poor vascularized organs, such as the joints, had partial or no benefit and ancillary therapies might be needed for patients. The results presented here support the idea that ERT started from birth leads to better treatment outcomes and should be considered whenever possible, a observation that gains relevance as newborn screening programs are being considered for MPS and other treatable lysosomal storage disorders. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Alglucosidase alfa enzyme replacement therapy as a therapeutic approach for glycogen storage disease type III.

    PubMed

    Sun, Baodong; Fredrickson, Keri; Austin, Stephanie; Tolun, Adviye A; Thurberg, Beth L; Kraus, William E; Bali, Deeksha; Chen, Yuan-Tsong; Kishnani, Priya S

    2013-02-01

    We investigated the feasibility of using recombinant human acid-α glucosidase (rhGAA, Alglucosidase alfa), an FDA approved therapy for Pompe dise