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Sample records for enzyme prodrug therapy

  1. Substrate Mediated Enzyme Prodrug Therapy

    PubMed Central

    Fejerskov, Betina; Zelikin, Alexander N.

    2012-01-01

    In this report, we detail Substrate Mediated Enzyme Prodrug Therapy (SMEPT) as a novel approach in drug delivery which relies on enzyme-functionalized cell culture substrates to achieve a localized conversion of benign prodrug(s) into active therapeutics with subsequent delivery to adhering cells or adjacent tissues. For proof-of-concept SMEPT, we use surface adhered micro-structured physical hydrogels based on poly(vinyl alcohol), β-glucuronidase enzyme and glucuronide prodrugs. We demonstrate enzymatic activity mediated by the assembled hydrogel samples and illustrate arms of control over rate of release of model fluorescent cargo. SMEPT was not impaired by adhering cells and afforded facile time - and dose – dependent uptake of the in situ generated fluorescent cargo by hepatic cells, HepG2. With the use of a glucuronide derivative of an anticancer drug, SN-38, SMEPT afforded a decrease in cell viability to a level similar to that achieved using parent drug. Finally, dose response was achieved using SMEPT and administration of judiciously chosen concentration of SN-38 glucuronide prodrug thus revealing external control over drug delivery using drug eluting surface. We believe that this highly adaptable concept will find use in diverse biomedical applications, specifically surface mediated drug delivery and tissue engineering. PMID:23152927

  2. Prodrugs of anthracyclines for use in antibody-directed enzyme prodrug therapy.

    PubMed

    Florent, J C; Dong, X; Gaudel, G; Mitaku, S; Monneret, C; Gesson, J P; Jacquesy, J C; Mondon, M; Renoux, B; Andrianomenjanahary, S; Michel, S; Koch, M; Tillequin, F; Gerken, M; Czech, J; Straub, R; Bosslet, K

    1998-09-10

    A series of new prodrugs of daunorubicin and doxorubicin which are candidates for antibody-directed enzyme prodrug therapy (ADEPT) is reported. These compounds (25a,b,c and 32a,b,c) have been designed to generate cytotoxic drugs after activation with beta-glucuronidase. As expected, recovery of the active drug was observed after enzymatic cleavage by Escherichia coli beta-glucuronidase as well as by a fusion protein which has been obtained from human beta-glucuronidase and humanized CEA-specific binding region. The six prodrugs are highly stable and are more than 100-fold less cytotoxic than doxorubicin against murine L1210 cell lines. The ortho-substituted phenyl carbamates 25a,b,c are better substrates for beta-glucuronidase than the corresponding para-substituted analogues. After taking into account additional factors such as stability in plasma and kinetics of enzymatic cleavage, we selected the o-nitro prodrug 25c for clinical trials. PMID:9733483

  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. Horseradish peroxidase-encapsulated chitosan nanoparticles for enzyme-prodrug cancer therapy.

    PubMed

    Cao, Xiaodan; Chen, Chao; Yu, Haijun; Wang, Ping

    2015-01-01

    Among various enzyme-based therapies, enzyme-prodrug therapy (EPT) promises minimized side effects in that it activates non-toxic prodrugs locally where the enzymes are placed. The success of such an approach requires high enzyme stability against both structural denaturation and potential immunogenicity. This work examines the efficiency of nanoparticles for enzyme protection in EPT applications. Specifically, horseradish peroxidase (HRP)-encapsulated chitosan nanoparticles (HRP-CSNP) were constructed and examined with respect to stability enhancement. HRP-CSNP retained enzyme activity and had improved stability at 37 °C in the presence of a denaturant, urea. The nanoparticles effectively bound to the surface of human breast cancer cell Bcap37 and led to over 80 % cell death when applied with a prodrug indole-3-acetic acid. PMID:25257586

  5. Nitroreductase gene-directed enzyme prodrug therapy: insights and advances toward clinical utility.

    PubMed

    Williams, Elsie M; Little, Rory F; Mowday, Alexandra M; Rich, Michelle H; Chan-Hyams, Jasmine V E; Copp, Janine N; Smaill, Jeff B; Patterson, Adam V; Ackerley, David F

    2015-10-15

    This review examines the vast catalytic and therapeutic potential offered by type I (i.e. oxygen-insensitive) nitroreductase enzymes in partnership with nitroaromatic prodrugs, with particular focus on gene-directed enzyme prodrug therapy (GDEPT; a form of cancer gene therapy). Important first indications of this potential were demonstrated over 20 years ago, for the enzyme-prodrug pairing of Escherichia coli NfsB and CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, it has become apparent that both the enzyme and the prodrug in this prototypical pairing have limitations that have impeded their clinical progression. Recently, substantial advances have been made in the biodiscovery and engineering of superior nitroreductase variants, in particular development of elegant high-throughput screening capabilities to enable optimization of desirable activities via directed evolution. These advances in enzymology have been paralleled by advances in medicinal chemistry, leading to the development of second- and third-generation nitroaromatic prodrugs that offer substantial advantages over CB1954 for nitroreductase GDEPT, including greater dose-potency and enhanced ability of the activated metabolite(s) to exhibit a local bystander effect. In addition to forging substantial progress towards future clinical trials, this research is supporting other fields, most notably the development and improvement of targeted cellular ablation capabilities in small animal models, such as zebrafish, to enable cell-specific physiology or regeneration studies. PMID:26431849

  6. 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. PMID:25820125

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

    PubMed Central

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

    2015-01-01

    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. PMID:25820125

  8. Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy.

    PubMed

    Hagen, Sven; Baumann, Tobias; Wagner, Hanna J; Morath, Volker; Kaufmann, Beate; Fischer, Adrian; Bergmann, Stefan; Schindler, Patrick; Arndt, Katja M; Müller, Kristian M

    2014-01-01

    The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT). PMID:24457557

  9. 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. PMID:25157916

  10. Recent progress in gene-directed enzyme prodrug therapy: an emerging cancer treatment.

    PubMed

    Both, Gerald W

    2009-08-01

    The principle of gene-directed enzyme prodrug therapy (GDEPT) has existed for many years but, while simple in concept, the effective practical application of this therapy has proven to be challenging. Improvements in the efficacy of GDEPT have been achieved principally through the choice and development of more effective vectors, by optimizing and controlling gene expression and by increasing the activity of the delivered enzyme through mutation. While innovation continues in this field, the pioneering GDEPT systems designed to treat glioma and prostate cancer have completed or are now entering late-stage clinical trials, respectively. As the pace of innovation in GDEPT technology far exceeds its clinical application, these initial products are anticipated to be replaced by next-generation biologicals. This review highlights recent progress in the strategies and development of GDEPT and summarizes the status of current clinical trials. With the first GDEPT product for treatment of resected gliomas poised to gain marketing approval, a new era in cancer gene medicine is emerging. PMID:19649987

  11. Direct retroviral delivery of human cytochrome P450 2B6 for gene-directed enzyme prodrug therapy of cancer.

    PubMed

    Kan, O; Griffiths, L; Baban, D; Iqball, S; Uden, M; Spearman, H; Slingsby, J; Price, T; Esapa, M; Kingsman, S; Kingsman, A; Slade, A; Naylor, S

    2001-07-01

    Human cytochrome P450 2B6 (CYP2B6) metabolizes the prodrug cyclophosphamide (CPA) to produce phosphoramide mustard that cross-links DNA leading to cell death. We have constructed a novel retroviral vector encoding CYP2B6 (designated "MetXia-P450") and used it to transduce the human tumor cell lines HT29 and T47D. MetXia-P450 transduction sensitised these cells to the cytotoxic effects of the prodrug CPA. Results from in vitro experiments demonstrated adverse effects on the clonogenic survival of cyclophosphamide-treated cells transduced with MetXia-P450. Cytotoxic activity accompanied by bystander effect was particularly evident in 3-D multicellular spheroid models suggesting that this in vitro system may be a more appropriate model for assessing the efficacy of gene directed-enzyme prodrug therapy (GDEPT). We have applied this approach in a clinically relevant gene therapy protocol on established subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450-based GDEPT strategy mediated by a direct retroviral gene transfer in vivo. PMID:11498768

  12. Targeted enzyme prodrug therapy for metastatic prostate cancer – a comparative study of L-methioninase, purine nucleoside phosphorylase, and cytosine deaminase

    PubMed Central

    2014-01-01

    Background Enzyme prodrug therapy shows promise for the treatment of solid tumors, but current approaches lack effective/safe delivery strategies. To address this, we previously developed three enzyme-containing fusion proteins targeted via annexin V to phosphatidylserine exposed on the tumor vasculature and tumor cells, using the enzymes L-methioninase, purine nucleoside phosphorylase, or cytosine deaminase. In enzyme prodrug therapy, the fusion protein is allowed to bind to the tumor before a nontoxic drug precursor, a prodrug, is introduced. Upon interaction of the prodrug with the bound enzyme, an anticancer compound is formed, but only in the direct vicinity of the tumor, thereby mitigating the risk of side effects while creating high intratumoral drug concentrations. The applicability of these enzyme prodrug systems to treating prostate cancer has remained unexplored. Additionally, target availability may increase with the addition of low dose docetaxel treatment to the enzyme prodrug treatment, but this effect has not been previously investigated. To this end, we examined the binding strength and the cytotoxic efficacy (with and without docetaxel treatment) of these enzyme prodrug systems on the human prostate cancer cell line PC-3. Results All three fusion proteins exhibited strong binding; dissociation constants were 0.572 nM for L-methioninase-annexin V (MT-AV), 0.406 nM for purine nucleoside phosphorylase-annexin V (PNP-AV), and 0.061 nM for cytosine deaminase-annexin V (CD-AV). MT-AV produced up to 99% cell death (p < 0.001) with limited cytotoxicity of the prodrug alone. PNP-AV with docetaxel created up to 78% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. CD-AV with docetaxel displayed up to 60% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. Docetaxel treatment created significant increases in cytotoxicity for PNP-AV and CD-AV. Conclusions Strong binding of fusion proteins to the prostate cancer cells

  13. A Fusion Protein of RGD4C and β-Lactamase Has a Favorable Targeting Effect in Its Use in Antibody Directed Enzyme Prodrug Therapy

    PubMed Central

    Wang, Hao; Zhou, Xiao-Liang; Long, Wei; Liu, Jin-Jian; Fan, Fei-Yue

    2015-01-01

    Antibody directed enzyme prodrug therapy (ADEPT) utilizing β-lactamase is a promising treatment strategy to enhance the therapeutic effect and safety of cytotoxic agents. In this method, a conjugate (antibody-β-lactamase fusion protein) is employed to precisely activate nontoxic cephalosporin prodrugs at the tumor site. A major obstacle to the clinical translation of this method, however, is the low catalytic activity and high immunogenicity of the wild-type enzymes. To overcome this challenge, we fused a cyclic decapeptide (RGD4C) targeting to the integrin with a β-lactamase variant with reduced immunogenicity which retains acceptable catalytic activity for prodrug hydrolysis. Here, we made a further investigation on its targeting effect and pharmacokinetic properties, the results demonstrated that the fusion protein retains a targeting effect on integrin positive cells and has acceptable pharmacokinetic characteristics, which benefits its use in ADEPT. PMID:25927583

  14. Silk-elastinlike protein polymers improve the efficacy of adenovirus thymidine kinase enzyme prodrug therapy of head and neck tumors

    PubMed Central

    Greish, Khaled; Frandsen, Jordan; Scharff, Stephanie; Gustafson, Joshua; Cappello, Joseph; Li, Daqing; O’Malley, Bert W.; Ghandehari, Hamidreza

    2010-01-01

    Background Adenoviral directed enzyme prodrug therapy is a promising approach for head and neck cancer gene therapy. Challenges with this approach however are transient gene expression and dissemination of viruses to distant organs. Methods We used recombinant silk-elastinlike protein copolymer (SELP) matrices for intratumoral delivery of adenoviruses containing both thymidine kinase-1, and luciferase genes in a nude mice model of JHU-022 head and neck tumor. Hydrogels made from two SELP analogues (47K and 815K) with similar silk to elastinlike block ratios but different block lengths were studied for intratumoral viral delivery. Tumor bearing mice were followed up for tumor progression and luciferase gene expression concomitantly for five weeks. Polymer’s safety was evaluated through body weight change, blood count, liver and kidney functions in addition to gross and microscopic histological examination. Results SELP 815K analogues efficiently controlled the duration and extent of transfection in tumors for up to 5 weeks with no detectable spread to the liver. About five-fold greater reduction in tumor volume was obtained with matrix-mediated delivery compared to intra-tumoral injection of adenoviruses in saline. SELP matrix proved safe in all injected mice compared to control group. Conclusion SELP- controlled gene delivery approach could potentially improve the anticancer activity of virus-mediated gene therapy while limiting viral spread to normal organs. PMID:20603862

  15. 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.

  16. Towards a ligand targeted enzyme prodrug therapy: single round panning of a beta-lactamase scaffold library on human cancer cells.

    PubMed

    Shukla, Girja S; Murray, Christopher J; Estabrook, Melodie; Shen, Guang-Ping; Schellenberger, Volker; Krag, David N

    2007-05-15

    A novel beta-lactamase scaffold library in which the target-binding moiety is built into the enzyme was generated using phage display technology. The binding element is composed of a fully randomized 8 amino acid loop inserted at position between Y34 and K37 on the outer surface of Enterobacter cloacae P99 cephalosporinase (beta-lactamase, E.C. 3.5.2.6) with all library members retaining catalytic activity. The frequency and diversity of amino acids distributions in peptide inserts from library clones were analyzed. The complexity of the randomized loop appears consistent with standards of other types of phage display library systems. The library was panned against SKBR3 human breast cancer cells in 1 round using rolling circle amplification of phage DNA to recover bound phage. Individual beta-lactamase clones, independent of phage, were rapidly assessed for their binding to SKBR3 cells using a simple high throughput screen based on cell-bound beta-lactamase activity. SKBR3 cell-binding beta-lactamase enzymes were also shown to bind specifically using an immunochemical method. Selected beta-lactamase clones were further studied for their protein expression, enzyme activity and binding to nontumor cell-lines. Overall, the approach outlined here offers the opportunity of rapidly selecting targeted beta-lactamase ligands that may have a potential for their use in enzyme prodrug therapy with cephalosporin-based prodrugs. It is expected that a similar approach will be useful in developing tumor-targeting molecules of several other enzyme candidates of cancer prodrug therapy. PMID:17285581

  17. The Engineered Thymidylate Kinase (TMPK)/AZT Enzyme-Prodrug Axis Offers Efficient Bystander Cell Killing for Suicide Gene Therapy of Cancer

    PubMed Central

    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. PMID:24194950

  18. 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. PMID:24194950

  19. 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. PMID:26072401

  20. Prodrug converting enzyme gene delivery by L. monocytogenes

    PubMed Central

    Stritzker, Jochen; Pilgrim, Sabine; Szalay, Aladar A; Goebel, Werner

    2008-01-01

    Background Listeria monocytogenes is a highly versatile bacterial carrier system for introducing protein, DNA and RNA into mammalian cells. The delivery of tumor antigens with the help of this carrier into tumor-bearing animals has been successfully carried out previously and it was recently reported that L. monocytogenes is able to colonize and replicate within solid tumors after local or even systemic injection. Methods Here we report on the delivery of two prodrug converting enzymes, purine-deoxynucleoside phosphorylase (PNP) and a fusion protein consisting of yeast cytosine deaminase and uracil phosphoribosyl transferase (FCU1) into cancer cells in culture by L. monocytogenes. Transfer of the prodrug converting enzymes was achieved by bacterium mediated transfer of eukaryotic expression plasmids or by secretion of the proteins directly into the host cell cytosol by the infecting bacteria. Results The results indicate that conversion of appropriate prodrugs to toxic drugs in the cancer cells occured after both procedures although L. monocytogenes-mediated bactofection proved to be more efficient than enzyme secretion 4T1, B16 and COS-1 tumor cells. Exchanging the constitutively PCMV-promoter with the melanoma specific P4xTETP-promoter resulted in melanoma cell-specific expression of the prodrug converting enzymes but reduced the efficiencies. Conclusion These experiments open the way for bacterium mediated tumor specific activation of prodrugs in live animals with tumors. PMID:18402662

  1. 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.

  2. Prodrugs of anthracyclines for chemotherapy via enzyme-monoclonal antibody conjugates.

    PubMed

    Gesson, J P; Jacquesy, J C; Mondon, M; Petit, P; Renoux, B; Andrianomenjanahary, S; Dufat-Trinh Van, H; Koch, M; Michel, S; Tillequin, F

    1994-10-01

    New prodrugs of daunorubicin, 1c, 1e and 2c, including a galactopyranosyl residue linked to the N-3' of the daunosaminyl moiety through substituted o- or p-benzyloxycarbonyl groups were synthesized. Their low cytotoxicity and high stability in plasma fulfil the conditions for antibody-directed enzyme prodrug therapy (ADEPT). Enzymatic hydrolysis using alpha-D-galactosidase gives rise to daunorubicin by subsequent self-elimination of the spacers. However, elimination clearly depends on the aromatic substitution pattern, as demonstrated especially by comparison with non-substituted analogues. PMID:7945725

  3. Substrate-competitive activity-based profiling of ester prodrug activating enzymes

    PubMed Central

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

    2015-01-01

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating pre-clinical 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 4-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 preclinical

  4. 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). PMID:25617724

  5. 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. PMID:26004323

  6. Adipose Tissue–derived Mesenchymal Stem Cells Expressing Prodrug-converting Enzyme Inhibit Human Prostate Tumor Growth

    PubMed Central

    Cavarretta, Ilaria T; Altanerova, Veronika; Matuskova, Miroslava; Kucerova, Lucia; Culig, Zoran; Altaner, Cestmir

    2009-01-01

    The ability of human adipose tissue–derived mesenchymal stem cells (AT-MSCs), engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT), to convert the relatively nontoxic 5-fluorocytosine (5-FC) into the highly toxic antitumor 5-fluorouracil (5-FU) together with their ability to track and engraft into tumors and micrometastases makes these cells an attractive tool to activate prodrugs directly within the tumor mass. In this study, we tested the feasibility and efficacy of these therapeutic cells to function as cellular vehicles of prodrug-activating enzymes in prostate cancer (PC) therapy. In in vitro migration experiments we have shown that therapeutic AT-MSCs migrated to all the prostate cell lines tested. In a pilot preclinical study, we observed that coinjections of human bone metastatic PC cells along with the transduced AT-MSCs into nude mice treated with 5-FC induced a complete tumor regression in a dose dependent manner or did not even allow the establishment of the tumor. More importantly, we also demonstrated that the therapeutic cells were effective in significantly inhibiting PC tumor growth after intravenous administration that is a key requisite for any clinical application of gene-directed enzyme prodrug therapies. PMID:19844197

  7. 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

  8. Boronic prodrug of endoxifen as an effective hormone therapy for breast cancer.

    PubMed

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

    2015-07-01

    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. This metabolic conversion is equally efficient in vivo as indicated in the pharmacokinetic study in mice. Moreover, when administered at the same dose, oral ZB483 afforded a 30- to 40-fold higher plasma level of endoxifen in mice than oral administration of endoxifen. 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

  9. ALA-Butyrate prodrugs for Photo-Dynamic Therapy

    NASA Astrophysics Data System (ADS)

    Berkovitch, G.; Nudelman, A.; Ehenberg, B.; Rephaeli, A.; Malik, Z.

    2010-05-01

    The use of 5-aminolevulinic acid (ALA) administration has led to many applications of photodynamic therapy (PDT) in cancer. However, the hydrophilic nature of ALA limits its ability to penetrate the cells and tissues, and therefore the need for ALA derivatives became an urgent research target. In this study we investigated the activity of novel multifunctional acyloxyalkyl ester prodrugs of ALA that upon metabolic hydrolysis release active components such as, formaldehyde, and the histone deacetylase inhibitory moiety, butyric acid. Evaluation of these prodrugs under photo-irradiation conditions showed that butyryloxyethyl 5-amino-4-oxopentanoate (ALA-BAC) generated the most efficient photodynamic destruction compared to ALA. ALA-BAC stimulated a rapid biosynthesis of protoporphyrin IX (PpIX) in human glioblastoma U-251 cells which resulted in generation of intracellular ROS, reduction of mitochondrial activity, leading to apoptotic and necrotic death of the cells. The apoptotic cell death induced by ALA / ALA-BAC followed by PDT equally activate intrinsic and extrinsic apoptotic signals and both pathways may occur simultaneously. The main advantage of ALA-BAC over ALA stems from its ability to induce photo-damage at a significantly lower dose than ALA.

  10. Enzyme Therapy: Current Perspectives.

    PubMed

    UmaMaheswari, Thiyagamoorthy; Hemalatha, Thiagarajan; Sankaranarayanan, Palavesam; Puvanakrishnan, Rengarajulu

    2016-01-01

    Enzymes control all metabolic processes in human system from simple digestion of food to highly complex immune response. Physiological reactions occuring in healthy individuals are disturbed when enzymes are deficient or absent. Enzymes are administered for normalizing biological function in certain pathologies. Initially, crude proteolytic enzymes were used for the treatment of gastrointestinal disorders. Recent advances have enabled enzyme therapy as a promising tool in the treatment of cardiovascular, oncological and hereditary diseases. Now, a spectrum of other diseases are also covered under enzyme therapy. But, the available information on the use of enzymes as therapeutic agents for different diseases is scanty. This review details the enzymes which have been used to treat various diseases/disorders. PMID:26891548

  11. The application of prodrug-based nano-drug delivery strategy in cancer combination therapy.

    PubMed

    Ge, Yanxiu; Ma, Yakun; Li, Lingbing

    2016-10-01

    Single drug therapy that leads to the multidrug resistance of cancer cells and severe side-effect is a thing of the past. Combination therapies that affect multiple signaling pathways have been the focus of recent active research. Due to the successful development of prodrug-based nano-drug delivery systems (P-N-DDSs), their use has been extended to combination therapy as drug delivery platforms. In this review, we focus specifically on the P-N-DDSs in the field of combination therapy including the combinations of prodrugs with different chemotherapeutic agents, other therapeutic agents, nucleic acid or the combination of different types of therapy (e.g. chemotherapy and phototherapy). The relevant examples of prodrug-based nanoparticulate drug delivery strategy in combination cancer therapy from the recent literature are discussed to demonstrate the feasibilities of relevant technology. PMID:27400243

  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. PMID:27342435

  14. 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

  15. 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. PMID:26518752

  16. Dual pH-responsive 5-aminolevulinic acid pseudopolyrotaxane prodrug micelles for enhanced photodynamic therapy.

    PubMed

    Tong, Hongxin; Wang, Yin; Li, Huan; Jin, Qiao; Ji, Jian

    2016-03-11

    Novel 5-aminolevulinic acid (ALA) pseudopolyrotaxane prodrug micelles with dual pH-responsive properties were prepared by the host-guest interaction of α-cyclodextrin (α-CD) and poly(ethylene glycol) (PEG). The micelles exhibited pH dependent cellular uptake and pH-sensitive ALA release, enabling enhanced photodynamic therapy. PMID:26882232

  17. 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.

  18. Potential use of radiolabeled glucuronide prodrugs with auger and/or alpha emitters in combined chemo- and radio-therapy of cancer.

    PubMed

    Unak, T

    2000-07-01

    Nowadays, the scientists from different disciplines have focused their attentions to new anticancer drug design for cancer chemotherapy. An effective anticancer drug should ensure the selective drug incorporation into the targeted tumor cells without principally incorporation into the normal cells. So, the targeted tumor cells can selectively be damaged by the cytotoxic effectiveness of the drug. The basic principles of drug design have involved "prodrug" concept, which means a chemical agent which is not itself active as an anticancer drug, but it can be transformed to an active form after its administration. Prodrugs can finally be activated onto the tumor cells by some kind of enzymes. In this context, the activation of glucuronide prodrugs by b-glucuronidase have a large potential applications in cancer chemotherapy. On the other hand, combined chemo- and radio-therapy of cancer (CCRTC) concept aims to combine the cytotoxicity of an aglycone with the radiotoxicity of an appropriate radionuclide on the same prodrug. So, the cytotoxic and radiotoxic effectiveness' will be able to be concentrated into the same tumor cell to increase obviously its damage. For experimental realization of this concept an effective anticancer prodrug should be radiolabeled with a radionuclide having high level of radiotoxic effectiveness such as Auger and/or alpha-emitter radionuclides. Iodine-125 and astatine-211 are very interesting radionuclides as being effective Auger and/or alpha-emitters. Briefly, the glucuronide prodrugs radiolabeled with iodine-125 or astatine-211 promise to be designed very effective anticancer agents in the future applications of cancer chemotherapy. PMID:10903386

  19. 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. PMID:26409000

  20. Human Enteric Microsomal CYP4F Enzymes O-Demethylate the Antiparasitic Prodrug Pafuramidine

    PubMed Central

    Wang, Michael Zhuo; Wu, Judy Qiju; Bridges, Arlene S.; Zeldin, Darryl C.; Kornbluth, Sally; Tidwell, Richard R.; Hall, James Edwin; Paine, Mary F.

    2008-01-01

    CYP4F enzymes, including CYP4F2 and CYP4F3B, were recently shown to be the major enzymes catalyzing the initial oxidative O-demethylation of the antiparasitic prodrug pafuramidine (DB289) by human liver microsomes. As suggested by a low oral bioavailability, DB289 could undergo first-pass biotransformation in the intestine, as well as in the liver. Using human intestinal microsomes (HIM), we characterized the enteric enzymes that catalyze the initial O-demethylation of DB289 to the intermediate metabolite, M1. M1 formation in HIM was catalyzed by cytochrome P450 (P450) enzymes, as evidenced by potent inhibition by 1-aminoben-zotriazole and the requirement for NADPH. Apparent Km and Vmax values ranged from 0.6 to 2.4 μM and from 0.02 to 0.89 nmol/min/mg protein, respectively (n = 9). Of the P450 chemical inhibitors evaluated, ketoconazole was the most potent, inhibiting M1 formation by 66%. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, inhibited M1 formation in a concentration-dependent manner (up to 95%). Immunoinhibition with an antibody raised against CYP4F2 showed concentration-dependent inhibition of M1 formation (up to 92%), whereas antibodies against CYP3A4/5 and CYP2J2 had negligible to modest effects. M1 formation rates correlated strongly with arachidonic acid ω-hydroxylation rates (r2 = 0.94, P < 0.0001, n = 12) in a panel of HIM that lacked detectable CYP4A11 protein expression. Quantitative Western blot analysis revealed appreciable CYP4F expression in these HIM, with a mean (range) of 7 (3–18) pmol/mg protein. We conclude that enteric CYP4F enzymes could play a role in the first-pass biotransformation of DB289 and other xenobiotics. PMID:17709372

  1. 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

  2. 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

  3. 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. PMID:26415543

  4. Engineering a Prostate-Specific Membrane Antigen–Activated Tumor Endothelial Cell Prodrug for Cancer Therapy

    PubMed Central

    Denmeade, Samuel R.; Mhaka, Annastasiah M.; Rosen, D. Marc; Brennen, W. Nathaniel; Dalrymple, Susan; Dach, Ingrid; Olesen, Claus; Gurel, Bora; DeMarzo, Angelo M.; Wilding, George; Carducci, Michael A.; Dionne, Craig A.; Møller, Jesper V.; Nissen, Poul; Christensen, S. Brøgger; Isaacs, John T.

    2013-01-01

    Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of solid tumors. We generated a prodrug, G202, consisting of a PSMA-specific peptide coupled to an analog of the potent SERCA pump inhibitor thapsigargin. G202 produced substantial tumor regression against a panel of human cancer xenografts in vivo at doses that were minimally toxic to the host. On the basis of these data, a phase 1 dose-escalation clinical trial has been initiated with G202 in patients with advanced cancer. PMID:22745436

  5. Synthesis of an enzyme-dependent prodrug and evaluation of its potential for colon targeting

    PubMed Central

    Pang, Yi-Nuo; Zhang, Yan; Zhang, Zhi-Rong

    2002-01-01

    AIM: To synthesize dexamethasone-succinate-dextran (DSD) conjugate and to evaluate the potentiality of DSD for the treatment of inflammatory bowel diseases. METHODS: Dexamethasone was attached to dextran (average molecular weight = 70400 Dalton) using succinate anhydride in an anhydrous environment catalyzed by 4-dimethylaminopyridine and 1,1’-carbonyldiimidazole. The chemical structure of DSD was identified by UV, IR and NMR, and the in vivo drug release behavior of this prodrug was investigated after oral administration of DSD suspension. RESULTS: The DSD conjugate was obtained in two steps and the content of dexamethasone in DSD was 11.28%. The dextran prodrug was stable in rat stomach and small intestine and negligibly absorbed from these tracts. Four to nine hours after the oral administration, most of the prodrug (> 95%) had moved to the cecum and colon, and was easily hydrolyzed by an endodextranase. Recover of dexamethasone from colon and cecum after administration of DSD conjugate was 6-12 folds higher than the recovery after administration of unmodified dexamethasone (t = 2.74, P < 0.05). The preferential release of free dexamethasone in cecum and colon over that in the small intestine was statistically significant (t = 2.27, P < 0.05). CONCLUSION: The results of this study indicate that dextran conjugates may be useful in selectively delivering glucocorticoids to the colon. PMID:12378641

  6. 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. PMID:16431120

  7. 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.

  8. 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

  9. 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

  10. Adenoviral vectors for prodrug activation-based gene therapy for cancer

    PubMed Central

    Doloff, Joshua C.; Waxman, David J.

    2013-01-01

    Cancer cell heterogeneity is a common feature - both between patients diagnosed with the same cancer and within an individual patient’s tumor - and leads to widely different response rates to cancer therapies and the potential for the emergence of drug resistance. Diverse therapeutic approaches have been developed to combat the complexity of cancer, including individual treatment modalities designed to target tumor heterogeneity. This review discusses adenoviral vectors and how they can be modified to replicate in a cancer-specific manner and deliver therapeutic genes under multi-tiered regulation to target tumor heterogeneity, including heterogeneity associated with cancer stem cell-like subpopulations. Strategies that allow for combination of prodrug-activation gene therapy with a novel replication-conditional, heterogeneous tumor-targeting adenovirus are discussed, as are the benefits of using adenoviral vectors as tumor-targeting oncolytic vectors. While the anticancer activity of many adenoviral vectors has been well established in preclinical studies, only limited successes have been achieved in the clinic, indicating a need for further improvements in activity, specificity, tumor cell delivery and avoidance of immunogenicity. PMID:23869779

  11. 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

  12. 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

  13. Combined Alloreactive CTL Cellular Therapy with Prodrug Activator Gene Therapy in a Model of Breast Cancer Metastatic to the Brain

    PubMed Central

    Hickey, Michelle J.; Malone, Colin C.; Erickson, Kate L.; Lin, Amy; Soto, Horacio; Ha, Edward T.; Kamijima, Shuichi; Inagaki, Akihito; Takahashi, Masamichi; Kato, Yuki; Kasahara, Noriyuki; Mueller, Barbara M.; Kruse, Carol A.

    2013-01-01

    Purpose Individual or combined strategies of cellular therapy with alloreactive cytotoxic T lymphocytes (alloCTL) and gene therapy employing retroviral replicating vectors (RRV) encoding a suicide prodrug activating gene were explored for the treatment of breast tumors metastatic to the brain. Experimental Design AlloCTL, sensitized to the human leukocyte antigens of MDA-MB-231 breast cancer cells, were examined in vitro for anti-tumor functionality toward breast cancer targets. RRV encoding the yeast cytosine deaminase (CD) gene was tested in vivo for virus spread, ability to infect, and kill breast cancer targets when exposed to 5-fluorocytosine (5-FC). Individual and combination treatments were tested in subcutaneous and intracranial xenograft models with 231BR, a brain tropic variant. Results AlloCTL preparations were cytotoxic, proliferated and produced interferon-gamma when coincubated with target cells displaying relevant HLA. In vivo, intratumorally-placed alloCTL trafficked through one established intracranial 231BR focus to another in contralateral brain and induced tumor cell apoptosis. RRV-CD efficiently spread in vivo, infected 231BR and induced their apoptosis upon 5-FC exposure. Subcutaneous tumor volumes were significantly reduced in alloCTL and/or gene therapy treated groups compared to control groups. Mice with established intracranial 231BR tumors treated with combined alloCTL and RRV-CD had a median survival of 97.5 days compared with single modalities (50–83 days); all experimental treatment groups survived significantly longer than sham-treated groups (median survivals 31.5 or 40 days) and exhibited good safety/toxicity profiles. Conclusion The results indicate combining cellular and suicide gene therapies is a viable strategy for the treatment of established breast tumors in the brain. PMID:23780889

  14. 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. PMID:26551869

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

    DOE PAGESBeta

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

    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

  16. 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.

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

    PubMed Central

    2015-01-01

    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. It also provides an opportunity for the wide application of upconverting red radiation in photonics and biophotonics. PMID:25291544

  18. Dendrimer Prodrugs.

    PubMed

    da Silva Santos, Soraya; Igne Ferreira, Elizabeth; Giarolla, Jeanine

    2016-01-01

    The main objective of this review is to describe the importance of dendrimer prodrugs in the design of new drugs, presenting numerous applications of these nanocomposites in the pharmaceutical field. Therefore, the use of dendrimer prodrugs as carrier for drug delivery, to improve pharmacokinetic properties of prototype, to promote drug sustained-release, to increase selectivity and, consequently, to decrease toxicity, are just some examples of topics that have been extensively reported in the literature, especially in the last decade. The examples discussed here give a panel of the growing interest dendrimer prodrugs have been evoking in the scientific community. PMID:27258239

  19. Imaging of enzyme replacement therapy using PET

    PubMed Central

    Phenix, Christopher P.; Rempel, Brian P.; Colobong, Karen; Doudet, Doris J.; Adam, Michael J.; Clarke, Lorne A.; Withers, Stephen G.

    2010-01-01

    Direct enzyme replacement therapy (ERT) has been introduced as a means to treat a number of rare, complex genetic conditions associated with lysosomal dysfunction. Gaucher disease was the first for which this therapy was applied and remains the prototypical example. Although ERT using recombinant lysosomal enzymes has been shown to be effective in altering the clinical course of Gaucher disease, Fabry disease, Hurler syndrome, Hunter syndrome, Maroteaux-Lamy syndrome, and Pompe disease, the recalcitrance of certain disease manifestations underscores important unanswered questions related to dosing regimes, tissue half-life of the recombinant enzyme and the ability of intravenously administered enzyme to reach critical sites of known disease pathology. We have developed an innovative method for tagging acid β-glucocerebrosidase (GCase), the recombinant enzyme formulated in Cerezyme® used to treat Gaucher disease, using an 18F-labeled substrate analogue that becomes trapped within the active site of the enzyme. Using micro-PET we show that the tissue distribution of injected enzyme can be imaged in a murine model and that the PET data correlate with tissue 18F counts. Further we show that PET imaging readily monitors pharmacokinetic changes effected by receptor blocking. The ability to 18F-label GCase to monitor the enzyme distribution and tissue half-life in vivo by PET provides a powerful research tool with an immediate clinical application to Gaucher disease and a clear path for application to other ERTs. PMID:20534487

  20. Nanoparticle-mediated delivery of a rapidly activatable prodrug of SN-38 for neuroblastoma therapy

    PubMed Central

    Alferiev, Ivan S.; Iyer, Radhika; Croucher, Jamie L.; Adamo, Richard F.; Zhang, Kehan; Mangino, Jennifer L.; Kolla, Venkatadri; Fishbein, Ilia; Brodeur, Garrett M.; Levy, Robert J.; Chorny, Michael

    2015-01-01

    Nanomedicine-based strategies have the potential to improve therapeutic performance of a wide range of anticancer agents. However, the successful implementation of nanoparticulate delivery systems requires the development of adequately sized nanocarriers delivering their therapeutic cargo to the target in a protected, pharmacologically active form. The present studies focused on a novel nanocarrier-based formulation strategy for SN-38, a topoisomerase I inhibitor with proven anticancer potential, whose clinical application is compromised by toxicity, poor stability and incompatibility with conventional delivery vehicles. SN-38 encapsulated in biodegradable sub-100 nm sized nanoparticles (NP) in the form of its rapidly activatable prodrug derivative with tocopherol succinate potently inhibited the growth of neuroblastoma cells in a dose- and exposure time-dependent manner, exhibiting a delayed response pattern distinct from that of free SN-38. In a xenograft model of neuroblastoma, prodrug-loaded NP caused rapid regression of established large tumors, significantly delayed tumor regrowth after treatment cessation and markedly extended animal survival. The NP formulation strategy enabled by a reversible chemical modification of the drug molecule offers a viable means for SN-38 delivery achieving sustained intratumoral drug levels and contributing to the potency and extended duration of antitumor activity, both prerequisites for effective treatment of neuroblastoma and other cancers. PMID:25770994

  1. Optimized human CYP4B1 in combination with the alkylator prodrug 4-ipomeanol serves as a novel suicide gene system for adoptive T-cell therapies.

    PubMed

    Roellecke, K; Virts, E L; Einholz, R; Edson, K Z; Altvater, B; Rossig, C; von Laer, D; Scheckenbach, K; Wagenmann, M; Reinhardt, D; Kramm, C M; Rettie, A E; Wiek, C; Hanenberg, H

    2016-07-01

    Engineering autologous or allogeneic T cells to express a suicide gene can control potential toxicity in adoptive T-cell therapies. We recently reported the development of a novel human suicide gene system that is based on an orphan human cytochrome P450 enzyme, CYP4B1, and the naturally occurring alkylator prodrug 4-ipomeanol. The goal of this study was to systematically develop a clinically applicable self-inactivating lentiviral vector for efficient co-expression of CYP4B1 as an ER-located protein with two distinct types of cell surface proteins, either MACS selection genes for donor lymphocyte infusions after allogeneic stem cell transplantation or chimeric antigen receptors for retargeting primary T cells. The U3 region of the myeloproliferative sarcoma virus in combination with the T2A site was found to drive high-level expression of our CYP4B1 mutant with truncated CD34 or CD271 as MACS suitable selection markers. This lentiviral vector backbone was also well suited for co-expression of CYP4B1 with a codon-optimized CD19 chimeric antigen receptor (CAR) construct. Finally, 4-ipomeanol efficiently induced apoptosis in primary T cells that co-express mutant CYP4B1 and the divergently located MACS selection and CAR genes. In conclusion, we here developed a clinically suited lentiviral vector that supports high-level co-expression of cell surface proteins with a potent novel human suicide gene. PMID:27092941

  2. 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

  3. 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

  4. 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

  5. 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. PMID:26974508

  6. Engineering of lipid prodrug-based, hyaluronic acid-decorated nanostructured lipid carriers platform for 5-fluorouracil and cisplatin combination gastric cancer therapy

    PubMed Central

    Qu, Chun-Ying; Zhou, Min; Chen, Ying-wei; Chen, Mei-mei; Shen, Feng; Xu, Lei-Ming

    2015-01-01

    Purpose The first-line chemotherapy treatment protocol for gastric cancer is combination chemotherapy of 5-fluorouracil (5-FU) and cisplatin (CDDP). The aim of this study was to engineer prodrug-based nanostructured lipid carriers (NLC) platform for codelivery of 5-FU and CDDP to enhance therapy and decrease toxicity. Methods First, 5-FU-stearic acid lipid conjugate was synthesized by two steps. Second, 5-FU-stearic acid prodrug and CDDP were loaded in NLC. Finally, hyaluronic acid (HA) was coated onto NLC surface. Average size, zeta potential, and drug loading capacity of NLC were evaluated. Human gastric cancer cell line BGC823 (BGC823 cells) was used for the testing of in vitro cytotoxicity assays. In vivo antitumor activity of NLC was evaluated in mice bearing BGC823 cells model. Results HA-coated 5-FU-stearic acid prodrug and CDDP-loaded NLC (HA-FU/C-NLC) showed a synergistic effect in combination therapy and displayed the greatest antitumor activity than all of the free drugs or uncoated NLC in vitro and in vivo. Conclusion This work reveals that HA-coated NLC could be used as a novel carrier to code-liver 5-FU and CDDP for gastric cancer therapy. HA-FU/C-NLC could be a promising targeted and combinational therapy in nanomedicine. PMID:26089667

  7. Prodrugs of reverse fosmidomycin analogues.

    PubMed

    Brücher, Karin; Gräwert, Tobias; Konzuch, Sarah; Held, Jana; Lienau, Claudia; Behrendt, Christoph; Illarionov, Boris; Maes, Louis; Bacher, Adelbert; Wittlin, Sergio; Mordmüller, Benjamin; Fischer, Markus; Kurz, Thomas

    2015-02-26

    Fosmidomycin inhibits IspC (Dxr, 1-deoxy-d-xylulose 5-phosphate reductoisomerase), a key enzyme in nonmevalonate isoprenoid biosynthesis that is essential in Plasmodium falciparum. The drug has been used successfully to treat malaria patients in clinical studies, thus validating IspC as an antimalarial target. However, improvement of the drug's pharmacodynamics and pharmacokinetics is desirable. Here, we show that the conversion of the phosphonate moiety into acyloxymethyl and alkoxycarbonyloxymethyl groups can increase the in vitro activity against asexual blood stages of P. falciparum by more than 1 order of magnitude. We also synthesized double prodrugs by additional esterification of the hydroxamate moiety. Prodrugs with modified hydroxamate moieties are subject to bioactivation in vitro. All prodrugs demonstrated improved antiplasmodial in vitro activity. Selected prodrugs and parent compounds were also tested for their cytotoxicity toward HeLa cells and in vivo in a Plasmodium berghei malaria model as well as in the SCID mouse P. falciparum model. PMID:25633870

  8. 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

  9. Modification of existing antibiotics in the form of precursor prodrugs that can be subsequently activated by nitroreductases of the target pathogen.

    PubMed

    Çelik, Ayhan; Yetiş, Gülden; Ay, Mehmet; Güngör, Tuğba

    2016-08-15

    The use of existing antibiotics in the form of prodrug followed by activation using enzymes of pathogenic origin could be a useful approach for antimicrobial therapy. To investigate this idea, a common antibiotic, sulfamethoxazole has been redesigned in the form of a prodrug by simple functional group replacement. Upon reductive activation by a type I nitroreductase from a pathogen, the drug displayed enhanced antimicrobial capacity. This strategy could improve the efficacy and selectively of antibiotics and reduce the incidence of resistance. PMID:27390065

  10. Molecular pathologies of and enzyme replacement therapies for lysosomal diseases.

    PubMed

    Sakuraba, Hitoshi; Sawada, Makoto; Matsuzawa, Fumiko; Aikawa, Sei-ichi; Chiba, Yasunori; Jigami, Yoshifumi; Itoh, Kohji

    2006-08-01

    Lysosomal diseases comprise a group of inherited disorders resulting from defects of lysosomal enzymes and their cofactors, and in many of them the nervous system is affected. Recently, enzyme replacement therapy with recombinant lysosomal enzymes has been clinically available for several lysosomal diseases. Such enzyme replacement therapies can improve non-neurological disorders but is not effective for neurological ones. In this review, we discuss the molecular pathologies of lysosomal diseases from the protein structural aspect, current enzyme replacement therapies, and attempts to develop enzyme replacement therapies effective for lysosomal diseases associated with neurological disorders, i.e., production of enzymes, brain-specific delivery and incorporation of lysosomal enzymes into cells. PMID:16918392

  11. Modern prodrug design for targeted oral drug delivery.

    PubMed

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

    2014-01-01

    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. PMID:25317578

  12. Controlled Au-Polymer Nanostructures for Multiphoton Imaging, Prodrug Delivery, and Chemo-Photothermal Therapy Platforms.

    PubMed

    Huang, Chih-Chia; Liu, Tzu-Ming

    2015-11-18

    We have successfully introduced a proton-induced controlled reaction of HAuCl4 and poly(styrene-alt-maleic acid) (PSMA) sodium salt to prepare triangular and multicore Au@polymer nanoparticles (NPs). The interparticle interactions in the core gave rise to an absorption band at the near-infrared wavelength. The near-infrared optical properties of the resulting Au-polymer nanostructures are highly stable in a physiological environment, which offered strong photo-to-thermal conversion by a moderate continuous-wave 808 nm laser and exhibited multiphoton fluorescence for imaging using a 1230 nm light excitation (femtosecond laser). Exposure of the carboxylate groups at the polymer shell made the surface structure of the Au multicore @polymer NPs directly conjugate Pt(II)-/Pt(IV)-based drugs, which possessed the elimination of the immediate toxicity over the short time and resulted in an anticancer effect after 3 days. A synergistic effect of the chemo-photothermal therapy showed a moderate hyperthermia assistance (<1 W/cm(2)) and better anticancer performance over time compared with the individual treatments. We demonstrated that such PSMA-based methodology not only enables a broad range of chemical material synthesis in the kinetic control to form Au nano-octahedrons and nanotriangles using Br(-)/I(-) ions additives but also could be extended to form Au/Fe3O4@polymer nanocomposites via proton-assisted PSMA self-assembly. PMID:26501876

  13. Co-Delivery of Cisplatin Prodrug and Chlorin e6 by Mesoporous Silica Nanoparticles for Chemo-Photodynamic Combination Therapy to Combat Drug Resistance.

    PubMed

    Zhang, Wei; Shen, Jianliang; Su, Hua; Mu, Ge; Sun, Jing-Hua; Tan, Cai-Ping; Liang, Xing-Jie; Ji, Liang-Nian; Mao, Zong-Wan

    2016-06-01

    Combination therapy shows great promise in circumventing cisplatin resistance. We report herein the development of a novel nanoscale drug delivery system (nDDS) based nanotherapeutic that combines chemotherapy and photodynamic therapy (PDT) into one single platform to achieve synergistic anticancer capacity to conquer cisplatin resistance. Mesoporous silica nanoparticle (MSNs) was used as the drug delivery vector to conjugate cisplatin prodrug and to load photosensitizer chlorin e6 (Ce6) to afford the dual drug loaded delivery system MSNs/Ce6/Pt. The hybrid nanoparticles have an average diameter of about 100 nm and slightly positive surface charge of about 18.2 mV. The MSNs/Ce6/Pt nanoparticles can be efficiently internalized by cells through endocytosis, thereby achieving much higher cellular Pt uptake than cisplatin in cisplatin-resistant A549R lung cancer cells. After 660 nm light irradiation (10 mW/cm(2)), the cellular reactive oxygen species (ROS) level in MSNs/Ce6/Pt treated cells was elevated dramatically. As a result of these properties, MSNs/Ce6/Pt exhibited very potent anticancer activity against A549R cells, giving a half-maximal inhibitory concentration (IC50) value for the combination therapy of 0.53 μM, much lower than that of cisplatin (25.1 μM). This study suggests the great potential of nDDS-based nanotherapeutic for combined chemo-photodynamic therapy to circumvent cisplatin resistance. PMID:27164222

  14. Enzyme replacement therapy for pancreatic insufficiency: present and future

    PubMed Central

    Fieker, Aaron; Philpott, Jessica; Armand, Martine

    2011-01-01

    Pancreatic enzyme replacement therapy is currently the mainstay of treatment for nutrient malabsorption secondary to pancreatic insufficiency. This treatment is safe and has few side effects. Data demonstrate efficacy in reducing steatorrhea and fat malabsorption. Effective therapy has been limited by the ability to replicate the physiologic process of enzyme delivery to the appropriate site, in general the duodenum, at the appropriate time. The challenges include enzyme destruction in the stomach, lack of adequate mixing with the chyme in the duodenum, and failing to deliver and activate at the appropriate time. Treatment is begun when clinically significant malabsorption occurs resulting in steatorrhea and weight loss. Treatment failure is addressed in a sequential fashion. Current research is aimed at studying new enzymes and delivery systems to improve the efficiency of action in the duodenum along with developing better means to monitor therapy. PMID:21753892

  15. Enzyme replacement therapy for Gaucher's disease: the early Canadian experience

    PubMed Central

    MacKenzie, J J; Amato, D; Clarke, J T

    1998-01-01

    BACKGROUND: The management of severe Gaucher's disease was dramatically improved by the development of enzyme replacement therapy. However, this treatment is very costly (currently about $21,000 per infusion for adults at the starting dose recommended by the manufacturer). The goal of this study was to determine how enzyme replacement therapy was being prescribed and financially supported in various parts of Canada. In addition, demographic and outcome information was elicited. METHODS: Prescribing physicians were identified through professional associations and with the help of the manufacturer of the enzyme preparations used for the treatment of Gaucher's disease. The physicians were surveyed by questionnaire in July 1995. The study included all patients in Canada who had received enzyme replacement therapy for Gaucher's disease before July 1, 1995. RESULTS: A total of 25 patients (15 children and 10 adults) with type 1 Gaucher's disease, the common nonneuronopathic variant of the disease, were receiving enzyme replacement therapy by the end of 1995. The indications for treatment included massive splenomegaly, growth failure, and severe bony, hematologic and pulmonary complications of the disease; no patients with mild disease were receiving treatment. Treatment regimens varied markedly (from 12 to 160 units of enzyme/kg per month). All the patients were reported to have responded well to therapy, based on serial measurements of hematologic indices, liver and spleen volumes, and numbers of bony crises as well as patients' subjective impressions. Financial support for therapy varied markedly from one province to another. None of the reporting physicians was aware of any patients with severe Gaucher's disease who were denied therapy as a result of inability to pay for the medication. Various agencies provided financial support for therapy, including both federal and provincial governments, private insurance carriers and the commercial supplier of the enzyme. In

  16. Sapropterin Hydrochloride: Enzyme Enhancement Therapy for Phenylketonuria

    PubMed Central

    Lachmann, Robin

    2011-01-01

    Phenylketonuria (PKU) is an inherited disorder of amino acid metabolism caused by deficiency of the enzyme phenylalanine hydroxylase (PAH). Historically PKU was a common genetic cause of severe learning difficulties and developmental delay, but with the introduction of newborn screening and early dietary management, it has become a treatable disease and people born with PKU should now have IQs and achievements similar to their peers. Dietary treatment, however, involves lifestyle changes that pervade most aspects of daily life for an individual and their family. A simple pharmacological treatment for PKU would have a great appeal. Sapropterin hydrochloride is a synthetic form of tetrahydrobiopterin, the cofactor for PAH. A proportion of mutant PAH enzymes show enhanced activity in the presence of pharmacological doses of sapropterin and, for some patients with milder forms of PKU, sapropterin can effectively lower plasma phenylalanine levels. This article discusses the potential place for sapropterin in the management of PKU and how this expensive orphan drug is being integrated into patient care in different healthcare systems. PMID:23148178

  17. Systemically Injectable Enzyme-Loaded Polyion Complex Vesicles as In Vivo Nanoreactors Functioning in Tumors.

    PubMed

    Anraku, Yasutaka; Kishimura, Akihiro; Kamiya, Mako; Tanaka, Sayaka; Nomoto, Takahiro; Toh, Kazuko; Matsumoto, Yu; Fukushima, Shigeto; Sueyoshi, Daiki; Kano, Mitsunobu R; Urano, Yasuteru; Nishiyama, Nobuhiro; Kataoka, Kazunori

    2016-01-11

    The design and construction of nanoreactors are important for biomedical applications of enzymes, but lipid- and polymeric-vesicle-based nanoreactors have some practical limitations. We have succeeded in preparing enzyme-loaded polyion complex vesicles (PICsomes) through a facile protein-loading method. The preservation of enzyme activity was confirmed even after cross-linking of the PICsomes. The cross-linked β-galactosidase-loaded PICsomes (β-gal@PICsomes) selectively accumulated in the tumor tissue of mice. Moreover, a model prodrug, HMDER-βGal, was successfully converted into a highly fluorescent product, HMDER, at the tumor site, even 4 days after administration of the β-gal@PICsomes. Intravital confocal microscopy showed continuous production of HMDER and its distribution throughout the tumor tissues. Thus, enzyme-loaded PICsomes are useful for prodrug activation at the tumor site and could be a versatile platform for enzyme delivery in enzyme prodrug therapy. PMID:26629778

  18. Management of chronic pancreatitis. Focus on enzyme replacement therapy.

    PubMed

    Dobrilla, G

    1989-01-01

    The goals of treatment with pancreatic extracts in patients with chronic relapsing pancreatitis are twofold: pain relief and control of maldigestion caused by exocrine pancreatic insufficiency. Experience with the use of pancreatic enzymes for analgesic purposes suggests that the less severe the pain, the greater the analgesic effect of these enzymes. However, the number of trials, as well as the number of patients treated, is fairly small and more studies in larger patient populations are needed. The use of pancreatic enzymes for maldigestion owing to exocrine pancreatic insufficiency which is secondary to chronic pancreatitis, pancreatectomy, cystic fibrosis, or GI bypass surgery incurs several problems. These problems are primarily caused by gastric inactivation of the enzymes, low enzyme activity of many commercial preparations and/or poor patient compliance. Treatment with conventional enzyme products (powdered extracts, enteric-coated tablets or capsules) has been disappointing. At best, results were inconsistent, showing a high degree of individual variation. The introduction of enzyme preparations in the form of pH-sensitive enteric-coated microspheres in hard gelatin capsules represents a significant advance. These microspheres are superior to conventional enzyme preparations in improving the symptoms of pancreatic insufficiency, particularly steatorrhea, where low doses of microspheres are as effective as large doses of conventional enzyme preparations. Steatorrhea, however, is rarely completely resolved. In cases refractory to therapy, treatment with the combination of pH-sensitive enteric-coated microspheres and H2-antagonists or prostaglandins has met with some success. PMID:2702247

  19. Synthesis and Biological Evaluation of a Novel Pentagastrin-Toxin Conjugate Designed for a Targeted Prodrug Mono-therapy of Cancer

    PubMed Central

    Tietze, Lutz F.; Panknin, Olaf; Krewer, Birgit; Major, Felix; Schuberth, Ingrid

    2008-01-01

    A novel carbamate prodrug 2 containing a pentagastrin moiety was synthesized. 2 was designed as a detoxified analogue of the highly cytotoxic natural antibiotic duocarmycin SA (1) for the use in a targeted prodrug monotherapy of cancers expressing cholecystokinin (CCK-B)/gastrin receptors. The synthesis of prodrug 2 was performed using a palladium-catalyzed carbonylation of bromide 6, followed by a radical cyclisation to give the pharmacophoric unit 10, coupling of 10 to the DNA-binding subunit 15 and transformation of the resulting seco-drug 3b into the carbamate 2 via addition of a pentagastrin moiety. PMID:19325786

  20. JCL Roundtable: enzyme replacement therapy for lipid storage disorders.

    PubMed

    Brown, W Virgil; Desnick, Robert J; Grabowski, Gregory A

    2014-01-01

    There are several inherited disorders that involve abnormal storage of lipids in tissues leading to severe compromise of organs. Sadly, these are often accompanied by lifelong morbidity and early mortality. Disorders such as Gaucher, Fabry, and lysosomal acid lipase deficiencies (Wolman and cholesteryl ester storage diseases) have been known for many years, and provide a difficult and frustrating set of problems for patients, their families, and their physicians. With recombinant methods of protein synthesis, it is now possible to literally replace the defective enzymes that underlie the basic pathophysiology of many such disorders. The delivery of these enzymes into the affected cells is possible because of their location in the lysosomes where the natural degradation of their lipid substrates occurs. I have asked 2 well-known investigators to join us for this Roundtable. These are professors who have been involved with the research that has made this type of therapy possible and who have participated in the clinical trials that demonstrated the value of enzyme replacement therapy. They are Dr. Robert Desnick, dean of Genetic and Genomic Medicine and professor and chairman emeritus of the Department of Genetics and Genomic Sciences at the Icahn School of Medicine at Mount Sinai in New York City, and Dr. Gregory Grabowski, professor of Microbiology, Biochemistry, and Pediatrics, at the University of Cincinnati College of Medicine. Dr. Grabowski recently retired from that school to become the chief science officer of Synageva, a company involved in producing enzymes for this type of therapy. PMID:25234559

  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. Rational Design of Antirheumatic Prodrugs Specific for Sites of Inflammation

    PubMed Central

    Onuoha, Shimobi C.; Ferrari, Mathieu; Sblattero, Daniele

    2015-01-01

    Objective Biologic drugs, such as the anti–tumor necrosis factor (anti‐TNF) antibody adalimumab, have represented a breakthrough in the treatment of rheumatoid arthritis. Yet, concerns remain over their lack of efficacy in a sizable proportion of patients and their potential for systemic side effects such as infection. Improved biologic prodrugs specifically targeted to the site of inflammation have the potential to alleviate current concerns surrounding biologic anticytokine therapies. The purpose of this study was to design, construct, and evaluate in vitro and ex vivo the targeting and antiinflammatory capacity of activatable bispecific antibodies. Methods Activatable dual variable domain (aDVD) antibodies were designed and constructed to target intercellular adhesion molecule 1 (ICAM‐1), which is up‐regulated at sites of inflammation, and anti‐TNF antibodies (adalimumab and infliximab). These bispecific molecules included an external arm that targets ICAM‐1 and an internal arm that comprises the therapeutic domain of an anti‐TNF antibody. Both arms were linked to matrix metalloproteinase (MMP)–cleavable linkers. The constructs were tested for their ability to bind and neutralize both in vitro and ex vivo targets. Results Intact aDVD constructs demonstrated significantly reduced binding and anti‐TNF activity in the prodrug formulation as compared to the parent antibodies. Human synovial fluid and physiologic concentrations of MMP enzyme were capable of cleaving the external domain of the antibody, revealing a fully active molecule. Activated antibodies retained the same binding and anti‐TNF inhibitory capacities as the parent molecules. Conclusion The design of a biologic prodrug with enhanced specificity for sites of inflammation (synovium) and reduced specificity for off‐target TNF is described. This construct has the potential to form a platform technology that is capable of enhancing the therapeutic index of drugs for the treatment of

  3. Bioengineering Novel Chimeric microRNA-34a for Prodrug Cancer Therapy: High-Yield Expression and Purification, and Structural and Functional Characterization.

    PubMed

    Wang, Wei-Peng; Ho, Pui Yan; Chen, Qiu-Xia; Addepalli, Balasubrahmanyam; Limbach, Patrick A; Li, Mei-Mei; Wu, Wen-Juan; Jilek, Joseph L; Qiu, Jing-Xin; Zhang, Hong-Jian; Li, Tianhong; Wun, Theodore; White, Ralph DeVere; Lam, Kit S; Yu, Ai-Ming

    2015-08-01

    Development of anticancer treatments based on microRNA (miRNA/miR) such as miR-34a replacement therapy is limited to the use of synthetic RNAs with artificial modifications. Herein, we present a new approach to a high-yield and large-scale biosynthesis, in Escherichia coli using transfer RNA (tRNA) scaffold, of chimeric miR-34a agent, which may act as a prodrug for anticancer therapy. The recombinant tRNA fusion pre-miR-34a (tRNA/mir-34a) was quickly purified to a high degree of homogeneity (>98%) using anion-exchange fast protein liquid chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-34a showed a favorable cellular stability while it was degradable by several ribonucleases. Deep sequencing and quantitative real-time polymerase chain reaction studies revealed that tRNA-carried pre-miR-34a was precisely processed to mature miR-34a within human carcinoma cells, and the same tRNA fragments were produced from tRNA/mir-34a and the control tRNA scaffold (tRNA/MSA). Consequently, tRNA/mir-34a inhibited the proliferation of various types of human carcinoma cells in a dose-dependent manner and to a much greater degree than the control tRNA/MSA, which was mechanistically attributable to the reduction of miR-34a target genes. Furthermore, tRNA/mir-34a significantly suppressed the growth of human non-small-cell lung cancer A549 and hepatocarcinoma HepG2 xenograft tumors in mice, compared with the same dose of tRNA/MSA. In addition, recombinant tRNA/mir-34a had no or minimal effect on blood chemistry and interleukin-6 level in mouse models, suggesting that recombinant RNAs were well tolerated. These findings provoke a conversation on producing biologic miRNAs to perform miRNA actions, and point toward a new direction in developing miRNA-based therapies. PMID:26022002

  4. Bioengineering Novel Chimeric microRNA-34a for Prodrug Cancer Therapy: High-Yield Expression and Purification, and Structural and Functional Characterization

    PubMed Central

    Wang, Wei-Peng; Ho, Pui Yan; Chen, Qiu-Xia; Addepalli, Balasubrahmanyam; Limbach, Patrick A.; Li, Mei-Mei; Wu, Wen-Juan; Jilek, Joseph L.; Qiu, Jing-Xin; Zhang, Hong-Jian; Li, Tianhong; Wun, Theodore; White, Ralph DeVere; Lam, Kit S.

    2015-01-01

    Development of anticancer treatments based on microRNA (miRNA/miR) such as miR-34a replacement therapy is limited to the use of synthetic RNAs with artificial modifications. Herein, we present a new approach to a high-yield and large-scale biosynthesis, in Escherichia coli using transfer RNA (tRNA) scaffold, of chimeric miR-34a agent, which may act as a prodrug for anticancer therapy. The recombinant tRNA fusion pre–miR-34a (tRNA/mir-34a) was quickly purified to a high degree of homogeneity (>98%) using anion-exchange fast protein liquid chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-34a showed a favorable cellular stability while it was degradable by several ribonucleases. Deep sequencing and quantitative real-time polymerase chain reaction studies revealed that tRNA-carried pre–miR-34a was precisely processed to mature miR-34a within human carcinoma cells, and the same tRNA fragments were produced from tRNA/mir-34a and the control tRNA scaffold (tRNA/MSA). Consequently, tRNA/mir-34a inhibited the proliferation of various types of human carcinoma cells in a dose-dependent manner and to a much greater degree than the control tRNA/MSA, which was mechanistically attributable to the reduction of miR-34a target genes. Furthermore, tRNA/mir-34a significantly suppressed the growth of human non–small-cell lung cancer A549 and hepatocarcinoma HepG2 xenograft tumors in mice, compared with the same dose of tRNA/MSA. In addition, recombinant tRNA/mir-34a had no or minimal effect on blood chemistry and interleukin-6 level in mouse models, suggesting that recombinant RNAs were well tolerated. These findings provoke a conversation on producing biologic miRNAs to perform miRNA actions, and point toward a new direction in developing miRNA-based therapies. PMID:26022002

  5. 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. PMID:26655063

  6. Theranostic Imaging of Cancer Gene Therapy.

    PubMed

    Sekar, Thillai V; Paulmurugan, Ramasamy

    2016-01-01

    Gene-directed enzyme prodrug therapy (GDEPT) is a promising therapeutic approach for treating cancers of various phenotypes. This strategy is independent of various other chemotherapeutic drugs used for treating cancers where the drugs are mainly designed to target endogenous cellular mechanisms, which are different in various cancer subtypes. In GDEPT an external enzyme, which is different from the cellular proteins, is expressed to convert the injected prodrug in to a toxic metabolite, that normally kill cancer cells express this protein. Theranostic imaging is an approach used to directly monitor the expression of these gene therapy enzymes while evaluating therapeutic effect. We recently developed a dual-GDEPT system where we combined mutant human herpes simplex thymidine kinase (HSV1sr39TK) and E. coli nitroreductase (NTR) enzyme, to improve therapeutic efficiency of cancer gene therapy by simultaneously injecting two prodrugs at a lower dose. In this approach we use two different prodrugs such as ganciclovir (GCV) and CB1954 to target two different cellular mechanisms to kill cancer cells. The developed dual GDEPT system was highly efficacious than that of either of the system used independently. In this chapter, we describe the complete protocol involved for in vitro and in vivo imaging of therapeutic cancer gene therapy evaluation. PMID:27424910

  7. 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. PMID:27060760

  8. Bioreductive prodrugs as cancer therapeutics: targeting tumor hypoxia

    PubMed Central

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

    2014-01-01

    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. PMID:23845143

  9. P450-dependent enzymes as targets for prostate cancer therapy.

    PubMed

    De Coster, R; Wouters, W; Bruynseels, J

    1996-01-01

    Metastatic prostate adenocarcinoma is a leading cause of cancer-related deaths among men. First line treatment is primarily aimed at blocking the synthesis and action of androgens. As primary endocrine treatment, androgen deprivation is usually achieved by orchidectomy or LHRH analogues, frequently combined with androgen receptor antagonists in order to block the residual adrenal androgens. However, nearly all the patients will eventually relapse. Available or potential second line therapies include, among others, alternative endocrine manipulations and chemotherapy. Cytochrome P450-dependent enzymes are involved in the synthesis and/or degradation of many endogenous compounds, such as steroids and retinoic acid. Some of these enzymes represent suitable targets for the treatment of prostate cancer. In first line therapy, inhibitors of the P450-dependent 17,20-lyase may achieve a maximal androgen ablation with a single drug treatment. Ketoconazole at high dose blocks both testicular and adrenal androgen biosynthesis but its side-effects, mainly gastric discomfort, limit its widespread use. A series of newly synthesized, more selective, steroidal 17,20-lyase inhibitors related to 17-(3-pyridyl)androsta-5,16-dien-3beta-ol, may open new perspectives in this field. In prostate cancer patients who relapse after surgical or medical castration, therapies aiming at suppressing the remaining adrenal androgen biosynthesis (ketoconazole) or producing a medical adrenalectomy (aminoglutethimide+hydrocortisone) have been used, but are becoming obsolete with the generalization of maximal androgen blockade in first line treatment. The role of inhibition of aromatase in prostate cancer therapy, which was postulated for aminoglutethimide, could not be confirmed by the use of more selective aromatase inhibitors, such as formestane. An alternative approach is represented by liarozole fumarate (LIA), a compound that blocks the P450-dependent catabolism of retinoic acid (RA). In vitro

  10. One-Step Way to Form Prodrug Micelles with High Amount Drug Loading.

    PubMed

    Zhang, Jing; Wu, Dan; Feng, Jie

    2016-06-01

    Prodrug micelles with high amount drug loading were obtained via one-step way. Antineoplastic drug doxorubicin (DOX), used as hydrophobic tail, was conjugated to hydrophilic head mPEG via hydrazone bonds, allowing drug release under intracellular condition. Free DOX was loaded into the hydrophobic core of micelles during the conjugation step simultaneously. Total drug content of the prodrug micelles was up to 61.2%. Endocytosis experiments confirmed that the prodrug micelles achieved good cellular-uptake ability. In vitro experiments indicated that the prodrug micelles showed better therapy efficacy than free drug in cancerous cells. PMID:27427600

  11. Enhanced cellular uptake and intracellular drug controlled release of VESylated gemcitabine prodrug nanocapsules.

    PubMed

    Fang, Yanfen; Du, Fang; Xu, Yanyun; Meng, Haijing; Huang, Jin; Zhang, Xiongwen; Lu, Wei; Liu, Shiyuan; Yu, Jiahui

    2015-04-01

    Gemcitabine, 2',2'-difluoro-2'-deoxycytidine (dFdC), is the first-line antitumor agent in the treatment of pancreatic tumors. However, it possesses certain drawbacks, such as poor biological half-life resulted from rapid metabolism and the induction of resistance, leading to its restricted therapeutic potential. With the purpose of overcoming the above drawbacks, we developed a novel VESylated gemcitabine (VES-dFdC) prodrug by coupling the N4-amino group of the pyrimidine ring of dFdC to the carboxylic group of vitamin E succinate (VES). The resulting amphiphilic compound could protect the N4-amino group of the pyrimidine ring of dFdC from being degraded by cytidine deaminase. What is more, the prodrug was able to form nanocapsules in aqueous media (similar to the structure of cytomembrane), confirmed by transmission electron microscope (TEM). Their average particle size is about 107 nm with zeta potential of -33.4 mV measured by dynamic light scattering (DLS). VES-dFdC nanocapsules showed accelerated accumulative drug release profile in simulated lysosome environment (sodium acetate buffer pH 5+cathepsin B, an enzyme in lysosome), due to the easily hydrolyzed property of amide bond by cathepsin B, while rather stable in PBS (pH 7.4) or sodium acetate buffer (pH 5.0) without cathepsin B, indicating their enhanced intracellular drug controlled release manner. Besides, VES-dFdC prodrug nanocapsules showed enhanced cellular uptake ability, and the amount of cellular uptake of the nanocapsules by the pancreatic cancer cell line BxPC-3 is seventy times higher than that of native gemcitabine in the first 1.5 h. Compared with free gemcitabine, VES-dFdC nanocapsules showed essentially increased growth inhibition activity against BxPC-3 cells, indicating its great potential as prodrug for pancreatic tumor therapy with improved antitumor activity. PMID:25746328

  12. Sphingolipid metabolism enzymes as targets for anticancer therapy.

    PubMed

    Kok, J W; Sietsma, H

    2004-05-01

    Treatment with anti-cancer agents in most cases ultimately results in apoptotic cell death of the target tumor cells. Unfortunately, tumor cells can develop multidrug resistance, e.g., by a reduced propensity to engage in apoptosis by which they become insensitive to multiple chemotherapeutics. Ceramide. the central molecule in cellular sphingolipid metabolism, has been recognized as an important mediator of apoptosis. Moreover, an increased cellular capacity for ceramide glycosylation has been identified as a novel multidrug resistance mechanism. Indeed, virtually all multidrug resistant cell types exhibit a deviating sphingolipid composition, most typically an increased level of glucosylceramide. Thus, the enzyme glucosylceramide synthase, which converts ceramide into glucosylceramide, has emerged as a potential target to increase apoptosis and decrease drug resistance of tumor cells. In addition, several other steps in the pathways of sphingolipid metabolism arc altered in multidrug resistant cells, opening a perspective on additional sphingolipid metabolism enzymes as targets for anti-cancer therapy. In this article, we present an overview of the current understanding concerning drug resistance-related changes in sphingolipid metabolism and how interference with this metabolism can be exploited to over come multidrug resistance. PMID:15134220

  13. High-dose enzyme replacement therapy in murine Hurler syndrome.

    PubMed

    Ou, Li; Herzog, Tyler; Koniar, Brenda L; Gunther, Roland; Whitley, Chester B

    2014-02-01

    Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease that is systemic, including progressive neurodegeneration, mental retardation and death before the age of 10 years. MPS I results from deficiency of α-L-iduronidase (IDUA) in lysosomes and subsequent accumulation of glycosaminoglycans (GAG). Clinical enzyme replacement therapy (ERT) with intravenous laronidase reverses some aspects of MPS I disease (e.g., hepatomegaly, splenomegaly, glycosaminoglycanuria) and ameliorates others (e.g., pulmonary function, cardiac disease, arthropathy, exercise tolerance). However, neurologic benefits are thought to be negligible because the blood-brain barrier (BBB) blocks enzyme from reaching the central nervous system (CNS). We considered the possibility that a very high dose of intravenous laronidase might be able to traverse the BBB in small quantities, and provide some metabolic correction in the brain. To address this question, high-dose laronidase was administered (11.6 mg/kg, once per week, 4 weeks) to adult MPS I mice. IDUA enzyme activity in the cortex of treated mice increased to 97% of that in wild type mice (p<0.01). GAG levels in cortex were reduced by 63% of that from untreated MPS I mice (p<0.05). Further, immunohistochemical analysis showed that treatment reduced secondary GM3-ganglioside accumulation in treated MPS I mice. Water T-maze tests showed that the learning abnormality in MPS I mice was reduced (p<0.0001). In summary, repeated, high-dose ERT facilitated laronidase transit across the BBB, reduced GAG accumulation within the CNS, and rescued cognitive impairment. PMID:24100243

  14. A novel anticancer theranostic pro-prodrug based on hypoxia and photo sequential control.

    PubMed

    Feng, Weipei; Gao, Chunyue; Liu, Wei; Ren, Huihui; Wang, Chao; Ge, Kun; Li, Shenghui; Zhou, Guoqiang; Li, Hongyan; Wang, Shuxiang; Jia, Guang; Li, Zhenhua; Zhang, Jinchao

    2016-08-01

    A novel anticancer pro-prodrug (GMC-CAE-NO2) with diagnosis and therapy functions based on hypoxia and photo sequential control was designed. It provides a platform for constructing theranostic pro-prodrugs to release active drugs controlled by hypoxic status and UV illumination. PMID:27379361

  15. 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. PMID:26335418

  16. 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

  17. [Enzyme replacement therapy in a patient with Pompe disease].

    PubMed

    Fujikawa, Yoshinao; Kinoshita, Satoru; Miyamoto, Yusaku; Nakayama, Tojo; Endo, Yusaku; Sasaki, Masayuki

    2007-09-01

    Pompe disease is a rare autosomal recessive disease caused by the deficiency of acid alpha-glucosidase (GAA), which is required for the degradation of lysosomal glycogen. Glycogen accumulation in heart, muscle and liver eventually leads to muscle weakness, hepatomegaly and cardiomegaly. Although an approved therapy does not exist, the efficacy of enzyme replacement therapy (ERT) has recently been reported in multinational trials in Europe and the US. Here, we present data on the efficacy of recombinant human acid alpha-glucosidase (rhGAA) (provided by Genzyme Corporation) in a patient with Pompe disease. At 5 months of age, motor delay (could not raise his head) and cardiomegaly were observed. A definite diagnosis of Pompe disease was made at 8 months of age after the accumulation of glycogen in a muscle biopsy specimen was observed. This was confirmed by low GAA activity. Since then, motor delay predominated and he was unable to sit independently by age 2.5 years. Every 2 weeks, 20 mg/kg of rhGAA was infused intravenously. To assess the effectiveness, chest X-ray, echocardiography and auditory brain response were recorded. The patient was administered rhGAA for 26 months from 2 years and 8 months of age. Following the initiation of ERT, hepatomegaly and cardiac function (ejection fraction) were rapidly improved and motor function was gradually improved. At 4 years and 10 months, the patient could walk with support. No adverse event has been observed. It can be concluded that ERT with rhGAA is an effective and safe regimen for this case. PMID:17879614

  18. 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

  19. Prodrugs of anthracycline antibiotics suited for tumor-specific activation.

    PubMed

    Azoulay, M; Florent, J C; Monneret, C; Gesson, J P; Jacquesy, J C; Tillequin, F; Koch, M; Bosslet, K; Czech, J; Hoffman, D

    1995-09-01

    The two novel prodrugs 4 and 11 have been prepared from tetra-O-acetyl-D-galactopyranose and doxorubicin in three and six steps, respectively. Their low cytotoxicity, high stability in plasma and, in the case of 11, efficient hydrolysis in the presence of alpha-galactosidase, fulfill preliminary conditions for their use in combination with monoclonal antibody-enzyme conjugates. PMID:7575986

  20. Prodrug Strategies for Paclitaxel.

    PubMed

    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

  1. 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

  2. Anti-Angiogenesis Therapy in the Vx2 Rabbit Cancer Model with a Lipase-cleavable Sn 2 Taxane Phospholipid Prodrug using αvβ3-Targeted Theranostic Nanoparticles

    PubMed Central

    Pan, Dipanjan; Schmieder, Anne H.; Wang, Kezheng; Yang, Xiaoxia; Senpan, Angana; Cui, Grace; Killgore, Kendall; Kim, Benjamin; Allen, John S.; Zhang, Huiying; Caruthers, Shelton D.; Shen, Baozhong; Wickline, Samuel A.; Lanza, Gregory M.

    2014-01-01

    In nanomedicine, the hydrophobic nature of paclitaxel has favored its incorporation into many nanoparticle formulations for anti-cancer chemotherapy. At lower doses taxanes are reported to elicit anti-angiogenic responses. In the present study, the facile synthesis, development and characterization of a new lipase-labile docetaxel prodrug is reported and shown to be an effective anti-angiogenic agent in vitro and in vivo. The Sn 2 phosphatidylcholine prodrug was stably incorporated into the lipid membrane of αvβ3-integrin targeted perfluorocarbon (PFC) nanoparticles (αvβ3-Dxtl-PD NP) and did not appreciably release during dissolution against PBS buffer or plasma over three days. Overnight exposure of αvβ3-Dxtl-PD NP to plasma spiked with phospholipase enzyme failed to liberate the taxane from the membrane until the nanoparticle integrity was compromised with alcohol. The bioactivity and efficacy of αvβ3-Dxtl-PD NP in endothelial cell culture was as effective as Taxol® or free docetaxel in methanol at equimolar doses over 96 hours. The anti-angiogenesis effectiveness of αvβ3-Dxtl-PD NP was demonstrated in the Vx2 rabbit model using MR imaging of angiogenesis with the same αvβ3-PFC nanoparticle platform. Nontargeted Dxtl-PD NP had a similar MR anti-angiogenesis response as the integrin-targeted agent, but microscopically measured decreases in tumor cell proliferation and increased apoptosis were detected only for the targeted drug. Equivalent dosages of Abraxane® given over the same treatment schedule had no effect on angiogenesis when compared to control rabbits receiving saline only. These data demonstrate that αvβ3-Dxtl-PD NP can reduce MR detectable angiogenesis and slow tumor progression in the Vx2 model, whereas equivalent systemic treatment with free taxane had no benefit. PMID:24723979

  3. 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

  4. Cytochrome P450-based cancer gene therapy: current status.

    PubMed

    Kan, On; Kingsman, Susan; Naylor, Stuart

    2002-12-01

    Results from a number of preclinical studies have demonstrated that a P450-based gene-directed enzyme prodrug therapy (GDEPT) strategy for the treatment of cancer is both safe and efficacious. This strategy has now moved forward into the clinic. At least two different approaches using different delivery methods (retroviral vector MetXia [Oxford BioMedica] and encapsulated P450 expressing cells), different cytochrome P450 isoforms (human CYP2B6 versus rat CYP2B1) and different prodrugs (cyclophosphamide [CPA] versus ifosfamide [IFA]) have concluded Phase I/II clinical trial with encouraging results. In the future, P450-based GDEPT can potentially be further enhanced by improved vectors for P450 gene delivery and disease-targeted promoters for focused gene expression at the target site. In addition, there is scope for developing synthetic P450s and their respective prodrugs to improve both enzyme kinetics and the profile of the active moiety. PMID:12517265

  5. 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. PMID:26004498

  6. 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

  7. Synthesis and Preclinical Evaluation of a Highly Improved Anticancer Prodrug Activated by Histone Deacetylases and Cathepsin L

    PubMed Central

    Ueki, Nobuhide; Wang, Wei; Swenson, Cooper; McNaughton, Caroline; Sampson, Nicole S.; Hayman, Michael J.

    2016-01-01

    Lack of absolute selectivity against cancer cells is a major limitation for current cancer therapies. In the previous study, we developed a prodrug strategy for selective cancer therapy using a masked cytotoxic agent puromycin [Boc-Lys(Ac)-Puromycin], which can be sequentially activated by histone deacetylases (HDACs) and cathepsin L (CTSL) to kill cancer cells expressing high levels of both enzymes. Despite the promise as a selective cancer therapy, its requirement of relatively high dosage could be a potential issue in the clinical setting. To address this issue, we aimed to further improve the overall efficacy of our prodrug strategy. Since the proteolytic cleavage by CTSL is the rate-limiting step for the drug activation, we sought to improve the substrate structure for CTSL activity by modifying the α-amino protecting group of lysine. Here we show that protection with Fmoc [Fmoc-Lys(Ac)-Puromycin] exhibits a marked improvement in overall anticancer efficacy compared to the original Boc-Lys(Ac)-Puromycin and this is mainly due to the highly efficient cellular uptake besides its improved substrate structure. Furthermore, to address a concern that the improved drug efficacy might direct high toxicity to the normal cells, we confirmed that Fmoc-Lys(Ac)-Puromycin still retains excellent cancer selectivity in vitro and no obvious systemic off-target toxicity in vivo. Thus our preclinical evaluation data presented here demonstrate that the Fmoc-Lys(Ac)-Puromycin exhibits substantially improved anticancer efficacy, further supporting our approach for the selective cancer therapy. PMID:27162551

  8. CYP4F Enzymes Are the Major Enzymes in Human Liver Microsomes That Catalyze the O-Demethylation of the Antiparasitic Prodrug DB289 [2,5-Bis(4-amidinophenyl)furan-bis-O-methylamidoxime

    PubMed Central

    Wang, Michael Zhuo; Saulter, Janelle Y.; Usuki, Etsuko; Cheung, Yen-Ling; Hall, Michael; Bridges, Arlene S.; Loewen, Greg; Parkinson, Oliver T.; Stephens, Chad E.; Allen, James L.; Zeldin, Darryl C.; Boykin, David W.; Tidwell, Richard R.; Parkinson, Andrew; Paine, Mary F.; Hall, James Edwin

    2007-01-01

    DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime] is biotransformed to the potent antiparasitic diamidine DB75 [2,5-bis(4-amidinophenyl) furan] by sequential oxidative O-demethylation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b5/NADH-cytochrome b5 reductase. Enzymes responsible for catalyzing the DB289 O-demethylation pathway have not been identified. We report an in vitro metabolism study to characterize enzymes in human liver microsomes (HLMs) that catalyze the initial O-demethylation of DB289 (M1 formation). Potent inhibition by 1-aminobenzotriazole confirmed that M1 formation is catalyzed by P450 enzymes. M1 formation by HLMs was NADPH-dependent, with a Km and Vmax of 0.5 μM and 3.8 nmol/min/mg protein, respectively. Initial screening showed that recombinant CYP1A1, CYP1A2, and CYP1B1 were efficient catalysts of M1 formation. However, none of these three enzymes was responsible for M1 formation by HLMs. Further screening showed that recombinant CYP2J2, CYP4F2, and CYP4F3B could also catalyze M1 formation. An antibody against CYP4F2, which inhibited both CYP4F2 and CYP4F3B, inhibited 91% of M1 formation by HLMs. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, effectively inhibited M1 formation by HLMs. Inhibition studies with ebastine and antibodies against CYP2J2 suggested that CYP2J2 was not involved in M1 formation by HLMs. Additionally, ketoconazole preferentially inhibited CYP4F2, but not CYP4F3B, and partially inhibited M1 formation by HLMs. We conclude that CYP4F enzymes (e.g., CYP4F2, CYP4F3B) are the major enzymes responsible for M1 formation by HLMs. These findings indicate that, in human liver, members of the CYP4F subfamily biotransform not only endogenous compounds but also xenobiotics. PMID:16997912

  9. 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 ...

  10. [DELAYED RESULTS OF ENZYME REPLACEMENT THERAPY, PRESCRIBED BY RESULTS OF 13C-TRIGLYCERIDE BREATH TEST].

    PubMed

    Chernyavskiy, V V; Gvozdetska, L S

    2015-01-01

    Maldigestion persists in most patients with chronic pancreatitis (CP). The objective lipase and amylase insufficiency diagnosis is needed to achieve an adequate clinical response to oral pancreatic enzyme substitution therapy. The novel data is presented in the article about the role of 13C-mixed triglyceride breath test as a tool for exocrine pancreatic insufficiency diagnosis, for evaluating fat malabsorbtion in CP patients. 135 patients were included in the investigation. Delayed results of enzyme replacement therapy were estimated after 1 and 2 year of surveillance. It has been shown, that partial recovery of exocrine pancreatic function is possible, and replacement therapy leads to patients nutritional status improving. Thus 13C-triglyceride breath test could be useful tool in clinical practice for CP diagnosis. The test make it possible to choose the initial pancreatic enzyme dosage and are beneficial during the treatment for pancreatic enzyme dose correction. PMID:26827447

  11. 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

  12. Trial watch - inhibiting PARP enzymes for anticancer therapy.

    PubMed

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

    2016-03-01

    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

  13. 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.

  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. Role of Nanotechnology for Enzyme Replacement Therapy in Lysosomal Diseases. A Focus on Gaucher's Disease.

    PubMed

    Martín-Banderas, L; Holgado, M A; Durán-Lobato, M; Infante, J J; Álvarez-Fuentes, J; Fernández-Arévalo, M

    2016-01-01

    Lysosomal storage diseases (LSDs) comprise a group of rare inherited chronic syndromes that cause deficiency of specific native enzymes within the lysosomes. The macromolecular compounds that are usually catabolized by lysosomal enzymes are accumulated within these organelles, causing progressive damage to tissues, skeleton and organs and, in several cases, the central nervous system (CNS). The damage caused by substrate accumulation finally results in physical deterioration, functional impairment and potential death. Up to date, the most promising therapy for most LSDs is enzyme-replacement therapy (ERT), which provides patients with the corresponding active enzyme. However, these enzymes do not have enough stability in blood, the treatment must be therefore periodically administrated by i.v. infusion under medical supervision, and immunogenicity issues are frequent. In addition, affected areas within the CNS, where the blood-brain barrier (BBB) is a major obstacle, cannot be reached by the enzymes. Nanotechnology can provide useful carriers to successfully protect and preserve enzymes, and transport them through the BBB towards brain locations. Several strategies based on targeting specific receptors on the BBB have led to nanoparticles that successfully carry sensitive molecules to the brain. Then, the main LSDs are described and a thorough review of nanotechnology strategies for brain delivery studied up to date is presented. PMID:26860997

  16. 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. PMID:18930676

  17. Modulating lipophilicity of rohitukine via prodrug approach: Preparation, characterization, and in vitro enzymatic hydrolysis in biorelevant media.

    PubMed

    Kumar, Vikas; Bharate, Sonali S; Vishwakarma, Ram A

    2016-09-20

    Rohitukine is a medicinally important natural product which has inspired the discovery of two anticancer clinical candidates. Rohitukine is highly hydrophilic in nature which hampers its oral bioavailability. Thus, herein our objective was to improve the drug-like properties of rohitukine via prodrug-strategy. Various ester prodrugs were synthesized and studied for solubility, lipophilicity, chemical stability and enzymatic hydrolysis in plasma/esterase. All prodrugs displayed lower aqueous solubility and improved lipophilicity compared with rohitukine, which was in accordance with the criteria of compounds in drug-discovery. The stability of synthesized prodrugs was evaluated in buffers at different pH, SGF, SIF, rat plasma and in esterase enzyme. The rate of hydrolysis in all incubation media was dependent primarily on the acyl promoieties. Hexanoyl ester prodrug of rohitukine, 3d, was stable under chemical conditions; however it was completely hydrolyzed to rohitukine, in plasma and in esterase in 4h. Hexanoate ester 3d appeared to be the most promising prodrug as it remained intact at gastric/intestinal pH and was completely transformed to the parent compound in plasma as desired for an ideal prodrug. The data presented herein, will help in designing prodrugs with desired physicochemical properties in future in structurally similar chemotypes. PMID:27422078

  18. Successful noninvasive ventilation and enzyme replacement therapy in an adult patient with morbus hunter.

    PubMed

    Westhoff, M; Litterst, P

    2012-01-01

    M. Hunter is characterized by an accumulation of mucopolysaccharides in cells, blood, and connective tissue as a consequence of a deficiency of the enzyme iduronate-2-sulfatase. Unlike enzyme replacement therapy with idursulfase in children, there is limited long-term experience in adult patients with Morbus Hunter.The case presented here describes the development of a man born in 1971 who was admitted to Hemer Lung Clinic in 2005 with severe obstructive sleep apnea, pulmonary functional impairment, and ventilatory failure (FEV 1: 0.8 L, VC: 1.0 L; pO(2): 52 mmHg; pCO(2): 81 mmHg, 6 MWT: 100 m). Initially, the patient received symptomatic treatment with noninvasive ventilation, which achieved a considerable improvement in pulmonary function and a normalization of blood gasses. Since February 2008, the patient received additional enzyme replacement therapy with idursulfase, which resulted in a further significant functional improvement (FEV1: 1.6; VC: 2.3 L; VO(2)max: 1,350 mL or 28.1 mL/kg body weight), in a normalization of prior elevated pulmonary artery pressures and also in impressive changes in the physiognomy and joint mobility. In November 2010, the polysomnography and nocturnal blood gas analysis without NIV showed only a mild obstructive sleep-related breathing disorder with no sign of hypoventilation. Therapy was changed to nocturnal CPAP therapy with a constant pressure of 6 cm H(2)O. Additional administration of oxygen was not required. With this therapy, the patient has been asymptomatic up to September 2011.Adult Hunter patients also benefit from enzyme replacement therapy and, in restrictive ventilatory defects with hypoventilation, from symptomatic therapy with noninvasive ventilation. PMID:23430920

  19. 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

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

    PubMed

    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

  1. 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

  2. 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

  3. 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. PMID:26684479

  4. Long Term Treatment with Enzyme Replacement Therapy in Patients with Fabry Disease.

    PubMed

    Oder, Daniel; Nordbeck, Peter; Wanner, Christoph

    2016-01-01

    Anderson-Fabry disease is a potentially life-threatening hereditary lysosomal storage disorder taking origin in over 1,000 known pathogenic mutations in the alpha-galactosidase A encoding gene. Over the past 15 years, intravenous replacement therapy of the deficient alpha agalsidase A enzyme has been well-established retarding the progression of a multisystemic disease and organ involvement. Despite this innovative treatment approach, premature deaths still do occur. The response to enzyme replacement therapy (ERT) varies considerably and appears to depend on gender, genotype (classic or later onset/non-classic), stage of disease or age and agalsidase inhibition by anti-agalsidase antibodies. Early ERT treatment at young age, a personalized approach, and adjunctive therapies for specific disease manifestations appear to impact on prognosis and are currently favored with the expectance of more effective intravenous and oral treatments in the short future. PMID:27576727

  5. A Screen for and Validation of Prodrug Antimicrobials

    PubMed Central

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

    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. PMID:24342644

  6. Acid ceramidase and the treatment of ceramide diseases: The expanding role of enzyme replacement therapy.

    PubMed

    Schuchman, Edward H

    2016-09-01

    Ceramides are a diverse group of sphingolipids that play important roles in many biological processes. Acid ceramidase (AC) is one key enzyme that regulates ceramide metabolism. Early research on AC focused on the fact that it is the enzyme deficient in the rare genetic disorder, Farber Lipogranulomatosis. Recent research has revealed that deficiency of the same enzyme is responsible for a rare form of spinal muscular atrophy associated with myoclonic epilepsy (SMA-PME). Due to their diverse role in biology, accumulation of ceramides also has been implicated in the pathobiology of many other common diseases, including infectious lung diseases, diabetes, cancers and others. This has revealed the potential of AC as a therapy for many of these diseases. This review will focus on the biology of AC and the potential role of this enzyme in the treatment of human disease. PMID:27155573

  7. Albumin-binding caspase-cleavable prodrug that is selectively activated in radiation exposed local tumor.

    PubMed

    Chung, Seung Woo; Choi, Jeong Uk; Lee, Beom Seok; Byun, Julia; Jeon, Ok-Cheol; Kim, Seong Who; Kim, In-San; Kim, Sang Yoon; Byun, Youngro

    2016-07-01

    Existence of the genomically and epigenomically diverse subclones in a tumor severely limits the therapeutic efficacy of targeted agents. To overcome such a limitation, we prepared a novel targeted prodrug, EMC-DEVD-S-DOX, which comprises two important features: radiation-induced apoptosis targeting and albumin-binding properties. In particular, the prodrug binds circulating albumin after intravenous administration and then activated by caspase-3, which is upregulated from apoptotic cells that responded to radiotherapy. The prodrug was designed to bind circulating albumin to extend half-life and facilitate tumor accumulation in order to increase the possibility of contacting caspase-3, which is only transiently upregulated during apoptosis. Our results showed that EMC-DEVD-S-DOX had a prolonged half-life with enhanced tumor accumulation, which clearly benefited the therapeutic effect of the prodrug. Also, agreeing with the in vitro studies that showed ignorable cytotoxic effect in the absence of caspase-3, the prodrug was effective only when combined with radiotherapy without any noticeable systemic toxicity in vivo. Due to the highly selective action of EMC-DEVD-S-DOX independent to the complex genomic profiles of tumor, the prodrug would overcome the limitation of current targeted therapy and potentiate radiotherapy in the clinical oncology. PMID:27085176

  8. Pharmacogenomics of drug metabolizing enzymes and transporters: implications for cancer therapy

    PubMed Central

    Li, Jing; Bluth, Martin H

    2011-01-01

    The new era of personalized medicine, which integrates the uniqueness of an individual with respect to the pharmacokinetics and pharmacodynamics of a drug, holds promise as a means to provide greater safety and efficacy in drug design and development. Personalized medicine is particularly important in oncology, whereby most clinically used anticancer drugs have a narrow therapeutic window and exhibit a large interindividual pharmacokinetic and pharmacodynamic variability. This variability can be explained, at least in part, by genetic variations in the genes encoding drug metabolizing enzymes, transporters, or drug targets. Understanding of how genetic variations influence drug disposition and action could help in tailoring cancer therapy based on individual’s genetic makeup. This review focuses on the pharmacogenomics of drug metabolizing enzymes and drug transporters, with a particular highlight of examples whereby genetic variations in the metabolizing enzymes and transporters influence the pharmacokinetics and/or response of chemotherapeutic agents. PMID:23226051

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

    PubMed

    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 [(13)C]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

  10. 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

  11. 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. PMID:22154081

  12. 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

  13. RTB Lectin: a novel receptor-independent delivery system for lysosomal enzyme replacement therapies

    PubMed Central

    Acosta, Walter; Ayala, Jorge; Dolan, Maureen C.; Cramer, Carole L.

    2015-01-01

    Enzyme replacement therapies have revolutionized patient treatment for multiple rare lysosomal storage diseases but show limited effectiveness for addressing pathologies in “hard-to-treat” organs and tissues including brain and bone. Here we investigate the plant lectin RTB as a novel carrier for human lysosomal enzymes. RTB enters mammalian cells by multiple mechanisms including both adsorptive-mediated and receptor-mediated endocytosis, and thus provides access to a broader array of organs and cells. Fusion proteins comprised of RTB and human α-L-iduronidase, the corrective enzyme for Mucopolysaccharidosis type I, were produced using a tobacco-based expression system. Fusion products retained both lectin selectivity and enzyme activity, were efficiently endocytosed into human fibroblasts, and corrected the disease phenotype of mucopolysaccharidosis patient fibroblasts in vitro. RTB-mediated delivery was independent of high-mannose and mannose-6-phosphate receptors, which are exploited for delivery of currently approved lysosomal enzyme therapeutics. Thus, the RTB carrier may support distinct in vivo pharmacodynamics with potential to address hard-to-treat tissues. PMID:26382970

  14. Synthesis and Biological Evaluation of Phosphate Prodrugs of 4-Phospho-d -erythronohydroxamic Acid, an Inhibitor of 6-Phosphogluconate Dehydrogenase

    PubMed Central

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

    2007-01-01

    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. PMID:17615587

  15. Early administration of enzyme replacement therapy for Pompe disease: short-term follow-up results.

    PubMed

    Hamdan, M A; Almalik, M H; Mirghani, H M

    2008-12-01

    Pompe disease (glycogen storage disease II, OMIM # 232300), is a hereditary lysosomal disorder. It is characterized by deficiency of acid alpha-glucosidase enzyme (acid maltase, GAA, OMIM *606800, EC 3.1.26.2), secondary to mutations in the GAA gene (HGNC:4065) on chromosome 17q25.2-q25.3. Absent enzyme activity in the infantile form of Pompe disease results in abnormal glycogen deposition in the skeletal, cardiac, and smooth muscles, leading to hypertrophic cardiomyopathy, feeding abnormalities, hypotonia, weakness, respiratory insufficiency, and ultimately death. Prenatal diagnosis is accomplished by enzyme assay, mutation analysis or electron microscopy of amniotic fluid cells or chorionic villus sample. However, these techniques may not always be available, and can result in perinatal morbidity and fetal loss. Early diagnosis of Pompe disease results in early institution of enzyme replacement therapy (ERT), which minimizes morbidity and prolongs survival. We report the case of a 35-week part-of-twin neonate, whose older sibling died earlier because of infantile Pompe disease. At 32 weeks of gestation, fetal echocardiography showed hypertrophic cardiomyopathy in twin 1, which persisted until birth at 35 weeks of gestation. Diagnosis was confirmed after birth by enzyme assay, and mutation analysis showing homozygosity for the sequence change 1327-2A>G (GAA intr 8). Administration of ERT at 18 h of age, resulted in normalization of cardiac abnormalities within 21 weeks of therapy, and normal neurodevelopmental assessment at 46 weeks, using Griffiths Mental Development Scales. To our knowledge, this is the youngest patient reported to receive ERT for Pompe disease, and the first report of prenatal diagnosis of Pompe disease by fetal echocardiography. PMID:19067231

  16. AB126. Enzyme replacement therapy in patient with mucopolysaccharidosis type I: a case report

    PubMed Central

    Can, Ngoc Thi Bich; Vu, Dung Chi; Le, Hang Thi Thuy; Nguyen, Khanh Ngoc; Bui, Huong Thi

    2015-01-01

    Background and objective Mucopolysaccharidosis I (MPS I) is a rare, recessively inherited, lysosomal storage disorder caused by deficiency on the enzyme α-L-iduronidase. This defect results in accumulation of heparan and dermatan sulfate in different tissues and organs due to a deficiency in the catabolism of glycosaminoglycans. The overall incidence of MPS I is 0.99-1.99/100,000 live births. Enzyme replacement therapy (ERT) with recombinant α-L-iduronidase (laronidase) has shown to significantl improve the quality of life in children. To describe clinical characteristics, enzyme activity and genetic finding in the first Vietnamese patient with MPS type I with aldurazyme replacement therapy. Methods Clinical features, biochemical finding, enzyme activity, mutation analysis and management in a 4 years 6-month-old girl was study. Based on analysis of a patient’s clinical symptoms associated with enzyme α-L-iduronidase activity measurement in leukocyte, the diagnosis of MPS type I was therefore made. Genomic DNAs were extracted from peripheral blood leukocytes from the patient and identify mutation of IDUA gene, 14 exons and their intronic boundaries of the IDUA gene were sequenced using genomic DNA from the patient. The patient has been treated with aldurazyme infusion every week with the dose of 0.58 mg/kg/week. Results A 4 years 6-month-old girl was presented with joint stiffness at 2 years old. She was admitted with the features of short status, coarse facial, corneal clouding, carpel tunnel syndrome and joint stiffness, kyphosis, abdominal distension, palpable liver at 3 cm below the costal margin, sleep disturbances/snoring. Laboratory showed: hearing lost at right ear in acoumetry, hepatosplenomegaly in ultrasound with right liver length of 117 mm, spleen length of 89 mm, a 6-minute walk test distance of 158.6 m, α-I-duronidase 0.43 nmoL/mg Prot/hrs (normal: 41.8±15.9), urine glycosaminoglycan (GAG) of 508.83 mg/g creatinine (normal: 10.74-112.02). PCR

  17. AB127. Enzyme replacement therapy in patient with mucopolysaccharidosis type II: a case report

    PubMed Central

    Can, Ngoc Thi Bich; Vu, Dung Chi; Le, Hang Thi Thuy; Nguyen, Khanh Ngoc; Bui, Huong Thi

    2015-01-01

    Background and objective Mucopolysaccharidosis (MPS) type II (Hunter syndrome) is an X-linked lysosomal storage disorder due to the deficit of iduronate 2-sulfatase, an enzyme catalysing the degradation of the glycosaminoglycans (GAG) dermatan- and heparan-sulfate. Treatment of the disease is mainly performed by enzyme replacement therapy (ERT) with idursulfase. This article aims to describe clinical characteristics, enzyme activity and genetic finding in the first Vietnamese patient with MPS type II treated with idursulfase (Elaprase) replacement therapy. Methods Clinical features, biochemical finding, enzyme activity, mutation analysis and management in a 4 years 6-month-old girl was study. Based on analysis of a patient’s clinical symptoms associated with enzyme iduronate-2-sulphate sulphatase activity measurement in plasma, the diagnosis of MPS type II was therefore made. Genomic DNAs were extracted from peripheral blood leukocytes from the patient and identify mutation of IDS gene, nine exons and their intronic boundaries of the IDS gene were sequenced using genomic DNA from the patient. The patient has been treated with Elaprase infusion every week with the dose of 0.05 mg/kg/week. Results A 34-month-old boy was presented with coarse facial at 24 months of age. He was admitted with the features of coarse facial, with frontal bossing, prominent supraorbital ridge, large nose and flat nasal bridge, widely spaced teeth, thickened gingival mucosa, and macroglossia, broadly built of the body habitus with a short neck, broad chest, and protuberant abdomen and Mongolian spots at the back and breech, joint finger stiffness, abdominal distension, palpable liver at 3 cm below the costal margin, sleep disturbances/snoring, mental development delay. Laboratory showed: hepatosplenomegaly in ultrasound with right liver length of 127 mm, spleen length of 93 mm, a 6-minute walk test distance of 240 m, DQ 55%, α iduronate sulphate: 0 nmoL/4 h/mL plasma (normal: 600

  18. Dual-Mode HDAC Prodrug for Covalent Modification and Subsequent Inhibitor Release

    PubMed Central

    2016-01-01

    Histone deacetylase inhibitors (HDACi) target abnormal epigenetic states associated with a variety of pathologies, including cancer. Here, the development of a prodrug of the canonical broad-spectrum HDACi suberoylanilide hydroxamic acid (SAHA) is described. Although hydroxamic acids are utilized universally in the development of metalloenzyme inhibitors, they are considered to be poor pharmacophores with reduced activity in vivo. We developed a prodrug of SAHA by appending a promoiety, sensitive to thiols, to the hydroxamic acid warhead (termed SAHA-TAP). After incubation of SAHA-TAP with an HDAC, the thiol of a conserved HDAC cysteine residue becomes covalently tagged with the promoiety, initiating a cascade reaction that leads to the release of SAHA. Mass spectrometry and enzyme kinetics experiments validate that the cysteine residue is covalently appended with the TAP promoiety. SAHA-TAP demonstrates cytotoxicity activity against various cancer cell lines. This strategy represents an original prodrug design with a dual mode of action for HDAC inhibition. PMID:25974739

  19. Antimalarial activity of prodrugs of N-branched acyclic nucleoside phosphonate inhibitors of 6-oxopurine phosphoribosyltransferases.

    PubMed

    Hocková, Dana; Janeba, Zlatko; Naesens, Lieve; Edstein, Michael D; Chavchich, Marina; Keough, Dianne T; Guddat, Luke W

    2015-09-01

    Acyclic nucleoside phosphonates (ANPs) that contain a 6-oxopurine base are good inhibitors of the human and Plasmodium falciparum 6-oxopurine phosphoribosyltransferases (PRTs), key enzymes of the purine salvage pathway. Chemical modifications, based on the crystal structures of several inhibitors in complex with the human PRTase, led to the design of a new class of inhibitors--the aza-ANPs. Because of the negative charges of the phosphonic acid moiety, their ability to cross cell membranes is, however, limited. Thus, phosphoramidate prodrugs of the aza-ANPs were prepared to improve permeability. These prodrugs arrest parasitemia with IC50 values in the micromolar range against Plasmodium falciparum-infected erythrocyte cultures (both chloroquine-sensitive and chloroquine-resistant Pf strains). The prodrugs exhibit low cytotoxicity in several human cell lines. Thus, they fulfill two essential criteria to qualify them as promising antimalarial drug leads. PMID:26275679

  20. Catalytic nucleic acid enzymes for the study and development of therapies in the central nervous system

    PubMed Central

    Tritz, Richard; Habita, Cellia; Robbins, Joan M.; Gomez, German G.; Kruse, Carol A.

    2005-01-01

    Summary Nucleic acid enzymes have been used with great success for studying natural processes in the central nervous system (CNS). We first provide information on the structural and enzymatic differences of various ribozymes and DNAzymes. We then discuss how they have been used to explore new therapeutic approaches for treating diseases of the CNS. They have been tested in various systems modeling retinitis pigmentosum, proliferative vitreoretinopathy, Alzheimer's disease, and malignant brain tumors. For these models, effective targets for nucleic acid enzymes have been readily identified and the rules for selecting cleavage sites have been well established. The bulk of studies, including those from our laboratory, have emphasized their use for gliomas. With the availability of multiple excellent animal models to test glioma treatments, good progress has been made in the initial testing of nucleic acid enzymes for brain tumor therapy. However, opportunities still exist to significantly improve the delivery and efficacy of ribozymes to achieve effective treatment. The future holds significant potential for the molecular targeting and therapy of eye diseases, neurodegenerative disorders, and brain tumors with these unique treatment agents. PMID:16467915

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

    PubMed

    Nair, Vasu; Okello, Maurice

    2015-01-01

    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. PMID:26184144

  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

    2015-09-01

    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. PMID:26252229

  3. Synthesis of Mevalonate- and Fluorinated Mevalonate Prodrugs and Their in vitro Human Plasma Stability

    PubMed Central

    Kang, Soosung; Watanabe, Mizuki; Jacobs, JC; Yamaguchi, Masaya; Dahesh, Samira; Nizet, Victor; Leyh, Thomas S.; Silverman, Richard B.

    2014-01-01

    The mevalonate pathway is essential for the production of many important molecules in lipid biosynthesis. Inhibition of this pathway is the mechanism of statin cholesterol-lowering drugs, as well as the target of drugs to treat osteoporosis, to combat parasites, and to inhibit tumor cell growth. Unlike the human mevalonate pathway, the bacterial pathway appears to be regulated by diphosphomevalonate (DPM). Enzymes in the mevalonate pathway act to produce isopentenyl diphosphate, the product of the DPM decarboxylase reaction, utilize phosphorylated (charged) intermediates, which are poorly bioavailable. It has been shown that fluorinated DPMs (6-fluoro- and 6,6,6-trifluoro-5-diphosphomevalonate) are excellent inhibitors of the bacterial pathway; however, highly charged DPM and analogues are not bioavailable. To increase cellular permeability of mevalonate analogues, we have synthesized various prodrugs of mevalonate and 6-fluoro- and 6,6,6-trifluoromevalonate that can be enzymatically transformed to the corresponding DPM or fluorinated DPM analogues by esterases or amidases. To probe the required stabilities as potentially bioavailable prodrugs, we measured the half-lives of esters, amides, carbonates, acetals, and ketal promoieties of mevalonate and the fluorinated mevalonate analogues in human blood plasma. Stability studies showed that the prodrugs are converted to the mevalonates in human plasma with a wide range of half-lives. These studies provide stability data for a variety of prodrug options having varying stabilities and should be very useful in the design of appropriate prodrugs of mevalonate and fluorinated mevalonates. PMID:25461893

  4. Self-Immolative Polycations as Gene Delivery Vectors and Prodrugs Targeting Polyamine Metabolism in Cancer

    PubMed Central

    2015-01-01

    Polycations are explored as carriers to deliver therapeutic nucleic acids. Polycations are conventionally pharmacological inert with the sole function of delivering therapeutic cargo. This study reports synthesis of a self-immolative polycation (DSS-BEN) based on a polyamine analogue drug N1,N11-bisethylnorspermine (BENSpm). The polycation was designed to function dually as a gene delivery carrier and a prodrug targeting dysregulated polyamine metabolism in cancer. Using a combination of NMR and HPLC, we confirm that the self-immolative polycation undergoes intracellular degradation into the parent drug BENSpm. The released BENSpm depletes cellular levels of spermidine and spermine and upregulates polyamine catabolic enzymes spermine/spermidine N1-acetyltransferase (SSAT) and spermine oxidase (SMO). The synthesized polycations form polyplexes with DNA and facilitate efficient transfection. Taking advantage of the ability of BENSpm to sensitize cancer cells to TNFα-induced apoptosis, we show that DSS-BEN enhances the cell killing activity of TNFα gene therapy. The reported findings validate DSS-BEN as a dual-function delivery system that can deliver a therapeutic gene and improve the outcome of gene therapy as a result of the intracellular degradation of DSS-BEN to BENSpm and the subsequent beneficial effect of BENSpm on dysregulated polyamine metabolism in cancer. PMID:25153488

  5. Assessment of stability, toxicity and immunogenicity of new polymeric nanoreactors for use in enzyme replacement therapy of MNGIE.

    PubMed

    De Vocht, Caroline; Ranquin, An; Willaert, Ronnie; Van Ginderachter, Jo A; Vanhaecke, Tamara; Rogiers, Vera; Versées, Wim; Van Gelder, Patrick; Steyaert, Jan

    2009-08-01

    The lack of a crucial metabolic enzyme can lead to accumulating substrate concentrations in the bloodstream and severe human enzyme deficiency diseases. Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE) is such a fatal genetic disorder, caused by a thymidine phosphorylase deficiency. Enzyme replacement therapy is a strategy where the deficient enzyme is administered intravenously in order to decrease the toxic substrate concentrations. Such a therapy is however not very efficient due to the fast elimination of the native enzyme from the circulation. In this study we evaluate the potential of using polymeric enzyme-loaded nanoparticles to improve the delivery of therapeutic enzymes. We constructed new 200-nanometer PMOXA-PDMS-PMOXA polymeric nanoparticles that encapsulate the enzyme thymidine phosphorylase. These particles are permeabilised for substrates and products by the reconstitution of the nucleoside-specific porin Tsx in their polymeric wall. We show that the obtained 'nanoreactors' are enzymatically active and stable in blood serum at 37 degrees C. Moreover, they do not provoke cytotoxicity when incubated with hepatocytes for 4 days, nor do they induce a macrophage-mediated inflammatory response ex vivo and in vivo. All data highlight the potential of such nanoreactors for their application in enzyme replacement therapy of MNGIE. PMID:19371766

  6. Recombinant arginine-degrading enzymes in metabolic anticancer therapy and bioanalytics.

    PubMed

    Stasyk, Oleh V; Boretsky, Yuriy R; Gonchar, Mykhailo V; Sibirny, Andriy A

    2015-03-01

    Tumor cells often exhibit specific metabolic defects due to the aberrations in oncogene-dependent regulatory and/or signaling pathways that distinguish them from normal cells. Among others, many malignant cells are deficient in biosynthesis of certain amino acids and concomitantly exhibit elevated sensitivity to deprivation of these amino acids. Although the underlying causes of such metabolic changes are still not fully understood, this feature of malignant cells is exploited in metabolic enzymotherapies based on single amino acid, e.g., arginine, deprivation. To achieve efficient arginine depletion in vivo, two recombinant enzymes, bacterial arginine deiminase and human arginase I have been evaluated and are undergoing further development. This review is aimed to summarize the current knowledge on the application of arginine-degrading enzymes as anticancer agents and as bioanalytical tools for arginine assays. The problems that have to be solved to optimize this therapy for clinical application are discussed. PMID:25231409

  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. Evaluating Aziridinyl Nitrobenzamide Compounds as Leishmanicidal Prodrugs

    PubMed Central

    Voak, Andrew A.; Seifert, Karin; Helsby, Nuala A.

    2014-01-01

    Many of the nitroaromatic agents used in medicine function as prodrugs and must undergo activation before exerting their toxic effects. In most cases, this is catalyzed by flavin mononucleotide (FMN)-dependent type I nitroreductases (NTRs), a class of enzyme absent from higher eukaryotes but expressed by bacteria and several eukaryotic microbes, including trypanosomes and Leishmania. Here, we utilize this difference to evaluate whether members of a library of aziridinyl nitrobenzamides have activity against Leishmania major. Biochemical screens using purified L. major NTR (LmNTR) revealed that compounds containing an aziridinyl-2,4-dinitrobenzyl core were effective substrates for the enzyme and showed that the 4-nitro group was important for this activity. To facilitate drug screening against intracellular amastigote parasites, we generated leishmanial cells that expressed the luciferase reporter gene and optimized a mammalian infection model in a 96-well plate format. A subset of aziridinyl-2,4-dinitrobenzyl compounds possessing a 5-amide substituent displayed significant growth-inhibitory properties against the parasite, with the most potent agents generating 50% inhibitory concentrations of <100 nM for the intracellular form. This antimicrobial activity was shown to be LmNTR specific since L. major NTR+/− heterozygote parasites were slightly resistance to most aziridinyl dinitrobenzyl agents tested. When the most potent leishmanicidal agents were screened against the mammalian cells in which the amastigote parasites were propagated, no growth-inhibitory effect was observed at concentrations of up to 100 μM. We conclude that the aziridinyl nitrobenzamides represent a new lead structure that may have the potential to treat leishmanial infections. PMID:24165190

  9. Enzyme replacement therapy in Hurler syndrome after failure of hematopoietic transplant

    PubMed Central

    Arranz, Leonor; Aldamiz-Echevarria, Luis

    2015-01-01

    The most severe form of Mucopolysaccharosidosis type I (MPS-I), Hurler syndrome, presents with progressive respiratory, cardiac and musculoskeletal symptoms and cognitive deterioration. Treatment includes enzyme replacement therapy (ERT) and/or hematopoietic stem cell transplantation (HSCT). We describe the case of an 8-year old boy with MPS-I, homozygous for W402X, treated at 10 months of age with HSCT and after failure of the transplant, with ERT during 2 years showing good results, including a positive neuropsychological development. PMID:26937401

  10. [Hurler syndrome. Early diagnosis and successful enzyme replacement therapy: a new therapeutic approach. Case report].

    PubMed

    Dupont, C; El Hachem, C; Harchaoui, S; Ribault, V; Amiour, M; Guillot, M; Maire, I; Froissart, R; Guffon-Fouilhoux, N

    2008-01-01

    Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder due to alpha-L-iduronidase deficiency. Its severe prognosis has been significantly improved by enzyme replacement therapy using recombinant human alpha-L-iduronidase (laronidase). We report the case of a boy who was diagnosed at 19 months of age with Hurler's disease, the most severe form of MPS I, and received thereafter a treatment by laronidase, resulting in clinical and biological improvement. The aim of this case report is to draw physicians' attention on the presenting signs of Hurler's disease, in order to enable an earlier diagnosis, increasing the treatment's benefits. PMID:18162380

  11. Enzyme replacement therapy in Hurler syndrome after failure of hematopoietic transplant.

    PubMed

    Arranz, Leonor; Aldamiz-Echevarria, Luis

    2015-06-01

    The most severe form of Mucopolysaccharosidosis type I (MPS-I), Hurler syndrome, presents with progressive respiratory, cardiac and musculoskeletal symptoms and cognitive deterioration. Treatment includes enzyme replacement therapy (ERT) and/or hematopoietic stem cell transplantation (HSCT). We describe the case of an 8-year old boy with MPS-I, homozygous for W402X, treated at 10 months of age with HSCT and after failure of the transplant, with ERT during 2 years showing good results, including a positive neuropsychological development. PMID:26937401

  12. Hydrogen sulfide prodrugs-a review.

    PubMed

    Zheng, Yueqin; Ji, Xingyue; Ji, Kaili; Wang, Binghe

    2015-09-01

    Hydrogen sulfide (H2S) is recognized as one of three gasotransmitters together with nitric oxide (NO) and carbon monoxide (CO). As a signaling molecule, H2S plays an important role in physiology and shows great potential in pharmaceutical applications. Along this line, there is a need for the development of H2S prodrugs for various reasons. In this review, we summarize different H2S prodrugs, their chemical properties, and some of their potential therapeutic applications. PMID:26579468

  13. Design and Synthesis of Phosphotyrosine Peptidomimetic Prodrugs

    PubMed Central

    Garrido-Hernandez, Hugo; Moon, Kyung D.; Geahlen, Robert L.; Borch, Richard F.

    2008-01-01

    A novel approach to the intracellular delivery of aryl phosphates has been developed that utilizes a phosphoramidate-based prodrug approach. The prodrugs contain an ester group that undergoes reductive activation intracellularly with concomitant expulsion of a phosphoramidate anion. This anion undergoes intramolecular cyclization and hydrolysis to generate aryl phosphate exclusively with a t1/2 = ∼ 20 min. Phosphoramidate prodrugs (8-10) of phosphate-containing peptidomimetics that target the SH2 domain were synthesized. Evaluation of these peptidomimetic prodrugs in a growth inhibition assay and, in a cell-based transcriptional assay, demonstrated that the prodrugs had IC50 values in the low micromolar range. Synthesis of phosphorodiamidate analogs containing a P-NH-Ar linker (16 – 18) was also carried out in the hope that the phosphoramidates released might be phosphatase-resistant. Comparable activation rates and cell-based activities were observed for these prodrugs, but the intermediate phosphoramidate dianion underwent spontaneous hydrolysis with a t1/2 = ∼ 30 min. PMID:16722656

  14. 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. PMID:17924344

  15. 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

  16. 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.

  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

    2016-01-01

    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. PMID:26623947

  18. Enzyme-Instructed Self-Assembly: A Multistep Process for Potential Cancer Therapy

    PubMed Central

    2015-01-01

    The central dogma of the action of current anticancer drugs is that the drug tightly binds to its molecular target for inhibition. The reliance on tight ligand–receptor binding, however, is also the major root of drug resistance in cancer therapy. In this article, we highlight enzyme-instructed self-assembly (EISA)—the integration of enzymatic transformation and molecular self-assembly—as a multistep process for the development of cancer therapy. Using apoptosis as an example, we illustrate that the combination of enzymatic transformation and self-assembly, in fact, is an inherent feature of apoptosis. After the introduction of EISA of small molecules in the context of supramolecular hydrogelation, we describe several key studies to underscore the promises of EISA for developing cancer therapy. Particularly, we will highlight that EISA allows one to develop approaches to target “undruggable” targets or “untargetable” features of cancer cells and provides the opportunity for simultaneously interacting with multiple targets. We envision that EISA, used separately or in combination with current anticancer therapeutics, will ultimately lead to a paradigm shift for developing anticancer medicine that inhibit multiple hallmark capabilities of cancer. PMID:25933032

  19. Enzyme-instructed self-assembly: a multistep process for potential cancer therapy.

    PubMed

    Zhou, Jie; Xu, Bing

    2015-06-17

    The central dogma of the action of current anticancer drugs is that the drug tightly binds to its molecular target for inhibition. The reliance on tight ligand-receptor binding, however, is also the major root of drug resistance in cancer therapy. In this article, we highlight enzyme-instructed self-assembly (EISA)-the integration of enzymatic transformation and molecular self-assembly-as a multistep process for the development of cancer therapy. Using apoptosis as an example, we illustrate that the combination of enzymatic transformation and self-assembly, in fact, is an inherent feature of apoptosis. After the introduction of EISA of small molecules in the context of supramolecular hydrogelation, we describe several key studies to underscore the promises of EISA for developing cancer therapy. Particularly, we will highlight that EISA allows one to develop approaches to target "undruggable" targets or "untargetable" features of cancer cells and provides the opportunity for simultaneously interacting with multiple targets. We envision that EISA, used separately or in combination with current anticancer therapeutics, will ultimately lead to a paradigm shift for developing anticancer medicine that inhibit multiple hallmark capabilities of cancer. PMID:25933032

  20. 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. PMID:26083278

  1. Outcome after three years of laronidase enzyme replacement therapy in a patient with Hurler syndrome.

    PubMed

    Thomas, J A; Jacobs, S; Kierstein, J; Van Hove, J

    2006-12-01

    Enzyme replacement therapy (ERT) with laronidase, recombinant alpha-L-iduronidase, for mucopolysaccharidosis type I (MPS I) has been clinically available since April 2003. Pre-approval studies were performed on patients with the more attenuated forms of MPS I, Hurler-Scheie and Scheie syndromes. The clinical efficacy of laronidase on the severe form of MPS I, Hurler syndrome, is not well known. We present a patient with Hurler syndrome who has been treated with laronidase for 3 years. Clinically, the patient demonstrated improvement in urinary glycosaminoglycan (GAG) levels and hepatomegaly, but continued to experience decline in respiratory status, musculoskeletal and spinal involvement, and developmental skills. Overall, the benefit of ERT with laronidase in advanced Hurler syndrome appeared to be minimal in this patient. PMID:17089217

  2. Amino Acid Carbamates As Prodrugs Of Resveratrol.

    PubMed

    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

  3. 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

  4. Selective Water-Soluble Gelatinase Inhibitor Prodrugs

    PubMed Central

    Gooyit, Major; Lee, Mijoon; Schroeder, Valerie A.; Ikejiri, Masahiro; Suckow, Mark A.; Mobashery, Shahriar; Chang, Mayland

    2011-01-01

    SB-3CT (1), a selective and potent thiirane-based gelatinase inhibitor, is effective in animal models of cancer metastasis and stroke; however, it is limited by poor aqueous solubility and extensive metabolism. We addressed these issues by blocking the primary site of metabolism and capitalizing on a prodrug strategy to achieve >5000-fold increased solubility. The amide prodrugs were quantitatively hydrolyzed in human blood to a potent gelatinase inhibitor, ND-322 (3). The arginyl amide prodrug (ND-478, 5d) was metabolically stable in mouse, rat, and human liver microsomes. Both 5d and 3 were non-mutagenic in the Ames II mutagenicity assay. The prodrug 5d showed moderate clearance of 0.0582 L/min/kg, remained mostly in the extracellular fluid compartment (Vd = 0.0978 L/kg), and had a terminal half-life of >4 h. The prodrug 5d had superior pharmacokinetic properties than 3, making the thiirane class of selective gelatinase inhibitors suitable for intravenous administration in treatment of acute gelatinase-dependent diseases. PMID:21866961

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

    PubMed

    Oz, Helieh S; Chen, Theresa S; Nagasawa, Herbert

    2007-08-01

    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-alpha), 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

  6. Enzyme replacement therapy of a novel humanized mouse model of globoid cell leukodystrophy.

    PubMed

    Matthes, Frank; Andersson, Claes; Stein, Axel; Eistrup, Carl; Fogh, Jens; Gieselmann, Volkmar; Wenger, David A; Matzner, Ulrich

    2015-09-01

    An inherited deficiency of β-galactosylceramidase (GALC) causes the lysosomal storage disease globoid cell leukodystrophy (GLD). The disease is characterized by the accumulation of the cytotoxic metabolite psychosine (galactosylsphingosine), causing rapid degeneration of myelinating cells. Most patients suffer from the infantile form of GLD with onset of disease between 3 and 6 months after birth and death by 2 years of age. The most widely used animal model of GLD, the twitcher mouse, presents with an even more rapid course of disease and death around 40 days of age. We have generated a novel "humanized" mouse model of GLD by inserting a human GALC cDNA containing an adult-onset patient mutation into the murine GALC gene. Humanized GALC mice exhibit pathological hallmarks of GLD including psychosine accumulation, neuroinflammation, CNS infiltration of macrophages, astrogliosis and demyelination. Residual GALC activities in mouse tissues are low and the mice display a median lifespan of 46 days. Due to the expression of the human transgene, the mice do not develop an immune response against rhGALC, rendering the animal model suitable for therapies based on human enzyme. Intravenously injected rhGALC was able to surmount the blood-brain barrier and was targeted to lysosomes of brain macrophages, astrocytes and neurons. High-dose enzyme replacement therapy started at postnatal day 21 reduced the elevated psychosine levels in the peripheral and central nervous system by 14-16%, but did not ameliorate neuroinflammation, demyelination and lifespan. These results may indicate that treatment must be started earlier before pathology occurs. PMID:25956830

  7. 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. PMID:22574107

  8. 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

  9. Predictors of Hepatitis B Cure Using Gene Therapy to Deliver DNA Cleavage Enzymes: A Mathematical Modeling Approach

    PubMed Central

    Schiffer, Joshua T.; Swan, Dave A.; Stone, Daniel; Jerome, Keith R.

    2013-01-01

    Most chronic viral infections are managed with small molecule therapies that inhibit replication but are not curative because non-replicating viral forms can persist despite decades of suppressive treatment. There are therefore numerous strategies in development to eradicate all non-replicating viruses from the body. We are currently engineering DNA cleavage enzymes that specifically target hepatitis B virus covalently closed circular DNA (HBV cccDNA), the episomal form of the virus that persists despite potent antiviral therapies. DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR-associated system 9 (Cas9) proteins, can disrupt specific regions of viral DNA. Because DNA repair is error prone, the virus can be neutralized after repeated cleavage events when a target sequence becomes mutated. DNA cleavage enzymes will be delivered as genes within viral vectors that enter hepatocytes. Here we develop mathematical models that describe the delivery and intracellular activity of DNA cleavage enzymes. Model simulations predict that high vector to target cell ratio, limited removal of delivery vectors by humoral immunity, and avid binding between enzyme and its DNA target will promote the highest level of cccDNA disruption. Development of de novo resistance to cleavage enzymes may occur if DNA cleavage and error prone repair does not render the viral episome replication incompetent: our model predicts that concurrent delivery of multiple enzymes which target different vital cccDNA regions, or sequential delivery of different enzymes, are both potentially useful strategies for avoiding multi-enzyme resistance. The underlying dynamics of cccDNA persistence are unlikely to impact the probability of cure provided that antiviral therapy is given concurrently during eradication trials. We conclude by describing experiments that can be used to validate the model, which will in turn

  10. Targeting histone methylation for cancer therapy: enzymes, inhibitors, biological activity and perspectives.

    PubMed

    Song, Yongcheng; Wu, Fangrui; Wu, Jingyu

    2016-01-01

    Post-translational methylation of histone lysine or arginine residues plays important roles in gene regulation and other physiological processes. Aberrant histone methylation caused by a gene mutation, translocation, or overexpression can often lead to initiation of a disease such as cancer. Small molecule inhibitors of such histone modifying enzymes that correct the abnormal methylation could be used as novel therapeutics for these diseases, or as chemical probes for investigation of epigenetics. Discovery and development of histone methylation modulators are in an early stage and undergo a rapid expansion in the past few years. A number of highly potent and selective compounds have been reported, together with extensive preclinical studies of their biological activity. Several compounds have been in clinical trials for safety, pharmacokinetics, and efficacy, targeting several types of cancer. This review summarizes the biochemistry, structures, and biology of cancer-relevant histone methylation modifying enzymes, small molecule inhibitors and their preclinical and clinical antitumor activities. Perspectives for targeting histone methylation for cancer therapy are also discussed. PMID:27316347

  11. 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

  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. Photoactivatable Prodrugs of Antimelanoma Agent Vemurafenib.

    PubMed

    Horbert, Rebecca; Pinchuk, Boris; Davies, Paul; Alessi, Dario; Peifer, Christian

    2015-09-18

    In this study, we report on novel photoactivatable caged prodrugs of vemurafenib. This kinase inhibitor was the first approved drug for the personalized treatment of BRAF-mutated melanoma and showed impressive results in clinical studies. However, the occurrence of severe side effects and drug resistance illustrates the urgent need for innovative therapeutic approaches. To conquer these limitations, we implemented photoremovable protecting groups into vemurafenib. In general, this caging concept provides spatial and temporal control over the activation of molecules triggered by ultraviolet light. Thus, higher inhibitor concentrations in tumor tissues might be reached with less systemic effects. Our study describes the first development of caged vemurafenib prodrugs useful as pharmacological tools. We investigated their photochemical characteristics and photoactivation. In vitro evaluation proved the intended loss-of-function and the light-dependent recovery of efficacy in kinase and cellular assays. The reported vemurafenib photo prodrugs represent a powerful biological tool for novel pharmacological approaches in cancer research. PMID:26061392

  14. Chemical Reactivity Window Determines Prodrug Efficiency toward Glutathione Transferase Overexpressing Cancer Cells.

    PubMed

    van Gisbergen, Marike W; Cebula, Marcus; Zhang, Jie; Ottosson-Wadlund, Astrid; Dubois, Ludwig; Lambin, Philippe; Tew, Kenneth D; Townsend, Danyelle M; Haenen, Guido R M M; Drittij-Reijnders, Marie-José; Saneyoshi, Hisao; Araki, Mika; Shishido, Yuko; Ito, Yoshihiro; Arnér, Elias S J; Abe, Hiroshi; Morgenstern, Ralf; Johansson, Katarina

    2016-06-01

    Glutathione transferases (GSTs) are often overexpressed in tumors and frequently correlated to bad prognosis and resistance against a number of different anticancer drugs. To selectively target these cells and to overcome this resistance we previously have developed prodrugs that are derivatives of existing anticancer drugs (e.g., doxorubicin) incorporating a sulfonamide moiety. When cleaved by GSTs, the prodrug releases the cytostatic moiety predominantly in GST overexpressing cells, thus sparing normal cells with moderate enzyme levels. By modifying the sulfonamide it is possible to control the rate of drug release and specifically target different GSTs. Here we show that the newly synthesized compounds, 4-acetyl-2-nitro-benzenesulfonyl etoposide (ANS-etoposide) and 4-acetyl-2-nitro-benzenesulfonyl doxorubicin (ANS-DOX), function as prodrugs for GSTA1 and MGST1 overexpressing cell lines. ANS-DOX, in particular, showed a desirable cytotoxic profile by inducing toxicity and DNA damage in a GST-dependent manner compared to control cells. Its moderate conversion of 500 nmol/min/mg, as catalyzed by GSTA1, seems hereby essential since the more reactive 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) (14000 nmol/min/mg) did not display a preference for GSTA1 overexpressing cells. DNS-DOX, however, effectively killed GSTP1 (20 nmol/min/mg) and MGST1 (450 nmol/min/mg) overexpressing cells as did the less reactive 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) in a MGST1-dependent manner (1.5 nmol/min/mg) as shown previously. Furthermore, we show that the mechanism of these prodrugs involves a reduction in GSH levels as well as inhibition of the redox regulatory enzyme thioredoxin reductase 1 (TrxR1) by virtue of their electrophilic sulfonamide moiety. TrxR1 is upregulated in many tumors and associated with resistance to chemotherapy and poor patient prognosis. Additionally, the prodrugs potentially acted as a general shuttle system for DOX, by overcoming resistance

  15. 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. PMID:24443373

  16. 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

  17. Comparison of hypoxia-activated prodrug evofosfamide (TH-302) and ifosfamide in preclinical non-small cell lung cancer models.

    PubMed

    Sun, Jessica D; Liu, Qian; Ahluwalia, Dharmendra; Ferraro, Damien J; Wang, Yan; Jung, Don; Matteucci, Mark D; Hart, Charles P

    2016-04-01

    Evofosfamide (TH-302) is a hypoxia-activated prodrug of the cytotoxin bromo-isophosphoramide. In hypoxic conditions Br-IPM is released and alkylates DNA. Ifosfamide is a chloro-isophosphoramide prodrug activated by hepatic Cytochrome P450 enzymes. Both compounds are used for the treatment of cancer. Ifosfamide has been approved by the FDA while evofosfamide is currently in the late stage of clinical development. The purpose of this study is to compare efficacy and safety profile of evofosfamide and ifosfamide in preclinical non-small cell lung cancer H460 xenograft models. Immunocompetent CD-1 mice and H460 tumor-bearing immunocompromised nude mice were used to investigate the safety profile. The efficacy of evofosfamide or ifosfamide, alone, and in combination with docetaxel or sunitinib was compared in ectopic and intrapleural othortopic H460 xenograft models in animals exposed to ambient air or different oxygen concentration breathing conditions. At an equal body weight loss level, evofosfamide showed greater or comparable efficacy in both ectopic and orthotopic H460 xenograft models. Evofosfamide, but not ifosfamide, exhibited controlled oxygen concentration breathing condition-dependent antitumor activity. However, at an equal body weight loss level, ifosfamide yielded severe hematologic toxicity when compared to evofosfamide, both in monotherapy and in combination with docetaxel. At an equal hematoxicity level, evofosfamide showed superior antitumor activity. These results indicate that evofosfamide shows superior or comparable efficacy and a favorable safety profile when compared to ifosfamide in preclinical human lung carcinoma models. This finding is consistent with multiple clinical trials of evofosfamide as a single agent, or in combination therapy, which demonstrated both anti-tumor activity and safety profile without severe myelosuppression. PMID:26818215

  18. Alkoxycarbonyloxyethyl ester prodrugs of FR900098 with improved in vivo antimalarial activity.

    PubMed

    Ortmann, Regina; Wiesner, Jochen; Reichenberg, Armin; Henschker, Dajana; Beck, Ewald; Jomaa, Hassan; Schlitzer, Martin

    2005-07-01

    FR900098 represents a derivative of the new antimalarial drug fosmidomycin with enhanced activity. The mechanism of action is the inhibition of the 1-desoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, an essential enzyme of the mevalonate independent pathway of isoprenoid biosynthesis. Prodrugs with increased oral activity in mice infected with the rodent malaria parasite Plasmodium vinckei were obtained by masking the phosphonate moiety of FR900098 as alkoxycarbonyloxyethyl esters. PMID:15996004

  19. Acyloxyalkyl ester prodrugs of FR900098 with improved in vivo anti-malarial activity.

    PubMed

    Ortmann, Regina; Wiesner, Jochen; Reichenberg, Armin; Henschker, Dajana; Beck, Ewald; Jomaa, Hassan; Schlitzer, Martin

    2003-07-01

    FR900098 represents an improved derivative of the new antimalarial drug fosmidomycin and acts through inhibition of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, an essential enzyme of the mevalonate independent pathway of isoprenoid biosynthesis. Prodrugs with increased activity after oral administration were obtained by chemical modification of the phosphonate moiety to yield acyloxyalkyl esters. The most successful compound demonstrated 2-fold increased activity in mice infected with the rodent malaria parasite Plasmodium vinckei. PMID:12798327

  20. 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. PMID:26923694

  1. 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. PMID:27008042

  2. ROS-activated anticancer prodrugs: a new strategy for tumor-specific damage

    PubMed Central

    Peng, Xiaohua; Gandhi, Varsha

    2013-01-01

    Targeting tumor cells is an important strategy to improve the selectivity of cancer therapies. With the advanced studies in cancer biology, we know that cancer cells are usually under increased oxidative stress. The high level of reactive oxygen species in cancer cells has been exploited for developing novel therapeutic strategies to preferentially kill cancer cells. Our group, amongst others, have used boronic acids/esters as triggers for developing ROS-activated anticancer prodrugs that target cancer cells. The selectivity was achieved by combining a specific reaction between boronates and H2O2 with the efficient masking of drug toxicity in the prodrug via boronates. Prodrugs activated via ferrocene-mediated oxidation have also been developed to improve the selectivity of anticancer drugs. We describe how the strategies of ROS-activation can be used for further development of new ROS-targeting prodrugs, eventually leading to novel approaches and/or combined technology for more efficient and selective treatment of cancers. PMID:22900465

  3. 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. PMID:21397538

  4. 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

  5. Targeting Carcinoma-Associated Fibroblasts Within the Tumor Stroma With a Fibroblast Activation Protein-Activated Prodrug

    PubMed Central

    2012-01-01

    : mean = 0.543mm3, 95% CI = 0.173 to 0.913mm3), respectively, on day 21 after therapy. Conclusions This study validates the proteolytic activity of FAP as a target for the activation of a systemically delivered cytotoxic prodrug and demonstrates that targeted killing of cells within the stromal compartment of the tumor microenvironment can produce a therapeutic response. PMID:22911669

  6. 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

  7. Dipeptidyl Peptidase IV as a Potential Target for Selective Prodrug Activation and Chemotherapeutic Action in Cancers

    PubMed Central

    2015-01-01

    The efficacy of chemotherapeutic drugs is often offset by severe side effects attributable to poor selectivity and toxicity to normal cells. Recently, the enzyme dipeptidyl peptidase IV (DPPIV) was considered as a potential target for the delivery of chemotherapeutic drugs. The purpose of this study was to investigate the feasibility of targeting chemotherapeutic drugs to DPPIV as a strategy to enhance their specificity. The expression profile of DPPIV was obtained for seven cancer cell lines using DNA microarray data from the DTP database, and was validated by RT-PCR. A prodrug was then synthesized by linking the cytotoxic drug melphalan to a proline-glycine dipeptide moiety, followed by hydrolysis studies in the seven cell lines with a standard substrate, as well as the glycyl-prolyl-melphalan (GP-Mel). Lastly, cell proliferation studies were carried out to demonstrate enzyme-dependent activation of the candidate prodrug. The relative RT-PCR expression levels of DPPIV in the cancer cell lines exhibited linear correlation with U95Av2 Affymetrix data (r2 = 0.94), and with specific activity of a standard substrate, glycine-proline-p-nitroanilide (r2 = 0.96). The significantly higher antiproliferative activity of GP-Mel in Caco-2 cells (GI50 = 261 μM) compared to that in SK-MEL-5 cells (GI50 = 807 μM) was consistent with the 9-fold higher specific activity of the prodrug in Caco-2 cells (5.14 pmol/min/μg protein) compared to SK-MEL-5 cells (0.68 pmol/min/μg protein) and with DPPIV expression levels in these cells. Our results demonstrate the great potential to exploit DPPIV as a prodrug activating enzyme for efficient chemotherapeutic drug targeting. PMID:25365774

  8. 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

  9. Respiratory mechanics in an infant with perinatal lethal hypophosphatasia treated with human recombinant enzyme replacement therapy.

    PubMed

    Rodriguez, Elena; Bober, Michael B; Davey, Lauren; Zamora, Arlene; Li Puma, Annelise B; Chidekel, Aaron; Shaffer, Thomas H

    2012-09-01

    Hypophosphatasia is a rare autosomal recessive disorder caused by deficient activity of tissue nonspecific alkaline phosphatase (TNSALP) and characterized by defective bone mineralization. In the perinatal lethal form, respiratory complications due to rachitic deformities of the thoracic cage and associated hypoplastic lungs are present. ENB-0040 is a bone-targeted human recombinant TNSALP fusion protein that aims to restore skeletal mineralization. The goal of this study was to characterize pulmonary and thoracic cage mechanics in an infant with the perinatal lethal form of hypophosphatasia under enzyme replacement therapy. Pulmonary function testing was performed on a preterm, 8-week-old patient with hypophosphatasia who was mechanically ventilated since birth because of severe chest wall insufficiency. The measurements consisted of respiratory impulse oscillation measurements (resistance and reactance), ventilatory mechanics (compliance and resistance), and thoracoabdominal motion (TAM) analysis. At baseline, chest wall compliance was 50% of normal, and the TAM indicated predominantly abdominal displacement. After 12 weeks of treatment, a consistent decrease in ventilator requirements and improvement in lung function and chest wall mechanics were observed and correlated with thoracic cage radiologic findings. Measurable changes in chest wall dynamics and respiratory mechanics using noninvasive technology were useful for respiratory management and therapeutic guidance of ENB-0040 treatment in this patient. PMID:22328548

  10. Progress in Enzyme Replacement Therapy in Glycogen Storage Disease Type II.

    PubMed

    Angelini, Corrado; Semplicini, Claudio; Tonin, Paola; Filosto, Massimiliano; Pegoraro, Elena; Sorarù, Gianni; Fanin, Marina

    2009-05-01

    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

  11. Efficacy of enzyme replacement therapy in an aggravated mouse model of metachromatic leukodystrophy declines with age.

    PubMed

    Matthes, Frank; Stroobants, Stijn; Gerlach, Debora; Wohlenberg, Claudia; Wessig, Carsten; Fogh, Jens; Gieselmann, Volkmar; Eckhardt, Matthias; D'Hooge, Rudi; Matzner, Ulrich

    2012-06-01

    Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a functional deficiency of arylsulfatase A (ASA). Previous studies in ASA-knockout mice suggested enzyme replacement therapy (ERT) to be a promising treatment option. The mild phenotype of ASA-knockout mice did, however, not allow to examine therapeutic responses of the severe neurological symptoms that dominate MLD. We, therefore, generated an aggravated MLD mouse model displaying progressive demyelination and reduced nerve conduction velocity (NCV) and treated it by weekly intravenous injections of 20 mg/kg recombinant human ASA for 16 weeks. To analyze the stage-dependent therapeutic effects, ERT was initiated in a presymptomatic, early and progressed disease stage, at age 4, 8 and 12 months, respectively. Brain sulfatide storage, NCV and behavioral alterations were improved only in early, but not in late, treated mice showing a clear age-dependent efficacy of treatment. Hematopoietic stem cell transplantation (HSCT) for late-onset variants is the only therapeutic option for MLD to date. ERT resembles a part of the HSCT rationale, which is based on ASA supply by donor cells. Beyond ERT, our results, therefore, corroborate the clinical observation that HSCT is only effective when performed in early stages of disease. PMID:22388935

  12. Enzyme replacement therapy prevents dental defects in a model of hypophosphatasia.

    PubMed

    McKee, M D; Nakano, Y; Masica, D L; Gray, J J; Lemire, I; Heft, R; Whyte, M P; Crine, P; Millán, J L

    2011-04-01

    Hypophosphatasia (HPP) occurs from loss-of-function mutation in the tissue-non-specific alkaline phosphatase (TNALP) gene, resulting in extracellular pyrophosphate accumulation that inhibits skeletal and dental mineralization. TNALP-null mice (Akp2(-/-)) phenocopy human infantile hypophosphatasia; they develop rickets at 1 week of age, and die before being weaned, having severe skeletal and dental hypomineralization and episodes of apnea and vitamin B(6)-responsive seizures. Delay and defects in dentin mineralization, together with a deficiency in acellular cementum, are characteristic. We report the prevention of these dental abnormalities in Akp2(-/-) mice receiving treatment from birth with daily injections of a mineral-targeting, human TNALP (sALP-FcD(10)). sALP-FcD(10) prevented hypomineralization of alveolar bone, dentin, and cementum as assessed by micro-computed tomography and histology. Osteopontin--a marker of acellular cementum--was immuno-localized along root surfaces, confirming that acellular cementum, typically missing or reduced in Akp2(-/-) mice, formed normally. Our findings provide insight concerning how acellular cementum is formed on tooth surfaces to effect periodontal ligament attachment to retain teeth in their osseous alveolar sockets. Furthermore, they provide evidence that this enzyme-replacement therapy, applied early in post-natal life--where the majority of tooth root development occurs, including acellular cementum formation--could prevent the accelerated tooth loss seen in individuals with HPP. PMID:21212313

  13. A duplex "Gemini" prodrug of naltrexone for transdermal delivery.

    PubMed

    Hammell, Dana C; Hamad, Mohamed; Vaddi, Haranath K; Crooks, Peter A; Stinchcomb, Audra L

    2004-06-18

    Transdermal naltrexone delivery is desirable in the treatment of narcotic dependence and alcoholism. The purpose of this study was to increase the delivery rate of naltrexone (NTX) across human skin by using a novel prodrug. A duplex "gemini" prodrug of naltrexone was synthesized and evaluated. In vitro human skin permeation rates of naltrexone and prodrug were measured using a flow-through diffusion cell system. Drug concentrations in the skin were quantitated at the end of the diffusion experiment. The prodrug was hydrolyzed on passing through the skin and appeared mainly as naltrexone in the receiver compartment. The prodrug provided a significantly higher naltrexone equivalent flux across human skin in vitro than naltrexone base. The naltrexone equivalent solubilities of naltrexone and the prodrug in the donor solution were not significantly different. No significant increase in drug concentration in the skin after prodrug treatment, as compared to naltrexone, was observed. The naltrexone equivalent permeability from the prodrug exceeded the permeability of naltrexone base by two-fold. Due to the design of this prodrug, toxicities associated with this compound should be nonexistent, because only naltrexone and carbon dioxide (carbonic acid) are released when the prodrug is cleaved. PMID:15196755

  14. Hepatic Enzyme Alterations in HIV Patients on Antiretroviral Therapy: A Case-Control Study in a Hospital Setting in Ghana

    PubMed Central

    Osakunor, Derick Nii Mensah; Obirikorang, Christian; Fianu, Vincent; Asare, Isaac; Dakorah, Mavis

    2015-01-01

    Background Diagnosing hepatic injury in HIV infection can be a herculean task for clinicians as several factors may be involved. In this study, we sought to determine the effects of antiretroviral therapy (ART) and disease progression on hepatic enzymes in HIV patients. Methods A case-control study conducted from January to May 2014 at the Akwatia Government Hospital, Eastern region, Ghana, The study included 209 HIV patients on ART (designated HIV-ART) and 132 ART-naive HIV patients (designated HIV-Controls). Data gathered included demography, clinical history and results of blood tests for hepatic enzymes. We employed the Fisher’s, Chi-square, unpaired t-test and Pearson’s correlation in analysis, using GraphPad Prism and SPSS. A P value < 0.05 was considered significant. Results Median CD4 lymphocyte count of HIV-ART participants (604.00 cells/mm3) was higher than that of HIV-Controls (491.50 cells/mm3; P = 0.0005). Mean values of ALP, ALT, AST and GGT did not differ between the two groups compared (P > 0.05). There was a significant positive correlation between hepatic enzymes (ALP, ALT, AST and GGT) for both groups (p < 0.01 each). Duration of ART correlated positively with ALT (p < 0.05). The effect size of disease progression on hepatic enzymes for both groups was small. Conclusion Antiretroviral therapy amongst this population has minimal effects on hepatic enzymes and does not suggest modifications in therapy. Hepatic injury may occur in HIV, even in the absence of ART and other traditional factors. Monitoring of hepatic enzymes is still important in HIV patients. PMID:26247879

  15. 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

  16. 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

  17. Enzyme Replacement Therapy in Mucopolysaccharidosis II Patients Under 1 Year of Age.

    PubMed

    Lampe, Christina; Atherton, Andrea; Burton, Barbara K; Descartes, Maria; Giugliani, Roberto; Horovitz, Dafne D G; Kyosen, Sandra O; Magalhães, Tatiana S P C; Martins, Ana Maria; Mendelsohn, Nancy J; Muenzer, Joseph; Smith, Laurie D

    2014-01-01

    Mucopolysaccharidosis (MPS) II, or Hunter syndrome, is a lysosomal storage disease characterized by multi-systemic involvement and a progressive clinical course. Enzyme replacement therapy with idursulfase has been approved in more than 50 countries worldwide; however, safety and efficacy data from clinical studies are currently only available for patients 1.4 years of age and older. Sibling case studies of infants with MPS I, II, and VI who initiated ERT in the first weeks or months of life have reported no new safety concerns and a more favorable clinical course for the sibling treated in infancy than for the later-treated sibling. Here we describe our experiences with a case series of eight MPS II patients for whom idursulfase treatment was initiated at under 1 year of age. The majority of the patients were diagnosed because of a family history of disease. All of the infants displayed abnormalities consistent with MPS II at diagnosis. The youngest age at treatment start was 10 days and the oldest was 6.5 months, with duration of treatment varying between 6 weeks and 5.5 years. No new safety concerns were observed, and none of the patients experienced an infusion-related reaction. All of the patients treated for more than 6 weeks showed improvements and/or stabilization of some somatic manifestations while on treatment. In some cases, caregivers made comparisons with other affected family members and reported that the early-treated patients experienced a less severe clinical course, although a lack of medical records for many family members precluded a rigorous comparison. PMID:24515576

  18. Effects of Enzyme Replacement Therapy Started Late in a Murine Model of Mucopolysaccharidosis Type I

    PubMed Central

    Pasqualim, Gabriela; Baldo, Guilherme; de Carvalho, Talita Giacomet; Tavares, Angela Maria Vicente; Giugliani, Roberto; Matte, Ursula

    2015-01-01

    Mucopolysaccharidosis type I (MPS I) is a progressive disorder caused by deficiency of α-L-iduronidase (IDUA), which leads to storage of heparan and dermatan sulphate. It is suggested that early enzyme replacement therapy (ERT) leads to better outcomes, although many patients are diagnosed late and don’t receive immediate treatment. This study aims to evaluate the effects of late onset ERT in a MPS I murine model. MPS I mice received treatment from 6 to 8 months of age (ERT 6–8mo) with 1.2mg laronidase/kg every 2 weeks and were compared to 8 months-old wild-type (Normal) and untreated animals (MPS I). ERT was effective in reducing urinary and visceral GAG to normal levels. Heart GAG levels and left ventricular (LV) shortening fraction were normalized but cardiac function was not completely improved. While no significant improvements were found on aortic wall width, treatment was able to significantly reduce heart valves thickening. High variability was found in behavior tests, with treated animals presenting intermediate results between normal and affected mice, without correlation with cerebral cortex GAG levels. Cathepsin D activity in cerebral cortex also did not correlate with behavior heterogeneity. All treated animals developed anti-laronidase antibodies but no correlation was found with any parameters analyzed. However, intermediary results from locomotion parameters analyzed are in accordance with intermediary levels of heart function, cathepsin D, activated glia and reduction of TNF-α expression in the cerebral cortex. In conclusion, even if started late, ERT can have beneficial effects on many aspects of the disease and should be considered whenever possible. PMID:25646802

  19. Adenosine Deaminase Enzyme Therapy Prevents and Reverses the Heightened Cavernosal Relaxation in Priapism

    PubMed Central

    Wen, Jiaming; Jiang, Xianzhen; Dai, Yingbo; Zhang, Yujin; Tang, Yuxin; Sun, Hong; Mi, Tiejuan; Kellems, Rodney E.; Blackburn, Michael R.; Xia, Yang

    2010-01-01

    Introduction Priapism featured with painful prolonged penile erection is dangerous and commonly seen in sickle cell disease (SCD). The preventive approaches or effective treatment options for the disorder are limited because of poor understanding of its pathogenesis. Recent studies have revealed a novel role of excess adenosine in priapism caused by heightened cavernosal relaxation, and therefore present an intriguing mechanism-based therapeutic possibility. Aim The aim of this study was to determine the therapeutic effects of adenosine deaminase (ADA) enzyme therapy to lower adenosine in priapism. Methods Both ADA-deficient mice and SCD transgenic (Tg) mice display priapism caused by excessive adenosine. Thus, we used these two distinct lines of mouse models of priapism as our investigative tools. Specifically, we treated both of these mice with different dosages of polyethylene glycol–modified ADA (PEG–ADA) to reduce adenosine levels in vivo. At the end points of the experiments, we evaluated the therapeutic effects of PEG–ADA treatment by measuring adenosine levels and monitoring the cavernosal relaxation. Main Outcome Measures Adenosine levels in penile tissues were measured by high-performance liquid chromatography, and cavernosal relaxation was quantified by electrical field stimulation (EFS)-induced corporal cavernosal strip (CCS) assays. Results We found that lowering adenosine levels in penile tissues by PEG–ADA treatment from birth in ADA-deficient mice prevented the increased EFS-induced CCS relaxation associated with priapism. Intriguingly, in both ADA-deficient mice and SCD Tg mice with established priapism, we found that normalization of adenosine levels in penile tissues by PEG–ADA treatment relieved the heightened EFS-induced cavernosal relaxation in priapism. Conclusions Our studies have identified that PEG–ADA is a novel, safe, and mechanism-based drug to prevent and correct excess adenosine-mediated increased cavernosal relaxation

  20. 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

  1. Long-term galsulfase enzyme replacement therapy in Taiwanese mucopolysaccharidosis VI patients: A case series

    PubMed Central

    Lin, Hsiang-Yu; Chuang, Chih-Kuang; Wang, Chung-Hsing; Chien, Yin-Hsiu; Wang, Yu-Mei; Tsai, Fuu-Jen; Chou, Yen-Yin; Lin, Shio Jean; Pan, Hui-Ping; Niu, Dau-Ming; Hwu, Wuh-Liang; Ke, Yu-Yuan; Lin, Shuan-Pei

    2016-01-01

    Background Information regarding the long-term outcome of enzyme replacement therapy (ERT) with recombinant human N-acetylgalactosamine 4-sulfatase (rhASB, galsulfase, Naglazyme®, BioMarin Pharmaceutical Inc.) for Taiwanese patients with mucopolysaccharidosis (MPS) VI is limited. Methods Nine Taiwanese patients with MPS VI (4 males and 5 females; age range, 1.4 to 21.1 years) treated with weekly intravenous infusions of galsulfase (1.0 mg/kg) in 5 medical centers in Taiwan were reviewed. A set of biochemical and clinical assessments were evaluated annually. Results After 6.2 to 11.2 years of galsulfase treatment, 6 patients experienced improvement over baseline in the 6-minute walk test by a mean of 150 m (59% change over time), and 3 patients also increased the 3-minute stair climb test by a mean of 60 steps (46%). In a manual dexterity test, 3 patients decreased the time required to pick up 10 coins and put the coins into a cup by 15 s (33%). Shoulder range of motion in all 9 patients improved, and Joint Pain and Stiffness Questionnaire scores improved by 0.42 points (21%). Four patients showed improved pulmonary function. Five patients had positive effects on cardiac-wall diameters. Four patients had improved cardiac diastolic function. Liver and spleen sizes as measured by abdominal ultrasonography remained the same or decreased in all 9 patients. However, the severity degree of valvular stenosis or regurgitation did not show improvement despite ERT. A mean overall 69% decrease in urinary glycosaminoglycan (GAG) excretion indicated a satisfactory biomarker response. Conclusions Long-term ERT was beneficial and safe for Taiwanese patients with MPS VI. This treatment reduced urinary GAG and had positive effects on a wide range of clinical functional assessments including endurance, mobility, joint function, pulmonary function, liver and spleen size, cardiac hypertrophy and diastolic dysfunction. PMID:27134829

  2. Prodrugs for transdermal drug delivery - trends and challenges.

    PubMed

    Ita, Kevin B

    2016-09-01

    Prodrugs continue to attract significant interest in the transdermal drug delivery field. These moieties can confer favorable physicochemical properties on transdermal drug delivery candidates. Alkyl chain lengthening, pegylation are some of the strategies used for prodrug synthesis. It is usually important to optimize partition coefficient, water and oil solubilities of drugs. In this review, progress made in the field of prodrugs for percutaneous penetration is highlighted and the challenges discussed. PMID:26878159

  3. 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.

  4. [Effectiveness of panzytrat--modern physiological enzyme preparation in complex therapy of pancreatic exocrine secretory insufficiency in cholelithiasis].

    PubMed

    Petukhov, V A; Mironov, A V; Semenov, Zh S; Ustinov, F S

    2009-01-01

    In the article the analysis of the survey with 102 patients with gallstone disease involved, 68 of whom underwent cholecystectomy and 34 were treated conservatively, is made. The content of fecal elastase 1 in stool was estimated for diagnostics of exocrine enzyme insufficiency of pancreas by immune-enzyme analysis. It was stated that 90% of patients possess secondary exocrine insufficiency of pancreas in case of gallstone disease. It is the result of complex metabolic liver abnormalities, portal and mesenterial haemodynamics, dysbiosis of large intestine which are the components of a syndrom of maldigestion and appear during gallstone disease progressing long time before hospitalization. Cholecystectomy doesn't eliminate enzyme insufficiency of pancreas. The effectiveness of using new physiological enzymatic drug Panzytrat in a complex therapy of a syndrom of maldigestion in case of gallstone disease is shown. PMID:19551960

  5. 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

  6. [Fospropofol: A new prodrug of propofol].

    PubMed

    Telletxea, S; Lauzirika, Z; Etxebarria, A; Ortega, L F

    2012-11-01

    The development of new propofol formulations has advanced rapidly in the last ten years with the achievement of the marketing a new prodrug of propofol: fospropofol, pharmacologically different from the original compound. It is a water soluble compound that requires metabolism of the prodrug to propofol, which leads to a time delay between its administration and the appearance of its pharmacological effect. Its pharmacokinetic and pharmacodynamic characteristics are different to the original formula. Due to its formulation it does not cause pain on intravenous injection, does not lead to hyperlipidaemia or excess bacterial growth. Although it is currently unavailable in Spain, it has been approved by the FDA (American Food and Drug Administration) for sedation in controlled care in diagnostic and therapeutic procedures in adults. It must only be administered by personnel qualified to administer anaesthesia, and the patients must be monitored throughout the whole procedure. PMID:22748853

  7. Enzyme replacement therapy prior to haematopoietic stem cell transplantation in Mucopolysaccharidosis Type I: 10year combined experience of 2 centres.

    PubMed

    Ghosh, Arunabha; Miller, Weston; Orchard, Paul J; Jones, Simon A; Mercer, Jean; Church, Heather J; Tylee, Karen; Lund, Troy; Bigger, Brian W; Tolar, Jakub; Wynn, Robert F

    2016-03-01

    Haematopoietic stem cell transplantation is the treatment of choice for the severe form of Mucopolysaccharidosis Type I, or Hurler syndrome. In many centres standard practice is to deliver enzyme replacement therapy alongside haematopoietic stem cell transplantation to improve the condition of the patient prior to transplant. We report the combined 10year experience of this approach in two paediatric metabolic and transplant centres. Of 81 patients who underwent a first transplant procedure for Hurler, 88% (71/81) survived and 81% (66/81) were alive and engrafted at a median follow-up of 46months (range 3-124months). The incidence of grade II-IV acute and any chronic graft versus host disease was 17% and 11% respectively. Urinary glycosaminoglycans were significantly reduced after a period of enzyme replacement therapy, and further reductions were seen at 13-24months and 25+months after transplantation. In several individuals with decreased cardiac contractility, an improvement of their condition during enzyme replacement therapy enabled them to undergo transplantation, with one individual receiving full intensity conditioning. PMID:26832957

  8. Potential of Host Defense Peptide Prodrugs as Neutrophil Elastase-Dependent Anti-Infective Agents for Cystic Fibrosis

    PubMed Central

    Humphreys, Hilary; Greene, Catherine M.; Fitzgerald-Hughes, Deirdre; Devocelle, Marc

    2014-01-01

    Host defense peptides (HDPs) are short antimicrobial peptides of the innate immune system. Deficiencies in HDPs contribute to enhanced susceptibility to infections, e.g., in cystic fibrosis (CF). Exogenous HDPs can compensate for these deficiencies, but their development as antimicrobials is limited by cytotoxicity. Three HDP prodrugs were designed so their net positive charge is masked by a promoiety containing a substrate for the enzyme neutrophil elastase (NE). This approach can confine activation to sites with high NE levels. Enzyme-labile peptides were synthesized, and their activation was investigated using purified NE. Susceptibilities of Pseudomonas aeruginosa to parent and prodrug peptides in the presence and absence of NE-rich CF human bronchoalveolar lavage (BAL) fluid and different NaCl concentrations were compared. The effect of the HDP promoiety on cytotoxicity was determined with cystic fibrosis bronchial epithelial (CFBE41o-) cells. NE in CF BAL fluids activated the HDP prodrugs, restoring bactericidal activity against reference and clinical isolates of P. aeruginosa. However, activation also required the addition of 300 mM NaCl. Under these conditions, the bactericidal activity levels of the HDP prodrugs differed, with pro-P18 demonstrating the greatest activity (90% to 100% of that of the parent, P18, at 6.25 μg/ml). Cytotoxic effects on CFBE41o- cells were reduced by the addition of the promoiety to HDPs. We demonstrate here for the first time the selective activation of novel HDP prodrugs by a host disease-associated enzyme at in vivo concentrations of the CF lung. This approach may lead to the development of novel therapeutic agents with low toxicity that are active under the challenging conditions of the CF lung. PMID:24277028

  9. Dual-Porosity Hollow Nanoparticles for the Immunoprotection and Delivery of Nonhuman Enzymes

    PubMed Central

    2015-01-01

    Although enzymes of nonhuman origin have been studied for a variety of therapeutic and diagnostic applications, their use has been limited by the immune responses generated against them. The described dual-porosity hollow nanoparticle platform obviates immune attack on nonhuman enzymes paving the way to in vivo applications including enzyme-prodrug therapies and enzymatic depletion of tumor nutrients. This platform is manufactured with a versatile, scalable, and robust fabrication method. It efficiently encapsulates macromolecular cargos filled through mesopores into a hollow interior, shielding them from antibodies and proteases once the mesopores are sealed with nanoporous material. The nanoporous shell allows small molecule diffusion allowing interaction with the large macromolecular payload in the hollow center. The approach has been validated in vivo using l-asparaginase to achieve l-asparagine depletion in the presence of neutralizing antibodies. PMID:24471767

  10. Use of a modified alpha-N-acetylgalactosaminidase in the development of enzyme replacement therapy for Fabry disease.

    PubMed

    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

    2009-11-01

    A modified alpha-N-acetylgalactosaminidase (NAGA) with alpha-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-alpha-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

  11. Non-inhibitory antibodies impede lysosomal storage reduction during enzyme replacement therapy of a lysosomal storage disease.

    PubMed

    Matzner, Ulrich; Matthes, Frank; Weigelt, Cecilia; Andersson, Claes; Eistrup, Carl; Fogh, Jens; Gieselmann, Volkmar

    2008-04-01

    Enzyme replacement therapy is a treatment option for several lysosomal storage disorders. We reported previously that treatment of a knockout mouse model of the sphingolipid storage disease metachromatic leukodystrophy (MLD) by intravenous injection of recombinant human arylsulfatase A (rhASA) reduces sulfatide storage and improves nervous system pathology and function. Here, we show that treated mice can develop anti-rhASA antibodies, which impede sulfatide clearance without inhibiting enzyme activity. The neutralizing effect of antibodies was reproduced in cell culture models of MLD by demonstrating that mouse immune serum reduces the ability of rhASA to clear sulfatide from cultured ASA-deficient Schwann and kidney cells. We show that reduced clearance is due to an antibody-mediated blockade of mannose 6-phosphate receptor-dependent enzyme uptake, retargeting of rhASA from sulfatide-storing cells to macrophages, intracellular misrouting of rhASA, and reduction of enzyme stability. Induction of immunotolerance to rhASA by transgenic expression of an active site mutant of human ASA restores sulfatide clearance in mice. The data indicate that the influence of non-inhibitory antibodies must be more intensively considered in evaluating the therapeutic efficacy of enzyme replacement in lysosomal storage disorders in general and in patients without cross-reacting material specifically. PMID:18360747

  12. 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

  13. Hydrophobic ion pairing of isoniazid using a prodrug approach.

    PubMed

    Zhou, Huiyu; Lengsfeld, Corinne; Claffey, David J; Ruth, James A; Hybertson, Brooks; Randolph, Theodore W; Ng, Ka-Yun; Manning, Mark C

    2002-06-01

    Inhalation therapy for infectious lung diseases, such as tuberculosis, is currently being explored, with microspheres being used to target alveolar macrophages. One method of drug encapsulation into polymeric microspheres to form hydrophobic ion-paired (HIP) complexes, and then coprecipitate the complex and polymer using supercritical fluid methodology. For the potent antituberculosis drug, isoniazid (isonicotinic acid hydrazide, INH), to be used in this fashion, it was modified into an ionizable form suitable for HIP. The charged prodrug, sodium isoniazid methanesulfonate (Na-INHMS), was then ion paired with hydrophobic cations, such as alkyltrimethylammonium or tetraalkylammonium. The logarithms of the apparent partition coefficients (log P') of various HIP complexes of INHMS display a roughly linear relationship with the numbers of carbon atoms in the organic counterions. The water solubility of the tetraheptylammonium-INHMS complex is about 220-fold lower than that of Na-INHMS, while the solubility in dichloromethane exceeds 10 mg/mL, which is sufficient for microencapsulation of the drug into poly(lactide) microspheres. The actual logarithm of the dichloromethane/water partition coefficient (log P) for tetraheptylammonium-INHMS is 1.55, compared to a value of - 1.8 for the sodium salt of INHMS. The dissolution kinetics of the tetraheptylammonium-INHMS complex in 0.9% aqueous solutions of NaCl was also investigated. Dissolution of tetraheptylammonium-INHMS exhibited a first-order time constant of about 0.28 min(-1), followed by a slower reverse ion exchange process to form Na-INHMS. The half-life of this HIP complex is on the order of 30 min, making the enhanced transport of the drug across biological barriers possible. This work represents the first use of a prodrug approach to introduce functionality that would allow HIP complex formation for a neutral molecule. PMID:12115849

  14. Aliskiren – an alternative to angiotensin-converting enzyme inhibitors or angiotensin receptor blockers in the therapy of arterial hypertension

    PubMed Central

    Hoffmann, Karolina; Bryl, Wiesław; Minczykowski, Andrzej

    2013-01-01

    There has been enormous progress in antihypertensive therapy over the last few decades. However, the management of arterial hypertension is still insufficient and more efforts are needed to improve both non-pharmacological and pharmacological treatment of this widely prevalent disease. Renin-angiotensin-aldosterone system (RAAS) inhibition is crucial both for blood pressure (BP) control and for prevention of organ damage or its development in patients with hypertension. Angiotensin-converting enzyme inhibitors and/or sartans block RAAS incompletely. Aliskiren is one of the novel drugs that has been introduced to antihypertensive therapy recently. Up to now no trial has confirmed that aliskiren is efficacious in reducing cardiovascular events. Double RAAS blockade with aliskiren was not always safe. This review article presents the current view on the place of aliskiren in the therapy of arterial hypertension. PMID:25276171

  15. Aliskiren - an alternative to angiotensin-converting enzyme inhibitors or angiotensin receptor blockers in the therapy of arterial hypertension.

    PubMed

    Zaporowska-Stachowiak, Iwona; Hoffmann, Karolina; Bryl, Wiesław; Minczykowski, Andrzej

    2014-08-29

    There has been enormous progress in antihypertensive therapy over the last few decades. However, the management of arterial hypertension is still insufficient and more efforts are needed to improve both non-pharmacological and pharmacological treatment of this widely prevalent disease. Renin-angiotensin-aldosterone system (RAAS) inhibition is crucial both for blood pressure (BP) control and for prevention of organ damage or its development in patients with hypertension. Angiotensin-converting enzyme inhibitors and/or sartans block RAAS incompletely. Aliskiren is one of the novel drugs that has been introduced to antihypertensive therapy recently. Up to now no trial has confirmed that aliskiren is efficacious in reducing cardiovascular events. Double RAAS blockade with aliskiren was not always safe. This review article presents the current view on the place of aliskiren in the therapy of arterial hypertension. PMID:25276171

  16. Allopurinol enhances the activity of hypoxanthine-guanine phosphoribosyltransferase in inflammatory bowel disease patients during low-dose thiopurine therapy: preliminary data of an ongoing series.

    PubMed

    Seinen, Margien L; de Boer, Nanne K H; Smid, Kees; van Asseldonk, Dirk P; Bouma, Gerd; van Bodegraven, Adriaan A; Peters, Godefridus J

    2011-12-01

    Thiopurines are crucial in the treatment of inflammatory bowel disease. The phenotype of pivotal metabolic enzymes determines whether thioguanine nucleotides (6-TGN) are generated in clinically sufficiently high levels. The first step in activation of thiopurine prodrugs to 6-TGN is catalysis by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Often, patients exhibit a clinically unfavorable metabolism, leading to discontinuation of conventional thiopurine therapy. The combination of allopurinol and low-dose thiopurine therapy may optimize this variant metabolism, presumably by affecting enzyme activities. We performed a prospective pharmacodynamic study to determine the effect of combination therapy on the activity of HGPRT. The activity of HGPRT and 6-TGN concentrations was measured in red blood cells during thiopurine monotherapy and after 4 weeks of combination therapy. The activity of HGPRT was also measured after 12 weeks of combination therapy. From the results, we conclude that combination therapy increases the activity of HGPRT and subsequently 6-TGN concentrations. PMID:22132961

  17. Feasibility of using an isolated intestinal segment as an artificial organ for enzyme replacement therapy.

    PubMed

    Shelt, D; Walton, D; Sato, P

    1982-01-01

    Guinea pigs fed an ascorbic acid-deficient diet develop scurvy because of the absence of the enzyme L-gulonolactone oxidase. In theory if this enzyme is provided and its substrate L-gulonolactone is present at adequate concentrations ascorbic acid will be synthesized and the development of scurvy prevented. Using this model we tested whether a viable segment of intestine could be used to contain the administered enzyme and act as an artificial organ for the production of ascorbic acid. A surgical procedure was developed to prepare an externalized pouch of intestine with its circulation left intact. When enzyme is inserted in this intestinal bag it is not toxic and not antigenic in some animals, whereas, enzyme injected intraperitoneally is clearly antigenic. Synthesis of ascorbic acid by this artificial organ could not, however, be detected by elevation of plasma concentrations of the vitamin. PMID:7104431

  18. Synthesis, pH-Dependent, and Plasma Stability of Meropenem Prodrugs for Potential Use Against Drug-Resistant Tuberculosis

    PubMed Central

    Teitelbaum, Aaron M.; Meissner, Anja; Harding, Ryan A.; Wong, Christopher A.; Aldrich, Courtney C.; Remmel, Rory P.

    2013-01-01

    Meropenem, a broad-spectrum parenteral β-lactam antibiotic, in combination with clavulanate has recently shown efficacy in patients with extensively drug-resistant tuberculosis. As a result of meropenem’s short half-life and lack of oral bioavailability, the development of an oral therapy is warranted for TB treatment in underserved countries where chronic parenteral therapy is impractical. To improve the oral absorption of meropenem, several alkyloxycarbonyloxyalkyl ester prodrugs with increased lipophilicity were synthesized and their stability in physiological aqueous solutions and guinea pig as well as human plasma was evaluated. The stability of prodrugs in aqueous solution at pH 6.0 and 7.4 was significantly dependent on the ester promoiety with the major degradation product identified as the parent compound meropenem. However, in simulated gastrointestinal fluid (pH 1.2) the major degradation product identified was ring-opened meropenem with the promoiety still intact, suggesting the gastrointestinal environment may reduce the absorption of meropenem prodrugs in vivo unless administered as an enteric-coated formulation. Additionally, the stability of the most aqueous stable prodrugs in guinea pig or human plasma was short, implying a rapid release of parent meropenem. PMID:23845282

  19. 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

  20. 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

  1. Potential efficacy of enzyme replacement and substrate reduction therapy in three siblings with Gaucher disease type III.

    PubMed

    Cox-Brinkman, J; van Breemen, M J; van Maldegem, B T; Bour, L; Donker, W E; Hollak, C E M; Wijburg, F A; Aerts, J M F G

    2008-12-01

    We report three siblings with Gaucher disease type III, born between 1992 and 2004. During this period, new developments resulted in different potential therapies, changing clinical practice. The two eldest siblings received enzyme replacement therapy (ERT) from the age of 24 and 5 months respectively, later followed by an increase in dosage. ERT was combined with substrate reduction therapy (SRT) from the ages of 12 and 8 years, respectively. In the youngest sibling the combination of high-dose ERT and SRT was given from the age of 5 months. The two eldest siblings showed significant neurological impairment from the age of 1.5 years, starting with a convergent strabismus and partial oculomotor apraxia, followed by cognitive decline and an abnormal EEG and BAER. In contrast, the neurological development in the youngest sibling is almost completely normal. At the age of 3 years, cognitive development, EEG and BAER are all normal. Disturbed saccadic eye movements, which were already present at the start of therapy, remained stable. In addition to the clinical efficacy, we report on the biochemical response to therapy. Based on our results, the combination of high-dose ERT and SRT should be considered as a possible therapeutic approach for GD III, especially if started at a young age. Further follow-up studies are necessary to explore the long-term therapeutic effects. PMID:18850301

  2. 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

  3. Zosuquidar and an albumin-binding prodrug of zosuquidar reverse multidrug resistance in breast cancer cells of doxorubicin and an albumin-binding prodrug of doxorubicin.

    PubMed

    Abu Ajaj, Khalid; Graeser, Ralph; Kratz, Felix

    2012-07-01

    The P-glycoprotein (P-gp) is a 170-kDa protein that acts as an energy dependent, transmembrane efflux pump and is encoded by the MDR1 gene. It has been shown to be responsible for multidrug resistance (MDR) in a defined subpopulation of breast cancer patients and thus represents a molecular target for circumventing MDR in this tumor indication. MDR modulators have been developed and demonstrated high selectivity for P-gp with inhibitory activities in the low nanomolar range. Although some objective responses were achieved in clinical trials, combination therapy with these MDR modulators, such as Ca2+ antagonists caused unacceptable toxicity. Targeting P-gp inhibitors to the tumor site is a mean to increase their therapeutic index, and in this context binding of tailor-made prodrugs to circulating albumin is an established technology to reduce the toxicity and enhance the efficacy of anticancer drugs. In this study, we consequently developed an acid-sensitive albumin-binding prodrug of the P-gp inhibitor zosuquidar (LY335979) in a two-step synthesis using a maleimide hydrazone linker system established in our laboratory that first introduces acetylbenzoic acid at the HO-group of zosuquidar followed by derivatization with 6-maleimidocaproyl hydrazide to form the acid-sensitive hydrazone bond. The maleimide group enables the prodrug to bind rapidly and selectively to the cysteine-34 position of endogenous albumin after intravenous administration. HPLC analysis demonstrated rapid albumin binding of the zosuquidar prodrug as well as the quantitative release of the acetylbenzoic ester derivative of zosuquidar at pH 5.0. Subsequently, its ability to circumvent MDR was tested in two doxorubicin-resistant breast carcinoma cell lines (MCF-7/ADR and MT-3/ADR). The MDR status of these cell lines can be reversed by zosuquidar which was confirmed in a rhodamine 123 assay using fluorescence microscopy and FACS analysis. Furthermore, zosuquidar as well its acid-sensitive albumin

  4. Synthesis, hydrolysis kinetics and pharmacological evaluation of aceclofenac prodrugs.

    PubMed

    Dhokchawle, Bharat V; Bhandari, Anil B

    2015-01-01

    The mutual prodrugs of aceclofenac with various naturally available antioxidants; menthol, thymol, eugenol, guiacol and vanillin have been synthesized by the DCC coupling method, purified and characterized by spectral data, as well as, partition coefficient, solubility and hydrolytic studies. The title compounds have more lipophilic character as compared to the parent moieties and good stability in acidic environment, which is prerequisite for the oral absorption of the drug. Under gastric as well as intestinal pH conditions these prodrugs showed variable susceptibility towards hydrolysis. The synthesized derivatives were evaluated for antiinflammatory, analgesic activities and ulcerogenic potential. Prodrug showed improved solubility in organic solvents, which implies lipophilic character of ester prodrugs and were also found to be chemically stable in acidic environment. The aceclofenac mutual prodrugs showed improved analgesic and anti-inflammatory activities with reduced ulcerogenicity. PMID:25403255

  5. 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.

  6. Phospholipid-Based Prodrugs for Drug Targeting in Inflammatory Bowel Disease: Computational Optimization and In-Vitro Correlation.

    PubMed

    Dahan, Arik; Ben-Shabat, Shimon; Cohen, Noa; Keinan, Shahar; Kurnikov, Igor; Aponick, Aaron; Zimmermann, Ellen M

    2016-01-01

    In inflammatory bowel disease (IBD) patients, the enzyme phospholipase A2 (PLA2) is overexpressed in the inflamed intestinal tissue, and hence may be exploited as a prodrug-activating enzyme allowing drug targeting to the site(s) of gut inflammation. The purpose of this work was to develop powerful modern computational approaches, to allow optimized a-priori design of phospholipid (PL) based prodrugs for IBD drug targeting. We performed simulations that predict the activation of PL-drug conjugates by PLA2 with both human and bee venom PLA2. The calculated results correlated well with in-vitro experimental data. In conclusion, a-priori drug design using a computational approach complements and extends experimentally derived data, and may improve resource utilization and speed drug development. PMID:27086789

  7. 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.

  8. 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. PMID:26233905

  9. 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. PMID:23218603

  10. 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. PMID:27019026

  11. Theranostic nanoparticles for enzyme-activatable fluorescence imaging and photodynamic/chemo dual therapy of triple-negative breast cancer

    PubMed Central

    Choi, Jaehee; Kim, Hyunjin

    2015-01-01

    Background Triple-negative breast cancer (TNBC) is a highly diverse group of cancers characterized by tumors that does not express estrogen and progesterone receptors, as well as human epidermal growth factor receptor 2 (HER2) gene expression. TNBC is associated with poor prognosis due to high rate of recurrence and distance metastasis, lack of response to hormonal or HER2-targeted therapies, and partial response to chemotherapy. Hence, development of new therapeutic strategies to overcome such limitations is of great importance. Here we describe the application of photosensitizer-conjugated and camptothecin (CPT)-encapsulated hyaluronic acid (HA) nanoparticles as enzyme-activatable theranostic nanoparticles (EATNP) for near-infrared (NIR) fluorescence imaging and photodynamic/chemo dual therapy of TNBC. Methods For the preparation of EATNPs, chlorin e6 (Ce6), a second generation photosensitizer, was covalently conjugated to a monomethoxy poly(ethylene glycol)-grafted HA backbone. Ce6-conjugated HA (Ce6-HA) formed self-assembled nanoparticles (i.e., Ce6-HA NPs) in an aqueous solution. Subsequently, CPT, a topoisomerase 1 inhibitor with remarkable anticancer efficacy but with low water solubility, was encapsulated inside the hydrophobic core of Ce6-HA NPs thereby forming EATNPs. Results Fluorescence and singlet oxygen generation (SOG) of EATNPs are quenched in its native state. Treatment of EATNPs with hyaluronidase (HAdase) induces enzyme concentration-dependent activation of NIR fluorescence and SOG. Moreover, HAdase-mediated degradation of the nanoparticles also triggers the release of CPT from the EATNPs. In vitro confocal microscopy and cytotoxicity tests confirmed that EATNPs were efficiently introduced into MDA-MB-231 TNBC cell line, thereby inducing better cytotoxicity than that by free CPT. Additional light irradiation onto the EATNP-treated cells significantly increased therapeutic efficacy in TNBC, which indicates that EATNP plays an important role in

  12. Aortic and Mitral Valve Involvement in Maroteaux-Lamy Syndrome VI: Surgical Implications in the Enzyme Replacement Therapy Era.

    PubMed

    Torre, Salvatore; Scarpelli, Mauro; Salviati, Alessandro; Buffone, Ebba; Faggian, Giuseppe; Luciani, Giovanni Battista

    2016-07-01

    Open-heart operations in patients with mucopolysaccharidoses are exceedingly rare and pose distinct clinical challenges. Few reports exist of valve replacement in type VI mucopolysaccharidosis, mostly entailing combined mitral and aortic valve replacement. Here reported is the case of a young woman with mitral and aortic valve disease, in whom the surgical procedure was confined to the aortic valve. The rationale behind this strategy, particularly in light of the benefits offered by specific enzyme replacement therapy of type VI mucopolysaccharidosis, is discussed. PMID:27343522

  13. Double ester prodrugs of FR900098 display enhanced in-vitro antimalarial activity.

    PubMed

    Wiesner, Jochen; Ortmann, Regina; Jomaa, Hassan; Schlitzer, Martin

    2007-12-01

    Fosmidomycin and FR900098 are inhibitors of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; IspC), a key enzyme of the mevalonate-independent isoprenoid biosynthesis pathway. We have determined the in-vitro antimalarial activity of two double ester prodrugs 2, 3 in direct comparison with the unmodified FR900098 1 against intraerythrocytic forms of Plasmodium falciparum. Temporarily masking the polar properties of the phosphonate moiety of the DXR inhibitor FR900098 1 enhanced not only its oral bioavailability but also the intrinsic activity of this series against the parasites. PMID:17994601

  14. 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. PMID:15787979

  15. Prodrugs of perzinfotel with improved oral bioavailability.

    PubMed

    Baudy, Reinhardt B; Butera, John A; Abou-Gharbia, Magid A; Chen, Hong; Harrison, Boyd; Jain, Uday; Magolda, Ronald; Sze, Jean Y; Brandt, Michael R; Cummons, Terri A; Kowal, Diane; Pangalos, Menelas N; Zupan, Bojana; Hoffmann, Matthew; May, Michael; Mugford, Cheryl; Kennedy, Jeffrey; Childers, Wayne E

    2009-02-12

    Previous studies with perzinfotel (1), a potent, selective, competitive NMDA receptor antagonist, showed it to be efficacious in inflammatory and neuropathic pain models. To increase the low oral bioavailability of 1 (3-5%), prodrug derivatives (3a-h) were synthesized and evaluated. The oxymethylene-spaced diphenyl analogue 3a demonstrated good stability at acidic and neutral pH, as well as in simulated gastric fluid. In rat plasma, 3a was rapidly converted to 1 via 2a. Pharmacokinetic studies indicated that the amount of systemic exposure of 1 produced by a 10 mg/kg oral dose of 3a was 2.5-fold greater than that produced by a 30 mg/kg oral dose of 1. Consistent with these results, 3a was significantly more potent and had a longer duration of activity than 1 following oral administration in a rodent model of inflammatory pain. Taken together, these results demonstrate that an oxymethylene-spaced prodrug approach increased the bioavailability of 1. PMID:19146418

  16. Comparative impact of AAV and enzyme replacement therapy on respiratory and cardiac function in adult Pompe mice

    PubMed Central

    Falk, Darin J; Soustek, Meghan S; Todd, Adrian Gary; Mah, Cathryn S; Cloutier, Denise A; Kelley, Jeffry S; Clement, Nathalie; Fuller, David D; Byrne, Barry J

    2015-01-01

    Pompe disease is an autosomal recessive genetic disorder characterized by a deficiency of the enzyme responsible for degradation of lysosomal glycogen (acid α-glucosidase (GAA)). Cardiac dysfunction and respiratory muscle weakness are primary features of this disorder. To attenuate the progressive and rapid accumulation of glycogen resulting in cardiorespiratory dysfunction, adult Gaa–/– mice were administered a single systemic injection of rAAV2/9-DES-hGAA (AAV9-DES) or bimonthly injections of recombinant human GAA (enzyme replacement therapy (ERT)). Assessment of cardiac function and morphology was measured 1 and 3 months after initiation of treatment while whole-body plethysmography and diaphragmatic contractile function was evaluated at 3 months post-treatment in all groups. Gaa–/– animals receiving either AAV9-DES or ERT demonstrated a significant improvement in cardiac function and diaphragmatic contractile function as compared to control animals. AAV9-DES treatment resulted in a significant reduction in cardiac dimension (end diastolic left ventricular mass/gram wet weight; EDMc) at 3 months postinjection. Neither AAV nor ERT therapy altered minute ventilation during quiet breathing (eupnea). However, breathing frequency and expiratory time were significantly improved in AAV9-DES animals. These results indicate systemic delivery of either strategy improves cardiac function but AAV9-DES alone improves respiratory parameters at 3 months post-treatment in a murine model of Pompe disease. PMID:26029718

  17. Effects of Chronic Swimming Training and Oestrogen Therapy on Coronary Vascular Reactivity and Expression of Antioxidant Enzymes in Ovariectomized Rats

    PubMed Central

    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.

    2013-01-01

    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. PMID:23755145

  18. Photobiomodulation Therapy Decreases Oxidative Stress in the Lung Tissue after Formaldehyde Exposure: Role of Oxidant/Antioxidant Enzymes.

    PubMed

    Silva Macedo, Rodrigo; Peres Leal, Mayara; Braga, Tarcio Teodoro; Barioni, Éric Diego; de Oliveira Duro, Stephanie; Ratto Tempestini Horliana, Anna Carolina; Câmara, Niels Olsen Saraiva; Marcourakis, Tânia; Farsky, Sandra Helena Poliselli; Lino-Dos-Santos-Franco, Adriana

    2016-01-01

    Formaldehyde is ubiquitous pollutant that induces oxidative stress in the lung. Several lung diseases have been associated with oxidative stress and their control is necessary. Photobiomodulation therapy (PBMT) has been highlighted as a promissory treatment, but its mechanisms need to be better investigated. Our objective was to evaluate the effects of PBMT on the oxidative stress generated by FA exposure. Male Wistar rats were submitted to FA exposure of 1% or vehicle (3 days) and treated or not with PBMT (1 and 5 h after each FA exposure). Rats treated only with laser were used as control. Twenty-four hours after the last FA exposure, we analyzed the effects of PBMT on the generation of nitrites and hydrogen peroxide, oxidative burst, glutathione reductase, peroxidase, S-transferase enzyme activities, the gene expression of nitric oxide, cyclooxygenase, superoxide dismutase, the catalase enzyme, and heme oxygenase-1. PBMT reduced the generation of nitrites and hydrogen peroxide and increased oxidative burst in the lung cells. A decreased level of oxidant enzymes was observed which were concomitantly related to an increased level of antioxidants. This study provides new information about the antioxidant mechanisms of PBMT in the lung and might constitute an important tool for lung disease treatment. PMID:27293324

  19. Photobiomodulation Therapy Decreases Oxidative Stress in the Lung Tissue after Formaldehyde Exposure: Role of Oxidant/Antioxidant Enzymes

    PubMed Central

    Braga, Tarcio Teodoro; Barioni, Éric Diego; de Oliveira Duro, Stephanie; Ratto Tempestini Horliana, Anna Carolina; Câmara, Niels Olsen Saraiva; Marcourakis, Tânia; Farsky, Sandra Helena Poliselli; Lino-dos-Santos-Franco, Adriana

    2016-01-01

    Formaldehyde is ubiquitous pollutant that induces oxidative stress in the lung. Several lung diseases have been associated with oxidative stress and their control is necessary. Photobiomodulation therapy (PBMT) has been highlighted as a promissory treatment, but its mechanisms need to be better investigated. Our objective was to evaluate the effects of PBMT on the oxidative stress generated by FA exposure. Male Wistar rats were submitted to FA exposure of 1% or vehicle (3 days) and treated or not with PBMT (1 and 5 h after each FA exposure). Rats treated only with laser were used as control. Twenty-four hours after the last FA exposure, we analyzed the effects of PBMT on the generation of nitrites and hydrogen peroxide, oxidative burst, glutathione reductase, peroxidase, S-transferase enzyme activities, the gene expression of nitric oxide, cyclooxygenase, superoxide dismutase, the catalase enzyme, and heme oxygenase-1. PBMT reduced the generation of nitrites and hydrogen peroxide and increased oxidative burst in the lung cells. A decreased level of oxidant enzymes was observed which were concomitantly related to an increased level of antioxidants. This study provides new information about the antioxidant mechanisms of PBMT in the lung and might constitute an important tool for lung disease treatment. PMID:27293324

  20. 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. PMID:23562162

  1. A macromolecular prodrug strategy for combinatorial drug delivery.

    PubMed

    Li, Nan-Nan; Lin, Jiantao; Gao, Di; Zhang, Li-Ming

    2014-03-01

    A novel macromolecular prodrug strategy was developed for the combinatorial delivery of two poorly water-soluble drugs, dexamethasone and doxorubicin. In this work, dexamethasone was firstly conjugated onto a water-soluble modified polysaccharide by an acid-labile hydrazone linkage. The resultant macromolecular prodrug had an amphiphilic character and could self-assemble into spherical polymeric micelles in aqueous system. With these micelles, doxorubicin was then encapsulated into their hydrophobic cores. For the conjugated dexamethasone and encapsulated doxorubicin, they could exhibit independent and acid-sensitive release characteristics. For the doxorubicin-loaded prodrug micelles, they were easily be internalized by living cells and showed obvious antitumor activity. PMID:24407691

  2. Effect of Antiviral Therapy on Serum Activity of Angiotensin Converting Enzyme in Patients with Chronic Hepatitis C

    PubMed Central

    Husic-Selimovic, Azra; Sofic, Amela; Huskic, Jasminko; Bulja, Deniz

    2016-01-01

    Introduction: Renin-angiotenzin system (RAS) is frequently activated in patients with chronic liver disease. Angiotenzin - II (AT-II), produced by angiotenzin converting enzyme (ACE), has many physiological effects, including an important role in liver fibrogenesis. Combined antiviral therapy with PEG-IFN and ribavirin besides its antiviral effect also leads to a reduction in liver parenchyma fibrosis. Aim of the study: Determining the value of ACE in serum of patients with chronic hepatitis C before and after combined antiviral therapy, as well as the value of ACE activities in sera of the control group. Materials and methods: We studied 50 patients treated at Gastroenterohepatology Department, in the time-period of four years. Value of ACE in serum was determined by Olympus AU 400 device, with application of kit “Infinity TN ACE Liquid Stable Reagent”. HCV RNA levels in sera were measured by real time PCR. HCV RNA test was performed with modular analysis of AMPLICOR and COBAS AMPLICOR HCV MONITOR test v2.0, which has proved infection and was used for quantification of the viruses and monitoring of the patients’ response to therapy. Liver histology was evaluated in accordance with the level of necroinflammation activity and stage of fibrosis. Results: Serum activities of ACE in chronic hepatitis C patients is statistically higher than the values in the control group (p=0.02). Antiviral therapy in chronic hepatitis C patients statistically decreases serum activities of ACE (p= 0.02) and indirectly affects fibrogenesis of the liver parenchyma. Correlation between ACE and ALT activity after the therapy was proved (0.3934). Conclusion: Our findings suggest that the activity of ACE in serum is a good indirect parameter of the liver damage, and could be used as an indirect prognostic factor of the level of liver parenchyma damage. Serum activity of ACE can be used as a parameter for non-invasive assessment of intensity of liver damage. PMID:27147779

  3. Immune response to enzyme replacement therapies in lysosomal storage diseases and the role of immune tolerance induction.

    PubMed

    Kishnani, Priya S; Dickson, Patricia I; Muldowney, Laurie; Lee, Jessica J; Rosenberg, Amy; Abichandani, Rekha; Bluestone, Jeffrey A; Burton, Barbara K; Dewey, Maureen; Freitas, Alexandra; Gavin, Derek; Griebel, Donna; Hogan, Melissa; Holland, Stephen; Tanpaiboon, Pranoot; Turka, Laurence A; Utz, Jeanine J; Wang, Yow-Ming; Whitley, Chester B; Kazi, Zoheb B; Pariser, Anne R

    2016-02-01

    The US Food and Drug Administration (FDA) and National Organization for Rare Disease (NORD) convened a public workshop titled "Immune Responses to Enzyme Replacement Therapies: Role of Immune Tolerance Induction" to discuss the impact of anti-drug antibodies (ADAs) on efficacy and safety of enzyme replacement therapies (ERTs) intended to treat patients with lysosomal storage diseases (LSDs). Participants in the workshop included FDA staff, clinicians, scientists, patients, industry, and advocacy group representatives. The risks and benefits of implementing prophylactic immune tolerance induction (ITI) to reduce the potential clinical impact of antibody development were considered. Complications due to immune responses to ERT are being recognized with increasing experience and lengths of exposure to ERTs to treat several LSDs. Strategies to mitigate immune responses and to optimize therapies are needed. Discussions during the workshop resulted in the identification of knowledge gaps and future areas of research, as well as the following proposals from the participants: (1) systematic collection of longitudinal data on immunogenicity to better understand the impact of ADAs on long-term clinical outcomes; (2) development of disease-specific biomarkers and outcome measures to assess the effect of ADAs and ITI on efficacy and safety; (3) development of consistent approaches to ADA assays to allow comparisons of immunogenicity data across different products and disease groups, and to expedite reporting of results; (4) establishment of a system to widely share data on antibody titers following treatment with ERTs; (5) identification of components of the protein that are immunogenic so that triggers and components of the immune responses can be targeted in ITI; and (6) consideration of early ITI in patients who are at risk of developing clinically relevant ADA that have been demonstrated to worsen treatment outcomes. PMID:26597321

  4. Towards the development of an enzyme replacement therapy for the metabolic disorder propionic acidemia.

    PubMed

    Darvish-Damavandi, Mahnaz; Ho, Han Kiat; Kang, Tse Siang

    2016-09-01

    Propionic acidemia (PA) is a life-threatening disease caused by the deficiency of a mitochondrial biotin-dependent enzyme known as propionyl coenzyme-A carboxylase (PCC). This enzyme is responsible for degrading the metabolic intermediate, propionyl coenzyme-A (PP-CoA), derived from multiple metabolic pathways. Currently, except for drastic surgical and dietary intervention that can only provide partial symptomatic relief, no other form of therapeutic option is available for this genetic disorder. Here, we examine a novel approach in protein delivery by specifically targeting and localizing our protein candidate of interest into the mitochondrial matrix of the cells. In order to test this concept of delivery, we have utilized cell penetrating peptides (CPPs) and mitochondria targeting sequences (MTS) to form specific fusion PCC protein, capable of translocating and localizing across cell membranes. In vitro delivery of our candidate fusion proteins, evaluated by confocal images and enzymatic activity assay, indicated effectiveness of this strategy. Therefore, it holds immense potential in creating a new paradigm in site-specific protein delivery and enzyme replacement therapeutic for PA. PMID:27504265

  5. N,N'-Dialkylaminoalkylcarbonyl (DAAC) prodrugs and aminoalkylcarbonyl (AAC) prodrugs of 4-hydroxyacetanilide and naltrexone with improved skin permeation properties.

    PubMed

    Devarajan-Ketha, H; Sloan, K B

    2011-07-01

    N,N'-Dialkylaminoalkylcarbonyl (DAAC) and aminoalkylcarbonyl (AAC) prodrugs of phenolic drugs acetaminophen (APAP) and naltrexone (NTX) are reported. The effects of incorporation of a basic amine group into the promoiety of an acyl prodrug of a phenolic drug on its skin permeation properties are also presented. DAAC-APAP prodrugs were synthesized via a three-step procedure starting with haloalkylcarbonyl esters which were reacted with five different amines: dimethylamine, diethylamine, dipropylamine, morpholine, and piperidine. The spacing between the amino group and the carbonyl group of the acyl group was 1-3 CH(2). After the hydrolysis of the ester, the carboxylic acid product was subsequently coupled with the parent drug via a dicyclohexyl carbodiimide (DCC) mediated coupling to yield the DAAC-APAP-HCl prodrugs in excellent yields. The AAC prodrugs were synthesized using commercially available Boc-protected amino acids using DCC or EDCI as coupling agents. The yields of the prodrugs synthesized using these two different methods have been compared. Half-lives (t(1/2)) of a few members of the DAAC and AAC series were measured in buffer (pH 6.0, 20mM). The members evaluated in hydrolysis experiments exhibit a t(1/2) range of 15-113min. Among AAC-APAP prodrugs, the isopropyl group in valinate-APAP-HCl exerted a steric effect that increased the t(1/2) value for this prodrug compared to alaninate-APAP-HCl or prolinate-APAP-HCl. The 2-morpholinylacetate-APAP prodrug was able to achieve twice the flux of APAP in in vitro diffusion cell experiments through hairless mouse skin. PMID:21616664

  6. [Outcome of two patients with Hurler's syndrome under enzyme replacement therapy with human recombinant alpha-L-iduronidase].

    PubMed

    Sardón, O; García Pardos, C; Mintegui, J; Pérez Ruiz, E; Coll, M J; Chabás, A; Olivé, T; Ruiz Benito, A

    2005-07-01

    We performed a prospective study of two patients with Hurler's syndrome (aged 4.8 years and 17 months at the beginning of the intervention) under enzyme replacement therapy with human recombinant alpha-L-iduronidase for 452 and 28 weeks respectively. The aim of this study was to analyze the safety and efficacy of the intervention during the treatment periods. Several diagnostic imaging tests, clinical examinations, and serial laboratory determinations were performed to demonstrate the effectiveness of the therapy in both patients. In patient 1 (a boy aged 4.8 years, homozygote W402X), the treatment was always intended to be palliative because of the advanced stage of the disease. In patient 2 (a 17-month-old girl, heterozygote W402X) the treatment was initiated early with subsequent clinical stabilization without acquisition of regressive factors. Bone marrow transplantation from an unrelated donor was successful. Currently, because of the lack of histocompatible bone marrow donors, transplantation of hematopoietic stem cells from umbilical cord blood or peripheral blood are being performed with satisfactory results. In the future, gene therapy may be able to prevent the diseases associated with Hurler's syndrome and halt the neurocognitive deterioration characteristic of these patients. PMID:15989873

  7. Melanoma cell therapy: Endothelial progenitor cells as shuttle of the MMP12 uPAR-degrading enzyme.

    PubMed

    Laurenzana, Anna; Biagioni, Alessio; D'Alessio, Silvia; Bianchini, Francesca; Chillà, Anastasia; Margheri, Francesca; Luciani, Cristina; Mazzanti, Benedetta; Pimpinelli, Nicola; Torre, Eugenio; Danese, Silvio; Calorini, Lido; Del Rosso, Mario; Fibbi, Gabriella

    2014-06-15

    The receptor for the urokinase-type plasminogen activator (uPAR) accounts for many features of cancer progression, and is therefore considered a target for anti-tumoral therapy. Only full length uPAR mediates tumor progression. Matrix-metallo-proteinase-12 (MMP12)-dependent uPAR cleavage results into the loss of invasion properties and angiogenesis. MMP12 can be employed in the field of "targeted therapies" as a biological drug to be delivered directly in patient's tumor mass. Endothelial Progenitor Cells (EPCs) are selectively recruited within the tumor and could be used as cellular vehicles for delivering anti-cancer molecules. The aim of our study is to inhibit cancer progression by engeneering ECFCs, a subset of EPC, with a lentivirus encoding the anti-tumor uPAR-degrading enzyme MMP12. Ex vivo manipulated ECFCs lost the capacity to perform capillary morphogenesis and acquired the anti-tumor and anti-angiogenetic activity. In vivo MMP12-engineered ECFCs cleaved uPAR within the tumor mass and strongly inhibited tumor growth, tumor angiogenesis and development of lung metastasis. The possibility to exploit tumor homing and activity of autologous MMP12-engineered ECFCs represents a novel way to combat melanoma by a "personalized therapy", without rejection risk. The i.v. injection of radiolabelled MMP12-ECFCs can thus provide a new theranostic approach to control melanoma progression and metastasis. PMID:25003596

  8. 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

  9. Platinum(iv) anticancer prodrugs - hypotheses and facts.

    PubMed

    Gibson, Dan

    2016-08-16

    In this manuscript we focus on Pt(iv) anticancer prodrugs. We explore the main working hypotheses for the design of effective Pt(iv) prodrugs and note the exceptions to the common assumptions that are prevalent in the field. Special attention was devoted to the emerging class of "dual action" Pt(iv) prodrugs, where bioactive ligands are conjugated to the axial positions of platinum in order to obtain orthogonal or complementary effects that will increase the efficacy of killing the cancer cells. We discuss the rationale behind the design of the "dual action" prodrugs and the results of the pharmacological studies obtained. Simultaneous release of two bioactive moieties inside the cancer cells often triggers several processes that together determine the fate of the cell. Pt(iv) complexes provide many opportunities for applying new concepts in targeting, synergistic cell killing and exploiting novel nanodelivery systems. PMID:27214873

  10. Azo-reductase activated budesodine prodrugs for colon targeting.

    PubMed

    Marquez Ruiz, Juan F; Kedziora, Kinga; O'Reilly, Mary; Maguire, Jacqueline; Keogh, Brian; Windle, Henry; Kelleher, Dermot P; Gilmer, John F

    2012-12-15

    Budesodine is a synthetic glurocorticoid that undergoes substantial first pass metabolism, limiting systemic exposure. Its use in treatment of inflammatory bowel disease would benefit from a targeting strategy that could lead to a local topical effect, improving safety and increasing anti-inflammatory efficacy. A two-step prodrug strategy involving azoreduction/cyclization that we developed previously for prednisolone is here applied with some variations to budesonide. The budesodine prodrugs were tested using an in vitro azoreductase assay simulating human colonic microflora. The kinetics of amino steroid ester cyclization and its pH dependence was also evaluated. The stability of the prodrugs systems in simulated human duodenal and gastric fluid was evaluated to determine the likelihood of intact intestinal transit. The propionic acid derived prodrug 3 undergoes rapid activation by Clostridium perfingens and its putative reduction product cyclizes with acceptable rapidity when synthesized independently. These properties of 3 suggest that it has potential in management of ulcerative colitis. PMID:23122819