Probabilistic hazard assessment for skin sensitization potency by dose–response modeling using feature elimination instead of quantitative structure–activity relationships

PubMed Central

McKim, James M.; Hartung, Thomas; Kleensang, Andre; Sá-Rocha, Vanessa

2016-01-01

Supervised learning methods promise to improve integrated testing strategies (ITS), but must be adjusted to handle high dimensionality and dose–response data. ITS approaches are currently fueled by the increasing mechanistic understanding of adverse outcome pathways (AOP) and the development of tests reflecting these mechanisms. Simple approaches to combine skin sensitization data sets, such as weight of evidence, fail due to problems in information redundancy and high dimension-ality. The problem is further amplified when potency information (dose/response) of hazards would be estimated. Skin sensitization currently serves as the foster child for AOP and ITS development, as legislative pressures combined with a very good mechanistic understanding of contact dermatitis have led to test development and relatively large high-quality data sets. We curated such a data set and combined a recursive variable selection algorithm to evaluate the information available through in silico, in chemico and in vitro assays. Chemical similarity alone could not cluster chemicals’ potency, and in vitro models consistently ranked high in recursive feature elimination. This allows reducing the number of tests included in an ITS. Next, we analyzed with a hidden Markov model that takes advantage of an intrinsic inter-relationship among the local lymph node assay classes, i.e. the monotonous connection between local lymph node assay and dose. The dose-informed random forest/hidden Markov model was superior to the dose-naive random forest model on all data sets. Although balanced accuracy improvement may seem small, this obscures the actual improvement in misclassifications as the dose-informed hidden Markov model strongly reduced "false-negatives" (i.e. extreme sensitizers as non-sensitizer) on all data sets. PMID:26046447

Further development of LLNA:DAE method as stand-alone skin-sensitization testing method and applied for evaluation of relative skin-sensitizing potency between chemicals.

PubMed

Yamashita, Kunihiko; Shinoda, Shinsuke; Hagiwara, Saori; Itagaki, Hiroshi

2015-04-01

To date, there has been no well-established local lymph node assay (LLNA) that includes an elicitation phase. Therefore, we developed a modified local lymph node assay with an elicitation phase (LLNA:DAE) to discriminate true skin sensitizers from chemicals that gave borderline positive results and previously reported this assay. To develop the LLNA:DAE method as a useful stand-alone testing method, we investigated the complete procedure for the LLNA:DAE method using hexyl cinnamic aldehyde (HCA), isoeugenol, and 2,4-dinitrochlorobenzene (DNCB) as test compounds. We defined the LLNA:DAE procedure as follows: in the dose-finding test, four concentrations of chemical applied to dorsum of the right ear on days 1, 2, and 3 and dorsum of both ears on day 10. Ear thickness and skin irritation score were measured on days 1, 3, 5, 10, and 12. Local lymph nodes were excised and weighed on day 12. The test dose for the primary LLNA:DAE study was selected as the dose that gave the highest left ear lymph node weight in the dose-finding study, or the lowest dose that produced a left ear lymph node of over 4 mg. This procedure was validated using nine different chemicals. Furthermore, qualitative relationship was observed between the degree of elicitation response in the left ear lymph node and the skin sensitizing potency of 32 chemicals tested in this study and the previous study. These results indicated that LLNA:DAE method was as first LLNA method that was able to evaluate the skin sensitizing potential and potency in elicitation response.

Probabilistic hazard assessment for skin sensitization potency by dose-response modeling using feature elimination instead of quantitative structure-activity relationships.

PubMed

Luechtefeld, Thomas; Maertens, Alexandra; McKim, James M; Hartung, Thomas; Kleensang, Andre; Sá-Rocha, Vanessa

2015-11-01

Supervised learning methods promise to improve integrated testing strategies (ITS), but must be adjusted to handle high dimensionality and dose-response data. ITS approaches are currently fueled by the increasing mechanistic understanding of adverse outcome pathways (AOP) and the development of tests reflecting these mechanisms. Simple approaches to combine skin sensitization data sets, such as weight of evidence, fail due to problems in information redundancy and high dimensionality. The problem is further amplified when potency information (dose/response) of hazards would be estimated. Skin sensitization currently serves as the foster child for AOP and ITS development, as legislative pressures combined with a very good mechanistic understanding of contact dermatitis have led to test development and relatively large high-quality data sets. We curated such a data set and combined a recursive variable selection algorithm to evaluate the information available through in silico, in chemico and in vitro assays. Chemical similarity alone could not cluster chemicals' potency, and in vitro models consistently ranked high in recursive feature elimination. This allows reducing the number of tests included in an ITS. Next, we analyzed with a hidden Markov model that takes advantage of an intrinsic inter-relationship among the local lymph node assay classes, i.e. the monotonous connection between local lymph node assay and dose. The dose-informed random forest/hidden Markov model was superior to the dose-naive random forest model on all data sets. Although balanced accuracy improvement may seem small, this obscures the actual improvement in misclassifications as the dose-informed hidden Markov model strongly reduced " false-negatives" (i.e. extreme sensitizers as non-sensitizer) on all data sets. Copyright © 2015 John Wiley & Sons, Ltd.

Gonadal hormones do not alter the development of antinociceptive tolerance to delta-9-tetrahydrocannabinol in adult rats

PubMed Central

Wakley, Alexa A; Wiley, Jenny L; Craft, Rebecca M

2015-01-01

The purpose of this study was to determine whether sex differences in the development of antinociceptive tolerance to delta-9-tetrahydrocannabinol (THC) are due to activational effects of gonadal hormones. Rats were sham-gonadectomized (sham-GDX) or gonadectomized (GDX). GDX females received no hormone replacement (GDX+0), estradiol (GDX+E2), progesterone (GDX+P4), or both (GDX+E2/P4). GDX male rats received no hormone (GDX+0) or testosterone (GDX+T). Two weeks later, antinociceptive potency of THC was determined (pre-chronic test) on the warm water tail withdrawal and paw pressure assays. Vehicle or a sex-specific THC dose (females, 5.7 mg/kg, males, 9.9 mg/kg) was administered twice-daily for 9 days, then the THC dose-effect curves were re-determined (post-chronic test). On the pre-chronic test (both assays), THC was more potent in sham-GDX females than males, and gonadectomy did not alter this sex difference. In GDX females, P4 significantly decreased THC’s antinociceptive potency, whereas E2 had no effect. In GDX males, T did not alter THC’s antinociceptive potency. After chronic THC treatment, THC’s antinociceptive potency was decreased more in sham-GDX females than males, on the tail withdrawal test; this sex difference in tolerance was not altered in GDX or hormone-treated groups. These results suggest that greater antinociceptive tolerance in females, which occurred despite females receiving 40% less THC than males, is not due to activational effects of gonadal hormones. PMID:25863271

Chemical applicability domain of the Local Lymph Node Assay (LLNA) for skin sensitisation potency. Part 2. The biological variability of the murine Local Lymph Node Assay (LLNA) for skin sensitisation.

PubMed

Roberts, David W; Api, Anne Marie; Aptula, Aynur O

2016-10-01

The Local Lymph Node Assay (LLNA) is the most common in vivo regulatory toxicology test for skin sensitisation, quantifying potency as the EC3, the concentration of chemical giving a threefold increase in thymidine uptake in the local lymph node. Existing LLNA data can, along with clinical data, provide useful comparator information on the potency of sensitisers. Understanding of the biological variability of data from LLNA studies is important for those developing non-animal based risk assessment approaches for skin allergy. Here an existing set of 94 EC3 values for 12 chemicals, all tested at least three times in the same vehicle have been analysed by calculating standard deviations (SD) for logEC3 values. The SDs range from 0.08 to 0.22. The overall SD for the 94 logEC3 values is 0.147. Thus the 95% confidence limits (2xSD) for LLNA EC3 values are within a factor of 2, comparable to those for physico-chemical measurements such as partition coefficients and solubility. The residual SDs of Quantitative Mechanistic Models (QMMs) based on physical organic chemistry parameters are similar to the overall SD of the LLNA, indicating that QMMs of this type are unlikely to be bettered for predictive accuracy. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparative reactivity of human IgE to cynomolgus monkey and human effector cells and effects on IgE effector cell potency

PubMed Central

Saul, Louise; Saul, Louise; Josephs, Debra H; Josephs, Debra H; Cutler, Keith; Cutler, Keith; Bradwell, Andrew; Bradwell, Andrew; Karagiannis, Panagiotis; Karagiannis, Panagiotis; Selkirk, Chris; Selkirk, Chris; Gould, Hannah J; Gould, Hannah J; Jones, Paul; Jones, Paul; Spicer, James F; Spicer, James F; Karagiannis, Sophia N; Karagiannis, Sophia N

2014-01-01

Background: Due to genetic similarities with humans, primates of the macaque genus such as the cynomolgus monkey are often chosen as models for toxicology studies of antibody therapies. IgE therapeutics in development depend upon engagement with the FcεRI and FcεRII receptors on immune effector cells for their function. Only limited knowledge of the primate IgE immune system is available to inform the choice of models for mechanistic and safety evaluations.   Methods: The recognition of human IgE by peripheral blood lymphocytes from cynomolgus monkey and man was compared. We used effector cells from each species in ex vivo affinity, dose-response, antibody-receptor dissociation and potency assays. Results: We report cross-reactivity of human IgE Fc with cynomolgus monkey cells, and comparable binding kinetics to peripheral blood lymphocytes from both species. In competition and dissociation assays, however, human IgE dissociated faster from cynomolgus monkey compared with human effector cells. Differences in association and dissociation kinetics were reflected in effector cell potency assays of IgE-mediated target cell killing, with higher concentrations of human IgE needed to elicit effector response in the cynomolgus monkey system. Additionally, human IgE binding on immune effector cells yielded significantly different cytokine release profiles in each species. Conclusion: These data suggest that human IgE binds with different characteristics to human and cynomolgus monkey IgE effector cells. This is likely to affect the potency of IgE effector functions in these two species, and so has relevance for the selection of biologically-relevant model systems when designing pre-clinical toxicology and functional studies. PMID:24492303

Beta-Endorphin: dissociation of receptor binding activity from analgesic potency.

PubMed

Li, C H; Tseng, L F; Ferrara, P; Yamashiro, D

1980-04-01

Biological activities of synthetic camel beta-endorphin and human beta-endorphin (beta h-EP) have been measured by the radioreceptor binding assay, using [Tyr27-3H]-beta h-EP as the primary ligand and by the tail-flick test for analgesic potency. Four synthetic analogs of beta h-EP, namely [Gly31]-beta h-EP-Gly-NH2, [Gly31]-beta h-EP-Gly-Gly-NH2, [Gln8,Gly31]-beta h-EP-Gly-Gly-NH2, and [CH3(CH2)4NH231]-beta h-EP, have also been assayed by the same procedures. Results indicate a clear dissociation of radioreceptor binding activity from analgesic potency.

Beta-Endorphin: dissociation of receptor binding activity from analgesic potency.

PubMed Central

Li, C H; Tseng, L F; Ferrara, P; Yamashiro, D

1980-01-01

Biological activities of synthetic camel beta-endorphin and human beta-endorphin (beta h-EP) have been measured by the radioreceptor binding assay, using [Tyr27-3H]-beta h-EP as the primary ligand and by the tail-flick test for analgesic potency. Four synthetic analogs of beta h-EP, namely [Gly31]-beta h-EP-Gly-NH2, [Gly31]-beta h-EP-Gly-Gly-NH2, [Gln8,Gly31]-beta h-EP-Gly-Gly-NH2, and [CH3(CH2)4NH231]-beta h-EP, have also been assayed by the same procedures. Results indicate a clear dissociation of radioreceptor binding activity from analgesic potency. PMID:6246537

Saccharomyces cerevisiae as a platform for assessing sphingolipid lipid kinase inhibitors

PubMed Central

Agah, Sayeh; Mendelson, Anna J.; Eletu, Oluwafunmilayo T.; Barkey-Bircann, Peter; Gesualdi, James

2018-01-01

Successful medicinal chemistry campaigns to discover and optimize sphingosine kinase inhibitors require a robust assay for screening chemical libraries and for determining rank order potencies. Existing assays for these enzymes are laborious, expensive and/or low throughput. The toxicity of excessive levels of phosphorylated sphingoid bases for the budding yeast, Saccharomyces cerevisiae, affords an assay wherein inhibitors added to the culture media rescue growth in a dose-dependent fashion. Herein, we describe our adaptation of a simple, inexpensive, and high throughput assay for assessing inhibitors of sphingosine kinase types 1 and 2 as well as ceramide kinase and for testing enzymatic activity of sphingosine kinase type 2 mutants. The assay was validated using recombinant enzymes and generally agrees with the rank order of potencies of existing inhibitors. PMID:29672528

Chemical applicability domain of the Local Lymph Node Assay (LLNA) for skin sensitisation potency. Part 3. Apparent discrepancies between LLNA and GPMT sensitisation potential: False positives or differences in sensitivity?

PubMed

Roberts, David W; Schultz, Terry W; Api, Anne Marie

2016-10-01

The Local Lymph Node Assay (LLNA) is the gold standard regulatory toxicology test for skin sensitisation along with the guinea pig maximisation test (GPMT). Compared with the GPMT, LLNA uses fewer animals, it is quantitative, and it gives a numerical prediction of potency. However several concerns have been raised with this assay, mainly related to false positives and false negatives. Over the years, many authors, including the developers of the assay, have presented cases where there have been discrepancies between the GMPT and LLNA results. Several theories have been put forward for these discrepancies, the main one being the "over-sensitivity" of the GPMT. This paper analyses the data from a systematic study, published in three papers from 2008 to 2011, covering several classes of chemicals, in particular unsaturated fatty acids, sugar surfactants and ethoxylated alcohols, with many cases of chemicals testing positive in the LLNA being negative in the GPMT. Based on consideration of reaction chemistry and structural alerts, it is concluded that these discrepancies are not LLNA false positives, but can be rationalised in terms of the different protocols of the assays. Copyright © 2016 Elsevier Inc. All rights reserved.

Potency under pressure: the impact of hydrostatic pressure on antigenic properties of influenza virus hemagglutinin.

PubMed

Eichelberger, Schafer L; Sultana, Ishrat; Gao, Jin; Getie-Kebtie, Melkamu; Alterman, Michail; Eichelberger, Maryna C

2013-11-01

Influenza vaccines are effective in protecting against illness and death caused by this seasonal pathogen. The potency of influenza vaccines is measured by single radial immunodiffusion (SRID) assay that quantifies antigenic forms of hemagglutinin (HA). Hydrostatic pressure results in loss of binding of influenza virus to red blood cells, but it is not known whether this infers loss of potency. Our goal was to determine the impact of pressure on HA antigenic structure. Viruses included in the 2010-2011 trivalent influenza vaccine were subjected to increasing number of cycles at 35,000 psi in a barocycler, and the impact of this treatment measured by determining hemagglutination units (HAU) and potency. Potency was assessed by SRID and immunogenicity in mice. After 25 cycles of pressure, the potency measured by SRID assay was below the limit of quantification for the H1N1 and B viruses used in our study, while the H3N2 component retained some potency that was lost after 50 pressure cycles. Pressure treatment also resulted in loss of HAU, but this did not strictly correlate with the potency value. Curiously, loss of potency was abrogated when influenza A, but not B, antigens were exposed to pressure in chicken egg allantoic fluid. Protection against pressure appeared to be mediated by specific interactions because addition of bovine serum albumin did not have the same effect. Our results show that pressure-induced loss of potency is strain dependent and suggests that pressure treatment may be useful for identifying vaccine formulations that improve HA stability. Published 2013. This article is U.S. Government work and in the public domain in the USA.

Skin sensitization potency of methyl methacrylate in the local lymph node assay: comparisons with guinea-pig data and human experience.

PubMed

Betts, Catherine J; Dearman, Rebecca J; Heylings, Jon R; Kimber, Ian; Basketter, David A

2006-09-01

There is compelling evidence that contact allergens differ substantially (by 4 or 5 orders of magnitude) with respect to their inherent skin-sensitizing potency. Relative potency can now be measured effectively using the mouse local lymph node assay (LLNA) and such data form the basis of risk assessment and risk management strategies. Such determinations also facilitate distinctions being drawn between the prevalence of skin sensitization to a particular contact allergen and inherent potency. The distinction is important because chemicals that are implicated as common causes of contact allergy are not necessarily potent sensitizers. One example is provided by nickel that is undoubtedly a common cause of allergic contact dermatitis, but is a comparatively weak sensitizer in predictive tests. In an attempt to explore other examples of contact allergens where there may exist a discrepancy between prevalence and potency, we describe here analyses conducted with methyl methacrylate (MMA). Results of LLNA studies have been interpreted in the context of historical clinical data on occupational allergic contact dermatitis associated with exposure to MMA.

The International Standard for Oxytetracycline

PubMed Central

Humphrey, J. H.; Lightbown, J. W.; Mussett, M. V.; Perry, W. L. M.

1955-01-01

The first attempt to set up an international standard for oxytetracycline, using oxytetracycline hydrochloride, failed because of difficulties in obtaining a preparation whose moisture content was uniform after distribution into ampoules. A preparation of dihydrate of oxytetracycline base was obtained instead, and was compared in an international collaborative assay with a sample of oxytetracycline hydrochloride, which was the current working standard of Chas. Pfizer & Co., Inc., USA. The results of the collaborative assay showed that the potency of the dihydrate was uniform, and that it was a suitable preparation for use as the International Standard. Evidence was obtained, however, that the reference preparation at the time of examination was less potent than had been originally supposed, and that it was hydrated. The potency of the proposed international standard was recalculated after allowance for water in the reference preparation, and the resulting biological potency agreed well with that to be expected on the basis of the physicochemical properties of the preparation. It was agreed, therefore, that the recalculated values should be used, and the preparation of oxytetracycline base dihydrate used in the collaborative assay is established as the International Standard for Oxytetracycline with a potency of 900 International Units per mg. PMID:13284563

Reproducibility of the anti-Factor Xa and anti-Factor IIa assays applied to enoxaparin solution.

PubMed

Martinez, Céline; Savadogo, Adama; Agut, Christophe; Anger, Pascal

2013-01-01

Enoxaparin is a widely used subcutaneously administered antithrombotic agent comprising a complex mixture of glycosaminoglycan chains. Owing to this complexity, its antithrombotic potency cannot be defined by physicochemical methods and is therefore evaluated using an enzymatic assay of anti-Xa and anti-IIa activity. Maintaining consistent anti-Xa activity in the final medicinal product allows physicians to ensure administration of the appropriate dosage to their patients. Bioassays are usually complex and display poorer reproducibility than physicochemical tests such as HPLC assays. Here, we describe the implementation of a common robotic platform and standard release potency testing procedures for enoxaparin sodium injection (Lovenox, Sanofi, Paris, France) products at seven quality control sites within Sanofi. Qualification and analytical procedures, as well as data handling, were optimized and harmonized to improve assay reproducibility. An inter-laboratory study was performed in routine-release conditions. The coefficients of variation for repeatability and reproducibility in assessments of anti-Xa activity were 1.0% and 1.2%, respectively. The tolerance interval in reproducibility precision conditions, expressed as percentage potency, was 96.8-103.2% of the drug product target of 10,000 IU/ml, comparing favorably with the United States of America Pharmacopeia specification (90-110%). The maximum difference between assays in two different laboratories is expected to be 4.1%. The reproducibility characteristics of anti-IIa activity assessments were found to be similar. These results demonstrate the effectiveness of the standardization process established and allow for further improvements to quality control in Lovenox manufacture. This process guarantees closeness between actual and target potencies, as exemplified by the results of release assays obtained during a three-year period. Copyright © 2013 Elsevier B.V. All rights reserved.

Establishment of a cell model for screening antibody drugs against rheumatoid arthritis with ADCC and CDC.

PubMed

Yan, Li; Hu, Rui; Tu, Song; Cheng, Wen-Jun; Zheng, Qiong; Wang, Jun-Wen; Kan, Wu-Sheng; Ren, Yi-Jun

2015-01-01

TNFα played a dominant role in the development and progression of rheumatoid arthritis (RA). Clinical trials proved the efficacies of anti-TNFα agents for curing RA. However, most researchers were concentrating on their abilities of neutralizing TNFα, the potencies of different anti-TNFα agents varied a lot due to the antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). For better understanding and differentiating the potentiality of various candidate anti-TNF reagents at the stage of new drug research and development, present study established a cell model expressing the transmembrane TNFα for usage in in vitro ADCC or CDC assay, meanwhile, the assay protocol described here could provide guidelines for screening macromolecular antibody drugs. A stable cell subline bearing transmembrane TNFα was first established by conventional transfection method, the expression of transmembrane TNFα was approved by flow cytometer, and the performance of the stable subline in ADCC and CDC assay was evaluated, using human peripheral blood mononuclear cells as effector cells, and Adalimumab as the anti-TNFα reagent. The stable cell subline demonstrated high level of surface expression of transmembrane TNFα, and Adalimumab exerted both ADCC and CDC effects on this cell model. In conclusion, the stable cell line we established in present research could be used in ADCC or CDC assay for screening antibody drugs, which would provide in-depth understanding of the potencies of candidate antibody drugs in addition to the traditional TNFα neutralizing assay.

USDA regulatory guidelines and practices for veterinary Leptospira vaccine potency testing.

PubMed

Srinivas, G B; Walker, A; Rippke, B

2013-09-01

Batch-release potency testing of leptospiral vaccines licensed by the United States Department of Agriculture (USDA) historically was conducted through animal vaccination-challenge models. The hamster vaccination-challenge assay was Codified in 1974 for bacterins containing Leptospira pomona, Leptospira icterohaemorrhagiae, and Leptospira canicola, and in 1975 for bacterins containing Leptospira grippotyphosa. In brief, 10 hamsters are vaccinated with a specified dilution of bacterin. After a holding period, the vaccinated hamsters, as well as nonvaccinated controls, are challenged with virulent Leptospira and observed for mortality. Eighty percent of vaccinated hamsters must survive in the face of a valid challenge. The high cost of the Codified tests, in terms of monetary expense and animal welfare, prompted the Center for Veterinary Biologics (CVB) to develop ELISA alternatives for them. Potency tests for other serogroups, such as Leptospira hardjo-bovis, that do not have Codified requirements for potency testing continue to be examined on a case-by-case basis. Published by Elsevier Ltd.

The local lymph node assay and the assessment of relative potency: status of validation.

PubMed

Basketter, David A; Gerberick, Frank; Kimber, Ian

2007-08-01

For the prediction of skin sensitization potential, the local lymph node assay (LLNA) is a fully validated alternative to guinea-pig tests. More recently, information from LLNA dose-response analyses has been used to assess the relative potency of skin sensitizing chemicals. These data are then deployed for risk assessment and risk management. In this commentary, the utility and validity of these relative potency measurements are reviewed. It is concluded that the LLNA does provide a valuable assessment of relative sensitizing potency in the form of the estimated concentration of a chemical required to produce a threefold stimulation of draining lymph node cell proliferation compared with concurrent controls (EC3 value) and that all reasonable validation requirements have been addressed successfully. EC3 measurements are reproducible in both intra- and interlaboratory evaluations and are stable over time. It has been shown also, by several independent groups, that EC3 values correlate closely with data on relative human skin sensitization potency. Consequently, the recommendation made here is that LLNA EC3 measurements should now be regarded as a validated method for the determination of the relative potency of skin sensitizing chemicals, a conclusion that has already been reached by a number of independent expert groups.

An Integrated View of Air Mutagenicity

EPA Science Inventory

The mutagenic potency of ambient air particulate material (PM) in the Salmonella mutagenicity assay (revertants/mg PM) varies only ~1 order of magnitude worldwide; however, the mutagenic potency of the air itself (revertants/m3 of air) varies ~5 orders of magnitude (IARC Monograp...

Development and validation of a quantitative competitive ELISA for potency testing of equine anti rabies sera with other potential use.

PubMed

Korimbocus, Jehanara; Dehay, Nicolas; Tordo, Noël; Cano, François; Morgeaux, Sylvie

2016-06-14

In case of a bite by a rabies infected animal, the World Health Organisation recommends a prophylactic treatment including the administration of Human Rabies Immunoglobulins (HRIGs) or highly purified F(ab')2 fragments produced from Equine Rabies Immunoglobulin (F(ab')2 - ERIGs). According to international regulation, quality control of F(ab')2 - ERIGs lots requires potency testing by the in vivo Mouse Neutralisation Test (MNT) prior marketing. However, the strategy of the 3Rs (Reduce, Refine, Replace) for animal testing required by the European Directive encourages the replacement of the in vivo potency test by an in vitro assay. In this context, a competitive ELISA method (c-ELISA) has been developed by the Agence Nationale de Sécurité du Médicament et des Produits de Santé where F(ab')2 - ERIGs are in competition with a monoclonal antibody recognizing the trimeric native form of the rabies glycoprotein. After a full validation study, the c-ELISA has been applied to commercial batches of F(ab')2 - ERIGs. A correlation study with the MNT demonstrated a similarity between the two methods (r=0.751). Moreover, the c-ELISA method which does not need any species specific reagent has been applied to HRIGs potency testing as an alternative method to Rapid Fluorescent Focus Inhibition Test (RFFIT), thus avoiding the handling of live rabies virus in BSL3 containment. In conclusion, the c-ELISA has shown its potential to replace MNT and possibly RFFIT for the quantification of rabies immunoglobulin. After optimisation it may be used for the quantification of rabies immunoglobulin in any animal species, notably for rabies immunogenicity assay in mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

Skin sensitisation: the Colipa strategy for developing and evaluating non-animal test methods for risk assessment.

PubMed

Maxwell, Gavin; Aeby, Pierre; Ashikaga, Takao; Bessou-Touya, Sandrine; Diembeck, Walter; Gerberick, Frank; Kern, Petra; Marrec-Fairley, Monique; Ovigne, Jean-Marc; Sakaguchi, Hitoshi; Schroeder, Klaus; Tailhardat, Magali; Teissier, Silvia; Winkler, Petra

2011-01-01

Allergic contact dermatitis is a delayed-type hypersensitivity reaction induced by small reactive chemicals (haptens). Currently, the sensitising potential and potency of new chemicals is usually characterised using data generated via animal studies, such as the local lymph node assay (LLNA). There are, however, increasing public and political concerns regarding the use of animals for the testing of new chemicals. Consequently, the development of in vitro, in chemico or in silico models for predicting the sensitising potential and/or potency of new chemicals is receiving widespread interest. The Colipa Skin Tolerance task force currently collaborates with and/or funds several academic research groups to expand our understanding of the molecular and cellular events occurring during the acquisition of skin sensitisation. Knowledge gained from this research is being used to support the development and evaluation of novel alternative approaches for the identification and characterisation of skin sensitizing chemicals. At present three non-animal test methods (Direct Peptide Reactivity Assay (DPRA), Myeloid U937 Skin Sensitisation Test (MUSST) and human Cell Line Activation Test (hCLAT)) have been evaluated in Colipa interlaboratory ring trials for their potential to predict skin sensitisation potential and were recently submitted to ECVAM for formal pre-validation. Data from all three test methods will now be used to support the study and development of testing strategy approaches for skin sensitiser potency prediction. This publication represents the current viewpoint of the cosmetics industry on the feasibility of replacing the need for animal test data for informing skin sensitisation risk assessment decisions.

Skin sensitization potency of isoeugenol and its dimers evaluated by a non-radioisotopic modification of the local lymph node assay and guinea pig maximization test.

PubMed

Takeyoshi, Masahiro; Iida, Kenji; Suzuki, Keiko; Yamazaki, Shunsuke

2008-05-01

Allergic contact dermatitis is the serious unwanted effect arising from the use of consumer products such as cosmetics. Isoeugenol is a fragrance chemical with spicy, carnation-like scent, is used in many kinds of cosmetics and is a well-known moderate human sensitizer. It was previously reported that the dimerization of eugenol yielded two types of dimer possessing different sensitization potencies. This study reports the differences in skin sensitization potencies for isoeugenol and two types of dimer, beta-O-4-dilignol and dehydrodiisoeugenol (DIEG), as evaluated by the non-radioisotopic local lymph node assay (non-RI LLNA) and guinea pig maximization test. In the guinea pig maximization test, isoeugenol, beta-O-4-dilignol and DIEG were classified as extreme, weak and moderate sensitizers, respectively. As for the results of non-RI LLNA, the EC3 for isoeugenol, beta-O-4-dilignol and DIEG were calculated as 12.7%, >30% and 9.4%, respectively. The two types of isoeugenol dimer showed different sensitizing activities similar to the case for eugenol dimers. A reduction of sensitization potency achieved by dimerization may lead to developing safer cosmetic ingredients. Isoeugenol dimers are not currently used for fragrance chemicals. However, the dimerization of isoeugenol may yield a promising candidate as a cosmetic ingredient with low sensitization risk. The data may also provide useful information for the structure-activity relationship (SAR) in skin sensitization. Copyright (c) 2007 John Wiley & Sons, Ltd.

Evaluating the Effect of Peptoid Lipophilicity on Antimicrobial Potency, Cytotoxicity, and Combinatorial Library Design.

PubMed

Turkett, Jeremy A; Bicker, Kevin L

2017-04-10

Growing prevalence of antibiotic resistant bacterial infections necessitates novel antimicrobials, which could be rapidly identified from combinatorial libraries. We report the use of the peptoid library agar diffusion (PLAD) assay to screen peptoid libraries against the ESKAPE pathogens, including the optimization of assay conditions for each pathogen. Work presented here focuses on the tailoring of combinatorial peptoid library design through a detailed study of how peptoid lipophilicity relates to antibacterial potency and mammalian cell toxicity. The information gleaned from this optimization was then applied using the aforementioned screening method to examine the relative potency of peptoid libraries against Staphylococcus aureus, Acinetobacter baumannii, and Enterococcus faecalis prior to and following functionalization with long alkyl tails. The data indicate that overall peptoid hydrophobicity and not simply alkyl tail length is strongly correlated with mammalian cell toxicity. Furthermore, this work demonstrates the utility of the PLAD assay in rapidly evaluating the effect of molecular property changes in similar libraries.

Development, validation and utility of an in vitro technique for assessment of potential clinical drug-drug interactions involving P-glycoprotein.

PubMed

Keogh, John P; Kunta, Jeevan R

2006-04-01

Regulatory interest is increasing for drug transporters generally and P-glycoprotein (Pgp) in particular, primarily in the area of drug-drug interactions. To aid in both identifying and discharging the potential liabilities associated with drug-transporter interactions, the pharmaceutical industry has a growing requirement for routine and robust non-clinical assays. An assay was designed, optimised and validated to determine the in vitro inhibitory potency of new chemical entities (NCEs) towards human Pgp-mediated transport. [3H]-Digoxin was established as a suitable probe substrate by investigating its characteristics in the in vitro system (MDCKII-MDR1 cells grown in 24-multiwell inserts). The inhibitory potencies (apparent IC50) of known Pgp inhibitors astemizole, GF120918, ketoconazole, itraconazole, quinidine, verapamil and quinine were determined over at least a 1000-fold concentration range. Validation was carried out using manual and automatic techniques. [3H]-Digoxin was found to be stable and have good mass balance in the system. In contrast to [A-->B] transport, [3H]-digoxin [B-->A] transport rates were readily measured with good reproducibility. There was no evidence of saturation of transport up to 10 microM digoxin and 30 nM digoxin was selected for routine assay use, reflecting clinical therapeutic concentrations. IC50 values ranged over approximately 100-fold with excellent reproducibility. Results from manual and automated versions were in close agreement. This method is suitable for routine use to assess the in vitro inhibitory potency of NCEs on Pgp-mediated digoxin transport. Comparison of IC50 values against clinical interaction profiles for the probe inhibitors indicated the in vitro assay is predictive of clinical digoxin-drug interactions mediated via Pgp.

Inducible indoleamine 2,3-dioxygenase 1 and programmed death ligand 1 expression as the potency marker for mesenchymal stromal cells.

PubMed

Guan, Qingdong; Li, Yun; Shpiruk, Tanner; Bhagwat, Swaroop; Wall, Donna A

2018-05-01

Establishment of a potency assay in the manufacturing of clinical-grade mesenchymal stromal cells (MSCs) has been a challenge due to issues of relevance to function, timeline and variability of responder cells. In this study, we attempted to develop a potency assay for MSCs. Clinical-grade bone marrow-derived MSCs were manufactured. The phenotype and immunosuppressive functions of the MSCs were evaluated based on the International Society for Cellular Therapy guidelines. Resting MSCs licensed by interferon (IFN)-γ exposure overnight were evaluated for changes in immune suppression and immune-relevant proteins. The relationship of immune-relevant protein expression with immunosuppression of MSCs was analyzed. MSC supressed third-party T-lymphocyte proliferation with high inter-donor and inter-test variability. The suppression of T-lymphocyte proliferation by IFN-γ-licensed MSCs correlated with that by resting MSCs. Many cellular proteins were up-regulated after IFN-γ exposure, including indoleamine 2,3-dioxygenase 1 (IDO-1), programmed death ligand 1 (PD-L1), vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and bone marrow stromal antigen 2 (BST-2). The expression levels of IDO-1 and PD-L1 on licensed MSCs, not VCAM-1, ICAM-1 or BST-2 on licensed MSCs, correlated with MSC suppression of third-party T-cell proliferation. A flow cytometry-based assay of MSCs post-IFN-γ exposure measuring expression of intracellular protein IDO-1 and cell surface protein PD-L1 captures two mechanisms of suppression and offers the potential of a relevant, rapid assay for MSC-mediated immune suppression that would fit with the manufacturing process. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

The local lymph node assay in 2014.

PubMed

Basketter, David A; Gerberick, G Frank; Kimber, Ian

2014-01-01

Toxicology endeavors to predict the potential of materials to cause adverse health (and environmental) effects and to assess the risk(s) associated with exposure. For skin sensitizers, the local lymph node assay was the first method to be fully and independently validated, as well as the first to offer an objective end point with a quantitative measure of sensitizing potency (in addition to hazard identification). Fifteen years later, it serves as the primary standard for the development of in vitro/in chemico/in silico alternatives.

A demonstration of the uncertainty in predicting the estrogenic ...

EPA Pesticide Factsheets

In vitro estrogen receptor assays are valuable screening tools for identifying environmental samples and chemicals that display estrogenic activity. However, in vitro potency cannot necessarily be extrapolated to estimates of in vivo potency because in vitro assays are currently unable to fully account for adsorption, distribution, metabolism, and excretion. To explore this issue, we calculated relative potency factors (RPF) for several chemicals and mixtures in the T47D-KBluc estrogen receptor transactivation assay. The in vitro RPF values were then used to predict rat uterotrophic assay responses following oral administration of individual chemicals and mixtures. 17β-estradiol (E2), 17α-ethinyl estradiol (EE2), benzyl-butyl phthalate (BBP), bisphenol-A (BPA), bisphenol-AF (BPAF), bisphenol-C (BPC), bisphenol-S (BPS), and methoxychlor (MET) were tested individually, while BPS+MET, BPAF+MET, and BPAF+BPC+BPS+EE2+MET were tested as equipotent mixtures. In vivo ED50 values for BPA, BPAF, and BPC were accurately predicted using in vitro data; however, E2 was less potent than predicted, BBP was a false positive, and BPS and MET were 76.6 and 368.3-fold more active in vivo than predicted from the in vitro potency assessment, respectively. Further, mixture ED50 values were more accurately predicted by the dose addition model using individual chemical in vivo uterotrophic data (0.7-1.5-fold difference from observed) than in vitro data (1.4-86.8-fold). Overall,

Embryonic vascular disruption adverse outcomes: Linking high-throughput signaling signatures with functional consequences.

PubMed

Ellis-Hutchings, Robert G; Settivari, Raja S; McCoy, Alene T; Kleinstreuer, Nicole; Franzosa, Jill; Knudsen, Thomas B; Carney, Edward W

2017-04-13

Embryonic vascular disruption is an important adverse outcome pathway (AOP) as chemical disruption of cardiovascular development induces broad prenatal defects. High-throughput screening (HTS) assays aid AOP development although linking in vitro data to in vivo apical endpoints remains challenging. This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. Both were identified as putative vascular disruptive compounds (pVDCs) in ToxCastDB and disrupted angiogenesis and embryogenesis in the functional assays. Differences were observed in potency and adverse effects: 5HPP-33 was embryolethal (WEC and ZET); TNP-470 produced caudal defects at lower concentrations. This study demonstrates how a tiered approach using HTS signatures and complex functional in vitro assays might be used to prioritize further in vivo developmental toxicity testing. Copyright © 2017 Elsevier Inc. All rights reserved.

Embryonic vascular disruption adverse outcomes: Linking high throughput signaling signatures with functional consequences.

PubMed

Ellis-Hutchings, Robert G; Settivari, Raja S; McCoy, Alene T; Kleinstreuer, Nicole; Franzosa, Jill; Knudsen, Thomas B; Carney, Edward W

2017-06-01

Embryonic vascular disruption is an important adverse outcome pathway (AOP) as chemical disruption of cardiovascular development induces broad prenatal defects. High throughput screening (HTS) assays aid AOP development although linking in vitro data to in vivo apical endpoints remains challenging. This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. Both were identified as putative vascular disruptive compounds (pVDCs) in ToxCastDB and disrupted angiogenesis and embryogenesis in the functional assays. Differences were observed in potency and adverse effects: 5HPP-33 was embryolethal (WEC and ZET); TNP-470 produced caudal defects at lower concentrations. This study demonstrates how a tiered approach using HTS signatures and complex functional in vitro assays might be used to prioritize further in vivo developmental toxicity testing. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparability of ELISA and toxin neutralization to measure immunogenicity of Protective Antigen in mice, as part of a potency test for anthrax vaccines.

PubMed

Parreiras, P M; Sirota, L A; Wagner, L D; Menzies, S L; Arciniega, J L

2009-07-16

Complexities of lethal challenge models have prompted the investigation of immunogenicity assays as potency tests of anthrax vaccines. An ELISA and a lethal toxin neutralization assay (TNA) were used to measure antibody response to Protective Antigen (PA) in mice immunized once with either a commercial or a recombinant PA (rPA) vaccine formulated in-house. Even though ELISA and TNA results showed correlation, ELISA results may not be able to accurately predict TNA results in this single immunization model.

Prenatal developmental toxicity testing of petroleum substances: Application of the mouse embryonic stem cell test (EST) to compare in vitro potencies with potencies observed in vivo.

PubMed

Kamelia, Lenny; Louisse, Jochem; de Haan, Laura; Rietjens, Ivonne M C M; Boogaard, Peter J

2017-10-01

Prenatal developmental toxicity (PDT) as observed with some petroleum substances (PS) has been associated with the presence of 3-7 ring polycyclic aromatic hydrocarbons (PAHs). In the present study, the applicability of ES-D3 cell differentiation assay of the EST to evaluate in vitro embryotoxicity potencies of PS and gas-to-liquid (GTL) products as compared to their in vivo potencies was investigated. DMSO-extracts of a range of PS, containing different amounts of PAHs, and GTL-products, which are devoid of PAHs, were tested in the ES-D3 cell proliferation and differentiation assays of the EST. The results show that PS inhibited the differentiation of ES-D3 cells into cardiomyocytes in a concentration-dependent manner at non-cytotoxic concentrations, and that their potency was proportional to their PAH content. In contrast, as expected, GTL-products did not inhibit ES-D3 cell viability or differentiation at all. The in vitro PDT potencies were compared to published in vivo PDT studies, and a good correlation was found between in vitro and in vivo results (R 2 =0.97). To conclude, our results support the hypothesis that PAHs are the primary inducers of the PDT in PS. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

The LLNA: A Brief Review of Recent Advances and Limitations

PubMed Central

Anderson, Stacey E.; Siegel, Paul D.; Meade, B. J.

2011-01-01

Allergic contact dermatitis is the second most commonly reported occupational illness, accounting for 10% to 15% of all occupational diseases. This highlights the importance of developing rapid and sensitive methods for hazard identification of chemical sensitizers. The murine local lymph node assay (LLNA) was developed and validated for the identification of low molecular weight sensitizing chemicals. It provides several benefits over other tests for sensitization because it provides a quantitative endpoint, dose-responsive data, and allows for prediction of potency. However, there are also several concerns with this assay including: levels of false positive responses, variability due to vehicle, and predictivity. This report serves as a concise review which briefly summarizes the progress, advances and limitations of the assay over the last decade. PMID:21747867

Potency assays for therapeutic live whole cell cancer vaccines.

PubMed

Petricciani, John; Egan, William; Vicari, Giuseppe; Furesz, John; Schild, Geoffrey

2007-04-01

Therapeutic cancer vaccines are under development with the goal of enhancing the body's immune response to cancer cells sufficient to arrest cancer cell growth. Among the various approaches being used are those based on whole tumor cells. Developing a suitable measure of the potency of such vaccines presents a significant challenge because neither cellular associated markers nor in vivo biological responses that are correlated with efficacy have been identified; nevertheless, manufacturers and regulatory agencies will need to develop methods to evaluate these products. At this moment, the challenge for manufacturers who are developing whole cell vaccines is to demonstrate batch-to-batch consistency for the vaccine used in clinical studies and to show that comparable vaccine batches have the same capacity to achieve an acceptable level of biological activity that may be related to efficacy. This is particularly challenging in that animal models to test that activity do not exist and direct serological or immunological correlates of clinical protection are not available because protection has not yet been established in clinical trials. In the absence of well-defined biological markers and tests for manufacturing consistency, manufacturers and regulators will need to rely heavily on a highly reproducible manufacturing process--the consistency of the process therefore becomes critical. In developing regulatory approaches to whole cell cancer vaccines, the experience from the field of infectious disease vaccines should be examined for general guidance. A framework that draws heavily on the field of infectious disease vaccines is presented and suggests that at this point in the development of this new class of products, it is reasonable to develop data on quantitative antigen expression as a measure of potency with the expectation that when clinical efficacy has been established it will confirm the appropriateness of this approach. But because this will not be known until the end of a pivotal trial, a bioassay should be considered and run in parallel. Several examples of bioassays are presented along with their advantages and disadvantages. The final selection of a potency assay for use in lot release of a commercializable therapeutic whole cell vaccine ultimately will depend on the totality of the data available at the time of approval by regulatory agencies. Based on information currently available, it is likely that quantitative antigen expression or a bioassay could be used to measure potency. If both are determined to be acceptable, the use of quantitative antigen expression could be considered for routine lot release, while the bioassay could be reserved for use as one of the elements in establishing comparability when manufacturing changes are being considered after approval.

Cytostatic versus cytocidal profiling of quinoline drug combinations via modified fixed-ratio isobologram analysis.

PubMed

Gorka, Alexander P; Jacobs, Lauren M; Roepe, Paul D

2013-09-18

Drug combination therapy is the frontline of malaria treatment. There is an ever-accelerating need for new, efficacious combination therapies active against drug resistant malaria. Proven drugs already in the treatment pipeline, such as the quinolines, are important components of current combination therapy and also present an attractive test bank for rapid development of new concepts. The efficacy of several drug combinations versus chloroquine-sensitive and chloroquine-resistant strains was measured using both cytostatic and cytocidal potency assays. These screens identify quinoline and non-quinoline pairs that exhibit synergy, additivity, or antagonism using the fixed-ratio isobologram method and find tafenoquine - methylene blue combination to be the most synergistic. Also, interestingly, for selected pairs, additivity, synergy, or antagonism defined by quantifying IC50 (cytostatic potency) does not necessarily predict similar behaviour when potency is defined by LD50 (cytocidal potency). These data further support an evolving new model for quinoline anti-malarials, wherein haem and haemozoin are the principle target for cytostatic activity, but may not be the only target relevant for cytocidal activity.

In vitro and in vivo potency of insulin analogues designed for clinical use.

PubMed

Vølund, A; Brange, J; Drejer, K; Jensen, I; Markussen, J; Ribel, U; Sørensen, A R; Schlichtkrull, J

1991-11-01

Analogues of human insulin designed to have improved absorption properties after subcutaneous injection have been prepared by recombinant DNA technology. Five rapidly absorbed analogues, being predominantly in mono- or di-meric states in the pharmaceutical preparation, and a hexameric analogue with very low solubility at neutral pH and slow absorption, were studied. Receptor binding assays with HEP-G2 cells showed overall agreement with mouse free adipocyte assays. Two analogues, B28Asp and A21Gly + B27Arg + B30Thr-NH2, had nearly the same molar in vitro potency as human insulin. Another two showed increased adipocyte potency and receptor binding, B10Asp 194% and 333% and A8His + B4His + B10Glu + B27His 575% and 511%, while B9Asp + B27Glu showed 29% and 18% and the B25Asp analogue only 0.12% and 0.05% potency. Bioassays in mice or rabbits of the analogues except B25Asp showed that they had the same in vivo potency as human insulin 1.00 IU = 6.00 nmol. Thus the variation had the same in vivo potency as human insulin 1.00 IU = 6.00 nmol. Thus the variation in in vivo potency reflects the differences in receptor binding affinity. Relative to human insulin a low concentration is sufficient for a high affinity analogue to produce a given receptor complex formation and metabolic response. In conclusion, human insulin and analogues with markedly different in vitro potencies were equipotent in terms of hypoglycaemic effect. This is in agreement with the concept that elimination of insulin from blood and its subsequent degradation is mediated by insulin receptors.

Otitis-Prone Children Produce Functional Antibodies to Pneumolysin and Pneumococcal Polysaccharides

PubMed Central

Wiertsema, Selma P.; Corscadden, Karli J.; Mateus, Tulia; Mullaney, Gemma L.; Zhang, Guicheng; Richmond, Peter C.; Thornton, Ruth B.

2016-01-01

ABSTRACT The pneumococcus is a major otitis media (OM) pathogen, but data are conflicting regarding whether otitis-prone children have impaired humoral immunity to pneumococcal antigens. We and others have shown that otitis-prone and healthy children have similar antibody titers to pneumococcal proteins and polysaccharides (vaccine and nonvaccine types); however, the quality of antibodies from otitis-prone children has not been investigated. Antibody function, rather than titer, is considered to be a better correlate of protection from pneumococcal disease. Therefore, we compared the capacities of antibodies from otitis-prone (cases) and healthy (controls) children to neutralize pneumolysin, the pneumococcal toxin currently in development as a vaccine antigen, and to opsonize pneumococcal vaccine and nonvaccine serotypes. A pneumolysin neutralization assay was conducted on cholesterol-depleted complement-inactivated sera from 165 cases and 61 controls. A multiplex opsonophagocytosis assay (MOPA) was conducted on sera from 20 cases and 20 controls. Neutralizing and opsonizing titers were calculated with antigen-specific IgG titers to determine antibody potency for pneumolysin, pneumococcal conjugate vaccine (PCV) polysaccharides, and non-PCV polysaccharides. There was no significant difference in antibody potencies between cases and controls for the antigens tested. Antipneumolysin neutralizing titers increased with the number of episodes of acute OM, but antibody potency did not. Pneumolysin antibody potency was lower in children colonized with pneumococci than in noncarriers, and this was significant for the otitis-prone group (P < 0.05). The production of functional antipneumococcal antibodies in otitis-prone children demonstrates that they respond to the current PCV and are likely to respond to pneumolysin-based vaccines as effectively as healthy children. PMID:28031178

Sulfamic and succinic acid derivatives of 25-OH-PPD and their activities to MCF-7, A-549, HCT-116, and BGC-823 cell lines.

PubMed

Zhou, Wu-Xi; Cao, Jia-Qing; Wang, Xu-De; Guo, Jun-Hui; Zhao, Yu-Qing

2017-02-15

In the search for new anti-tumor agents with higher potency than our previously identified compound 1 (25-OH-PPD, 25-hydroxyprotopanaxadiol), 12 novel sulfamic and succinic acid derivatives that could improve water solubility and contribute to good drug potency and pharmacokinetic profiles were designed and synthesized. Their in vitro anti-tumor activities in MCF-7, A-549, HCT-116, and BGC-823 cell lines and one normal cell line were tested by standard MTT assay. Results showed that compared with compound 1, compounds 2, 3, and 7 exhibited higher cytotoxic activity on A-549 and BGC-823 cell lines, together with lower toxicity in the normal cell. In particular, compound 2 exhibited the best anti-tumor activity in the in vitro assays, which may provide valuable data for the research and development of new anti-tumor agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Use of the mouse jumping test for estimating antagonistic potencies of morphine antagonists.

PubMed

Cowan, A

1976-03-01

The potencies of 19 reference morphine antagonists have been compared in a modified version of the mouse jumping test. Mice were each implanted subcutaneously with one 75 mg pellet of morphine. Antagonist challenge took place 72 h later and the incidence of repetitive vertical-jumping was monitored over 1 h. A high Pearson correlation coefficient (r = 0.997) was found between quantitative assays based on the total number of jumps per mouse and quantal assays based on mice jumping at least 6 times. A comparison of relative potencies obtained with the mouse test and with non-withdrawn morphine-dependent monkeys gave a Spearman rank order coefficient of 0.91 while a similar comparison with values obtained with the guinea-pig isolated ileum preparation also gave a high correlation coefficient (r= 0.92). Whereas it is difficult to assess the antagonistic component of buprenorphine and cyclorphan with the ileum preparation, both compounds can be satisfactorily assayed in the mouse jumping test. The reported antagonistic properties of ketocyclazocine and profadol could not be confirmed in the mouse model.

Accounting Artifacts in High-Throughput Toxicity Assays.

PubMed

Hsieh, Jui-Hua

2016-01-01

Compound activity identification is the primary goal in high-throughput screening (HTS) assays. However, assay artifacts including both systematic (e.g., compound auto-fluorescence) and nonsystematic (e.g., noise) complicate activity interpretation. In addition, other than the traditional potency parameter, half-maximal effect concentration (EC50), additional activity parameters (e.g., point-of-departure, POD) could be derived from HTS data for activity profiling. A data analysis pipeline has been developed to handle the artifacts and to provide compound activity characterization with either binary or continuous metrics. This chapter outlines the steps in the pipeline using Tox21 glucocorticoid receptor (GR) β-lactamase assays, including the formats to identify either agonists or antagonists, as well as the counter-screen assays for identifying artifacts as examples. The steps can be applied to other lower-throughput assays with concentration-response data.

Validation of an indirect ELISA to detect antibodies against BoHV-1 in bovine and guinea-pig serum samples using ISO/IEC 17025 standards.

PubMed

Parreño, Viviana; Romera, S Alejandra; Makek, Lucia; Rodriguez, Daniela; Malacari, Darío; Maidana, Silvina; Compaired, Diego; Combessies, Gustavo; Vena, María Marta; Garaicoechea, Lorena; Wigdorovitz, Andrés; Marangunich, Laura; Fernandez, Fernando

2010-10-01

Two ELISAs to quantify antibodies to BoHV-1 in the sera of cattle and immunized guinea pigs were developed and validated using ISO/IEC 17025 standards. The cut-off value of the assay was established at 20% positivity of a high positive control for screening of cattle. Using this threshold, the assay properly classified the OIE bovine reference sera EU1, EU2 and EU3. For vaccine potency testing, a cut-off of 40% was selected for both species. The reliability of the assays, given by their diagnostic sensitivity and specificity, using the threshold of 40% was 89.7% and 100%, respectively, for bovines and 94.9% and 100% for guinea pigs, respectively. There was almost perfect agreement between the ELISA and virus neutralization results. In addition, after vaccination, there was a good correlation between the neutralizing and ELISA antibody titers of the serum from the same bovine or guinea pig, sampled at 60 and 30 days post-vaccination, respectively (R(bovine)=0.88, R(guinea pig)=0.92; p<0.0001). A similar correlation was observed when analyzing the mean antibody titers of groups of vaccinated animals (R(bovine)=0.95 and R(guinea pig)=0.97; p<0.0001), indicating the relevance of the ELISAs for batch to batch vaccine potency testing in the target species and in the laboratory animal model. The intermediate precision of the assays expressed as the relative coefficient of variation (CV) of the positive control assayed over a 3-year period in the same laboratory was 22.2% for bovines and 23.1% for guinea pigs. The reproducibility of both techniques obtained in inter-laboratory assays was CV=12.4% for bovines and CV approximately 0 for guinea pigs, which met the requirements of the OIE (CV<30%). The validated ELISAs represent important methods for vaccine potency testing and for controlling BoHV-1 infections. Copyright (c) 2010 Elsevier B.V. All rights reserved.

The noninvasive mouse ear swelling assay. II. Testing the contact sensitizing potency of fragrances.

PubMed

Thorne, P S; Hawk, C; Kaliszewski, S D; Guiney, P D

1991-11-01

The noninvasive mouse ear swelling assay (MESA) for contact allergy testing was evaluated using fragrance components and complex fragrance mixtures. The test materials represented weak sensitizers and nonsensitizers. Two versions of the MESA were investigated. Both were noninvasive and utilized only topical abdominal dosing and ear challenge with single applications in BALB/cBy mice. The vit A MESA differed from the regular MESA only in that mice were maintained on a diet with 17-fold higher levels of vitamin A (vit A) acetate beginning 3 weeks prior to induction. Sensitization reactions were determined by measuring the mean increase in ear swelling over baseline at 24, 48 and 72 hr postexposure. Irritation dose-response curves facilitated choosing a high nonirritating challenge dose. Sensitization dose-response curves were developed for cinnamaldehyde (CINN) and a complex fragrance mixture, F-16. From these curves, the SD50 was determined. This value represents the dose which sensitized half the animals and serves to rank the potency of compounds for allergic contact dermatitis and to compare values among different assays. The SD50 for CINN was 21.6% while the SD50vit A for F-16 was 26.6%. The other fragrance, isoeugenol (ISOE), and fragrance mixtures, F-07 and F-22, were also found to be weak sensitizers in the MESA and vit A MESA. The results in the MESA for CINN and ISOE were in the range observed with guinea pig test protocols but showed that the MESA was more sensitive than human test protocols. Two of the fragrance mixtures tested in the MESA gave comparable results in the Buehler guinea pig assay. However, the third (F-22) was negative in the Buehler assay and the MESA, but positive in the vit A MESA. The results of this work with weak sensitizers and the companion study (Thorne et al., 1991) with potent sensitizers at low doses illustrate that the noninvasive MESA is as sensitive as many standard guinea pig assays. In addition, it is easier and much less expensive to perform. The vit A MESA has the sensitivity and predictive power needed to test compounds and mixtures for contact sensitizing potency.

Benzothiophene inhibitors of MK2. Part 2: Improvements in kinase selectivity and cell potency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, David R.; Meyers, Marvin J.; Kurumbail, Ravi G.

2010-10-01

Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of inhibitors bound to MK2.

Assessment of pyrrolizidine alkaloid-induced toxicity in an in vitro screening model.

PubMed

Li, Yan Hong; Kan, Winnie Lai Ting; Li, Na; Lin, Ge

2013-11-25

Pyrrolizidine alkaloids (PAs) are a group of heterocyclic phytotoxins present in a wide range of plants. The consumption of PA-containing medicinal herbs or PA-contaminated foodstuffs has long been reported to cause human hepatotoxicity. However, the degrees of hepatotoxicity of different PAs are unknown, which makes it difficult to determine a universal threshold of toxic dose of individual PAs for safe regulation of PA-containing natural products. The aim of the present study is to develop a simple and convenient in vitro model to assess the hepatotoxicity of different PAs. Six common cytotoxicity assays were used to evaluate the hepatotoxicity of different PAs in human hepatocellular carcinoma HepG2 cells. The combination of MTT and bromodeoxyuridine incorporation (BrdU) assays demonstrated to be a suitable method to evaluate the toxic potencies of various PAs in HepG2 cells, and the results indicated that otonecine-type PA (clivorine: IC₂₀=0.013 ± 0.004 mM (MTT), 0.066 ± 0.031 mM (BrdU)) exhibited significantly higher cytotoxic and anti-proliferative effects than retronecine-type PA (retrorsine: IC₂₀=0.27 ± 0.07 mM (MTT), 0.19 ± 0.03 mM (BrdU)). While as expected, the known less toxic platyphylline-type PA (platyphylline: IC₂₀=0.85 ± 0.11 mM (MTT), 1.01 ± 0.40 mM (BrdU)) exhibited significantly less toxicity. The different cytotoxic and anti-proliferative potencies of various PAs in the same retronecine-type could also be discriminated by using the combined MTT and BrdU assays. In addition, the developed assays were further utilized to test alkaloid extract of Gynura segetum, a senecionine and seneciphylline-containing herb, the overall cytotoxicity of two PAs in the extract was comparable to that of these two PAs tested individually. Using the developed in vitro model, the cytotoxicity of different PAs and the extract of a PA-containing herb were investigated in parallel in one system, and their different hepatotoxic potencies were determined and directly compared for the first time. The results suggested that the developed model has a great potential to be applied for the quick screening of the toxicity of PAs and PA-containing natural products. © 2013 Elsevier Ireland Ltd. All rights reserved.

Identification of Thyroid Receptor Ant/Agonists in Water Sources Using Mass Balance Analysis and Monte Carlo Simulation

PubMed Central

Shi, Wei; Wei, Si; Hu, Xin-xin; Hu, Guan-jiu; Chen, Cu-lan; Wang, Xin-ru; Giesy, John P.; Yu, Hong-xia

2013-01-01

Some synthetic chemicals, which have been shown to disrupt thyroid hormone (TH) function, have been detected in surface waters and people have the potential to be exposed through water-drinking. Here, the presence of thyroid-active chemicals and their toxic potential in drinking water sources in Yangtze River Delta were investigated by use of instrumental analysis combined with cell-based reporter gene assay. A novel approach was developed to use Monte Carlo simulation, for evaluation of the potential risks of measured concentrations of TH agonists and antagonists and to determine the major contributors to observed thyroid receptor (TR) antagonist potency. None of the extracts exhibited TR agonist potency, while 12 of 14 water samples exhibited TR antagonistic potency. The most probable observed antagonist equivalents ranged from 1.4 to 5.6 µg di-n-butyl phthalate (DNBP)/L, which posed potential risk in water sources. Based on Monte Carlo simulation related mass balance analysis, DNBP accounted for 64.4% for the entire observed antagonist toxic unit in water sources, while diisobutyl phthalate (DIBP), di-n-octyl phthalate (DNOP) and di-2-ethylhexyl phthalate (DEHP) also contributed. The most probable observed equivalent and most probable relative potency (REP) derived from Monte Carlo simulation is useful for potency comparison and responsible chemicals screening. PMID:24204563

Establishment of a biological reference preparation for hepatitis A vaccine (inactivated, non-adsorbed).

PubMed

Stalder, J; Costanzo, A; Daas, A; Rautmann, G; Buchheit, K-H

2010-04-01

A reference standard calibrated in International Units (IU) is needed for the in vitro potency assay of hepatitis A vaccines prepared by formalin-inactivation of purified hepatitis A virus grown in cell cultures. Thus, a project was launched by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to establish one or more non-adsorbed inactivated hepatitis A vaccine reference preparation(s) as working standard(s), calibrated against the 1st International Standard (IS), for the in vitro potency assay (ELISA) of all vaccines present on the European market. Four non-adsorbed liquid preparations of formalin-inactivated hepatitis A antigen with a known antigen content were obtained from 3 manufacturers as candidate Biological Reference Preparations (BRPs). Thirteen laboratories participated in the collaborative study. They were asked to use an in vitro ELISA method adapted from a commercially available kit for the detection of antibodies to hepatitis A virus. In-house validated assays were to be run in parallel, where available. Some participants also included commercially available hepatitis A vaccines in the assays, after appropriate desorption. During the collaborative study, several participants using the standard method were faced with problems with some of the most recent lots of the test kits. Due to these problems, the standard method did not perform satisfactorily and a high number of assays were invalid, whereas the in-house methods appeared to perform better. Despite this, the overall mean results of the valid assays using both methods were in agreement. Nonetheless, it was decided to base the assignment of the potency values on the in-house methods only. The results showed that all candidate BRPs were suitable for the intended purpose. However, based on availability of the material and on the results of end-product testing, 2 candidate reference preparations, Samples C and D, were selected. Both were from the same batch but filled on different days; no statistically significant difference in potency was observed. They were thus combined in 1 single batch. The candidate preparation (Sample C/D) was adopted at the June 2009 session of the European Pharmacopoeia (Ph. Eur.) Commission as the Ph. Eur. BRP batch 1 for hepatitis A vaccine (inactivated, non-adsorbed), with an assigned potency of 12 IU/ml for in vitro antigen content assays. Accelerated degradation studies have been initiated. The preliminary data show that the BRP is stable at the recommended storage temperature (< -50 degrees C). The BRP will be monitored at regular intervals throughout its lifetime.

Sample preparation composite and replicate strategy case studies for assay of solid oral drug products.

PubMed

Nickerson, Beverly; Harrington, Brent; Li, Fasheng; Guo, Michele Xuemei

2017-11-30

Drug product assay is one of several tests required for new drug products to ensure the quality of the product at release and throughout the life cycle of the product. Drug product assay testing is typically performed by preparing a composite sample of multiple dosage units to obtain an assay value representative of the batch. In some cases replicate composite samples may be prepared and the reportable assay value is the average value of all the replicates. In previously published work by Harrington et al. (2014) [5], a sample preparation composite and replicate strategy for assay was developed to provide a systematic approach which accounts for variability due to the analytical method and dosage form with a standard error of the potency assay criteria based on compendia and regulatory requirements. In this work, this sample preparation composite and replicate strategy for assay is applied to several case studies to demonstrate the utility of this approach and its application at various stages of pharmaceutical drug product development. Copyright © 2017 Elsevier B.V. All rights reserved.

The murine local lymph node assay: regulatory and potency considerations under REACH.

PubMed

McGarry, Helen F

2007-09-05

From June 2007, new chemicals legislation on the registration, evaluation, authorization and restriction of chemicals (REACH) will come into force across the European Union. This will require the submission of data on human health effects of chemicals, including chemical safety assessments which will require measurements of potency. For skin sensitization hazard identification, REACH states that the first-choice in vivo assay is the local lymph node assay (LLNA). This test has also been the UK competent authority's preferred test for skin sensitization since 2002, and has now replaced guinea pig tests in dossiers submitted to it under the Notification of New Substances Regulations. Advantages of the LLNA over guinea pig tests include improvements in animal welfare, a more scientific approach to hazard identification, and the inclusion of a dose-response element in the endpoint, which enables an estimation of potency. However, notifiers to the UK competent authority have sometimes been reluctant to use the assay because of concerns over false-positive reactions. Across Europe, these concerns have been heightened in the lead-up to the introduction of REACH, since the use of in vivo alternatives to the LLNA will require scientific justification. This review will address some of these concerns from a regulatory perspective.

Bridging the Gap Between Validation and Implementation of Non-Animal Veterinary Vaccine Potency Testing Methods.

PubMed

Dozier, Samantha; Brown, Jeffrey; Currie, Alistair

2011-11-29

In recent years, technologically advanced high-throughput techniques have been developed that replace, reduce or refine animal use in vaccine quality control tests. Following validation, these tests are slowly being accepted for use by international regulatory authorities. Because regulatory acceptance itself has not guaranteed that approved humane methods are adopted by manufacturers, various organizations have sought to foster the preferential use of validated non-animal methods by interfacing with industry and regulatory authorities. After noticing this gap between regulation and uptake by industry, we began developing a paradigm that seeks to narrow the gap and quicken implementation of new replacement, refinement or reduction guidance. A systematic analysis of our experience in promoting the transparent implementation of validated non-animal vaccine potency assays has led to the refinement of our paradigmatic process, presented here, by which interested parties can assess the local regulatory acceptance of methods that reduce animal use and integrate them into quality control testing protocols, or ensure the elimination of peripheral barriers to their use, particularly for potency and other tests carried out on production batches.

Quantitative relationship between the local lymph node assay and human skin sensitization assays.

PubMed

Schneider, K; Akkan, Z

2004-06-01

The local lymph node assay (LLNA) is a new test method which allows for the quantitative assessment of sensitizing potency in the mouse. Here, we investigate the quantitative correlation between results from the LLNA and two human sensitization tests--specifically, human repeat insult patch tests (HRIPTs) and human maximization tests (HMTs). Data for 57 substances were evaluated, of which 46 showed skin sensitizing properties in human tests, whereas 11 yielded negative results in humans. For better comparability data from mouse and human tests were transformed to applied doses per skin area, which ranged over four orders of magnitude for the substances considered. Regression analysis for the 46 human sensitizing substances revealed a significant positive correlation between the LLNA and human tests. The correlation was better between LLNA and HRIPT data (n=23; r=0.77) than between LLNA and HMT data (n=38; r=0.65). The observed scattering of data points is related to various uncertainties, in part associated with insufficiencies of data from older HMT studies. Predominantly negative results in the LLNA for another 11 substances which showed no skin sensitizing activity in human maximization tests further corroborate the correspondence between LLNA and human tests. Based on this analysis, the LLNA can be considered a reliable basis for relative potency assessments for skin sensitizers. Proposals are made for the regulatory exploitation of the LLNA: four potency groups can be established, and assignment of substances to these groups according to the outcome of the LLNA can be used to characterize skin sensitizing potency in substance-specific assessments. Moreover, based on these potency groups, a more adequate consideration of sensitizing substances in preparations becomes possible. It is proposed to replace the current single concentration limit for skin sensitizers in preparations, which leads to an all or nothing classification of a preparation as sensitizing to skin ("R43") in the European Union, by differentiated concentration limits derived from the limits for the four potency groups.

Estimating skin sensitization potency from a single dose LLNA.

PubMed

Roberts, David W

2015-04-01

Skin sensitization is an important aspect of safety assessment. The mouse local lymph node assay (LLNA) developed in the 1990 s is an in vivo test used for skin sensitization hazard identification and characterization. More recently a reduced version of the LLNA (rLLNA) has been developed as a means of identifying, but not quantifying, sensitization hazard. The work presented here is aimed at enabling rLLNA data to be used to give quantitative potency information that can be used, inter alia, in modeling and read-across approaches to non-animal based potency estimation. A probit function has been derived enabling estimation of EC3 from a single dose. This has led to development of a modified version of the rLLNA, whereby as a general principle the SI value at 10%, or at a lower concentration if 10% is not testable, is used to calculate the EC3. This version of the rLLNA has been evaluated against a selection of chemicals for which full LLNA data are available, and has been shown to give EC3 values in good agreement with those derived from the full LLNA. Copyright © 2015 Elsevier Inc. All rights reserved.

Skin sensitizers differentially regulate signaling pathways in MUTZ-3 cells in relation to their individual potency

PubMed Central

2014-01-01

Background Due to the recent European legislations posing a ban of animal tests for safety assessment within the cosmetic industry, development of in vitro alternatives for assessment of skin sensitization is highly prioritized. To date, proposed in vitro assays are mainly based on single biomarkers, which so far have not been able to classify and stratify chemicals into subgroups, related to risk or potency. Methods Recently, we presented the Genomic Allergen Rapid Detection (GARD) assay for assessment of chemical sensitizers. In this paper, we show how the genome wide readout of GARD can be expanded and used to identify differentially regulated pathways relating to individual chemical sensitizers. In this study, we investigated the mechanisms of action of a range of skin sensitizers through pathway identification, pathway classification and transcription factor analysis and related this to the reactive mechanisms and potency of the sensitizing agents. Results By transcriptional profiling of chemically stimulated MUTZ-3 cells, 33 canonical pathways intimately involved in sensitization to chemical substances were identified. The results showed that metabolic processes, cell cycling and oxidative stress responses are the key events activated during skin sensitization, and that these functions are engaged differently depending on the reactivity mechanisms of the sensitizing agent. Furthermore, the results indicate that the chemical reactivity groups seem to gradually engage more pathways and more molecules in each pathway with increasing sensitizing potency of the chemical used for stimulation. Also, a switch in gene regulation from up to down regulation, with increasing potency, was seen both in genes involved in metabolic functions and cell cycling. These observed pathway patterns were clearly reflected in the regulatory elements identified to drive these processes, where 33 regulatory elements have been proposed for further analysis. Conclusions This study demonstrates that functional analysis of biomarkers identified from our genomics study of human MUTZ-3 cells can be used to assess sensitizing potency of chemicals in vitro, by the identification of key cellular events, such as metabolic and cell cycling pathways. PMID:24517095

Dioxin-like activity of brominated dioxins as individual compounds or mixtures in in vitro reporter gene assays with rat and mouse hepatoma cell lines.

PubMed

Suzuki, G; Nakamura, M; Michinaka, C; Tue, N M; Handa, H; Takigami, H

2017-10-01

In vitro reporter gene assays detecting dioxin-like compounds have been developed and validated since the middle 1990's, and applied to the determination of dioxin-like activities in various samples for their risk management. Data on characterizing the potency of individual brominated dioxins and their activity in mixture with chlorinated dioxins are still limited on the cell-based assay. This study characterized the dioxin-like activities of the 32 brominated dioxins, such as polybrominated dibenzo-p-dioxins, polybrominated dibenzofurans (PBDFs), coplanar polybrominated biphenyls, mixed halogenated dibenzo-p-dioxins and dibenzofurans (PXDFs), as a sole component or in a mixture by DR-CALUX (dioxin-responsive chemically activated luciferase expression) using the rat hepatoma H4IIE cell line and XDS-CALUX (xenobiotic detection systems-chemically activated luciferase expression) assays using the mouse hepatoma H1L6.1 cell line. The 2,3,7,8-TCDD-relative potencies (REPs) of most of the brominated dioxins were within a factor of 10 of the WHO toxicity equivalency factor (WHO-TEF) for the chlorinated analogues. The REPs of a few PXDFs were an order of magnitude higher than the corresponding WHO-TEFs, indicating their toxicological importance. Results with reconstituted mixtures suggest that the activity of brominated and chlorinated dioxins in both CALUX assays was dose-additive. Thus, obtained results indicated the applicability of the CALUX assays as screening tools of brominated dioxins together with their chlorinated analogues. Copyright © 2017 Elsevier Ltd. All rights reserved.

Establishment of alternative potency test for botulinum toxin type A using compound muscle action potential (CMAP) in rats.

PubMed

Torii, Yasushi; Goto, Yoshitaka; Nakahira, Shinji; Ginnaga, Akihiro

2014-11-01

The biological activity of botulinum toxin type A has been evaluated using the mouse intraperitoneal (ip) LD50 test. This method requires a large number of mice to precisely determine toxin activity, and, as such, poses problems with regard to animal welfare. We previously developed a compound muscle action potential (CMAP) assay using rats as an alternative method to the mouse ip LD50 test. In this study, to evaluate this quantitative method of measuring toxin activity using CMAP, we assessed the parameters necessary for quantitative tests according to ICH Q2 (R1). This assay could be used to evaluate the activity of the toxin, even when inactive toxin was mixed with the sample. To reduce the number of animals needed, this assay was set to measure two samples per animal. Linearity was detected over a range of 0.1-12.8 U/mL, and the measurement range was set at 0.4-6.4 U/mL. The results for accuracy and precision showed low variability. The body weight was selected as a variable factor, but it showed no effect on the CMAP amplitude. In this study, potency tests using the rat CMAP assay of botulinum toxin type A demonstrated that it met the criteria for a quantitative analysis method. Copyright © 2014 Elsevier Ltd. All rights reserved.

A new ELISA for determination of potency in snake antivenoms.

PubMed

Rial, A; Morais, V; Rossi, S; Massaldi, H

2006-09-15

A competitive ELISA for potency determination of bothropic equine antivenom was developed and compared to the conventional in vivo ED(50) assay, with the aim of partially substituting the in vivo assay in the monitoring of antivenom immunoglobulin levels. On this purpose, blood samples were taken at different times during and after the immunization protocol of the lot of horses used for production of snake antivenom at the Instituto de Higiene, Uruguay. Both the competitive ELISA and the ED(50) assay were performed on those samples. In addition, a group of five commercial pepsin-digested antivenoms were tested by both methods. A significant (P<0.001) correlation (Pearson's r=0.957) was found between the ELISA titres and the corresponding ED(50) values, indicating that the in vitro test can estimate the neutralizing antibody capacity of the sera as well as the in vivo assay. By means of this new ELISA, it was found that the immunized animals maintained good venom antibody titres, in the order of 20-50% of the maximum achieved, even 10 month after the end of the immunization schedule. The main advantage of our ELISA design is its ability to correctly estimate the neutralization capacity of crude hyperimmune plasma and antivenom sera independently of their antibody composition in terms of whole IgG or F(ab')(2) fragment.

A functional and thromboelastometric-based micromethod for assessing crotoxin anticoagulant activity and antiserum relative potency against Crotalus durissus terrificus venom.

PubMed

Prezoto, B C; Tanaka-Azevedo, A M; Marcelino, J R; Tashima, A K; Nishiduka, E S; Kapronezai, J; Mota, J O; Rocha, M M T; Serino-Silva, C; Oguiura, N

2018-06-15

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies on traditional rodent in vivo lethality assay. ED 50 and LD 50 assays require large quantities of venoms and antivenoms, and besides leading to animal suffering. Therefore, in vitro tests should be introduced for assessing antivenom neutralizing capacity in intermediary steps of antivenom production. This task is facilitated when one key lethal toxin is identified. A good example is crotoxin, a β-neurotoxin phospholipase A 2 -like toxin that presents anticoagulant activity in vitro and is responsible for the lethality of venoms of Crotalus durissus snakes. By using rotational thromboelastometry, we reported recently one sensitive coagulation assay for assessing relative potency of the anti-bothropic serum in neutralizing procoagulant activity of Bothrops jararaca venom upon recalcified factor-XII-deficient chicken plasma samples (CPS). In this study, we stablished conditions for determining relative potency of four batches of the anti-crotalic serum (ACS) (antagonist) in inactivating crotoxin anticoagulant activity in CPS (target) simultaneously treated with one classical activator of coagulation (agonists). The correlation coefficient (r) between values related the ACS potency in inactivating both in vitro crotoxin anticoagulant activity and the in vivo lethality of whole venom (ED 50 ) was 0.94 (p value < 0.05). In conclusion, slowness in spontaneous thrombin/fibrin generation even after recalcification elicit time lapse sufficient for elaboration of one dose-response curve to pro- or anti-coagulant agonists in CPS. We propose this methodology as an alternative and sensitive assay for assessing antivenom neutralizing ability in plasma of immunized horses as well as for in-process quality control. Copyright © 2018 Elsevier Ltd. All rights reserved.

Ricin detection: tracking active toxin.

PubMed

Bozza, William P; Tolleson, William H; Rivera Rosado, Leslie A; Zhang, Baolin

2015-01-01

Ricin is a plant toxin with high bioterrorism potential due to its natural abundance and potency in inducing cell death. Early detection of the active toxin is essential for developing appropriate countermeasures. Here we review concepts for designing ricin detection methods, including mechanism of action of the toxin, advantages and disadvantages of current detection assays, and perspectives on the future development of rapid and reliable methods for detecting ricin in environmental samples. Published by Elsevier Inc.

EDQM biological reference preparation for rabies vaccine (inactivated) for veterinary use.

PubMed

Daas, A; Bruckner, L; Milne, C

2015-01-01

Rabies is a deadly zoonotic disease. Control of rabies in animals by vaccination is an important strategy to protect humans from infection and control the spread of the disease. Requirements for the quality control of rabies vaccines (inactivated) for veterinary use include an in vivo quantitative potency determination as outlined in the Ph. Eur. monograph 0451. Performance of this assay requires a reference preparation calibrated in International Units (IU). A European Pharmacopeia (Ph. Eur.) Biological Reference Preparation (BRP) for rabies vaccines (inactivated) for veterinary use, calibrated in IU, has been established for this purpose. Due to the dwindling stocks of the current batch (batch 4) of Ph. Eur. BRP for rabies vaccines (inactivated) for veterinary use, a collaborative study was run as part of the EDQM Biological Standardisation Programme to establish BRP batch 5. Ten laboratories, including Official Medicines Control Laboratories and manufacturers, participated. The candidate BRP5 was assayed against the 6(th) International Standard for rabies vaccine using the in vivo vaccination-challenge assay (monograph 0451) to assign a potency value. The candidate was also compared to BRP batch 4 to establish continuity. Taking into account the results from the comparisons a potency of 10 IU/vial was assigned and in March 2015 the Ph. Eur. Commission adopted the material as Ph. Eur. BRP for rabies vaccines (inactivated) for veterinary use batch 5. In addition to the in vivo assay 3 laboratories tested the candidate material using their in-house in vitro assays for information.

Kinome-wide selectivity profiling of ATP-competitive mammalian target of rapamycin (mTOR) inhibitors and characterization of their binding kinetics.

PubMed

Liu, Qingsong; Kirubakaran, Sivapriya; Hur, Wooyoung; Niepel, Mario; Westover, Kenneth; Thoreen, Carson C; Wang, Jinhua; Ni, Jing; Patricelli, Matthew P; Vogel, Kurt; Riddle, Steve; Waller, David L; Traynor, Ryan; Sanda, Takaomi; Zhao, Zheng; Kang, Seong A; Zhao, Jean; Look, A Thomas; Sorger, Peter K; Sabatini, David M; Gray, Nathanael S

2012-03-23

An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 μM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.

Otitis-Prone Children Produce Functional Antibodies to Pneumolysin and Pneumococcal Polysaccharides.

PubMed

Kirkham, Lea-Ann S; Wiertsema, Selma P; Corscadden, Karli J; Mateus, Tulia; Mullaney, Gemma L; Zhang, Guicheng; Richmond, Peter C; Thornton, Ruth B

2017-03-01

The pneumococcus is a major otitis media (OM) pathogen, but data are conflicting regarding whether otitis-prone children have impaired humoral immunity to pneumococcal antigens. We and others have shown that otitis-prone and healthy children have similar antibody titers to pneumococcal proteins and polysaccharides (vaccine and nonvaccine types); however, the quality of antibodies from otitis-prone children has not been investigated. Antibody function, rather than titer, is considered to be a better correlate of protection from pneumococcal disease. Therefore, we compared the capacities of antibodies from otitis-prone (cases) and healthy (controls) children to neutralize pneumolysin, the pneumococcal toxin currently in development as a vaccine antigen, and to opsonize pneumococcal vaccine and nonvaccine serotypes. A pneumolysin neutralization assay was conducted on cholesterol-depleted complement-inactivated sera from 165 cases and 61 controls. A multiplex opsonophagocytosis assay (MOPA) was conducted on sera from 20 cases and 20 controls. Neutralizing and opsonizing titers were calculated with antigen-specific IgG titers to determine antibody potency for pneumolysin, pneumococcal conjugate vaccine (PCV) polysaccharides, and non-PCV polysaccharides. There was no significant difference in antibody potencies between cases and controls for the antigens tested. Antipneumolysin neutralizing titers increased with the number of episodes of acute OM, but antibody potency did not. Pneumolysin antibody potency was lower in children colonized with pneumococci than in noncarriers, and this was significant for the otitis-prone group ( P < 0.05). The production of functional antipneumococcal antibodies in otitis-prone children demonstrates that they respond to the current PCV and are likely to respond to pneumolysin-based vaccines as effectively as healthy children. Copyright © 2017 Kirkham et al.

Effects of Anticholinesterases on Catalysis and Induced Conformational Change of the Peripheral Anionic Site of Murine Acetylcholinesterase

PubMed Central

Tong, Fan; Islam, Rafique M.; Carlier, Paul R.; Ma, Ming; Ekström, Fredrik; Bloomquist, Jeffrey R.

2013-01-01

Conventional insecticides targeting acetylcholinesterase (AChE) typically show high mammalian toxicities and because there is resistance to these compounds in many insect species, alternatives to established AChE inhibitors used for pest control are needed. Here we used a fluorescence method to monitor interactions between various AChE inhibitors and the AChE peripheral anionic site, which is a novel target for new insecticides acting on this enzyme. The assay uses thioflavin-T as a probe, which binds to the peripheral anionic site of AChE and yields an increase in fluorescent signal. Three types of AChE inhibitors were studied: catalytic site inhibitors (carbamate insecticides, edrophonium, and benzylpiperidine), peripheral site inhibitors (tubocurarine, ethidium bromide, and propidium iodide), and bivalent inhibitors (donepezil, BW284C51, and a series of bis(n)-tacrines). All were screened on murine AChE to compare and contrast changes of peripheral site conformation in the TFT assay with catalytic inhibition. All the inhibitors reduced thioflavin-T fluorescence in a concentration-dependent manner with potencies (IC50) ranging from 8 nM for bis(6)-tacrine to 159 μM for benzylpiperidine. Potencies in the fluorescence assay were correlated well with their potencies for enzyme inhibition (R2 = 0.884). Efficacies for reducing thioflavin-T fluorescence ranged from 23–36% for catalytic site inhibitors and tubocurarine to near 100% for ethidium bromide and propidium iodide. Maximal efficacies could be reconciled with known mechanisms of interaction of the inhibitors with AChE. When extended to pest species, we anticipate these findings will assist in the discovery and development of novel, selective bivalent insecticides acting on AChE. PMID:24003261

The Concentrations but Not the Components of Particulate Material and Secondary Transformation Products May Account for Much of the Variation in Air Mutagenicity

EPA Science Inventory

The mutagenic potency of ambient air PM in the Salmonella mutagenicity assay (rev/mg PM) varies only ~1 order of magnitude worldwide; however, the mutagenic potency of the air itself (rev/m3 of air) varies ~5 orders of magnitude (IARC Monograph Vol 109, 2016). Thus, the componen...

Establishment of hepatitis A vaccine (inactivated, non-adsorbed) BRP batches 2 and 3.

PubMed

Morgeaux, S; Manniam, I; Variot, P; Buchheit, K H; Daas, A; Wierer, M; Costanzo, A

2015-01-01

The current hepatitis A vaccine (HAV), inactivated, non-adsorbed, European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) is used for the in vitro potency assay of HAV as prescribed by the Ph. Eur. general chapter 2.7.14 Assay of hepatitis A vaccine. This reference preparation was calibrated in 2008 through an international collaborative study and was assigned a potency of 12 IU/mL. During use of this BRP it appeared to be inapplicable in certain cases due to a low nominal antigen content. Consequently, the European Directorate for the Quality of Medicines and HealthCare (EDQM) established replacement batches for this BRP, calibrated against the 1(st) WHO International Standard (IS) for HAV (inactivated), using the standard in vitro ELISA (enzyme-linked immunosorbent assay) method validated previously. The results of the study showed that the candidate BRPs were suitable for the intended purpose, and following completion of the study, they were adopted in November 2014 by the Ph. Eur. Commission as HAV (inactivated, non-adsorbed) BRP batches 2 and 3, with an assigned potency of 1350 IU/mL, for in vitro antigen content determination by ELISA. As the amount of material in each vial largely exceeds the amount required for the performance of a single assay, the BRPs are to be aliquoted by users as single-use aliquots and refrozen below -50 °C prior to their use as reference preparations.

Synthesis, chemical reactivity as Michael acceptors, and biological potency of monocyclic cyanoenones, novel and highly potent anti-inflammatory and cytoprotective agents.

PubMed

Zheng, Suqing; Santosh Laxmi, Y R; David, Emilie; Dinkova-Kostova, Albena T; Shiavoni, Katherine H; Ren, Yanqing; Zheng, Ying; Trevino, Isaac; Bumeister, Ronald; Ojima, Iwao; Wigley, W Christian; Bliska, James B; Mierke, Dale F; Honda, Tadashi

2012-05-24

Novel monocyclic cyanoenones examined to date display unique features regarding chemical reactivity as Michael acceptors and biological potency. Remarkably, in some biological assays, the simple structure is more potent than pentacyclic triterpenoids (e.g., CDDO and bardoxolone methyl) and tricycles (e.g., TBE-31). Among monocyclic cyanoenones, 1 is a highly reactive Michael acceptor with thiol nucleophiles. Furthermore, an important feature of 1 is that its Michael addition is reversible. For the inhibition of NO production, 1 shows the highest potency. Notably, its potency is about three times higher than CDDO, whose methyl ester (bardoxolone methyl) is presently in phase III clinical trials. For the induction of NQO1, 1 also demonstrated the highest potency. These results suggest that the reactivity of these Michael acceptors is closely related to their biological potency. Interestingly, in LPS-stimulated macrophages, 1 causes apoptosis and inhibits secretion of TNF-α and IL-1β with potencies that are higher than those of bardoxolone methyl and TBE-31.

Pharmacological characterization of a fluorescent uptake assay for the noradrenaline transporter.

PubMed

Haunsø, Anders; Buchanan, Dawn

2007-04-01

The noradrenaline transporter (NET) is a Na(+)/Cl(-) dependent monoamine transporter that mediates rapid clearance of noradrenaline from the synaptic cleft, thereby terminating neuronal signaling. NET is an important target for drug development and is known to be modulated by many psychoactive compounds, including psychostimulants and antidepressants. Here, the authors describe the development and pharmacological characterization of a nonhomogeneous fluorescent NET uptake assay using the compound 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP(+)). Data presented show that the pharmacology of both the classic radiolabeled (3)H-noradrenaline- and ASP(+)-based uptake assays are comparable, with an excellent correlation between potency obtained for known modulators of NET (r = 0.95, p < 0.0001). Furthermore, the fluorescent uptake assay is highly reproducible and has sufficiently large Z' values to be amenable for high-throughput screening (HTS). The advantage of this assay is compatibility with both 96- and 384-well formats and lack of radioactivity usage. Thus, the authors conclude that the assay is an inexpensive, viable approach for the identification and pharmacological profiling of small-molecule modulators of the monoamine transporter NET and may be amenable for HTS.

A practical approach for the validation of sterility, endotoxin and potency testing of bone marrow mononucleated cells used in cardiac regeneration in compliance with good manufacturing practice.

PubMed

Soncin, Sabrina; Lo Cicero, Viviana; Astori, Giuseppe; Soldati, Gianni; Gola, Mauro; Sürder, Daniel; Moccetti, Tiziano

2009-09-08

Main scope of the EU and FDA regulations is to establish a classification criterion for advanced therapy medicinal products (ATMP). Regulations require that ATMPs must be prepared under good manufacturing practice (GMP). We have validated a commercial system for the determination of bacterial endotoxins in compliance with EU Pharmacopoeia 2.6.14, the sterility testing in compliance with EU Pharmacopoeia 2.6.1 and a potency assay in an ATMP constituted of mononucleated cells used in cardiac regeneration. For the potency assay, cells were placed in the upper part of a modified Boyden chamber containing Endocult Basal Medium with supplements and transmigrated cells were scored. The invasion index was expressed as the ratio between the numbers of invading cells relative to cell migration through a control insert membrane. For endotoxins, we used a commercially available system based on the kinetic chromogenic LAL-test. Validation of sterility was performed by direct inoculation of TSB and FTM media with the cell product following Eu Ph 2.6.1 guideline. The calculated MVD and endotoxin limit were 780x and 39 EU/ml respectively. The 1:10 and 1:100 dilutions were selected for the validation. For sterility, all the FTM cultures were positive after 3 days. For TSB cultures, Mycetes and B. subtilis were positive after 5 and 3 days respectively. The detection limit was 1-10 colonies. A total of four invasion assay were performed: the calculated invasion index was 28.89 +/- 16.82% (mean +/- SD). We have validated a strategy for endotoxin, sterility and potency testing in an ATMP used in cardiac regeneration. Unlike pharmaceutical products, many stem-cell-based products may originate in hospitals where personnel are unfamiliar with the applicable regulations. As new ATMPs are developed, the regulatory framework is likely to evolve. Meanwhile, existing regulations provide an appropriate structure for ensuring the safety and efficacy of the next generation of ATMPs. Personnel must be adequately trained on relevant methods and their application to stem-cell-based products.

Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate.

PubMed

Schmitt, Martin L; Ladwein, Kathrin I; Carlino, Luca; Schulz-Fincke, Johannes; Willmann, Dominica; Metzger, Eric; Schilcher, Pierre; Imhof, Axel; Schüle, Roland; Sippl, Wolfgang; Jung, Manfred

2014-07-01

Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry. © 2014 Society for Laboratory Automation and Screening.

Streamlined system for purifying and quantifying a diverse library of compounds and the effect of compound concentration measurements on the accurate interpretation of biological assay results.

PubMed

Popa-Burke, Ioana G; Issakova, Olga; Arroway, James D; Bernasconi, Paul; Chen, Min; Coudurier, Louis; Galasinski, Scott; Jadhav, Ajit P; Janzen, William P; Lagasca, Dennis; Liu, Darren; Lewis, Roderic S; Mohney, Robert P; Sepetov, Nikolai; Sparkman, Darren A; Hodge, C Nicholas

2004-12-15

As part of an overall systems approach to generating highly accurate screening data across large numbers of compounds and biological targets, we have developed and implemented streamlined methods for purifying and quantitating compounds at various stages of the screening process, coupled with automated "traditional" storage methods (DMSO, -20 degrees C). Specifically, all of the compounds in our druglike library are purified by LC/MS/UV and are then controlled for identity and concentration in their respective DMSO stock solutions by chemiluminescent nitrogen detection (CLND)/evaporative light scattering detection (ELSD) and MS/UV. In addition, the compound-buffer solutions used in the various biological assays are quantitated by LC/UV/CLND to determine the concentration of compound actually present during screening. Our results show that LC/UV/CLND/ELSD/MS is a widely applicable method that can be used to purify, quantitate, and identify most small organic molecules from compound libraries. The LC/UV/CLND technique is a simple and sensitive method that can be easily and cost-effectively employed to rapidly determine the concentrations of even small amounts of any N-containing compound in aqueous solution. We present data to establish error limits for concentration determination that are well within the overall variability of the screening process. This study demonstrates that there is a significant difference between the predicted amount of soluble compound from stock DMSO solutions following dilution into assay buffer and the actual amount present in assay buffer solutions, even at the low concentrations employed for the assays. We also demonstrate that knowledge of the concentrations of compounds to which the biological target is exposed is critical for accurate potency determinations. Accurate potency values are in turn particularly important for drug discovery, for understanding structure-activity relationships, and for building useful empirical models of protein-ligand interactions. Our new understanding of relative solubility demonstrates that most, if not all, decisions that are made in early discovery are based upon missing or inaccurate information. Finally, we demonstrate that careful control of compound handling and concentration, coupled with accurate assay methods, allows the use of both positive and negative data in analyzing screening data sets for structure-activity relationships that determine potency and selectivity.

Implementation of a Fluorescence-Based Screening Assay Identifies Histamine H3 Receptor Antagonists Clobenpropit and Iodophenpropit as Subunit-Selective N-Methyl-d-Aspartate Receptor Antagonists

PubMed Central

Hansen, Kasper B.; Mullasseril, Praseeda; Dawit, Sara; Kurtkaya, Natalie L.; Yuan, Hongjie; Vance, Katie M.; Orr, Anna G.; Kvist, Trine; Ogden, Kevin K.; Le, Phuong; Vellano, Kimberly M.; Lewis, Iestyn; Kurtkaya, Serdar; Du, Yuhong; Qui, Min; Murphy, T. J.; Snyder, James P.; Bräuner-Osborne, Hans

2010-01-01

N-Methyl-d-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca2+-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism. PMID:20197375

High-performance liquid chromatographic method for potency determination of amoxicillin in commercial preparations and for stability studies.

PubMed Central

Hsu, M C; Hsu, P W

1992-01-01

A reversed-phase column liquid chromatographic method was developed for the assay of amoxicillin and its preparations. The linear calibration range was 0.2 to 2.0 mg/ml (r = 0.9998), and recoveries were generally greater than 99%. The high-performance liquid chromatographic assay results were compared with those obtained from a microbiological assay of bulk drug substance and capsule, injection, and granule formulations containing amoxicillin and degraded amoxicillin. At the 99% confidence level, no significant intermethod differences were noted for the paired results. Commercial formulations were also analyzed, and the results obtained by the proposed method closely agreed with those found by the microbiological method. The results indicated that the proposed method is a suitable substitute for the microbiological method for assays and stability studies of amoxicillin preparations. PMID:1416827

Comparative In Vitro Toxicity Evaluation of Heavy Metals (Lead, Cadmium, Arsenic, and Methylmercury) on HT-22 Hippocampal Cell Line.

PubMed

Karri, Venkatanaidu; Kumar, Vikas; Ramos, David; Oliveira, Eliandre; Schuhmacher, Marta

2018-07-01

Heavy metals are considered some of the most toxic environmental pollutants. Exposure to heavy metals including lead (Pb), cadmium (Cd), arsenic (As), and methyl mercury (MeHg) has long been known to cause damage to human health. Many recent studies have supported the hippocampus as the major target for these four metals for inflicting cognitive dysfunction. In the present study, we proposed hippocampal relevant in vitro toxicity of Pb, Cd, As, and MeHg in HT-22 cell line. This study reports, initially, cytotoxic effects in acute, subchronic, chronic exposures. We further investigated the mechanistic potency of DNA damage and apoptosis damage with the observed cytotoxicity. The genotoxicity and apoptosis were measured by using the comet assay, annexin-V FTIC / propidium iodide (PI) assay, respectively. The results of cytotoxicity assay clearly demonstrated significant concentration and time-dependent effects on HT-22 cell line. The genotoxic and apoptosis effects also concentration-dependent fashion with respect to their potency in the range of IC 10 -IC 30, maximal level of damage observed in MeHg. In conclusion, the obtained result suggests concentration and potency-dependent response; the maximal level of toxicity was observed in MeHg. These novel findings support that Pb, Cd, As, and MeHg induce cytotoxic, genotoxic, and apoptotic effects on HT-22 cells in potency-dependent manner; MeHg> As> Cd> Pb. Therefore, the toxicity of Pb, Cd, As, and MeHg could be useful for knowing the common underlying molecular mechanism, and also for estimating the mixture impacts on HT-22 cell line.

Efficacy of simple short-term in vitro assays for predicting the potential of metal oxide nanoparticles to cause pulmonary inflammation.

PubMed

Lu, Senlin; Duffin, Rodger; Poland, Craig; Daly, Paul; Murphy, Fiona; Drost, Ellen; Macnee, William; Stone, Vicki; Donaldson, Ken

2009-02-01

There has been concern regarding risks from inhalation exposure to nanoparticles (NPs). The large number of particles requiring testing means that alternative approaches to animal testing are needed. We set out to determine whether short-term in vitro assays that assess intrinsic oxidative stress potential and membrane-damaging potency of a panel of metal oxide NPs can be used to predict their inflammogenic potency. For a panel of metal oxide NPs, we investigated intrinsic free radical generation, oxidative activity in an extracellular environment, cytotoxicity to lung epithelial cells, hemolysis, and inflammation potency in rat lungs. All exposures were carried out at equal surface area doses. Only nickel oxide (NiO) and alumina 2 caused significant lung inflammation when instilled into rat lungs at equal surface area, suggesting that these two had extra surface reactivity. We observed significant free radical generation with 4 of 13 metal oxides, only one of which was inflammogenic. Only 3 of 13 were significantly hemolytic, two of which were inflammogenic. Potency in generating free radicals in vitro did not predict inflammation, whereas alumina 2 had no free radical activity but was inflammogenic. The hemolysis assay was correct in predicting the proinflammatory potential of 12 of 13 of the particles examined. Using a battery of simple in vitro tests, it is possible to predict the inflammogenicity of metal oxide NPs, although some false-positive results are likely. More research using a larger panel is needed to confirm the efficacy and generality of this approach for metal oxide NPs.

Skin sensitisation, vehicle effects and the local lymph node assay.

PubMed

Basketter, D A; Gerberick, G F; Kimber, I

2001-06-01

Accurate risk assessment in allergic contact dermatitis is dependent on the successful prospective identification of chemicals which possess the ability to behave as skin sensitisers, followed by appropriate measurement of the relative ability to cause sensitisation; their potency. Tools for hazard identification have been available for many years; more recently, a novel approach to the quantitative assessment of potency--the derivation of EC3 values in the local lymph node assay (LLNA)--has been described. It must be recognised, however, that these evaluations of chemical sensitisers also may be affected by the vehicle matrix in which skin exposure occurs. In this article, our knowledge of this area is reviewed and potential mechanisms through which vehicle effects may occur are detailed. Using the LLNA as an example, it is demonstrated that the vehicle may have little impact on the accuracy of basic hazard identification; the data also therefore support the view that testing ingredients in specific product formulations is not warranted for hazard identification purposes. However, the effect on potency estimations is of greater significance. Although not all chemical allergens are affected similarly, for certain substances a greater than 10-fold vehicle-dependent change in potency is observed. Such data are vital for accurate risk assessment. Unfortunately, it does not at present appear possible to predict notionally the effect of the vehicle matrix on skin sensitising potency without recourse to direct testing, for example by estimation of LLNA EC3 data, which provides a valuable tool for this purpose.

Graphical method for comparative statistical study of vaccine potency tests.

PubMed

Pay, T W; Hingley, P J

1984-03-01

Producers and consumers are interested in some of the intrinsic characteristics of vaccine potency assays for the comparative evaluation of suitable experimental design. A graphical method is developed which represents the precision of test results, the sensitivity of such results to changes in dosage, and the relevance of the results in the way they reflect the protection afforded in the host species. The graphs can be constructed from Producer's scores and Consumer's scores on each of the scales of test score, antigen dose and probability of protection against disease. A method for calculating these scores is suggested and illustrated for single and multiple component vaccines, for tests which do or do not employ a standard reference preparation, and for tests which employ quantitative or quantal systems of scoring.

Arylimidamide-Azole Combinations Against Leishmaniasis

DTIC Science & Technology

2015-09-01

potency of posaconazole in an amastigote macrophage assay2, the only azole to demonstrate activity in vitro against CL species, showed variable activity ...ranging from no activity observed against L. panamensis and L. guyanensis to modest activity against L. tropica to potent activity against L. major...species, and the potency is variable; while posaconazole is active against Old World CL species such as L. major and L. tropica it is not active

Alcohol's Effects on Lipid Bilayer Properties

PubMed Central

Ingólfsson, Helgi I.; Andersen, Olaf S.

2011-01-01

Alcohols are known modulators of lipid bilayer properties. Their biological effects have long been attributed to their bilayer-modifying effects, but alcohols can also alter protein function through direct protein interactions. This raises the question: Do alcohol's biological actions result predominantly from direct protein-alcohol interactions or from general changes in the membrane properties? The efficacy of alcohols of various chain lengths tends to exhibit a so-called cutoff effect (i.e., increasing potency with increased chain length, which that eventually levels off). The cutoff varies depending on the assay, and numerous mechanisms have been proposed such as: limited size of the alcohol-protein interaction site, limited alcohol solubility, and a chain-length-dependent lipid bilayer-alcohol interaction. To address these issues, we determined the bilayer-modifying potency of 27 aliphatic alcohols using a gramicidin-based fluorescence assay. All of the alcohols tested (with chain lengths of 1–16 carbons) alter the bilayer properties, as sensed by a bilayer-spanning channel. The bilayer-modifying potency of the short-chain alcohols scales linearly with their bilayer partitioning; the potency tapers off at higher chain lengths, and eventually changes sign for the longest-chain alcohols, demonstrating an alcohol cutoff effect in a system that has no alcohol-binding pocket. PMID:21843475

PRELIMINARY ASSESSMENTS OF IN VITRO PHARMACOKINETIC DATA AND EXPOSURE INFORMATION FOR THE TOXCAST PHASE II CHEMICALS

EPA Science Inventory

Momentum has been growing in Toxicology to assess the utility of high-throughput screening (HTS) assays in the determination of chemical testing priorities. However, in vitro potencies determined in these assays do not consider in vivo bioavailability, clearance or exposure estim...

Comparing BMD‐derived genotoxic potency estimations across variants of the transgenic rodent gene mutation assay

PubMed Central

Johnson, George E.; Battaion, Hannah L.; Slob, Wout; Gollapudi, B.

2017-01-01

There is growing interest in quantitative analysis of in vivo genetic toxicity dose‐response data, and use of point‐of‐departure (PoD) metrics such as the benchmark dose (BMD) for human health risk assessment (HHRA). Currently, multiple transgenic rodent (TGR) assay variants, employing different rodent strains and reporter transgenes, are used for the assessment of chemically‐induced genotoxic effects in vivo. However, regulatory issues arise when different PoD values (e.g., lower BMD confidence intervals or BMDLs) are obtained for the same compound across different TGR assay variants. This study therefore employed the BMD approach to examine the ability of different TGR variants to yield comparable genotoxic potency estimates. Review of over 2000 dose‐response datasets identified suitably‐matched dose‐response data for three compounds (ethyl methanesulfonate or EMS, N‐ethyl‐N‐nitrosourea or ENU, and dimethylnitrosamine or DMN) across four commonly‐used murine TGR variants (Muta™Mouse lacZ, Muta™Mouse cII, gpt delta and BigBlue® lacI). Dose‐response analyses provided no conclusive evidence that TGR variant choice significantly influences the derived genotoxic potency estimate. This conclusion was reliant upon taking into account the importance of comparing BMD confidence intervals as opposed to directly comparing PoD values (e.g., comparing BMDLs). Comparisons with earlier works suggested that with respect to potency determination, tissue choice is potentially more important than choice of TGR assay variant. Scoring multiple tissues selected on the basis of supporting toxicokinetic information is therefore recommended. Finally, we used typical within‐group variances to estimate preliminary endpoint‐specific benchmark response (BMR) values across several TGR variants/tissues. We discuss why such values are required for routine use of genetic toxicity PoDs for HHRA. Environ. Mol. Mutagen. 58:632–643, 2017. © 2017 Her Majesty the Queen in Right of Canada. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc. PMID:28945287

A simple and rapid Hepatitis A Virus (HAV) titration assay based on antibiotic resistance of infected cells: evaluation of the HAV neutralization potency of human immune globulin preparations

PubMed Central

Konduru, Krishnamurthy; Virata-Theimer, Maria Luisa; Yu, Mei-ying W; Kaplan, Gerardo G

2008-01-01

Background Hepatitis A virus (HAV), the causative agent of acute hepatitis in humans, is an atypical Picornaviridae that grows poorly in cell culture. HAV titrations are laborious and time-consuming because the virus in general does not cause cytopathic effect and is detected by immunochemical or molecular probes. Simple HAV titration assays could be developed using currently available viral construct containing selectable markers. Results We developed an antibiotic resistance titration assay (ARTA) based on the infection of human hepatoma cells with a wild type HAV construct containing a blasticidin (Bsd) resistance gene. Human hepatoma cells infected with the HAV-Bsd construct survived selection with 2 μg/ml of blasticidin whereas uninfected cells died within a few days. At 8 days postinfection, the color of the pH indicator phenol red in cell culture media correlated with the presence of HAV-Bsd-infected blasticidin-resistant cells: an orange-to-yellow color indicated the presence of growing cells whereas a pink-to-purple color indicated that the cells were dead. HAV-Bsd titers were determined by an endpoint dilution assay based on the color of the cell culture medium scoring orange-to-yellow wells as positive and pink-to-purple wells as negative for HAV. As a proof-of-concept, we used the ARTA to evaluate the HAV neutralization potency of two commercially available human immune globulin (IG) preparations and a WHO International Standard for anti-HAV. The three IG preparations contained comparable levels of anti-HAV antibodies that neutralized approximately 1.5 log of HAV-Bsd. Similar neutralization results were obtained in the absence of blasticidin by an endpoint dilution ELISA at 2 weeks postinfection. Conclusion The ARTA is a simple and rapid method to determine HAV titers without using HAV-specific probes. We determined the HAV neutralization potency of human IG preparations in 8 days by ARTA compared to the 14 days required by the endpoint dilution ELISA. The ARTA reduced the labour, time, and cost of HAV titrations making it suitable for high throughput screening of sera and antivirals, determination of anti-HAV antibodies in human immune globulin preparations, and research applications that involve the routine evaluation of HAV titers. PMID:19094229

ICECAP: an integrated, general-purpose, automation-assisted IC50/EC50 assay platform.

PubMed

Li, Ming; Chou, Judy; King, Kristopher W; Jing, Jing; Wei, Dong; Yang, Liyu

2015-02-01

IC50 and EC50 values are commonly used to evaluate drug potency. Mass spectrometry (MS)-centric bioanalytical and biomarker labs are now conducting IC50/EC50 assays, which, if done manually, are tedious and error-prone. Existing bioanalytical sample preparation automation systems cannot meet IC50/EC50 assay throughput demand. A general-purpose, automation-assisted IC50/EC50 assay platform was developed to automate the calculations of spiking solutions and the matrix solutions preparation scheme, the actual spiking and matrix solutions preparations, as well as the flexible sample extraction procedures after incubation. In addition, the platform also automates the data extraction, nonlinear regression curve fitting, computation of IC50/EC50 values, graphing, and reporting. The automation-assisted IC50/EC50 assay platform can process the whole class of assays of varying assay conditions. In each run, the system can handle up to 32 compounds and up to 10 concentration levels per compound, and it greatly improves IC50/EC50 assay experimental productivity and data processing efficiency. © 2014 Society for Laboratory Automation and Screening.

Generation of cell lines for drug discovery through random activation of gene expression: application to the human histamine H3 receptor.

PubMed

Song, J; Doucette, C; Hanniford, D; Hunady, K; Wang, N; Sherf, B; Harrington, J J; Brunden, K R; Stricker-Krongrad, A

2005-06-01

Target-based high-throughput screening (HTS) plays an integral role in drug discovery. The implementation of HTS assays generally requires high expression levels of the target protein, and this is typically accomplished using recombinant cDNA methodologies. However, the isolated gene sequences to many drug targets have intellectual property claims that restrict the ability to implement drug discovery programs. The present study describes the pharmacological characterization of the human histamine H3 receptor that was expressed using random activation of gene expression (RAGE), a technology that over-expresses proteins by up-regulating endogenous genes rather than introducing cDNA expression vectors into the cell. Saturation binding analysis using [125I]iodoproxyfan and RAGE-H3 membranes revealed a single class of binding sites with a K(D) value of 0.77 nM and a B(max) equal to 756 fmol/mg of protein. Competition binding studies showed that the rank order of potency for H3 agonists was N(alpha)-methylhistamine approximately (R)-alpha- methylhistamine > histamine and that the rank order of potency for H3 antagonists was clobenpropit > iodophenpropit > thioperamide. The same rank order of potency for H3 agonists and antagonists was observed in the functional assays as in the binding assays. The Fluorometic Imaging Plate Reader assays in RAGE-H3 cells gave high Z' values for agonist and antagonist screening, respectively. These results reveal that the human H3 receptor expressed with the RAGE technology is pharmacologically comparable to that expressed through recombinant methods. Moreover, the level of expression of the H3 receptor in the RAGE-H3 cells is suitable for HTS and secondary assays.

Bisphenol A alternatives bisphenol S and bisphenol F interfere with thyroid hormone signaling pathway in vitro and in vivo.

PubMed

Zhang, Yin-Feng; Ren, Xiao-Min; Li, Yuan-Yuan; Yao, Xiao-Fang; Li, Chuan-Hai; Qin, Zhan-Fen; Guo, Liang-Hong

2018-06-01

The wide use of the alternatives to bisphenol A (BPA) has raised concerns about their potential toxicities. Considering the disrupting activity of BPA on thyroid hormone (TH) signaling, we investigated whether bisphenol S (BPS) and bisphenol F (BPF), two leading alternatives, could interfere with TH signaling pathway using a series of assays in vitro and in vivo. In the fluorescence competitive binding assay, we found BPS and BPF, like BPA, bound to TH receptors (TRα and TRβ), with the binding potencies an order of magnitude lower than BPA (BPA > BPF > BPS). Molecular docking data also show their binding potencies to TRs. In the coactivator recruitment assay, BPS and BPF recruited coactivator to TRβ but not TRα, with weaker potencies than BPA. Correspondingly, agonistic actions of the three bisphenols in the absence or presence of T3 were observed in the TR-mediated reporter gene transcription assay. Also, all the three bisphenols induced TH-dependent GH3 cell proliferation, whereas BPA and BPF inhibited T3 induction in the presence of T3. As for in vivo assay, the three bisphenols like T3 induced TH-response gene transcription in Pelophylax nigromaculatus tadpoles, but in the presence of T3 altered T3-induced gene transcription in a biphasic concentration-response manner. These results for the first time demonstrate that BPS and BPF, like BPA, have potential to interfere with TH signaling pathway, i.e., they generally activate TH signaling in the absence of T3, but in the presence of TH, display agonistic or/and antagonistic actions under certain condition. Our study highlights the potential risks of BPS and BPF as BPA alternatives. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hazard assessment of nitrosamine and nitramine by-products of amine-based CCS: alternative approaches.

PubMed

Buist, H E; Devito, S; Goldbohm, R A; Stierum, R H; Venhorst, J; Kroese, E D

2015-04-01

Carbon capture and storage (CCS) technologies are considered vital and economic elements for achieving global CO2 reduction targets, and is currently introduced worldwide (for more information on CCS, consult for example the websites of the International Energy Agency (http://www.iea.org/topics/ccs/) and the Global CCS Institute (http://www.globalccsinstitute.com/)). One prominent CCS technology, the amine-based post-combustion process, may generate nitrosamines and their related nitramines as by-products, the former well known for their potential mutagenic and carcinogenic properties. In order to efficiently assess the carcinogenic potency of any of these by-products this paper reviews and discusses novel prediction approaches consuming less time, money and animals than the traditionally applied 2-year rodent assay. For this, available animal carcinogenicity studies with N-nitroso compounds and nitramines have been used to derive carcinogenic potency values, that were subsequently used to assess the predictive performance of alternative prediction approaches for these chemicals. Promising cancer prediction models are the QSARs developed by the Helguera group, in vitro transformation assays, and the in vivo initiation-promotion, and transgenic animal assays. All these models, however, have not been adequately explored for this purpose, as the number of N-nitroso compounds investigated is yet too limited, and therefore further testing with relevant N-nitroso compounds is needed. Copyright © 2015. Published by Elsevier Inc.

A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the LD50 of polychlorinated biphenyls in avian species.

PubMed

Manning, Gillian E; Farmahin, Reza; Crump, Doug; Jones, Stephanie P; Klein, Jeff; Konstantinov, Alex; Potter, Dave; Kennedy, Sean W

2012-09-15

Birds differ in sensitivity to the embryotoxic effects of polychlorinated biphenyls (PCBs), which complicates environmental risk assessments for these chemicals. Recent research has shown that the identities of amino acid residues 324 and 380 in the avian aryl hydrocarbon receptor 1 (AHR1) ligand binding domain (LBD) are primarily responsible for differences in avian species sensitivity to selected dibenzo-p-dioxins and furans. A luciferase reporter gene (LRG) assay was developed in our laboratory to measure AHR1-mediated induction of a cytochrome P450 1A5 reporter gene in COS-7 cells transfected with different avian AHR1 constructs. In the present study, the LRG assay was used to measure the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and PCBs 126, 77, 105 and 118 on luciferase activity in COS-7 cells transfected with AHR1 constructs representative of 86 avian species in order to predict their sensitivity to PCB-induced embryolethality and the relative potency of PCBs in these species. The results of the LRG assay indicate that the identity of amino acid residues 324 and 380 in the AHR1 LBD are the major determinants of avian species sensitivity to PCBs. The relative potency of PCBs did not differ greatly among AHR1 constructs. Luciferase activity was significantly correlated with embryolethality data obtained from the literature (R(2)≥0.87, p<0.0001). Thus, the LRG assay in combination with the knowledge of a species' AHR1 LBD sequence can be used to predict PCB-induced embryolethality in potentially any avian species of interest without the use of lethal methods on a large number of individuals. Copyright © 2012 Elsevier Inc. All rights reserved.

The second international standard for polymyxin B.

PubMed

Lightbown, J W; Thomas, A H; Grab, B; Outschoorn, A S

1973-01-01

Since supplies of the first International Standard for Polymyxin B were exhausted, it was replaced by a second international standard the potency of which was estimated from the results of a collaborative assay carried out by 5 laboratories in 4 countries. The wide variations in the results probably resulted from difficulties experienced in handling the first international standard. The potency finally agreed upon by the collaborating laboratories, on the basis of the overall mean values obtained after rejection of the most discrepant assays, was 8 403 IU/mg. That value was accepted by the WHO Expert Committee on Biological Standardization (1970), which consequently defined the International Unit of polymyxin B as the activity contained in 0.000119 mg of the second international standard.

The second international standard for polymyxin B*

PubMed Central

Lightbown, J. W.; Thomas, A. H.; Grab, B.; Outschoorn, A. S.

1973-01-01

Since supplies of the first International Standard for Polymyxin B were exhausted, it was replaced by a second international standard the potency of which was estimated from the results of a collaborative assay carried out by 5 laboratories in 4 countries. The wide variations in the results probably resulted from difficulties experienced in handling the first international standard. The potency finally agreed upon by the collaborating laboratories, on the basis of the overall mean values obtained after rejection of the most discrepant assays, was 8 403 IU/mg. That value was accepted by the WHO Expert Committee on Biological Standardization (1970), which consequently defined the International Unit of polymyxin B as the activity contained in 0.000119 mg of the second international standard. PMID:4350877

Maintenance dosing for sublingual immunotherapy by prominent European allergen manufacturers expressed in bioequivalent allergy units.

PubMed

Larenas-Linnemann, Désirée; Esch, Robert; Plunkett, Greg; Brown, Shannon; Maddox, Daniel; Barnes, Charles; Constable, Derek

2011-11-01

Sublingual immunotherapy (SLIT) has become established in Europe, and its efficacy is being evaluated in the United States. The doses used for SLIT in Europe today are difficult to evaluate, because each manufacturer expresses the potency of its extracts differently. To compare in vitro European SLIT maintenance solutions against US licensed standardized allergenic extract concentrates and to determine the monthly SLIT doses delivered expressed in bioequivalent allergy units ([B]AU). We studied Dermatophagoides pteronyssinus, timothy grass pollen, cat (hair) and short ragweed pollen allergen extracts. The SLIT maintenance solutions of 4 leading European manufacturers and standardized concentrate extracts of 3 US manufacturers were analyzed with the following assays: protein content, relative potency (immunoglobulin E [IgE]-binding enzyme-linked immunosorbent assay [ELISA] inhibition) and major allergen content. The relative monthly allergen dose in (B)AU was calculated for each recommended SLIT schedule. Relative potency was approximately 10 times higher for US concentrate standardized extracts-which are meant to be diluted-than for European SLIT maintenance solutions of D pteronyssinus and timothy grass pollen. For cat (hair) and short ragweed pollen, the difference was less. Measurements of relative potency and major allergen content correlated well. In our assays, European mite extracts contain a very low quantity of Der p 2 compared with US mites. Recommended SLIT doses in Europe vary widely among the manufacturers, but are consistently lower (Eur1) or higher (Eur4) over all four allergens tested. SLIT efficacy probably depends on additional factors apart from the exact dose. SLIT dose finding studies should be done for each product. Copyright © 2011 American College of Allergy. Published by Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsukamoto, Ikuko, E-mail: tukamoto@med.kagawa-u.ac.jp; Sakakibara, Norikazu; Maruyama, Tokumi

Research highlights: {yields} A novel nucleic acid analogue (2Cl-C.OXT-A, m.w. 284) showed angiogenic potency. {yields} It stimulated the tube formation, proliferation and migration of HUVEC in vitro. {yields} 2Cl-C.OXT-A induced the activation of ERK1/2 and MEK in HUVEC. {yields} Angiogenic potency in vivo was confirmed in CAM assay and rabbit cornea assay. {yields} A synthesized small angiogenic agent would have great clinical therapeutic value. -- Abstract: A novel nucleic acid analogue (2Cl-C.OXT-A) significantly stimulated tube formation of human umbilical endothelial cells (HUVEC). Its maximum potency at 100 {mu}M was stronger than that of vascular endothelial growth factor (VEGF), a positivemore » control. At this concentration, 2Cl-C.OXT-A moderately stimulated proliferation as well as migration of HUVEC. To gain mechanistic insights how 2Cl-C.OXT-A promotes angiogenic responses in HUVEC, we performed immunoblot analyses using phospho-specific antibodies as probes. 2Cl-C.OXT-A induced robust phosphorylation/activation of MAP kinase ERK1/2 and an upstream MAP kinase kinase MEK. Conversely, a MEK inhibitor PD98059 abolished ERK1/2 activation and tube formation both enhanced by 2Cl-C.OXT-A. In contrast, MAP kinase responses elicited by 2Cl-C.OXT-A were not inhibited by SU5416, a specific inhibitor of VEGF receptor tyrosine kinase. Collectively these results suggest that 2Cl-C.OXT-A-induces angiogenic responses in HUVEC mediated by a MAP kinase cascade comprising MEK and ERK1/2, but independently of VEGF receptor tyrosine kinase. In vivo assay using chicken chorioallantoic membrane (CAM) and rabbit cornea also suggested the angiogenic potency of 2Cl-C.OXT-A.« less

An Extended Structure–Activity Relationship of Nondioxin-Like PCBs Evaluates and Supports Modeling Predictions and Identifies Picomolar Potency of PCB 202 Towards Ryanodine Receptors

PubMed Central

Feng, Wei; Zheng, Jing; Dong, Yao; Li, Xueshu; Lehmler, Hans-Joachim; Pessah, Isaac N.

2017-01-01

Nondioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine-sensitive Ca2+ channels (RyRs) and this activation has been associated with neurotoxicity in exposed animals. RyR-active congeners follow a distinct structure–activity relationship and a quantitative structure–activity relationship (QSAR) predicts that a large number of PCBs likely activate the receptor, which requires validation. Additionally, previous structural based conclusions have been established using receptor ligand binding assays but the impact of varying PCB structures on ion channel gating behavior is not understood. We used [3H]Ryanodine ([3H]Ry) binding to assess the RyR-activity of 14 previously untested PCB congeners evaluating the predictability of the QSAR. Congeners determined to display widely varying potency were then assayed with single channel voltage clamp analysis to assess direct influences on channel gating kinetics. The RyR-activity of individual PCBs assessed in in vitro assays followed the general pattern predicted by the QSAR but binding and lipid bilayer experiments demonstrated higher potency than predicted. Of the 49 congeners tested to date, tetra-ortho PCB 202 was found to be the most potent RyR-active congener increasing channel open probability at 200 pM. Shifting meta-substitutions to the para-position resulted in a > 100-fold reduction in potency as seen with PCB 197. Non-ortho PCB 11 was found to lack activity at the receptor supporting a minimum mono-ortho substitution for PCB RyR activity. These findings expand and support previous SAR assessments; where out of the 49 congeners tested to date 42 activate the receptor demonstrating that the RyR is a sensitive and common target of PCBs. PMID:27655348

The Third International Standard for Corticotrophin

PubMed Central

Bangham, D. R.; Mussett, M. V.; Stack-Dunne, M. P.

1962-01-01

At its meeting in September 1957, the WHO Expert Committee on Biological Standardization agreed with the recommendation of the International Conference on Corticotrophin, held in July 1957, that a new international standard for corticotrophin should be set up, since the Second International Standard was made from crude material and was unsuitable for the assay of the purer preparations of corticotrophin now in general clinical use. In this paper, the authors describe the steps taken to establish the Third International Standard for Corticotrophin, from the preparation and international collaborative assay of the new material to the choice of the ”subcutaneous assay” for deriving the potency. The clinical and pharmacological implications of this choice are discussed. Since the preparation, characterization and exact quantitative assay of standards for corticotrophin are so difficult, several batches of approximately 3500 ampoules were prepared in a similar way from the same material to serve as an international Working Standard. Samples from two batches were included in the collaborative assay and found to have the same potency as the Third Standard. Sufficient ampoules of the Working Standard are available for use as national and laboratory standards. PMID:13966359

A human induced pluripotent stem cell-based in vitro assay predicts developmental toxicity through a retinoic acid receptor-mediated pathway for a series of related retinoid analogues.

PubMed

Palmer, Jessica A; Smith, Alan M; Egnash, Laura A; Colwell, Michael R; Donley, Elizabeth L R; Kirchner, Fred R; Burrier, Robert E

2017-10-01

The relative developmental toxicity potency of a series of retinoid analogues was evaluated using a human induced pluripotent stem (iPS) cell assay that measures changes in the biomarkers ornithine and cystine. Analogue potency was predicted, based on the assay endpoint of the ornithine/cystine (o/c) ratio, to be all-trans-retinoic acid>TTNPB>13-cis-retinoic acid≈9-cis-retinoic acid>acitretin>etretinate>retinol. These rankings correlate with in vivo data and demonstrate successful application of the assay to rank a series of related toxic and non-toxic compounds. The retinoic acid receptor α (RARα)-selective antagonist Ro 41-5253 inhibited the cystine perturbation caused by all-trans-retinoic acid, TTNPB, 13-cis-retinoic acid, 9-cis-retinoic acid, and acitretin. Ornithine was altered independent of RARα in all retinoids except acitretin. These results suggest a role for an RARα-mediated mechanism in retinoid-induced developmental toxicity through altered cystine metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

Immuno-detection of cleaved SNAP-25 from differentiated mouse embryonic stem cells provides a sensitive assay for determination of botulinum A toxin and antitoxin potency.

PubMed

Yadirgi, G; Stickings, P; Rajagopal, S; Liu, Y; Sesardic, D

2017-12-01

Botulinum toxin type A is a causative agent of human botulism. Due to high toxicity and ease of production it is classified by the Centres for Disease Control and Prevention as a category A bioterrorism agent. The same serotype, BoNT/A, is also the most widely used in pharmaceutical preparations for treatment of a diverse range of neuromuscular disorders. Traditionally, animals are used to confirm the presence and activity of toxin and to establish neutralizing capabilities of countermeasures in toxin neutralization tests. Cell based assays for BoNT/A have been reported as the most viable alternative to animal models, since they are capable of reflecting all key steps (binding, translocation, internalization and cleavage of intracellular substrate) involved in toxin activity. In this paper we report preliminary development of a simple immunochemical method for specifically detecting BoNT/A cleaved intracellular substrate, SNAP-25, in cell lysates of neurons derived from mouse embryonic stem cells. The assay offers sensitivity of better than 0.1LD50/ml (3fM) which is not matched by other functional assays, including the mouse bioassay, and provides serotype specificity for quantitative detection of BoNT/A and anti-BoNT/A antitoxin. Subject to formal validation, the method described here could potentially be used as a substitute for the mouse bioassay to measure potency and consistency of therapeutic products. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Discovery of Novel Irreversible Inhibitors of Interleukin (IL)-2-inducible Tyrosine Kinase (Itk) by Targeting Cysteine 442 in the ATP Pocket

PubMed Central

Harling, John D.; Deakin, Angela M.; Campos, Sébastien; Grimley, Rachel; Chaudry, Laiq; Nye, Catherine; Polyakova, Oxana; Bessant, Christina M.; Barton, Nick; Somers, Don; Barrett, John; Graves, Rebecca H.; Hanns, Laura; Kerr, William J.; Solari, Roberto

2013-01-01

IL-2-inducible tyrosine kinase (Itk) plays a key role in antigen receptor signaling in T cells and is considered an important target for anti-inflammatory drug discovery. In order to generate inhibitors with the necessary potency and selectivity, a compound that targeted cysteine 442 in the ATP binding pocket and with an envisaged irreversible mode of action was designed. We incorporated a high degree of molecular recognition and specific design features making the compound suitable for inhaled delivery. This study confirms the irreversible covalent binding of the inhibitor to the kinase by x-ray crystallography and enzymology while demonstrating potency, selectivity, and prolonged duration of action in in vitro biological assays. The biosynthetic turnover of the kinase was also examined as a critical factor when designing irreversible inhibitors for extended duration of action. The exemplified Itk inhibitor demonstrated inhibition of both TH1 and TH2 cytokines, was additive with fluticasone propionate, and inhibited cytokine release from human lung fragments. Finally, we describe an in vivo pharmacodynamic assay that allows rapid preclinical development without animal efficacy models. PMID:23935099

New cytotoxic benzosuberene analogs. Synthesis, molecular modeling and biological evaluation.

PubMed

Chen, Zecheng; Maderna, Andreas; Sukuru, Sai Chetan K; Wagenaar, Melissa; O'Donnell, Christopher J; Lam, My-Hanh; Musto, Sylvia; Loganzo, Frank

2013-12-15

In this Letter we describe the synthesis and biological evaluation of new benzosuberene analogs with structural modifications on the B-ring. The focus was initially to probe the chemical space around the B-ring C-8 position. This position was readily available for derivatization chemistry using our recently developed new synthesis for this compound class. Furthermore, we describe two new B-ring analogs, one containing a diene and the other a cyclic ether group. Both new analogs show excellent potencies in tumor cell proliferation assays. In addition, we describe molecular modeling studies that provide a binding rationale for reference compound 8 in the colchicine binding site using the known colchicine crystal structure. We also examine whether the cell based potency data obtained with selected new analogs are supported by modeling results. Copyright © 2013 Elsevier Ltd. All rights reserved.

The opioid receptor triple agonist DPI-125 produces analgesia with less respiratory depression and reduced abuse liability.

PubMed

Yi, Shou-Pu; Kong, Qing-Hong; Li, Yu-Lei; Pan, Chen-Ling; Yu, Jie; Cui, Ben-Qiang; Wang, Ying-Fei; Wang, Guan-Lin; Zhou, Pei-Lan; Wang, Li-Li; Gong, Ze-Hui; Su, Rui-Bin; Shen, Yue-Hai; Yu, Gang; Chang, Kwen-Jen

2017-07-01

Opioid analgesics remain the first choice for the treatment of moderate to severe pain, but they are also notorious for their respiratory depression and addictive effects. This study focused on the pharmacology of a novel opioid receptor mixed agonist DPI-125 and attempted to elucidate the relationship between the δ-, μ- and κ-receptor potency ratio and respiratory depression and abuse liability. Five diarylmethylpiperazine compounds (DPI-125, DPI-3290, DPI-130, KUST202 and KUST13T02) were selected for this study. PKA fluorescence redistribution assays in CHO cells individually expressing δ-, μ- or κ-receptors were used to measure the agonist potency. The respiratory safety profiles were estimated in rats by the ratio of ED 50 (pCO 2 increase)/ED 50 (antinociception). The abuse liability of DPI-125 was evaluated with a self-administration model in rhesus monkeys. The observed agonist potencies of DPI-125 for δ-, μ- and κ-opioid receptors were 4.29±0.36, 11.10±3.04, and 16.57±4.14 nmol/L, respectively. The other four compounds were also mixed agonists with varying potencies. DPI-125 exhibited a high respiratory safety profile, clearly related to its high δ-receptor potency. The ratio of the EC 50 potencies for the μ- and δ-receptors was found to be positively correlated with the respiratory safety ratio. DPI-125 has similar potencies for μ- and κ-receptors, which is likely the reason for its reduced abuse potential. Our results demonstrate that the opioid receptor mixed agonist DPI-125 is safer and less addictive than traditional μ-agonist analgesics. These findings suggest that the development of δ>μ∼κ opioid receptor mixed agonists is feasible, and such compounds could represent a promising class of potent analgesics with wider therapeutic windows.

Non-animal sensitization testing: state-of-the-art.

PubMed

Vandebriel, Rob J; van Loveren, Henk

2010-05-01

Predictive tests to identify the sensitizing properties of chemicals are carried out using animals. In the European Union timelines for phasing out many standard animal tests were established for cosmetics. Following this policy, the new European Chemicals Legislation (REACH) favors alternative methods, if validated and appropriate. In this review the authors aim to provide a state-of-the art overview of alternative methods (in silico, in chemico, and in vitro) to identify contact and respiratory sensitizing capacity and in some occasions give a measure of potency. The past few years have seen major advances in QSAR (quantitative structure-activity relationship) models where especially mechanism-based models have great potential, peptide reactivity assays where multiple parameters can be measured simultaneously, providing a more complete reactivity profile, and cell-based assays. Several cell-based assays are in development, not only using different cell types, but also several specifically developed assays such as three-dimenionally (3D)-reconstituted skin models, an antioxidant response reporter assay, determination of signaling pathways, and gene profiling. Some of these assays show relatively high sensitivity and specificity for a large number of sensitizers and should enter validation (or are indeed entering this process). Integrating multiple assays in a decision tree or integrated testing system is a next step, but has yet to be developed. Adequate risk assessment, however, is likely to require significantly more time and efforts.

Structure-activity relationship of ortho- and meta-phenol based LFA-1 ICAM inhibitors.

PubMed

Lin, Edward Yin-Shiang; Guckian, Kevin M; Silvian, Laura; Chin, Donovan; Boriack-Sjodin, P Ann; van Vlijmen, Herman; Friedman, Jessica E; Scott, Daniel M

2008-10-01

LFA-1 ICAM inhibitors based on ortho- and meta-phenol templates were designed and synthesized by Mitsunobu chemistry. The selection of targets was guided by X-ray co-crystal data, and led to compounds which showed an up to 30-fold increase in potency over reference compound 1 in the LFA-1/ICAM1-Ig assay. The most active compound exploited a new hydrogen bond to the I-domain and exhibited subnanomolar potency.

Further evaluation of quantitative structure--activity relationship models for the prediction of the skin sensitization potency of selected fragrance allergens.

PubMed

Patlewicz, Grace Y; Basketter, David A; Pease, Camilla K Smith; Wilson, Karen; Wright, Zoe M; Roberts, David W; Bernard, Guillaume; Arnau, Elena Giménez; Lepoittevin, Jean-Pierre

2004-02-01

Fragrance substances represent a very diverse group of chemicals; a proportion of them are associated with the ability to cause allergic reactions in the skin. Efforts to find substitute materials are hindered by the need to undertake animal testing for determining both skin sensitization hazard and potency. One strategy to avoid such testing is through an understanding of the relationships between chemical structure and skin sensitization, so-called structure-activity relationships. In recent work, we evaluated 2 groups of fragrance chemicals -- saturated aldehydes and alpha,beta-unsaturated aldehydes. Simple quantitative structure-activity relationship (QSAR) models relating the EC3 values [derived from the local lymph node assay (LLNA)] to physicochemical properties were developed for both sets of aldehydes. In the current study, we evaluated an additional group of carbonyl-containing compounds to test the predictive power of the developed QSARs and to extend their scope. The QSAR models were used to predict EC3 values of 10 newly selected compounds. Local lymph node assay data generated for these compounds demonstrated that the original QSARs were fairly accurate, but still required improvement. Development of these QSAR models has provided us with a better understanding of the potential mechanisms of action for aldehydes, and hence how to avoid or limit allergy. Knowledge generated from this work is being incorporated into new/improved rules for sensitization in the expert toxicity prediction system, deductive estimation of risk from existing knowledge (DEREK).

The local lymph node assay and skin sensitization testing.

PubMed

Kimber, Ian; Dearman, Rebecca J

2010-01-01

The mouse local lymph node assay (LLNA) is a method for the identification and characterization of skin sensitization hazards. In this context the method can be used both to identify contact allergens, and also determine the relative skin sensitizing potency as a basis for derivation of effective risk assessments.The assay is based on measurement of proliferative responses by draining lymph node cells induced following topical exposure of mice to test chemicals. Such responses are known to be causally and quantitatively associated with the acquisition of skin sensitization and therefore provide a relevant marker for characterization of contact allergic potential.The LLNA has been the subject of exhaustive evaluation and validation exercises and has been assigned Organization for Economic Cooperation and Development (OECD) test guideline 429. Herein we describe the conduct and interpretation of the LLNA.

Selective enhancement of fentanyl-induced antinociception by the delta agonist SNC162 but not by ketamine in rhesus monkeys: Further evidence supportive of delta agonists as candidate adjuncts to mu opioid analgesics.

PubMed

Banks, Matthew L; Folk, John E; Rice, Kenner C; Negus, S Stevens

2010-12-01

Mu-opioid receptor agonists such as fentanyl are effective analgesics, but their clinical use is limited by untoward effects. Adjunct medications may improve the effectiveness and/or safety of opioid analgesics. This study compared interactions between fentanyl and either the noncompetitive N-methyl-D-aspartate (NMDA) glutamate receptor antagonist ketamine or the delta-opioid receptor agonist SNC162 [(+)-4-[(alphaR)-alpha-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-phenyl)methyl]-N,N-diethylbenzamide] in two behavioral assays in rhesus monkeys. An assay of thermal nociception evaluated tail-withdrawal latencies from water heated to 50 and 54°C. An assay of schedule-controlled responding evaluated response rates maintained under a fixed-ratio 30 schedule of food presentation. Effects of each drug alone and of three mixtures of ketamine+fentanyl (22:1, 65:1, 195:1 ketamine/fentanyl) or SNC162+fentanyl (59:1, 176:1, 528:1 SNC162/fentanyl) were evaluated in each assay. All drugs and mixtures dose-dependently decreased rates of food-maintained responding, and drug proportions in the mixtures were based on relative potencies in this assay. Ketamine and SNC162 were inactive in the assay of thermal antinociception, but fentanyl and all mixtures produced dose-dependent antinociception. Drug interactions were evaluated using dose-addition and dose-ratio analysis. Dose-addition analysis revealed that interactions for all ketamine/fentanyl mixtures were additive in both assays. SNC162/fentanyl interactions were usually additive, but one mixture (176:1) produced synergistic antinociception at 50°C. Dose-ratio analysis indicated that ketamine failed to improve the relative potency of fentanyl to produce antinociception vs. rate suppression, whereas two SNC162/fentanyl mixtures (59:1 and 176:1) increased the relative potency of fentanyl to produce antinociception. These results suggest that delta agonists may produce more selective enhancement than ketamine of mu agonist-induced antinociception. Copyright © 2010 Elsevier Inc. All rights reserved.

Synthesis and Agonistic Activity at the GPR35 of 5,6-Dihydroxyindole-2-carboxylic Acid Analogues

PubMed Central

2012-01-01

5,6-Dihydroxyindole-2-carboxylic acid (DHICA), an intermediate of melanin synthesis and an eumelanin building block, was recently discovered to be a GPR35 agonist with moderate potency. Here, we report the synthesis and pharmacological characterization of a series of DHICA analogues against GPR35 using both label-free dynamic mass redistribution and Tango β-arrestin translocation assays. This led to identification of novel GPR35 agonists with improved potency and/or having biased agonism. PMID:24900508

Hallmarks of pluripotency.

PubMed

De Los Angeles, Alejandro; Ferrari, Francesco; Xi, Ruibin; Fujiwara, Yuko; Benvenisty, Nissim; Deng, Hongkui; Hochedlinger, Konrad; Jaenisch, Rudolf; Lee, Soohyun; Leitch, Harry G; Lensch, M William; Lujan, Ernesto; Pei, Duanqing; Rossant, Janet; Wernig, Marius; Park, Peter J; Daley, George Q

2015-09-24

Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance.

Fluorescent Trimethoprim Conjugate Probes To Assess Drug Accumulation in Wild Type and Mutant Escherichia coli

PubMed Central

2016-01-01

Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment. PMID:27737551

A Sensitive in Vitro High-Throughput Screen To Identify Pan-filoviral Replication Inhibitors Targeting the VP35–NP Interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Gai; Nash, Peter J.; Johnson, Britney

The 2014 Ebola outbreak in West Africa, the largest outbreak on record, highlighted the need for novel approaches to therapeutics targeting Ebola virus (EBOV). Within the EBOV replication complex, the interaction between polymerase cofactor, viral protein 35 (VP35), and nucleoprotein (NP) is critical for viral RNA synthesis. We recently identified a peptide at the N-terminus of VP35 (termed NPBP) that is sufficient for interaction with NP and suppresses EBOV replication, suggesting that the NPBP binding pocket can serve as a potential drug target. Here we describe the development and validation of a sensitive high-throughput screen (HTS) using a fluorescence polarizationmore » assay. Initial hits from this HTS include the FDA-approved compound tolcapone, whose potency against EBOV infection was validated in a nonfluorescent secondary assay. High conservation of the NP–VP35 interface among filoviruses suggests that this assay has the capacity to identify pan-filoviral inhibitors for development as antivirals.« less

The comparative performance of the single intradermal test and the single intradermal comparative tuberculin test in Irish cattle, using tuberculin PPD combinations of differing potencies.

PubMed

Good, M; Clegg, T A; Costello, E; More, S J

2011-11-01

In national bovine tuberculosis (BTB) control programmes, testing is generally conducted using a single source of bovine purified protein derivative (PPD) tuberculin. Alternative tuberculin sources should be identified as part of a broad risk management strategy as problems of supply or quality cannot be discounted. This study was conducted to compare the impact of different potencies of a single bovine PPD tuberculin on the field performance of the single intradermal comparative tuberculin test (SICTT) and single intradermal test (SIT). Three trial potencies of bovine PPD tuberculin, as assayed in naturally infected bovines, namely, low (1192IU/dose), normal (6184IU/dose) and high (12,554IU/dose) were used. Three SICTTs (using) were conducted on 2102 animals. Test results were compared based on reactor-status and changes in skin-thickness at the bovine tuberculin injection site. There was a significant difference in the number of reactors detected using the high and low potency tuberculins. In the SICTT, high and low potency tuberculin detected 40% more and 50% fewer reactors, respectively, than normal potency tuberculin. Furthermore, use of the low potency tuberculin in the SICTT failed to detect 20% of 35 animals with visible lesions, and in the SIT 11% of the visible lesion animals would have been classified as negative. Tuberculin potency is critical to the performance of both the SICTT and SIT. Tuberculin of different potencies will affect reactor disclosure rates, confounding between-year or between-country comparisons. Independent checks of tuberculin potency are an important aspect of quality control in national BTB control programmes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Development of a μO-Conotoxin Analogue with Improved Lipid Membrane Interactions and Potency for the Analgesic Sodium Channel NaV1.8*

PubMed Central

Deuis, Jennifer R.; Dekan, Zoltan; Inserra, Marco C.; Lee, Tzong-Hsien; Aguilar, Marie-Isabel; Craik, David J.; Lewis, Richard J.; Alewood, Paul F.; Mobli, Mehdi; Schroeder, Christina I.; Henriques, Sónia Troeira; Vetter, Irina

2016-01-01

The μO-conotoxins MrVIA, MrVIB, and MfVIA inhibit the voltage-gated sodium channel NaV1.8, a well described target for the treatment of pain; however, little is known about the residues or structural elements that define this activity. In this study, we determined the three-dimensional structure of MfVIA, examined its membrane binding properties, performed alanine-scanning mutagenesis, and identified residues important for its activity at human NaV1.8. A second round of mutations resulted in (E5K,E8K)MfVIA, a double mutant with greater positive surface charge and greater affinity for lipid membranes compared with MfVIA. This analogue had increased potency at NaV1.8 and was analgesic in the mouse formalin assay. PMID:27026701

Positive allosteric modulators of the human sweet taste receptor enhance sweet taste

PubMed Central

Servant, Guy; Tachdjian, Catherine; Tang, Xiao-Qing; Werner, Sara; Zhang, Feng; Li, Xiaodong; Kamdar, Poonit; Petrovic, Goran; Ditschun, Tanya; Java, Antoniette; Brust, Paul; Brune, Nicole; DuBois, Grant E.; Zoller, Mark; Karanewsky, Donald S.

2010-01-01

To identify molecules that could enhance sweetness perception, we undertook the screening of a compound library using a cell-based assay for the human sweet taste receptor and a panel of selected sweeteners. In one of these screens we found a hit, SE-1, which significantly enhanced the activity of sucralose in the assay. At 50 μM, SE-1 increased the sucralose potency by >20-fold. On the other hand, SE-1 exhibited little or no agonist activity on its own. SE-1 effects were strikingly selective for sucralose. Other popular sweeteners such as aspartame, cyclamate, and saccharin were not enhanced by SE-1 whereas sucrose and neotame potency were increased only by 1.3- to 2.5-fold at 50 μM. Further assay-guided chemical optimization of the initial hit SE-1 led to the discovery of SE-2 and SE-3, selective enhancers of sucralose and sucrose, respectively. SE-2 (50 μM) and SE-3 (200 μM) increased sucralose and sucrose potencies in the assay by 24- and 4.7-fold, respectively. In human taste tests, 100 μM of SE-1 and SE-2 allowed for a reduction of 50% to >80% in the concentration of sucralose, respectively, while maintaining the sweetness intensity, and 100 μM SE-3 allowed for a reduction of 33% in the concentration of sucrose while maintaining the sweetness intensity. These enhancers did not exhibit any sweetness when tasted on their own. Positive allosteric modulators of the human sweet taste receptor could help reduce the caloric content in food and beverages while maintaining the desired taste. PMID:20173092

Test battery with the human cell line activation test, direct peptide reactivity assay and DEREK based on a 139 chemical data set for predicting skin sensitizing potential and potency of chemicals.

PubMed

Takenouchi, Osamu; Fukui, Shiho; Okamoto, Kenji; Kurotani, Satoru; Imai, Noriyasu; Fujishiro, Miyuki; Kyotani, Daiki; Kato, Yoshinao; Kasahara, Toshihiko; Fujita, Masaharu; Toyoda, Akemi; Sekiya, Daisuke; Watanabe, Shinichi; Seto, Hirokazu; Hirota, Morihiko; Ashikaga, Takao; Miyazawa, Masaaki

2015-11-01

To develop a testing strategy incorporating the human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA) and DEREK, we created an expanded data set of 139 chemicals (102 sensitizers and 37 non-sensitizers) by combining the existing data set of 101 chemicals through the collaborative projects of Japan Cosmetic Industry Association. Of the additional 38 chemicals, 15 chemicals with relatively low water solubility (log Kow > 3.5) were selected to clarify the limitation of testing strategies regarding the lipophilic chemicals. Predictivities of the h-CLAT, DPRA and DEREK, and the combinations thereof were evaluated by comparison to results of the local lymph node assay. When evaluating 139 chemicals using combinations of three methods based on integrated testing strategy (ITS) concept (ITS-based test battery) and a sequential testing strategy (STS) weighing the predictive performance of the h-CLAT and DPRA, overall similar predictivities were found as before on the 101 chemical data set. An analysis of false negative chemicals suggested a major limitation of our strategies was the testing of low water-soluble chemicals. When excluded the negative results for chemicals with log Kow > 3.5, the sensitivity and accuracy of ITS improved to 97% (91 of 94 chemicals) and 89% (114 of 128). Likewise, the sensitivity and accuracy of STS to 98% (92 of 94) and 85% (111 of 129). Moreover, the ITS and STS also showed good correlation with local lymph node assay on three potency classifications, yielding accuracies of 74% (ITS) and 73% (STS). Thus, the inclusion of log Kow in analysis could give both strategies a higher predictive performance. Copyright © 2015 John Wiley & Sons, Ltd.

Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors.

PubMed

Mukherjee, Abhisek; Syeb, Kathleen; Concannon, John; Callegari, Keri; Soto, Claudio; Glicksman, Marcie A

2015-01-01

Currently there is no effective treatment available for major neurodegenerative disorders associated to protein misfolding, including Alzheimer's and Parkinson's disease. One of most promising therapeutic approaches under development focuses on inhibiting the misfolding and aggregation pathway. However, it is likely that by the time clinical symptoms appear, there is a large accumulation of misfolded aggregates and a very substantial damage to the brain. Thus, it seems that at the clinical stage of the disease it is necessary also to develop strategies aiming to prevent the neuronal damage produced by already formed misfolded aggregates. Chronic activation of calcineurin (CaN), a type IIB phosphatase, has been implicated as a pivotal molecule connecting synaptic loss and neuronal damage to protein misfolding. The fact that the crystal structure of CaN is also well established makes it an ideal target for drug discovery. CaN activity assays for High Throughput Screening (HTS) reported so far are based on absorbance. In this article we report the development of a fluorescent quenching based CaN activity assay suitable for robotic screening of large chemical libraries to find novel inhibitors. The assay yielded a Z score of 0.84 with coefficient of variance ≤ 15%. Our results also show that this assay can be used to identify CaN inhibitors with a wide range of potencies.

Enterocin A Mutants Identified by Saturation Mutagenesis Enhance Potency Towards Vancomycin-Resistant Enterococci

PubMed Central

McClintock, Maria K.; Kaznessis, Yiannis N.; Hackel, Benjamin J.

2016-01-01

Vancomycin-resistant Enterococci infections are a significant clinical problem. One proposed solution is to use probiotics, such as lactic acid bacteria, to produce antimicrobial peptides at the site of infection. Enterocin A, a class 2a bacteriocin, exhibits inhibitory activity against E. faecium and E. faecalis, which account for 86% of vancomycin-resistant Enterococci infections. In this study, we aimed to engineer enterocin A mutants with enhanced potency within a lactic acid bacterial production system. Peptide mutants resulting from saturation mutagenesis at sites A24 and T27 were efficiently screened in a 96-well plate assay for inhibition of pathogen growth. Several mutants exhibit increased potency relative to wild-type enterocin A in both liquid- and solid-medium growth assays. In particular, A24P and T27G exhibit enhanced inhibition of multiple strains of E. faecium and E. faecalis, including clinically isolated vancomycin-resistant strains. A24P and T27G enhance killing of E. faecium 8 by 13±3- and 18±4-fold, respectively. The engineered enterocin A/lactic acid bacteria systems offer significant potential to combat antibiotic-resistant infections. PMID:26191783

Enterocin A mutants identified by saturation mutagenesis enhance potency towards vancomycin-resistant Enterococci.

PubMed

McClintock, Maria K; Kaznessis, Yiannis N; Hackel, Benjamin J

2016-02-01

Vancomycin-resistant Enterococci infections are a significant clinical problem. One proposed solution is to use probiotics, such as lactic acid bacteria, to produce antimicrobial peptides at the site of infection. Enterocin A, a class 2a bacteriocin, exhibits inhibitory activity against E. faecium and E. faecalis, which account for 86% of vancomycin-resistant Enterococci infections. In this study, we aimed to engineer enterocin A mutants with enhanced potency within a lactic acid bacterial production system. Peptide mutants resulting from saturation mutagenesis at sites A24 and T27 were efficiently screened in a 96-well plate assay for inhibition of pathogen growth. Several mutants exhibit increased potency relative to wild-type enterocin A in both liquid- and solid-medium growth assays. In particular, A24P and T27G exhibit enhanced inhibition of multiple strains of E. faecium and E. faecalis, including clinically isolated vancomycin-resistant strains. A24P and T27G enhance killing of E. faecium 8 by 13 ± 3- and 18 ± 4-fold, respectively. The engineered enterocin A/lactic acid bacteria systems offer significant potential to combat antibiotic-resistant infections. © 2015 Wiley Periodicals, Inc.

Synthesis and Evaluation of Orexin-1 Receptor Antagonists with Improved Solubility and CNS Permeability.

PubMed

Perrey, David A; Decker, Ann M; Zhang, Yanan

2018-03-21

Orexins are hypothalamic neuropeptides playing important roles in many functions including the motivation of addictive behaviors. Blockade of the orexin-1 receptor has been suggested as a potential strategy for the treatment of drug addiction. We have previously reported OX 1 receptor antagonists based on the tetrahydroisoquinoline scaffold with excellent OX 1 potency and selectivity; however, these compounds had high lipophilicity (clogP > 5) and low to moderate solubility. In an effort to improve their properties, we have designed and synthesized a series of analogues where the 7-position substituents known to favor OX 1 potency and selectivity were retained, and groups of different nature were introduced at the 1-position where substitution was generally tolerated as demonstrated in previous studies. Compound 44 with lower lipophilicity (clogP = 3.07) displayed excellent OX 1 potency ( K e = 5.7 nM) and selectivity (>1,760-fold over OX 2 ) in calcium mobilization assays. In preliminary ADME studies, 44 showed excellent kinetic solubility (>200 μM), good CNS permeability ( P app = 14.7 × 10 -6 cm/sec in MDCK assay), and low drug efflux (efflux ratio = 3.3).

Skin sensitization potency and cross-reactivity of p-phenylenediamine and its derivatives evaluated by non-radioactive murine local lymph node assay and guinea-pig maximization test.

PubMed

Yamano, Tetsuo; Shimizu, Mitsuru

2009-04-01

p-Phenylenediamine (PPD)-related chemicals have been used as antioxidants in rubber products, and many cases of contact dermatitis caused by these chemicals have been reported. The aim of this study was to investigate relative sensitizing potency and cross-reactivity among PPD derivatives. Five PPD derivatives, p-aminodiphenylamine (PADPA), N,N'-diphenyl-p-phenylenediamine (DPPD), N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD), N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (DMBPPD), N-(1-methylheptyl)-N'-phenyl-p-phenylenediamine (MHPPD), and the core chemical PPD were evaluated for their sensitizing potency and cross-reactivity using the non-radioactive murine local lymph node assay (LLNA) and the guinea-pig maximization test (GPMT). PPD and all the derivatives were identified as primary sensitizers in both tests. The order of potency in the LLNA was as follows: IPPD and PADPA > PPD > DMBPPD and MHPPD > DPPD. In the GPMT, all six groups of animals sensitized with one of these chemicals cross-reacted to four other derivatives. Specifically, the five groups that have a common basic PADPA structure, that is PADPA, DPPD, IPPD, DMBPPD, and MHPPD, all reacted to each other at almost the same scores, while none of them reacted to PPD. The cross-reactivity profile found in the study was to some extent different from that in previous human data, where distinction between cross-reaction and concomitant primary sensitization is not always clear.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goebel, Carsten, E-mail: goebel.c.1@pg.com; Troutman, John; Hennen, Jenny

The strong sensitizing potencies of the most important primary intermediates of oxidative hair dyes, p-phenylenediamine (PPD) and p-toluylenediamine (PTD, i.e. 2-methyl-PPD) are well established. They are considered as the key sensitizers in hair dye allergic contact dermatitis. While modification of their molecular structure is expected to alter their sensitizing properties, it may also impair their color performance. With introduction of a methoxymethyl side chain we found the primary intermediate 2-methoxymethyl-p-phenylenediamine (ME-PPD) with excellent hair coloring performance but significantly reduced sensitizing properties compared to PPD and PTD: In vitro, ME-PPD showed an attenuated innate immune response when analyzed for its proteinmore » reactivity and dendritic cell activation potential. In vivo, the effective concentration of ME-PPD necessary to induce an immune response 3-fold above vehicle control (EC3 value) in the local lymph node assay (LLNA) was 4.3%, indicating a moderate skin sensitizing potency compared to values of 0.1 and 0.17% for PPD and PTD, respectively. Finally, assessing the skin sensitizing potency of ME-PPD under consumer hair dye usage conditions through a quantitative risk assessment (QRA) indicated an allergy induction risk negligible compared to PPD or PTD. - Highlights: • Methoxymethyl side chain in p-phenylenediamine reduces its strong skin sensitizing properties. • Reduced protein reactivity and dendritic cell activation. • Reduced skin sensitizing potency in local lymph node assay (LLNA). • Negligible allergy induction risk under hair dye usage conditions.« less

Evaluation of radioisotopic and non-radioisotopic versions of local lymph node assays for subcategorization of skin sensitizers compliant to UN GHS rev 4.

PubMed

Ha, Soojin; Ahn, Il Young; Kim, Da-Eun; Lee, Jong Kwon; Sohn, Soojung; Jung, Mi-Sook; Heo, Yong; Omori, Takashi; Bae, SeungJin; Lim, Kyung-Min

2017-04-01

Recently UN GHS has introduced the sub-categorization of skin sensitizers for which ECt (concentration estimated to induce stimulation index above threshold) of the murine local lymph node assay (LLNA) is used as criteria. Non-radioisotopic variants of LLNA, LLNA: DA, LLNA: BrdU-ELISA, LNCC and LLNA: BrdU-FCM were developed yet their utilities for potency sub-categorization are not established. Here we assessed the agreement of LLNA variants with LLNA or human data in potency sub-categorization for 22 reference substances of OECD TG429. Concordance of sub-categorization with LLNA was highest for LLNA: BrdU-FCM(91%, κ = 0.833, weighted kappa) followed by LLNA: BrdU-ELISA (82%, κ = 0.744) and LLNA: DA (73%, κ = 0.656) whereas LNCC only showed a modest association (64%, κ = 0.441). With human data, LLNA agreed best (77%) followed by LLNA: DA and LLNA: BrdU-FCM(73%), LLNA: BrdU-ELISA (68%) and LNCC(55%). Bland-Altman plot revealed that ECt's of LLNA variants largely agreed with LLNA where most values fell within 95% limit of agreement. Correlation between ECt's of LLNA and LLNA variants were high except for LNCC(pair-wise with LLNA, LLNA: DA, r = 0.848, LLNA: BrdU-ELISA, r = 0.744, LLNA: BrdU-FCM, r=0.786, and LNCC, r = 0.561 by Pearson). Collectively, these results demonstrated that LLNA variants exhibit performance comparable to LLNA in the potency sub-categorization although additional substances shall be analyzed in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel enzyme-linked immuno-sorbent assay (ELISA) for the quantification of total and free polysaccharide in Haemophilus influenzae b-Tetanus toxoid conjugate vaccines in monovalent and combined vaccine formulations.

PubMed

Saydam, Manolya; Rigsby, Peter; Mawas, Fatme

2014-01-01

Current Haemophilus influenzae b conjugate vaccines (Hib), which are made of purified capsular polysaccharide (poly-ribosyl-ribitol-phosphate; PRP) conjugated to a carrier protein, are almost completely evaluated by physico-chemical methods to ensure the integrity and stability of the vaccine and consistency of manufacture of batches. The absence of a potency assay makes the quantification of total PRP content (in SI units) and of % free polysaccharide in final fills or bulk components of Hib vaccines critical release tests for both manufacturers and national control authorities. Here we describe a simple and sensitive Enzyme-Linked Immuno-sorbent Assay (ELISA) which has been developed to quantify total and free PRP content in Hib-TT vaccine alone or when in combination with other vaccines. The assay is robust, specific and highly sensitive. Copyright © 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Silibinin derivatives as anti-prostate cancer agents: Synthesis and cell-based evaluations.

PubMed

Vue, Bao; Zhang, Sheng; Zhang, Xiaojie; Parisis, Konstantinos; Zhang, Qiang; Zheng, Shilong; Wang, Guangdi; Chen, Qiao-Hong

2016-02-15

This study aims to systematically explore the alkylation effect of 7-OH in silibinin and 2,3-dehydrosilibinin on the antiproliferative potency toward three prostate cancer cell lines. Eight 7-O-alkylsilibinins, eight 7-O-alkyl-2,3-dehydrosilibinins, and eight 3,7-O-dialkyl-2,3-dehydrosilibinins have been synthesized from commercially available silibinin for the in vitro cell-based evaluation. The WST-1 cell proliferation assay indicates that nineteen out of twenty-four silibinin derivatives have significantly improved antiproliferative potency when compared with silibinin. 7-O-Methylsilibinin (2) and 7-O-ethylsilibinin (3) have been identified as the most potent compounds with 98- and 123-fold enhanced potency against LNCaP human androgen-dependent prostate cancer cell line. Among 2,3-dehydrosilibinin derivatives, 7-O-methyl-2,3-dehydrosilibinin (10) and 7-O-ethyl-2,3-dehydrosilibinin (11) have been identified as the optimal compounds with the highest potency towards both androgen-dependent LNCaP and androgen-independent PC-3 prostate cancer cell lines. 7-O-Ethyl-2,3-dehydrosilibinin (11) was demonstrated to arrest PC-3 cell cycle at the G0/G1 phase and to induce PC-3 cell apoptosis. The findings in this study suggest that antiproliferative potency of silibinin and 2,3-dehydrosilibinin can be appreciably enhanced through suitable chemical modifications on the phenolic hydroxyl group at C-7 and that introduction of a chemical moiety with the potential to improve bioavailability through a linker to 7-OH in silibinin and 2,3-dehydrosilibinin would be a feasible strategy for the development of silibinin derivatives as anti-prostate cancer agents. Published by Elsevier Masson SAS.

Application of a high-throughput relative chemical stability assay to screen therapeutic protein formulations by assessment of conformational stability and correlation to aggregation propensity.

PubMed

Rizzo, Joseph M; Shi, Shuai; Li, Yunsong; Semple, Andrew; Esposito, Jessica J; Yu, Shenjiang; Richardson, Daisy; Antochshuk, Valentyn; Shameem, Mohammed

2015-05-01

In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

A homogeneous cell-based assay for measurement of endogenous paraoxonase 1 activity.

PubMed

Ahmad, Syed; Carter, Jade J; Scott, John E

2010-05-01

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. Thus, there is significant interest in identifying nutritional and pharmacological enhancers of PON1 activity. To identify such compounds, we developed a rapid homogeneous assay to detect endogenous cell-associated PON1 activity. PON1 activity was measured by the simple addition of fluorigenic PON1 substrate DEPFMU to live Huh7 cells in medium and monitoring change in fluorescence. A specific PON1 inhibitor, 2-hydroxyquinoline, was used to confirm that the observed activity was due to PON1. The assay was optimized and characterized with regard to time course, substrate and sodium chloride concentration, number of cells, and tolerance to dimethyl sulfoxide and serum. Aspirin, quercetin, and simvastatin are compounds reported to increase PON1 expression. Consistent with the literature and Western blot data, these compounds enhanced PON1 activity in this assay with comparable efficacies and potencies. A known toxic compound did not increase assay signal. This assay method also detected PON1 activity in normal hepatocytes. Thus, a novel homogeneous assay for detection of endogenous PON1 expression has been developed and is amenable to high-throughput screening for the identification of small molecules that enhance PON1 expression. 2010 Elsevier Inc. All rights reserved.

Water-soluble derivatives of 25-OCH3-PPD and their anti-proliferative activities.

PubMed

Zhou, Wu-Xi; Sun, Yuan-Yuan; Yuan, Wei-Hui; Zhao, Yu-Qing

2017-05-01

(20R)-25-Methoxyl-dammarane-3β,12β,20-triol (25-OCH 3 -PPD, AD-1) is a dammarane-type sapogenin showing anti-tumor potential. In the search for new anti-tumor agents with higher potency than our previously identified compound 25-OCH 3 -PPD, 11 novel sulfamic acid and diacid derivatives that could improve water solubility and contribute to good drug potency and pharmacokinetic profiles were designed and synthesized. Their in vitro anti-tumor activities in MCF-7, A-549, HCT-116, and BGC-823 cell lines and one normal cell line were tested by standard MTT assay. Results showed that compared with compound 25-OCH 3 -PPD, compounds 1, 4, and 5 exhibited higher cytotoxic activity on almost all cell lines, together with lower toxicity in the normal cell. In particular, compound 1 exhibited the best anti-tumor activity in the in vitro assays. The water solubility of 25-OCH 3 -PPD and its derivatives was tested and the results showed that the solubility of 25-OCH 3 -PPD sulfamic acid and diacid derivatives were better than that of 25-OCH 3 -PPD in water, which may provide valuable data for the research and development of new anti-tumor agents. Copyright © 2017 Elsevier Inc. All rights reserved.

Immunologic monitoring of cancer vaccine therapy: results of a workshop sponsored by the Society for Biological Therapy.

PubMed

Keilholz, Ulrich; Weber, Jeffrey; Finke, James H; Gabrilovich, Dmitry I; Kast, W Martin; Disis, Mary L; Kirkwood, John M; Scheibenbogen, Carmen; Schlom, Jeff; Maino, Vernon C; Lyerly, H Kim; Lee, Peter P; Storkus, Walter; Marincola, Franceso; Worobec, Alexandra; Atkins, Michael B

2002-01-01

The Society for Biological Therapy held a Workshop last fall devoted to immune monitoring for cancer immunotherapy trials. Participants included members of the academic and pharmaceutical communities as well as the National Cancer Institute and the Food and Drug Administration. Discussion focused on the relative merits and appropriate use of various immune monitoring tools. Six breakout groups dealt with assays of T-cell function, serologic and proliferation assays to assess B cell and T helper cell activity, and enzyme-linked immunospot assay, tetramer, cytokine flow cytometry, and reverse transcription polymerase chain reaction assays of T-cell immunity. General conclusions included: (1) future vaccine studies should be designed to determine whether T-cell dysfunction (tumor-specific and nonspecific) correlated with clinical outcome; (2) tetramer-based assays yield quantitative but not functional data (3) enzyme-linked immunospot assays have the lowest limit of detection (4) cytokine flow cytometry have a higher limit of detection than enzyme-linked immunospot assay, but offer the advantages of speed and the ability to identify subsets of reactive cells; (5) antibody tests are simple and accurate and should be incorporated to a greater extent in monitoring plans; (6) proliferation assays are imprecise and should not be emphasized in future studies; (7) the reverse transcription polymerase chain reaction assay is a promising research approach that is not ready for widespread application; and (8)there is a critical need to validate these assays as surrogates for vaccine potency and clinical effect. Current data and opinion support the use of a functional assay like the enzyme-linked immunospot assay or cytokine flow cytometry in combination with a quantitative assay like tetramers for immune monitoring. At present, assays appear to be most useful as measures of vaccine potency. Careful immune monitoring in association with larger scale clinical trials ultimately may enable the correlation of monitoring results with clinical benefit.

An Extended Structure-Activity Relationship of Nondioxin-Like PCBs Evaluates and Supports Modeling Predictions and Identifies Picomolar Potency of PCB 202 Towards Ryanodine Receptors.

PubMed

Holland, Erika B; Feng, Wei; Zheng, Jing; Dong, Yao; Li, Xueshu; Lehmler, Hans-Joachim; Pessah, Isaac N

2017-01-01

Nondioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine-sensitive Ca 2+  channels (RyRs) and this activation has been associated with neurotoxicity in exposed animals. RyR-active congeners follow a distinct structure-activity relationship and a quantitative structure-activity relationship (QSAR) predicts that a large number of PCBs likely activate the receptor, which requires validation. Additionally, previous structural based conclusions have been established using receptor ligand binding assays but the impact of varying PCB structures on ion channel gating behavior is not understood. We used [ 3 H]Ryanodine ([ 3 H]Ry) binding to assess the RyR-activity of 14 previously untested PCB congeners evaluating the predictability of the QSAR. Congeners determined to display widely varying potency were then assayed with single channel voltage clamp analysis to assess direct influences on channel gating kinetics. The RyR-activity of individual PCBs assessed in in vitro assays followed the general pattern predicted by the QSAR but binding and lipid bilayer experiments demonstrated higher potency than predicted. Of the 49 congeners tested to date, tetra-ortho PCB 202 was found to be the most potent RyR-active congener increasing channel open probability at 200 pM. Shifting meta-substitutions to the para-position resulted in a > 100-fold reduction in potency as seen with PCB 197. Non-ortho PCB 11 was found to lack activity at the receptor supporting a minimum mono-ortho substitution for PCB RyR activity. These findings expand and support previous SAR assessments; where out of the 49 congeners tested to date 42 activate the receptor demonstrating that the RyR is a sensitive and common target of PCBs. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Fragment-wise design of inhibitors to 3C proteinase from enterovirus 71.

PubMed

Wu, Caiming; Zhang, Lanjun; Li, Peng; Cai, Qixu; Peng, Xuanjia; Yin, Ke; Chen, Xinsheng; Ren, Haixia; Zhong, Shilin; Weng, Yuwei; Guan, Yi; Chen, Shuhui; Wu, Jinzhun; Li, Jian; Lin, Tianwei

2016-06-01

Enterovirus 71 (EV71) is a causative agent of hand, foot and mouth disease (HFMD), which can spread its infection to central nervous and other systems with severe consequence. A key factor in the replication of EV71 is its 3C proteinase (3C(pro)), a significant drug target. Peptidomimetics were employed as inhibitors of this enzyme for developing antivirals. However, the peptide bonds in these peptidomimetics are a source of low bioavailability due to their susceptibility to protease digestion. To produce non-peptidomimetic inhibitors by replacing these peptide bonds, it would be important to gain better understanding on the contribution of each component to the interaction and potency. A series of compounds of different lengths targeting 3C(pro) and having an α,β-unsaturated ester as the warhead were synthesized and their interactions with the enzyme were evaluated by complex structure analyses and potency assays for a better understanding on the relationship between potency and evolution of interaction. The P2 moiety of the compound would need to be oriented to interact in the S2 site in the substrate binding cleft and the P3-P4 moieties were required to generate sufficient potency. A hydrophobic terminal group will benefit the cellular uptake and improve the activity in vivo. The data presented here provide a basis for designing a new generation of non-peptidomimetics to target EV71 3C(pro). Copyright © 2016 Elsevier B.V. All rights reserved.

Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay.

PubMed

Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco

2011-04-01

The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF.

Testing for developmental neurotoxicity using a battery of in vitro assays for key cellular events in neurodevelopment.

PubMed

Harrill, Joshua A; Freudenrich, Theresa; Wallace, Kathleen; Ball, Kenneth; Shafer, Timothy J; Mundy, William R

2018-04-05

Medium- to high-throughput in vitro assays that recapitulate the critical processes of nervous system development have been proposed as a means to facilitate rapid testing and identification of chemicals which may affect brain development. In vivo neurodevelopment is a complex progression of distinct cellular processes. Therefore, batteries of in vitro assays that model and quantify effects on a variety of neurodevelopmental processes have the potential to identify chemicals which may affect brain development at different developmental stages. In the present study, the results of concentration-response screening of 67 reference chemicals in a battery of high content imaging and microplate reader-based assays that evaluate neural progenitor cell proliferation, neural proginitor cell apoptosis, neurite initiation/outgrowth, neurite maturation and synaptogenesis are summarized and compared. The assay battery had a high degree of combined sensitivity (87%) for categorizing chemicals known to affect neurodevelopment as active and a moderate degree of combined specificity (71%) for categorizing chemicals not associated with affects on neurodevelopment as inactive. The combined sensitivity of the assay battery was higher compared to any individual assay while the combined specificity of the assay battery was lower compared to any individual assay. When selectivity of effects for a neurodevelopmental endpoint as compared to general cytotoxicity was taken into account, the combined sensitivity of the assay battery decreased (68%) while the combined specificity increased (93%). The identity and potency of chemicals identified as active varied across the assay battery, underscoring the need for use of a combination of diverse in vitro models to comprehensively screen chemicals and identify those which potentially affect neurodevelopment. Overall, these data indicate that a battery of assays which address many different processes in nervous system development may be used to identify potential developmental neurotoxicants and to distinguish specific from generalized cytotoxic effects with a high degree of success. Published by Elsevier Inc.

Development of a central nervous system axonal myelination assay for high throughput screening.

PubMed

Lariosa-Willingham, Karen D; Rosler, Elen S; Tung, Jay S; Dugas, Jason C; Collins, Tassie L; Leonoudakis, Dmitri

2016-04-22

Regeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system. We established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency. We have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.

Modification of sample processing for the Limulus amebocyte lysate assay enhances detection of inflammogenic endotoxin in intact bacteria and organic dust.

PubMed

Hoppe Parr, Kimberly A; Hađina, Suzana; Kilburg-Basnyat, Brita; Wang, Yifang; Chavez, Dulce; Thorne, Peter S; Weiss, Jerrold P

2017-04-01

The pro-inflammatory potency and causal relationship with asthma of inhaled endotoxins have underscored the importance of accurately assessing the endotoxin content of organic dusts. The Limulus amebocyte lysate (LAL) assay has emerged as the preferred assay, but its ability to measure endotoxin in intact bacteria and organic dusts with similar sensitivity as purified endotoxin is unknown. We used metabolically radiolabeled Neisseria meningitidis and both rough and smooth Escherichia coli to compare dose-dependent activation in the LAL with purified endotoxin from these bacteria and shed outer membrane (OM) blebs. Labeled [ 14 C]-3-OH-fatty acids were used to quantify the endotoxin content of the samples. Purified meningococcal and E. coli endotoxins and OM blebs displayed similar specific activity in the LAL assay to the purified LPS standard. In contrast, intact bacteria exhibited fivefold lower specific activity in the LAL assay but showed similar MD-2-dependent potency as purified endotoxin in inducing acute airway inflammation in mice. Pre-treatment of intact bacteria and organic dusts with 0.1 M Tris-HCl/10 mM EDTA increased by fivefold the release of endotoxin. These findings demonstrate that house dust and other organic dusts should be extracted with Tris/EDTA to more accurately assess the endotoxin content and pro-inflammatory potential of these environmental samples.

Modification of Sample Processing for the Limulus Amebocyte Lysate Assay Enhances Detection of Inflammogenic Endotoxin in Intact Bacteria and Organic Dust

PubMed Central

Hoppe Parr, Kimberly A.; Hađina, Suzana; Kilburg-Basnyat, Brita; Wang, Yifang; Chavez, Dulce; Thorne, Peter S.; Weiss, Jerrold P.

2018-01-01

The pro-inflammatory potency and causal relationship with asthma of inhaled endotoxins have underscored the importance of accurately assessing the endotoxin content of organic dusts. The Limulus Amebocyte Lysate (LAL) assay has emerged as the preferred assay but its ability to measure endotoxin in intact bacteria and organic dusts with similar sensitivity as purified endotoxin is unknown. We used metabolically radiolabeled Neisseria meningitidis and both rough and smooth Escherichia coli to compare dose-dependent activation in the LAL with purified endotoxin from these bacteria and shed outer membrane (OM) blebs. Bacteria labeled with [14C]-3-OH-fatty acids were used to quantify the endotoxin content of the samples. Purified meningococcal and E. coli endotoxins and OM blebs displayed similar specific activity in the LAL assay to the purified LPS standard. In contrast, intact bacteria exhibited 5-fold lower specific activity in the LAL assay but showed similar MD-2-dependent potency as purified endotoxin in inducing acute airway inflammation in mice. Pretreatment of intact bacteria and organic dusts with 0.1M Tris-HCl/10mM EDTA increased by 5-fold the release of endotoxin. These findings demonstrate that house dust and other organic dusts should be extracted with Tris/EDTA to more accurately assess the endotoxin content and pro-inflammatory potential of these environmental samples. PMID:28359219

Kinetic microplate bioassays for relative potency of antibiotics improved by partial Least Square (PLS) regression.

PubMed

Francisco, Fabiane Lacerda; Saviano, Alessandro Morais; Almeida, Túlia de Souza Botelho; Lourenço, Felipe Rebello

2016-05-01

Microbiological assays are widely used to estimate the relative potencies of antibiotics in order to guarantee the efficacy, safety, and quality of drug products. Despite of the advantages of turbidimetric bioassays when compared to other methods, it has limitations concerning the linearity and range of the dose-response curve determination. Here, we proposed to use partial least squares (PLS) regression to solve these limitations and to improve the prediction of relative potencies of antibiotics. Kinetic-reading microplate turbidimetric bioassays for apramacyin and vancomycin were performed using Escherichia coli (ATCC 8739) and Bacillus subtilis (ATCC 6633), respectively. Microbial growths were measured as absorbance up to 180 and 300min for apramycin and vancomycin turbidimetric bioassays, respectively. Conventional dose-response curves (absorbances or area under the microbial growth curve vs. log of antibiotic concentration) showed significant regression, however there were significant deviation of linearity. Thus, they could not be used for relative potency estimations. PLS regression allowed us to construct a predictive model for estimating the relative potencies of apramycin and vancomycin without over-fitting and it improved the linear range of turbidimetric bioassay. In addition, PLS regression provided predictions of relative potencies equivalent to those obtained from agar diffusion official methods. Therefore, we conclude that PLS regression may be used to estimate the relative potencies of antibiotics with significant advantages when compared to conventional dose-response curve determination. Copyright © 2016 Elsevier B.V. All rights reserved.

A new series of estrogen receptor modulators that display selectivity for estrogen receptor beta.

PubMed

Henke, Brad R; Consler, Thomas G; Go, Ning; Hale, Ron L; Hohman, Dana R; Jones, Stacey A; Lu, Amy T; Moore, Linda B; Moore, John T; Orband-Miller, Lisa A; Robinett, R Graham; Shearin, Jean; Spearing, Paul K; Stewart, Eugene L; Turnbull, Philip S; Weaver, Susan L; Williams, Shawn P; Wisely, G Bruce; Lambert, Millard H

2002-12-05

A series of 1,3,5-triazine-based estrogen receptor (ER) modulators that are modestly selective for the ERbeta subtype are reported. Compound 1, which displayed modest potency and selectivity for ERbeta vs ERalpha, was identified via high-throughput screening utilizing an ERbeta SPA-based binding assay. Subsequent analogue preparation resulted in the identification of compounds such as 21 and 43 that display 25- to 30-fold selectivity for ERbeta with potencies in the 10-30 nM range. These compounds profile as full antagonists at ERbeta and weak partial agonists at ERalpha in a cell-based reporter gene assay. In addition, the X-ray crystal structure of compound 15 complexed with the ligand binding domain of ERbeta has been solved and was utilized in the design of more conformationally restrained analogues such as 31 in an attempt to increase selectivity for the ERbeta subtype.

Structure-function relations of heparin-mimetic sulfated xylan oligosaccharides: inhibition of human immunodeficiency virus-1 infectivity in vitro.

PubMed

Stone, A L; Melton, D J; Lewis, M S

1998-07-01

Heparins/heparan sulfates modulate the function of proteins and cell membranes in numerous biological systems including normal and disease processes in humans. Heparin has been used for many years as an anticoagulant, and anticoagulant heparin-mimetics were developed several decades ago by chemical sulfation of non-mammalian polysaccharides, e.g., an antithrombotic sulfated xylan. This pharmaceutical, which comprises a mixture of sulfated oligoxylans, also mimics most other biological actions of natural heparins in vitro, including inhibition of the human immunodeficiency virus, but the molecular basis for these actions has been unclear. Here, numerous Components of the sulfated oligoxylan mixture were isolated and when bioassayed in the case of anti-HIV-1 infectivity revealed that a structural specificity underlines the capacity of sulfated xylan to inhibit HIV-1, rather than a non-specific mechanism. Components were isolated by chromatographic fractionation through Bio-Gel P10 in 0.5 M ammonium bicarbonate. This fractionation revealed an elution range associated with apparent molecular weights of approximately 22000 to <1500 relative to standard heparin and heparan sulfates and newly prepared sulfated oligosaccharide standards. Components were characterized by metachromatic absorption spectroscopy, ultracentrifugation, GlcA analysis, and potency against HIV-1 infectivity, both in the tetrazolium cytotoxicity assay and in syncytium-forming assays, in CD4-lymphocytes. Structural specificity was indicated by the differential potencies exhibited by the Components: Highest activity (cytotoxicity) was exhibited by Components in the chromatographic region > or = approximately 5500 in mass (50% effective (inhibitory) concentration = 0.5-0.7 microg ml(-1) in the first fractionation series, and 0.1-0.5 microg ml(-1) in a second series). The potency declined sharply below approximately 5400 in mass, but with an exception; a second structure exhibiting relatively high potency eluted among low-mass oligosaccharides which had an average size of approximately a nonomer. Components displayed differential potencies also against the syncytium-forming infectivity of HIV-1. The high potency against syncytium-formation was retained by Components down to a minimum size of about 4500 in mass, smaller than the > or = approximately 5400 required above. One in ten of the beta1,4-linked xyloses in the native xylan are substituted with a monomeric alpha1,2 DGlcA branch. We have speculated that pharmaceutical actions of sulfated xylan might be related to structures involving the alpha-D linked substituents and this was examined using a space-filling model of a sulfated octaxylan and by analyses of Components for GlcA content. Understanding structure/function relations in the heparin-like actions of these agents would be of general significance for the careful examination of their potential clinical usefulness in many human processes modulated by heparins, including AIDS.

Studies of structure-activity relationship on plant polyphenol-induced suppression of human liver cancer cells.

PubMed

Loa, Jacky; Chow, Pierce; Zhang, Kai

2009-05-01

To study anticancer activities of 68 plant polyphenols with different backbone structures and various substitutions and to analyze the structure-activity relationships. Antiproliferative activity of 68 plant polyphenols on human liver cancer cells were screened by the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide method. Structure-activity relationships were analyzed by comparison of their activities with selected structures. Cell cycle progression was assayed by flow cytometry analysis and apoptosis was analyzed by DNA fragment assay. Based on their backbone structures, 68 polyphenols were sub-classed to flavonoids (chalcones, flavanones, flavones and isoflavones), chromones and coumarins. The order of their potency to suppress the human liver cancer cells is chalcones > flavones > chromones > isoflavones > flavanones > coumarins. Chalcones comprise the most potent group with IC(50) values ranging from 21.69 to 197 microM. Top nine most potent chalcones in the group have hydroxylation at 2'-carbon position in B-ring. Flavones ranked second in their potencies. Quercetin, 4-hydroxyflavone and luteolin are three hydroxyflavones with highest potencies in this group. Their IC(50) values are 30.81, 39.29 and 71.17 microM, respectively. Chromones, isoflavones, flavanones and coumarins showed much lower potencies when compared to the first two groups with IC(50) ranges of 61 to >400, 131 to >400, 138 to >400 and 360.85 to >400 microM, respectively. In mechanistic studies, the most potent chalcone, 2,2'-dihydroxychalcone could induce G2/M arrest and then apoptosis of the cancer cells. An analysis of structure-activity relationship showed that following structures are required for their inhibitory potencies on human liver cancer cells: (1) of the six sub-classes of the polyphenols tested, the unique backbone structure of chalcones with a open C-ring; (2) within the chalcone group, hydroxyl substitution at 2'-carbon of B-ring; (3) hydroxyl substitution at 3'-carbon in B-ring of flavones. However, some other structures were found to decrease their potencies: e.g. substitutions by sugar moieties in flavones. These data are valuable for design and modification of new polyphenols, which could be potential antiproliferative agents of cancer cells.

Evaluation of a recombinant yeast cell estrogen screening assay.

PubMed Central

Coldham, N G; Dave, M; Sivapathasundaram, S; McDonnell, D P; Connor, C; Sauer, M J

1997-01-01

A wide range of chemicals with diverse structures derived from plant and environmental origins are reported to have hormonal activity. The potential for appreciable exposure of humans to such substances prompts the need to develop sensitive screening methods to quantitate and evaluate the risk to the public. Yeast cells transformed with plasmids encoding the human estrogen receptor and an estrogen responsive promoter linked to a reporter gene were evaluated for screening compounds for estrogenic activity. Relative sensitivity to estrogens was evaluated by reference to 17 beta-estradiol (E2) calibration curves derived using the recombinant yeast cells, MCF-7 human breast cancer cells, and a prepubertal mouse uterotrophic bioassay. The recombinant yeast cell bioassay (RCBA) was approximately two and five orders of magnitude more sensitive to E2 than MCF-7 cells and the uterotrophic assay, respectively. The estrogenic potency of 53 chemicals, including steroid hormones, synthetic estrogens, environmental pollutants, and phytoestrogens, was measured using the RCBA. Potency values produced with the RCBA relative to E2 (100) included estrone (9.6), diethylstilbestrol (74.3), tamoxifen (0.0047), alpha-zearalanol (1.3), equol (0.085), 4-nonylphenol (0.005), and butylbenzyl phathalate (0.0004), which were similar to literature values but generally higher than those produced by the uterotrophic assay. Exquisite sensitivity, absence of test compound biotransformation, ease of use, and the possibility of measuring antiestrogenic activity are important attributes that argue for the suitability of the RCBA in screening for potential xenoestrogens to evaluate risk to humans, wildlife, and the environment. Images Figure 1. Figure 2. Figure 3. Figure 4. PMID:9294720

Potency values from the local lymph node assay: application to classification, labelling and risk assessment.

PubMed

Loveless, S E; Api, A-M; Crevel, R W R; Debruyne, E; Gamer, A; Jowsey, I R; Kern, P; Kimber, I; Lea, L; Lloyd, P; Mehmood, Z; Steiling, W; Veenstra, G; Woolhiser, M; Hennes, C

2010-02-01

Hundreds of chemicals are contact allergens but there remains a need to identify and characterise accurately skin sensitising hazards. The purpose of this review was fourfold. First, when using the local lymph node assay (LLNA), consider whether an exposure concentration (EC3 value) lower than 100% can be defined and used as a threshold criterion for classification and labelling. Second, is there any reason to revise the recommendation of a previous ECETOC Task Force regarding specific EC3 values used for sub-categorisation of substances based upon potency? Third, what recommendations can be made regarding classification and labelling of preparations under GHS? Finally, consider how to integrate LLNA data into risk assessment and provide a rationale for using concentration responses and corresponding no-effect concentrations. Although skin sensitising chemicals having high EC3 values may represent only relatively low risks to humans, it is not possible currently to define an EC3 value below 100% that would serve as an appropriate threshold for classification and labelling. The conclusion drawn from reviewing the use of distinct categories for characterising contact allergens was that the most appropriate, science-based classification of contact allergens according to potency is one in which four sub-categories are identified: 'extreme', 'strong', 'moderate' and 'weak'. Since draining lymph node cell proliferation is related causally and quantitatively to potency, LLNA EC3 values are recommended for determination of a no expected sensitisation induction level that represents the first step in quantitative risk assessment. 2009 Elsevier Inc. All rights reserved.

Assessment of toxic potency of complex mixtures of PAHs from Lincoln Creek, Milwaukee, WI

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villeneuve, D.; Crunkilton, R.; DeVita, W.

1995-12-31

An assay of cytochrome P4501A catalytic activity in PLHC-1 fish hepatoma cells was used to evaluate the toxic potency of dialysates from triolein filled semipermeable polymeric membrane devices (SPMDS) exposed for variable durations and under various flow regimes to water from Lincoln Creek. Toxic potency was expressed as 2,3,7,8 tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQ) calculated from bioassay results. Dose-dependent responses in measured ethoxyresorufin-o-deethylase (EROD) activity of PLHC-1 cells exposed to SPMD dialysates were shown. Toxic potency of dialysates, expressed as bioassay derived TCDD equivalents, increased with duration of SPMD exposure in Lincoln Creek from 2.0 pg/uL for a 2 day exposure tomore » 19.5 pg/uL for a 30 day exposure. This corresponded to an increase in dialysate polycyclic aromatic hydrocarbon (PAH) concentration from 8.82 ug/g after a 2 day exposure to 24.14 ug/g after 30 days. Dialysates from SPMDs exposed to Lincoln Creek stormflow had higher toxic potencies and total PAH concentrations than those exposed to baseflow only, These results suggest that levels of PAH contamination, particularly those associated with stormflow, in Lincoln Creek have potential to accumulate in fish to levels significant enough to elicit a measurable biological response (cytochrome P4501 A induction) at a potency level approaching 0.08% that of TCDD.« less

Comparative analysis of skin sensitization potency of acrylates (methyl acrylate, ethyl acrylate, butyl acrylate, and ethylhexyl acrylate) using the local lymph node assay.

PubMed

Dearman, Rebecca J; Betts, Catherine J; Farr, Craig; McLaughlin, James; Berdasco, Nancy; Wiench, Karin; Kimber, Ian

2007-10-01

There are currently available no systematic experimental data on the skin sensitizing properties of acrylates that are of relevance in occupational settings. Limited information from previous guinea-pig tests or from the local lymph node assay (LLNA) is available; however, these data are incomplete and somewhat contradictory. For those reasons, we have examined in the LLNA 4 acrylates: butyl acrylate (BA), ethyl acrylate (EA), methyl acrylate (MA), and ethylhexyl acrylate (EHA). The LLNA data indicated that all 4 compounds have some potential to cause skin sensitization. In addition, the relative potencies of these acrylates were measured by derivation from LLNA dose-response analyses of EC3 values (the effective concentration of chemical required to induce a threefold increase in proliferation of draining lymph node cells compared with control values). On the basis of 1 scheme for the categorization of skin sensitization potency, BA, EA, and MA were each classified as weak sensitizers. Using the same scheme, EHA was considered a moderate sensitizer. However, it must be emphasized that the EC3 value for this chemical of 9.7% is on the borderline between moderate (<10%) and weak (>10%) categories. Thus, the judicious view is that all 4 chemicals possess relatively weak skin sensitizing potential.

Comparative in vitro study of the antimicrobial activities of different commercial antibiotic products of vancomycin

PubMed Central

2011-01-01

Background One of the most critical problems about antimicrobial therapy is the increasing resistance to antibiotics. Previous studies have shown that there is a direct relation between erroneous prescription, dosage, route, duration of the therapy and the antibiotics resistance. Other important point is the uncertainty about the quality of the prescribed medicines. Some physicians believe that generic drugs are not as effective as innovator ones, so it is very important to have evidence that shows that all commercialized drugs are suitable for therapeutic use. Methods Microbial assays were used to establish the potency, the Minimal Inhibitory Concentrations (MICs), the Minimal Bactericidal Concentration (MBCs), the critical concentrations, and the production of spontaneous mutants that are resistant to vancomycin. Results The microbial assay was validated in order to determine the Vancomycin potency of the tasted samples. All the products showed that have potency values between 90 - 115% (USP requirement). The products behave similarly because the MICs, The MBCs, the critical concentrations, the critical concentrations ratios between standard and samples, and the production of spontaneous mutants don't have significant differences. Conclusions All products analyzed by microbiological tests, show that both trademarks and generics do not have statistical variability and the answer of antimicrobial activity Show also that they are pharmaceutical equivalents. PMID:21777438

Comparative in vitro study of the antimicrobial activities of different commercial antibiotic products of vancomycin.

PubMed

Diaz, Jorge A; Silva, Edelberto; Arias, Maria J; Garzón, María

2011-07-21

One of the most critical problems about antimicrobial therapy is the increasing resistance to antibiotics. Previous studies have shown that there is a direct relation between erroneous prescription, dosage, route, duration of the therapy and the antibiotics resistance. Other important point is the uncertainty about the quality of the prescribed medicines. Some physicians believe that generic drugs are not as effective as innovator ones, so it is very important to have evidence that shows that all commercialized drugs are suitable for therapeutic use. Microbial assays were used to establish the potency, the Minimal Inhibitory Concentrations (MICs), the Minimal Bactericidal Concentration (MBCs), the critical concentrations, and the production of spontaneous mutants that are resistant to vancomycin. The microbial assay was validated in order to determine the Vancomycin potency of the tasted samples. All the products showed that have potency values between 90 - 115% (USP requirement). The products behave similarly because the MICs, The MBCs, the critical concentrations, the critical concentrations ratios between standard and samples, and the production of spontaneous mutants don't have significant differences. All products analyzed by microbiological tests, show that both trademarks and generics do not have statistical variability and the answer of antimicrobial activity Show also that they are pharmaceutical equivalents.

Potency and selectivity of carprofen enantiomers for inhibition of bovine cyclooxygenase in whole blood assays.

PubMed

Brentnall, Claire; Cheng, Zhangrui; McKellar, Quintin A; Lees, Peter

2012-12-01

Whole blood in vitro assays were used to determine the potency and selectivity of carprofen enantiomers for inhibition of the isoforms of cyclooxygenase (COX), COX-1 and COX-2, in the calf. S(+)-carprofen possessed preferential activity for COX-2 inhibition but, because the slopes of inhibition curves differed, the COX-1:COX-2 inhibition ratio decreased from 9.04:1 for inhibitory concentration (IC)10 to 1.84:1 for IC95. R(-) carprofen inhibited COX-2 preferentially only for low inhibition of the COX isoforms (IC10 COX-1:COX-2=6.63:1), whereas inhibition was preferential for COX-1 for a high level of inhibition (IC95 COX-1:COX-2=0.20:1). S(+) carprofen was the more potent inhibitor of COX isoforms; potency ratios S(+):R(-) carprofen were 11.6:1 for IC10 and 218:1 for IC90. Based on serum concentrations of carprofen enantiomers obtained after administration of a therapeutic dose of 1.4 mg/kg to calves subcutaneously, S(+)-carprofen concentrations exceeded the in vitro IC80 COX-2 value for 32 h and the IC20 for COX-1 for 33 h. The findings are discussed in relation to efficacy and safety of carprofen in calves. Copyright © 2012 Elsevier Ltd. All rights reserved.

Structure-activity relationships of rationally designed AMACR 1A inhibitors.

PubMed

Yevglevskis, Maksims; Lee, Guat L; Nathubhai, Amit; Petrova, Yoana D; James, Tony D; Threadgill, Michael D; Woodman, Timothy J; Lloyd, Matthew D

2018-04-30

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the 'racemisation' reaction and the difficulties in the separation of epimeric products; consequently few inhibitors have been described and no structure-activity relationship study has been performed. This paper describes the first structure-activity relationship study, in which a series of 23 known and potential rational AMACR inhibitors were evaluated. AMACR was potently inhibited (IC 50  = 400-750 nM) by ibuprofenoyl-CoA and derivatives. Potency was positively correlated with inhibitor lipophilicity. AMACR was also inhibited by straight-chain and branched-chain acyl-CoA esters, with potency positively correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold more potent inhibitors than decanoyl-CoA, demonstrating the importance of the 2-methyl group for effective inhibition. Elimination substrates and compounds with modified acyl-CoA cores were also investigated, and shown to be potent inhibitors. These results are the first to demonstrate structure-activity relationships of rational AMACR inhibitors and that potency can be predicted by acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric assay for thorough inhibitor characterisation. Copyright © 2018 Elsevier Inc. All rights reserved.

Development of Tricyclic Hydroxy-1H-pyrrolopyridine-trione Containing HIV-1 Integrase Inhibitors

PubMed Central

Zhao, Xue Zhi; Maddali, Kasthuraiah; Metifiot, Mathieu; Smith, Steven J.; Vu, B. Christie; Marchand, Christophe; Hughes, Stephen H.; Pommier, Yves; Burke, Terrence R.

2011-01-01

New tricyclic HIV-1 integrase (IN) inhibitors were prepared that combined structural features of bicyclic pyrimidinones with recently disclosed 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones. This combination resulted in the introduction of a nitrogen into the aryl ring and the addition of a fused third ring to our previously described inhibitors. The resulting analogues showed low micromolar inhibitory potency in in vitro HIV-1 integrase assays, with good selectivity for strand transfer relative to 3′-processing. PMID:21493066

Novel orally active growth hormone secretagogues.

PubMed

Hansen, T K; Ankersen, M; Hansen, B S; Raun, K; Nielsen, K K; Lau, J; Peschke, B; Lundt, B F; Thøgersen, H; Johansen, N L; Madsen, K; Andersen, P H

1998-09-10

A novel class of growth hormone-releasing compounds with a molecular weight in the range from 500 to 650 has been discovered. The aim of this study was to obtain growth hormone secretagogues with oral bioavailability. By a rational approach we were able to reduce the size of the lead compound ipamorelin (4) and simultaneously to reduce hydrogen-bonding potential by incorporation of backbone isosters while retaining in vivo potency in swine. A rat pituitary assay was used for screening of all compounds and to evaluate which compounds should be tested further for in vivo potency in swine and oral bioavailability, fpo, in dogs. Most of the tested compounds had fpo in the range of 10-55%. In vivo potency in swine after iv dosing is reported, and ED50 was found to be 30 nmol/kg of body weight for the most potent compound.

Optimized Target Residence Time: Type I1/2 Inhibitors for p38α MAP Kinase with Improved Binding Kinetics through Direct Interaction with the R-Spine.

PubMed

Wentsch, Heike K; Walter, Niklas M; Bührmann, Mike; Mayer-Wrangowski, Svenja; Rauh, Daniel; Zaman, Guido J R; Willemsen-Seegers, Nicole; Buijsman, Rogier C; Henning, Melanie; Dauch, Daniel; Zender, Lars; Laufer, Stefan

2017-05-02

Skepinone-L was recently reported to be a p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, this class of compounds still act as fully ATP-competitive Type I binders which, furthermore, suffer from short residence times at the enzyme. We herein describe a further development with the first Type I1/2 binders for p38α MAP kinase. Type I1/2 inhibitors interfere with the R-spine, inducing a glycine flip and occupying both hydrophobic regions I and II. This design approach leads to prolonged target residence time, binding to both the active and inactive states of the kinase, excellent selectivity, excellent potency on the enzyme level, and low nanomolar activity in a human whole blood assay. This promising binding mode is proven by X-ray crystallography. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Unique DC-SIGN clustering activity of a small glycomimetic: A lesson for ligand design.

PubMed

Sutkeviciute, Ieva; Thépaut, Michel; Sattin, Sara; Berzi, Angela; McGeagh, John; Grudinin, Sergei; Weiser, Jörg; Le Roy, Aline; Reina, Jose J; Rojo, Javier; Clerici, Mario; Bernardi, Anna; Ebel, Christine; Fieschi, Franck

2014-06-20

DC-SIGN is a dendritic cell-specific C-type lectin receptor that recognizes highly glycosylated ligands expressed on the surface of various pathogens. This receptor plays an important role in the early stages of many viral infections, including HIV, which makes it an interesting therapeutic target. Glycomimetic compounds are good drug candidates for DC-SIGN inhibition due to their high solubility, resistance to glycosidases, and nontoxicity. We studied the structural properties of the interaction of the tetrameric DC-SIGN extracellular domain (ECD), with two glycomimetic antagonists, a pseudomannobioside (1) and a linear pseudomannotrioside (2). Though the inhibitory potency of 2, as measured by SPR competition experiments, was 1 order of magnitude higher than that of 1, crystal structures of the complexes within the DC-SIGN carbohydrate recognition domain showed the same binding mode for both compounds. Moreover, when conjugated to multivalent scaffolds, the inhibitory potencies of these compounds became uniform. Combining isothermal titration microcalorimetry, analytical ultracentrifugation, and dynamic light scattering techniques to study DC-SIGN ECD interaction with these glycomimetics revealed that 2 is able, without any multivalent presentation, to cluster DC-SIGN tetramers leading to an artificially overestimated inhibitory potency. The use of multivalent scaffolds presenting 1 or 2 in HIV trans-infection inhibition assay confirms the loss of potency of 2 upon conjugation and the equal efficacy of chemically simpler compound 1. This study documents a unique case where, among two active compounds chemically derived, the compound with the lower apparent activity is the optimal lead for further drug development.

Synthesis of Fucosylated Chondroitin Sulfate Glycoclusters: A Robust Route to New Anticoagulant Agents.

PubMed

Zhang, Xiao; Yao, Wang; Xu, Xiaojiang; Sun, Huifang; Zhao, Jinhua; Meng, Xiangbao; Wu, Mingyi; Li, Zhongjun

2018-02-01

Fucosylated chondroitin sulfate (FuCS) is a structurally distinct glycosaminoglycan with excellent anticoagulant activity. Studies show that FuCS and its depolymerized fragments exhibit a different anticoagulant mechanism from that of heparin derivatives, with decreased risks of adverse effects and bleeding. However, further exploitation has been hindered by the scarcity of structurally defined oligosaccharides. Herein, facile method is reported for the synthesis of the repeating trisaccharide unit of FuCS based on the degradation of chondroitin sulfate polymers. A series of simplified FuCS glycomimetics that have highly tunable structures, controllable branches, and defined sulfation motifs were generated by copper-catalyzed alkyne-azide cycloaddition. Remarkable improvement in activated partial thromboplastin time (APTT) assay activities was observed as the branches increased, but no significant influences were observed for prothrombin time (PT) and thrombin time (TT) assay activities. Further FXase inhibition tests suggested that glycoclusters 33 b-40 b selectively inhibited intrinsic anticoagulant activities, but had little effect on the extrinsic and common coagulation pathways. Notably, glycoclusters with the 2,4-di-O-sulfated fucosyl residue displayed the most potency, which was in consistent with that of natural polysaccharides. These FuCS clusters demonstrated potency to mimic linear glycosaminoglycans and offer a new framework for the development of novel anticoagulant agents. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Establishment of the 2nd Korean national biological reference standard for blood coagulation factor VIII:C concentrate.

PubMed

Lee, Naery; Seo, Ji Suk; Kim, Jae Ok; Ban, Sang Ja

2017-05-01

Since the 1st Korean national biological reference standard for factor (F)VIII concentrate, established in 2001, has shown declining potency, we conducted this study to replace this standard with a 2nd Korean national biological reference standard for blood coagulation FVIII concentrate. The candidate materials for the 2nd standard were prepared in 8000 vials with 10 IU/ml of target potency, according to the approved manufacturing process of blood coagulation Factor VIII:C Monoclonal Antibody-purified, Freeze-dried Human Blood Coagulation Factor VIII:C. Potency was evaluated by one-stage clotting and chromogenic methods and the stability was confirmed to meet the specifications during a period of 73 months. Since the potencies obtained by the two methods differed significantly (P < 0.015), the values were determined separately according to the geometric means (8.9 and 7.4 IU/vial, respectively). The geometric coefficients of interlaboratory variability were 3.4% and 7.6% by the one-stage clotting and chromogenic assays, respectively. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

The Assessment of Parameters Affecting the Quality of Cord Blood by the Appliance of the Annexin V Staining Method and Correlation with CFU Assays

PubMed Central

Radke, Teja Falk; Barbosa, David; Duggleby, Richard Charles; Saccardi, Riccardo; Querol, Sergio; Kögler, Gesine

2013-01-01

The assessment of nonviable haematopoietic cells by Annexin V staining method in flow cytometry has recently been published by Duggleby et al. Resulting in a better correlation with the observed colony formation in methylcellulose assays than the standard ISHAGE protocol, it presents a promising method to predict cord blood potency. Herein, we applied this method for examining the parameters during processing which potentially could affect cord blood viability. We could verify that the current standards regarding time and temperature are sufficient, since no significant difference was observed within 48 hours or in storage at 4°C up to 26°C. However, the addition of DMSO for cryopreservation alone leads to an inevitable increase in nonviable haematopoietic stem cells from initially 14.8% ± 4.3% to at least 30.6% ± 5.5%. Furthermore, CFU-assays with varied seeding density were performed in order to evaluate the applicability as a quantitative method. The results revealed that only in a narrow range reproducible clonogenic efficiency (ClonE) could be assessed, giving at least a semiquantitative estimation. We conclude that both Annexin V staining method and CFU-assays with defined seeding density are reliable means leading to a better prediction of the final potency. Especially Annexin V, due to its fast readout, is a practical tool for examining and optimising specific steps in processing, while CFU-assays add a functional confirmation. PMID:23533443

A new series of highly potent growth hormone-releasing peptides derived from ipamorelin.

PubMed

Ankersen, M; Johansen, N L; Madsen, K; Hansen, B S; Raun, K; Nielsen, K K; Thogersen, H; Hansen, T K; Peschke, B; Lau, J; Lundt, B F; Andersen, P H

1998-09-10

A new series of GH secretagogues derived from ipamorelin is described. In an attempt to obtain oral bioavailability, by reducing the size and the number of potential hydrogen-bonding sites of the compounds, a strategy using the peptidomimetic fragment 3-(aminomethyl)benzoic acid and sequential backbone N-methylations was applied. Several compounds from this series release GH with high in vitro potency and efficacy in a rat pituitary cell assay and high in vivo potency and efficacy in anesthetized rats. The tetrapeptide NNC 26-0235 (3-(aminomethyl)benzoyl-D-2Nal-N-Me-D-Phe-Lys-NH2) shows, following iv administration, comparable in vivo potency to ipamorelin, GHRP-2, and GHRP-6 with an ED50 in swine at 2 nmol/kg. NNC 26-0235 demonstrated a 10% oral bioavailability in dogs, and NNC 26-0235 and ipamorelin were able to increase basal GH level by more than 10-fold after oral administration of a dose of 1.8 and 2.7 mg/kg, respectively. The tripeptide NNC 26-0323 (3-(aminomethyl)benzoic acid-N-Me-D-2Nal-N-Me-D-Phe-ol) which showed moderate in vitro potency but lacked in vivo potency demonstrated a 20% oral bioavailability in rats.

Quantitative measurement of cell membrane receptor internalization by the nanoluciferase reporter: Using the G protein-coupled receptor RXFP3 as a model.

PubMed

Liu, Yu; Song, Ge; Shao, Xiao-Xia; Liu, Ya-Li; Guo, Zhan-Yun

2015-02-01

Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence to date. In the present work, we developed NanoLuc as a sensitive bioluminescent reporter to measure quantitatively the internalization of cell membrane receptors, based on the pH dependence of the reporter activity. The G protein-coupled receptor RXFP3, the cognate receptor of relaxin-3/INSL7, was used as a model receptor. We first generated stable HEK293T cells that inducibly coexpressed a C-terminally NanoLuc-tagged human RXFP3 and a C-terminally enhanced green fluorescent protein (EGFP)-tagged human RXFP3. The C-terminal EGFP-tag and NanoLuc-tag had no detrimental effects on the ligand-binding potency and intracellular trafficking of RXFP3. Based on the fluorescence of the tagged EGFP reporter, the ligand-induced RXFP3 internalization was visualized directly under a fluorescence microscope. Based on the bioluminescence of the tagged NanoLuc reporter, the ligand-induced RXFP3 internalization was measured quantitatively by a convenient bioluminescent assay. Coexpression of an EGFP-tagged inactive [E141R]RXFP3 had no detrimental effect on the ligand-binding potency and ligand-induced internalization of the NanoLuc-tagged wild-type RXFP3, suggesting that the mutant RXFP3 and wild-type RXFP3 worked independently. The present bioluminescent internalization assay could be extended to other G protein-coupled receptors and other cell membrane receptors to study ligand-receptor and receptor-receptor interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization of potent and selective iodonium-class inhibitors of NADPH oxidases.

PubMed

Lu, Jiamo; Risbood, Prabhakar; Kane, Charles T; Hossain, Md Tafazzal; Anderson, Larry; Hill, Kimberly; Monks, Anne; Wu, Yongzhong; Antony, Smitha; Juhasz, Agnes; Liu, Han; Jiang, Guojian; Harris, Erik; Roy, Krishnendu; Meitzler, Jennifer L; Konaté, Mariam; Doroshow, James H

2017-11-01

The NADPH oxidases (NOXs) play a recognized role in the development and progression of inflammation-associated disorders, as well as cancer. To date, several NOX inhibitors have been developed, through either high throughput screening or targeted disruption of NOX interaction partners, although only a few have reached clinical trials. To improve the efficacy and bioavailability of the iodonium class NOX inhibitor diphenylene iodonium (DPI), we synthesized 36 analogs of DPI, focusing on improved solubility and functionalization. The inhibitory activity of the analogs was interrogated through cell viability and clonogenic studies with a colon cancer cell line (HT-29) that depends on NOX for its proliferative potential. Lack of altered cellular respiration at relevant iodonium analog concentrations was also demonstrated. Additionally, inhibition of ROS generation was evaluated with a luminescence assay for superoxide, or by Amplex Red® assay for H 2 O 2 production, in cell models expressing specific NOX isoforms. DPI and four analogs (NSCs 740104, 751140, 734428, 737392) strongly inhibited HT-29 cell growth and ROS production with nanomolar potency in a concentration-dependent manner. NSC 737392 and 734428, which both feature nitro functional groups at the meta position, had >10-fold higher activity against ROS production by cells that overexpress dual oxidase 2 (DUOX2) than the other compounds examined (IC 50 ≈200-400nM). Based on these results, we synthesized and tested NSC 780521 with optimized potency against DUOX2. Iodonium analogs with anticancer activity, including the first generation of targeted agents with improved specificity against DUOX2, may provide a novel therapeutic approach to NOX-driven tumors. Published by Elsevier Inc.

[Methods of testing inactivated antirabies vaccines].

PubMed

Nedosekov, V V; Vishniakov, I F; Gruzdev, K N

2001-01-01

Methods for evaluating the potency of inactivated rabies vaccines are reviewed. Shortcomings of the traditional NIH method and advantages of modern rapid immunological in vitro methods (antibody binding test, radial immunodiffusion test, enzyme linked immunoadsorbent assay) for estimation of antigenic activity of vaccines are discussed.

Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells.

PubMed

Masaki, Hideki; Kato-Itoh, Megumi; Umino, Ayumi; Sato, Hideyuki; Hamanaka, Sanae; Kobayashi, Toshihiro; Yamaguchi, Tomoyuki; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Nakauchi, Hiromitsu

2015-09-15

Functional assay limitations are an emerging issue in characterizing human pluripotent stem cells (PSCs). With rodent PSCs, chimera formation using pre-implantation embryos is the gold-standard assay of pluripotency (competence of progeny to differentiate into all three germ layers). In human PSCs (hPSCs), however, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-human or human-animal chimeras. To circumvent this issue, we developed a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay) that enables the development and observation of embryos up to headfold stage. The assay uses mouse pre-implantation embryos and rat, monkey and human PSCs to create interspecies chimeras cultured in vitro to the early egg-cylinder stage. Intra- and interspecific chimera assays with rodent PSC lines were performed to confirm the consistency of results in vitro and in vivo. The behavior of chimeras developed in vitro appeared to recapitulate that of chimeras developed in vivo; that is, PSC-derived cells survived and were integrated into the epiblast of egg-cylinder-stage embryos. This indicates that the interspecific in vitro chimera assay is useful in evaluating the chimera-forming ability of rodent PSCs. However, when human induced PSCs (both conventional and naïve-like types) were injected into mouse embryos and cultured, some human cells survived but were segregated; unlike epiblast-stage rodent PSCs, they never integrated into the epiblast of egg-cylinder-stage embryos. These data suggest that the mouse-human interspecies in vitro chimera assay does not accurately reflect the early developmental potential/process of hPSCs. The use of evolutionarily more closely related species as host embryos might be necessary to evaluate the developmental potency of hPSCs. © 2015. Published by The Company of Biologists Ltd.

Survey of the mutagenicity of surface water, sediments, and drinking water from the Penobscot Indian Nation.

PubMed

Warren, Sarah H; Claxton, Larry D; Diliberto, Janet; Hughes, Thomas J; Swank, Adam; Kusnierz, Daniel H; Marshall, Valerie; DeMarini, David M

2015-02-01

U.S. Environmental Protection Agency (US EPA) Regional Applied Research Effort (RARE) projects address the effects of environmental pollutants in a particular region on the health of the population in that region. This report is part of a RARE project that addresses this for the Penobscot Indian Nation (PIN), Penobscot Island, Maine, U.S., where the Penobscot River has had fish advisories for many years due to high levels of mercury. We used the Salmonella mutagenicity assay with strains TA100, TA98, YG1041, and YG1042 with and without metabolic activation to assess the mutagenic potencies of organic extracts of the Penobscot River water and sediment, as well as drinking-water samples, all collected by the PIN Department of Natural Resources. The source water for the PIN drinking water is gravel-packed groundwater wells adjacent to the Penobscot River. Most samples of all extracts were either not mutagenic or had low to moderate mutagenic potencies. The average mutagenic potencies (revertants/L-equivalent) were 337 for the drinking-water extracts and 177 for the river-water extracts; the average mutagenic potency for the river-sediment extracts was 244 revertants(g-equivalent)(-1). This part of the RARE project showed that extracts of the Penobscot River water and sediments and Penobscot drinking water have little to no mutagenic activity that might be due to the classes of compounds that the Salmonella mutagenicity assay detects, such as polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs (nitroarenes), and aromatic amines. This study is the first to examine the mutagenicity of environmental samples from a tribal nation in the U.S. Published by Elsevier Ltd.

A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin.

PubMed

Menchon, Grégory; Prota, Andrea E; Lucena-Agell, Daniel; Bucher, Pascal; Jansen, Rolf; Irschik, Herbert; Müller, Rolf; Paterson, Ian; Díaz, J Fernando; Altmann, Karl-Heinz; Steinmetz, Michel O

2018-05-29

Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on β-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer.

Design and Synthesis of Selective Estrogen Receptor beta Agonists and Their Pharmacology

NASA Astrophysics Data System (ADS)

Perera, K. L. Iresha Sampathi

Estrogens (17beta-estradiol, E2) have garnered considerable attention in influencing cognitive process in relation to phases of the menstrual cycle, aging and menopausal symptoms. However, hormone replacement therapy can have deleterious effects leading to breast and endometrial cancer, predominantly mediated by estrogen receptor-alpha (ERalpha) the major isoform present in the mammary gland and uterus. Further evidence supports a dominant role of estrogen receptor-beta (ERbeta) for improved cognitive effects such as enhanced hippocampal signaling and memory consolidation via estrogen activated signaling cascades. Creation of the ERbeta selective ligands is challenging due to high structural similarity of both receptors. Thus far, several ERbeta selective agonists have been developed, however, none of these have made it to clinical use due to their lower selectivity or considerable side effects. The research in this dissertation involved the design of non-steroidal ERbeta selective agonists for hippocampal memory consolidation. The step-wise process to achieve the ultimate goal of this research includes: (1) design and synthesis of (4-hydroxyphenyl)cyclohexyl or cycloheptyl derivatives, (2) in vitro biological evaluation of synthesized compounds to identify highly potent and selective candidates, and (3) in vivo biological evaluation of selected candidates for hippocampal memory consolidation. Several (4-hydroxyphenyl)cyclohexyl or cycloheptyl derivatives were synthesized having structural alterations on both aromatic and cyclohexyl/heptyl ring scaffolds. ERbeta agonist potency was initially evaluated in TR-FRET ERbeta ligand binding assay and compounds having high potency were re-evaluated in functional cell based assays for potency and ERbeta vs. ERalpha selectivity. Two compounds from each series, ISP 163-PK4 and ISP 358-2 were identified as most selective ERbeta agonists. Both compounds revealed high metabolic stability, solubility and no cross reactivity towards other nuclear receptors. In vivo efficiency of ISP 358-2 was evaluated in ovariectomized mice (C57BL/6) with object recognition (OR) and object placement (OP) tasks. The results indicate improved memory consolidation at 100 pg/ hemisphere and 0.5 mg/Kg via DH infusion and IP injection respectively. The information learned from this project serves as a foundation for development of other cycloheptyl/hexyl based ERbeta agonists or antagonists having acceptable pharmacological profiles.

Structure-activity relationships of phenothiazines and related drugs for inhibition of protein kinase C.

PubMed

Aftab, D T; Ballas, L M; Loomis, C R; Hait, W N

1991-11-01

Phenothiazines are known to inhibit the activity of protein kinase C. To identify structural features that determine inhibitory activity against the enzyme, we utilized a semiautomated assay [Anal. Biochem. 187:84-88 (1990)] to compare the potency of greater than 50 phenothiazines and related compounds. Potency was decreased by trifluoro substitution at position 2 on the phenothiazine nucleus and increased by quinoid structures on the nucleus. An alkyl bridge of at least three carbons connecting the terminal amine to the nucleus was required for activity. Primary amines and unsubstituted piperazines were the most potent amino side chains. We selected 7,8-dihydroxychlorpromazine (DHCP) (IC50 = 8.3 microM) and 2-chloro-9-(3-[1-piperazinyl]propylidene)thioxanthene (N751) (IC50 = 14 microM) for further study because of their potency and distinct structural features. Under standard (vesicle) assay conditions, DHCP was noncompetitive with respect to phosphatidylserine and a mixed-type inhibitor with respect to ATP. N751 was competitive with respect to phosphatidylserine and noncompetitive with respect to ATP. Using the mixed micelle assay, DHCP was a competitive inhibitor with respect to both phosphatidylserine and ATP. DHCP was selective for protein kinase C compared with cAMP-dependent protein kinase, calmodulin-dependent protein kinase type II, and casein kinase. N751 was more potent against protein kinase C compared with cAMP-dependent protein kinase and casein kinase but less potent against protein kinase C compared with calmodulin-dependent protein kinase type II. DHCP was analyzed for its ability to inhibit different isoenzymes of protein kinase C, and no significant isozyme selectivity was detected. These data provide important information for the rational design of more potent and selective inhibitors of protein kinase C.

Generation of Tg(cyp1a:gfp) Transgenic Zebrafish for Development of a Convenient and Sensitive In Vivo Assay for Aryl Hydrocarbon Receptor Activity.

PubMed

Xu, Hongyan; Li, Caixia; Li, Yan; Ng, Grace Hwee Boon; Liu, Chunsheng; Zhang, Xiaoyan; Gong, Zhiyuan

2015-12-01

Both dioxins/dioxin-like compounds and polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants and cause multiple adverse health effects on human and wildlife. Cyp1a is the most commonly used biomarker induced by these pollutants through activation of the aryl hydrocarbon receptor (AhR) pathway. Here we generated Tg(cyp1a:gfp) transgenic zebrafish for establishing a convenient in vivo assay for analysing these xenobiotic compounds. The Tg(cyp1a:gfp) larvae at 4 day post-fertilization were tested with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and GFP induction was observed mainly in the kidney, liver and gut. Similar GFP expression was also induced strongly by two dioxin-like chemicals, co-planar polychlorinated biphenyl (PCB126) and polychlorinated dibenzo-p-furan (PeCDF) and relatively weakly by two PAHs, 3-methylcholanthrene (3-MC) and benzo[a]pyrene (BAP). The lowest observed effective concentration (LOEC) of TCDD was estimated to be ∼1 pM and the EC50 (effective concentration to induce GFP in 50 % of Tg(cyp1a:gfp) larvae) was ∼10 pM. PCB126 and PeCDF had ∼10× lower potencies in GFP induction than TCDD, while the potencies for 3-MC and BAP were at least 1000× lower. The sensitivity of Tg(cyp1a:gfp) larvae to respond TCDD was also favourable compared to that of ethoxyresorufin-O-deethylase (EROD) assay in both zebrafish larvae and adult livers. As GFP-based assay in transgenic zebrafish can be easily accommodated in multi-well dishes, the Tg(cyp1a:gfp) zebrafish should provide not only a valuable biomonitoring tool for aquatic contaminants but also a potential high-throughput chemical screening platform for identification of new AhR agonists.

Local lymph node assay (LLNA) for detection of sensitization capacity of chemicals.

PubMed

Gerberick, G Frank; Ryan, Cindy A; Dearman, Rebecca J; Kimber, Ian

2007-01-01

The local lymph node assay (LLNA) is a murine model developed to evaluate the skin sensitization potential of chemicals. The LLNA is an alternative approach to traditional guinea pig methods and in comparison provides important animal welfare benefits. The assay relies on measurement of events induced during the induction phase of skin sensitization, specifically lymphocyte proliferation in the draining lymph nodes which is a hallmark of a skin sensitization response. Since its introduction the LLNA has been the subject of extensive evaluation on a national and international scale, and has been successfully validated and incorporated worldwide into regulatory guidelines. Experience gained in recent years has demonstrated that adherence to published procedures and guidelines for the LLNA (e.g., with respect to dose and vehicle selection) is critical for the successful conduct and eventual interpretation of the data. In addition to providing a robust method for skin sensitization hazard identification, the LLNA has proven very useful in assessing the skin sensitizing potency of test chemicals, and this has provided invaluable information to risk assessors. The primary method to make comparisons of the relative potency of chemical sensitizers is to use linear interpolation to estimate the concentration of chemical required to induce a stimulation index of three relative to concurrent vehicle-treated controls (EC3). In certain situations where there are available less than optimal dose response data a log-linear extrapolation method can be used to estimate an EC3 value which can reduce significantly the need for repeat testing of chemicals. The LLNA, when conducted according to published guidelines, provides a robust method for skin sensitization testing that not only provides reliable hazard identification information but also data necessary for effective risk assessment and risk management.

DC-159a Shows Inhibitory Activity against DNA Gyrases of Mycobacterium leprae.

PubMed

Yamaguchi, Tomoyuki; Yokoyama, Kazumasa; Nakajima, Chie; Suzuki, Yasuhiko

2016-09-01

Fluoroquinolones are a class of antibacterial agents used for leprosy treatment. Some new fluoroquinolones have been attracting interest due to their remarkable potency that is reportedly better than that of ofloxacin, the fluoroquinolone currently recommended for treatment of leprosy. For example, DC-159a, a recently developed 8-methoxy fluoroquinolone, has been found to be highly potent against various bacterial species. Nonetheless, the efficacy of DC-159a against Mycobacterium leprae is yet to be examined. To gather data that can support highly effective fluoroquinolones as candidates for new remedies for leprosy treatment, we conducted in vitro assays to assess and compare the inhibitory activities of DC-159a and two fluoroquinolones that are already known to be more effective against M. leprae than ofloxacin. The fluoroquinolone-inhibited DNA supercoiling assay using recombinant DNA gyrases of wild type and ofloxacin-resistant M. leprae revealed that inhibitory activities of DC-159a and sitafloxacin were at most 9.8- and 11.9-fold higher than moxifloxacin. Also the fluoroquinolone-mediated cleavage assay showed that potencies of those drugs were at most 13.5- and 9.8-fold higher than moxifloxacin. In addition, these two drugs retained their inhibitory activities even against DNA gyrases of ofloxacin-resistant M. leprae. The results indicated that DC-159a and sitafloxacin are more effective against wild type and mutant M. leprae DNA gyrases than moxifloxacin, suggesting that these antibacterial drugs can be good candidates that may supersede current fluoroquinolone remedies. DC-159a in particular is very promising because it is classified in a subgroup of fluoroquinolones that is known to be less likely to cause adverse effects. Our results implied that DC-159a is well worth further investigation to ascertain its in vivo effectiveness and clinical safety for humans.

In Vitro Assays for the Discovery of PCSK9 Autoprocessing Inhibitors.

PubMed

Salowe, Scott P; Zhang, Lei; Zokian, Hratch J; Gesell, Jennifer J; Zink, Deborah L; Wiltsie, Judyann; Ai, Xi; Kavana, Michael; Pinto, Shirly

2016-12-01

PCSK9 plays a significant role in regulating low-density lipoprotein (LDL) cholesterol levels and has become an important drug target for treating hypercholesterolemia. Although a member of the serine protease family, PCSK9 only catalyzes a single reaction, the autocleavage of its prodomain. The maturation of the proprotein is an essential prerequisite for the secretion of PCSK9 to the extracellular space where it binds the LDL receptor and targets it for degradation. We have found that a construct of proPCSK9 where the C-terminal domain has been truncated has sufficient stability to be expressed and purified from Escherichia coli for the in vitro study of autoprocessing. Using automated Western analysis, we demonstrate that autoprocessing exhibits the anticipated first-order kinetics. A high-throughput time-resolved fluorescence resonance energy transfer assay for autocleavage has been developed using a PCSK9 monoclonal antibody that is sensitive to the conformational changes that occur upon maturation of the proprotein. Kinetic theory has been developed that describes the behavior of both reversible and irreversible inhibitors of autocleavage. The analysis of an irreversible lactone inhibitor validates the expected relationship between potency and the reaction end point. An orthogonal liquid chromatography-mass spectrometry assay has also been implemented for the confirmation of hits from the antibody-based assays.

A novel BRET-based binding assay for interaction studies of relaxin family peptide receptor 3 with its ligands.

PubMed

Wang, Jia-Hui; Shao, Xiao-Xia; Hu, Meng-Jun; Wei, Dian; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

2017-05-01

Relaxin family peptide receptor 3 (RXFP3) is an A-class G protein-coupled receptor that is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. To study its interaction with various ligands, we developed a novel bioluminescence resonance energy transfer (BRET)-based binding assay using the brightest NanoLuc as an energy donor and a newly developed cyan-excitable orange fluorescent protein (CyOFP) as an energy acceptor. An engineered CyOFP without intrinsic cysteine residues but with an introduced cysteine at the C-terminus was overexpressed in Escherichia coli and chemically conjugated to the A-chain N-terminus of an easily labeled chimeric R3/I5 peptide via an intermolecular disulfide linkage. After the CyOFP-conjugated R3/I5 bound to a shortened human RXFP3 (removal of 33 N-terminal residues) fused with the NanoLuc reporter at the N-terminus, high BRET signals were detected. Saturation binding and real-time binding assays demonstrated that this BRET pair retained high binding affinity with fast association/dissociation. Using this BRET pair, binding potencies of various ligands with RXFP3 were conveniently measured through competition binding assays. Thus, the novel BRET-based binding assay facilitates interaction studies of RXFP3 with various ligands. The engineered CyOFP without intrinsic cysteine residues may also be applied to other BRET-based binding assays in future studies.

The lethality test used for estimating the potency of antivenoms against Bothrops asper snake venom: pathophysiological mechanisms, prophylactic analgesia, and a surrogate in vitro assay.

PubMed

Chacón, Francisco; Oviedo, Andrea; Escalante, Teresa; Solano, Gabriela; Rucavado, Alexandra; Gutiérrez, José María

2015-01-01

The potency of antivenoms is assessed by analyzing the neutralization of venom-induced lethality, and is expressed as the Median Effective Dose (ED50). The present study was designed to investigate the pathophysiological mechanisms responsible for lethality induced by the venom of Bothrops asper, in the experimental conditions used for the evaluation of the neutralizing potency of antivenoms. Mice injected with 4 LD50s of venom by the intraperitoneal route died within ∼25 min with drastic alterations in the abdominal organs, characterized by hemorrhage, increment in plasma extravasation, and hemoconcentration, thus leading to hypovolemia and cardiovascular collapse. Snake venom metalloproteinases (SVMPs) play a predominat role in lethality, as judged by partial inhibition by the chelating agent CaNa2EDTA. When venom was mixed with antivenom, there was a venom/antivenom ratio at which hemorrhage was significantly reduced, but mice died at later time intervals with evident hemoconcentration, indicating that other components in addition to SVMPs also contribute to plasma extravasation and lethality. Pretreatment with the analgesic tramadol did not affect the outcome of the neutralization test, thus suggesting that prophylactic (precautionary) analgesia can be introduced in this assay. Neutralization of lethality in mice correlated with neutralization of in vitro coagulant activity in human plasma. Copyright © 2014 Elsevier Ltd. All rights reserved.

Investigation of the dermal sensitization potential of various essential oils in the local lymph node assay.

PubMed

Lalko, J; Api, A M

2006-05-01

Essential oils are commonly used fragrance ingredients. The oils themselves are complex mixtures, which may contain naturally occurring contact sensitizers. The local lymph node assay was used to evaluate the dermal sensitization potential of basil, citronella, clove leaf, geranium, litsea cubeba, lemongrass, and palmarosa oils. Three of the major components--citral, eugenol, and geraniol--were included to investigate any difference in sensitization potential arising from their exposure in a mixture. Each fragrance material was tested at five concentration ranging from 2.5% to 50% w/v in 1:3 ethanol:diethyl phthalate. The stimulation index (SI) values were calculated for each dose level, an SI > or = 3 was considered a positive response. The estimated concentration (EC3) required to elicit a positive was calculated and taken as a measure of relative potency. The EC3 values and potency classification for basil, clove leaf, litsea cubeba, lemongrass and palmarosa oils were calculated to be <2.5% (> or = moderate), 7.1% (weak), 8.4% (weak), 6.5% (weak) and 9.6% (weak), respectively. Citronella and geranium oils were negative. The individual components citral, eugenol and geraniol resulted in EC3 values of 6.3%, 5.4% and 11.4%, respectively. In general, the potency of each essential oil did not differ significantly from that observed for its main individual component.

WHO Working Group on revision of the Manual of Laboratory Methods for Testing DTP Vaccines-Report of two meetings held on 20-21 July 2006 and 28-30 March 2007, Geneva, Switzerland.

PubMed

Corbel, Michael J; Das, Rose Gaines; Lei, Dianliang; Xing, Dorothy K L; Horiuchi, Yoshinobu; Dobbelaer, Roland

2008-04-07

This report reflects the discussion and conclusions of a WHO group of experts from National Regulatory Authorities (NRAs), National Control Laboratories (NCLs), vaccine industries and other relevant institutions involved in standardization and control of diphtheria, tetanus and pertussis vaccines (DTP), held on 20-21 July 2006 and 28-30 March 2007, in Geneva Switzerland for the revision of WHO Manual for quality control of DTP vaccines. Taking into account recent developments and standardization in quality control methods and the revision of WHO recommendations for D, T, P vaccines, and a need for updating the manual has been recognized. In these two meetings the current situation of quality control methods in terms of potency, safety and identity tests for DTP vaccines and statistical analysis of data were reviewed. Based on the WHO recommendations and recent validation of testing methods, the content of current manual were reviewed and discussed. The group agreed that the principles to be observed in selecting methods included identifying those critical for assuring safety, efficacy and quality and which were consistent with WHO recommendations/requirements. Methods that were well recognized but not yet included in current Recommendations should be taken into account. These would include in vivo and/or in vitro methods for determining potency, safety testing and identity. The statistical analysis of the data should be revised and updated. It was noted that the mouse based assays for toxoid potency were still quite widely used and it was desirable to establish appropriate standards for these to enable the results to be related to the standard guinea pig assays. The working group was met again to review the first drafts and to input further suggestions or amendments to the contributions of the drafting groups. The revised manual was to be finalized and published by WHO.

Embryotoxic and pharmacologic potency ranking of six azoles in the rat whole embryo culture by morphological and transcriptomic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dimopoulou, Myrto, E-mail: myrto.dimopoulou@wur.nl

Differential gene expression analysis in the rat whole embryo culture (WEC) assay provides mechanistic insight into the embryotoxicity of test compounds. In our study, we hypothesized that comparative analysis of the transcriptomes of rat embryos exposed to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole) could lead to a better mechanism-based understanding of their embryotoxicity and pharmacological action. For evaluating embryotoxicity, we applied the total morphological scoring system (TMS) in embryos exposed for 48 h. The compounds tested showed embryotoxicity in a dose-response fashion. Functional analysis of differential gene expression after 4 h exposure at the ID{sub 10} (effectivemore » dose for 10% decreased TMS), revealed the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the retinoic acid (RA) pathway, albeit in a differential way. Flusilazole, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. Dose-dependent analysis of the effects of flusilazole revealed that the RA pathway related genes were already differentially expressed at low dose levels while the sterol pathway showed strong regulation at higher embryotoxic doses, suggesting that this pathway is less predictive for the observed embryotoxicity. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by flusilazole. In summary, the rat WEC assay in combination with transcriptomics could add a mechanistic insight into the embryotoxic potency ranking and pharmacological mode of action of the tested compounds. - Highlights: • Embryonic exposure to azoles revealed concentration-dependent malformations. • Transcriptomics could enhance the mechanistic knowledge of embryotoxicants. • Retinoic acid gene set identifies early embryotoxic responses to azoles. • Toxic versus pharmacologic potency determines functional efficacy.« less

A novel mixed phospholipid functionalized monolithic column for early screening of drug induced phospholipidosis risk.

PubMed

Zhao, XiangLong; Chen, WeiJia; Liu, ZhengHua; Guo, JiaLiang; Zhou, ZhengYin; Crommen, Jacques; Moaddel, Ruin; Jiang, ZhengJin

2014-11-07

Drug-induced phospholipidosis (PLD) is characterized by the excessive accumulation of phospholipids, resulting in multilamellar vesicle structure within lysosomes. In the present study, a novel mixed phospholipid functionalized monolithic column was developed for the first time through a facile one-step co-polymerization approach. The phospholipid composition of the monolith can be adjusted quantitatively and accurately to mimic the mixed phospholipid environment of different biomembranes on a solid matrix. The mixed phospholipid functionalized monolith as a promising immobilized artificial membrane technique was used to study drug-phospholipid interaction. Scanning electron microscopy, elemental analysis, FT-IR spectra, ζ-potential analysis and micro-HPLC were carried out to characterize the physicochemical properties and separation performance of the monolith. Mechanism studies revealed that both hydrophobic and electrostatic interactions play an important role in the retention of analytes. The ratio of their contributions to retention can be easily manipulated by adjusting the composition of the mixed phospholipids, in order to better mimic the interaction between drugs and cell membrane. The obtained mixed phospholipid functionalized monolithic columns were applied to the screening of drug-induced PLD potency. Data from 79 drugs on the market demonstrated that the chromatographic hydrophobicity index referring to the mixed phospholipid functionalized monolith at pH 7.4 (CHI IAM7.4) for the selected drugs were highly correlated with the drug-induced PLD potency data obtained from other in vivo or in vitro assays. Moreover, the effect of the acidic phospholipid phosphatidylserine proportion on prediction accuracy was also investigated. The monolith containing 20% phosphatidylserine and 80% phosphatidylcholine exhibited the best prediction ability for the drug-induced PLD potency of the tested compounds. This research has led to the successful development of a novel and facile approach to prepare a mixed phospholipids functionalized monolith, which offers a reliable, cost-effective and high-throughput screening tool for early prediction of the PLD potency of drug candidates. Copyright © 2014 Elsevier B.V. All rights reserved.

Bridging the Gap Between Validation and Implementation of Non-Animal Veterinary Vaccine Potency Testing Methods

PubMed Central

Dozier, Samantha; Brown, Jeffrey; Currie, Alistair

2011-01-01

Simple Summary Many vaccines are tested for quality in experiments that require the use of large numbers of animals in procedures that often cause significant pain and distress. Newer technologies have fostered the development of vaccine quality control tests that reduce or eliminate the use of animals, but the availability of these newer methods has not guaranteed their acceptance by regulators or use by manufacturers. We discuss a strategic approach that has been used to assess and ultimately increase the use of non-animal vaccine quality tests in the U.S. and U.K. Abstract In recent years, technologically advanced high-throughput techniques have been developed that replace, reduce or refine animal use in vaccine quality control tests. Following validation, these tests are slowly being accepted for use by international regulatory authorities. Because regulatory acceptance itself has not guaranteed that approved humane methods are adopted by manufacturers, various organizations have sought to foster the preferential use of validated non-animal methods by interfacing with industry and regulatory authorities. After noticing this gap between regulation and uptake by industry, we began developing a paradigm that seeks to narrow the gap and quicken implementation of new replacement, refinement or reduction guidance. A systematic analysis of our experience in promoting the transparent implementation of validated non-animal vaccine potency assays has led to the refinement of our paradigmatic process, presented here, by which interested parties can assess the local regulatory acceptance of methods that reduce animal use and integrate them into quality control testing protocols, or ensure the elimination of peripheral barriers to their use, particularly for potency and other tests carried out on production batches. PMID:26486625

Pharmacological evaluation of a novel series of urea, thiourea and guanidine derivatives as P2X7 receptor antagonists.

PubMed

Wong, Erick C N; Reekie, Tristan A; Werry, Eryn L; O'Brien-Brown, James; Bowyer, Sarah L; Kassiou, Michael

2017-06-01

We report on P2X 7 receptor antagonists based on a lead adamantly-cyanoguanidine-aryl moiety. We have investigated the importance of the central cyanoguanidine moiety by replacing it with urea, thiourea or guanidine moieties. We have also investigated the linker length between the central moiety and the aryl portion. All compounds were assessed for their inhibitory potency in a pore-formation dye uptake assay at the P2X 7 receptor. None of the compounds resulted in an improved potency illustrating the importance of the cyanoguanidine moiety in this chemotype. Copyright © 2017 Elsevier Ltd. All rights reserved.

In silico optimization of a fragment-based hit yields biologically active, high-efficiency inhibitors for glutamate racemase.

PubMed

Whalen, Katie L; Chau, Anthony C; Spies, M Ashley

2013-10-01

A novel lead compound for inhibition of the antibacterial drug target, glutamate racemase (GR), was optimized for both ligand efficiency and lipophilic efficiency. A previously developed hybrid molecular dynamics-docking and scoring scheme, FERM-SMD, was used to predict relative potencies of potential derivatives prior to chemical synthesis. This scheme was successful in distinguishing between high- and low-affinity binders with minimal experimental structural information, saving time and resources in the process. In vitro potency was increased approximately fourfold against GR from the model organism, B. subtilis. Lead derivatives show two- to fourfold increased antimicrobial potency over the parent scaffold. In addition, specificity toward B. subtilis over E. coli and S. aureus depends on the substituent added to the parent scaffold. Finally, insight was gained into the capacity for these compounds to reach the target enzyme in vivo using a bacterial cell wall lysis assay. The outcome of this study is a novel small-molecule inhibitor of GR with the following characteristics: Ki=2.5 μM, LE=0.45 kcal mol(-1) atom(-1), LiPE=6.0, MIC50=260 μg mL(-1) against B. subtilis, EC50, lysis=520 μg mL(-1) against B. subtilis. Copyright © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Addressing the selectivity and toxicity of antiviral nucleosides.

PubMed

Feng, Joy Y

2018-01-01

Nucleoside and nucleotide analogs have played significant roles in antiviral therapies and are valued for their impressive potency and high barrier to resistance. They have been approved for treatment of herpes simplex virus-1, HIV, HBV, HCV, and influenza, and new drugs are being developed for the treatment of RSV, Ebola, coronavirus MERS, and other emerging viruses. However, this class of compounds has also experienced a high attrition rate in clinical trials due to toxicity. In this review, we discuss the utility of different biochemical and cell-based assays and provide recommendations for assessing toxicity liability before entering animal toxicity studies.

A Simplified and Rapid Screening Assay using Zebrafish to Assess Cardiac Effects of Air Pollution-derived Particulate Matter

EPA Science Inventory

Comparative toxicity assessment of particulate matter (PM) from different sources will potentially inform the understanding of regional differences in PM-induced cardiac health effects by identifying PM sources linked to highest potency components. Conventional low-throughput in...

Discovery of an Oxybenzylglycine Based Peroxisome Proliferator Activated Receptor Alpha Selective

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, J.; Kennedy, L; Shi, Y

2010-01-01

An 1,3-oxybenzylglycine based compound 2 (BMS-687453) was discovered to be a potent and selective peroxisome proliferator activated receptor (PPAR) {alpha} agonist, with an EC{sub 50} of 10 nM for human PPAR{alpha} and {approx}410-fold selectivity vs human PPAR{gamma} in PPAR-GAL4 transactivation assays. Similar potencies and selectivity were also observed in the full length receptor co-transfection assays. Compound 2 has negligible cross-reactivity against a panel of human nuclear hormone receptors including PPAR{delta}. Compound 2 demonstrated an excellent pharmacological and safety profile in preclinical studies and thus was chosen as a development candidate for the treatment of atherosclerosis and dyslipidemia. The X-ray cocrystalmore » structures of the early lead compound 12 and compound 2 in complex with PPAR{alpha} ligand binding domain (LBD) were determined. The role of the crystal structure of compound 12 with PPAR{alpha} in the development of the SAR that ultimately resulted in the discovery of compound 2 is discussed.« less

Arylimidamide-Azole Combinations against Leishmaniasis

DTIC Science & Technology

2016-09-01

This compound will be selected for further in vivo testing in Q1 of Year 3. 3. Accomplishments: The efficacy of 3 arylimidamide compounds was...of this compound will take place in Q1 of Year 3. 4. IMPACT: The search for an orally bioavailable arylimidamide analogue with efficacy against...macrophage assay2 against L. major. One of the 4 compounds tested, AA2- 160, showed potency in this assay. This compound will be selected for toxicity testing and in vivo efficacy testing in Q1 of Year 3.

Engineering high-potency R-spondin adult stem cell growth factors.

PubMed

Warner, Margaret L; Bell, Tufica; Pioszak, Augen A

2015-01-01

Secreted R-spondin proteins (RSPOs1-4) function as adult stem cell growth factors by potentiating Wnt signaling. Simultaneous binding of distinct regions of the RSPO Fu1-Fu2 domain module to the extracellular domains (ECDs) of the LGR4 G protein-coupled receptor and the ZNRF3 transmembrane E3 ubiquitin ligase regulates Wnt receptor availability. Here, we examine the molecular basis for the differing signaling strengths of RSPOs1-4 using purified RSPO Fu1-Fu2, LGR4 ECD, and ZNRF3 ECD proteins in Wnt signaling and receptor binding assays, and we engineer novel high-potency RSPOs. RSPO2/3/4 had similar signaling potencies that were stronger than that of RSPO1, whereas RSPO1/2/3 had similar efficacies that were greater than that of RSPO4. The RSPOs bound LGR4 with affinity rank order RSPO4 > RSPO2/3 > RSPO1 and ZNRF3 with affinity rank order RSPO2/3 > > RSPO1 > RSPO4. An RSPO2-4 chimera combining RSPO2 ZNRF3 binding with RSPO4 LGR4 binding was a "Superspondin" that exhibited enhanced ternary complex formation and 10-fold stronger signaling potency than RSPO2 and efficacy equivalent to RSPO2. An RSPO4-1 chimera combining RSPO4 ZNRF3 binding with RSPO1 LGR4 binding was a "Poorspondin" that exhibited signaling potency similar to RSPO1 and efficacy equivalent to RSPO4. Conferring increased ZNRF3 binding upon RSPO4 with amino acid substitutions L56F, I58L, and I63M enhanced its signaling potency and efficacy. Our results reveal the molecular basis for RSPOs1-4 activity differences and suggest that signaling potency is determined by ternary complex formation ability, whereas efficacy depends on ZNRF3 recruitment. High-potency RSPOs may be of value for regenerative medicine and/or therapeutic applications. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

A sensitive WST-8-based bioassay for PEGylated granulocyte colony stimulating factor using the NFS-60 cell line.

PubMed

Tiwari, Krishna; Wavdhane, Madan; Haque, Shafiul; Govender, Thavendran; Kruger, Hendrik G; Mishra, Maheshwari K; Chandra, Ramesh; Tiwari, Dileep

2015-06-01

Granulocyte colony stimulating factor (G-CSF) has been commonly used to treat neutropenia caused by chemotherapy, radiotherapy, and organ transplants. Improved in vitro efficacy of G-CSF has already been observed by conjugating it to polyethylene glycol (PEG). The in vivo bioassay using tetrazolium dye with the NFS-60 cell line has been recommended for G-CSF but no such monographs are available for PEGylated G-CSF in pharmacopeias. In the present study, the assay recommended for G-CSF was evaluated for its suitability to PEGylated G-CSF. The generally used MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-based assay was compared with a bioassay employing a water-soluble tetrazolium dye, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium], using NFS-60 cells at a concentration of 7 × 10(5) cells/ml against 800 IU/ml of PEGylated G-CSF at 24, 48, 72, and 72 h time points to determine the efficacy of PEGylated G-CSF. Further, the optimized WST-8 dye-based assay was used to test the potency of various commercially available PEGylated G-CSF preparations. The results demonstrated enhanced sensitivity of the WST-8-based assay over the conventional MTS-based assay for determining the potency of PEGylated G-CSF using the NFS-60 cell line. Our study demonstrates the potential application of WST-8-based bioassays for other biotherapeutic proteins of human and veterinary interest.

Potential application of the consistency approach for vaccine potency testing.

PubMed

Arciniega, J; Sirota, L A

2012-01-01

The Consistency Approach offers the possibility of reducing the number of animals used for a potency test. However, it is critical to assess the effect that such reduction may have on assay performance. Consistency of production, sometimes referred to as consistency of manufacture or manufacturing, is an old concept implicit in regulation, which aims to ensure the uninterrupted release of safe and effective products. Consistency of manufacture can be described in terms of process capability, or the ability of a process to produce output within specification limits. For example, the standard method for potency testing of inactivated rabies vaccines is a multiple-dilution vaccination challenge test in mice that gives a quantitative, although highly variable estimate. On the other hand, a single-dilution test that does not give a quantitative estimate, but rather shows if the vaccine meets the specification has been proposed. This simplified test can lead to a considerable reduction in the number of animals used. However, traditional indices of process capability assume that the output population (potency values) is normally distributed, which clearly is not the case for the simplified approach. Appropriate computation of capability indices for the latter case will require special statistical considerations.

Characteristics of recombinantly expressed rat and human histamine H3 receptors.

PubMed

Wulff, Birgitte S; Hastrup, Sven; Rimvall, Karin

2002-10-18

Human and rat histamine H(3) receptors were recombinantly expressed and characterized using receptor binding and a functional cAMP assay. Seven of nine agonists had similar affinities and potencies at the rat and human histamine H(3) receptor. S-alpha-methylhistamine had a significantly higher affinity and potency at the human than rat receptor, and for 4-[(1R*,2R*)-2-(5,5-dimethyl-1-hexynyl)cyclopropyl]-1H-imidazole (Perceptin) the preference was the reverse. Only two of six antagonists had similar affinities and potencies at the human and the rat histamine H(3) receptor. Ciproxifan, thioperamide and (1R*,2R*)-trans-2-imidazol-4 ylcyclopropyl) (cyclohexylmethoxy) carboxamide (GT2394) had significantly higher affinities and potencies at the rat than at the human histamine H(3) receptor, while for N-(4-chlorobenzyl)-N-(7-pyrrolodin-1-ylheptyl)guanidine (JB98064) the preference was the reverse. All antagonists also showed potent inverse agonism properties. Iodoproxyfan, Perceptin, proxyfan and GR175737, compounds previously described as histamine H(3) receptor antagonists, acted as full or partial agonists at both the rat and the human histamine H(3) receptor. Copyright 2002 Elsevier Science B.V.

Study of the effect of thiols on the vasodilatory potency of S-nitrosothiols by using a modified aortic ring assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giustarini, Daniela, E-mail: giustarini@unisi.it; Tsikas, Dimitrios, E-mail: tsikas.dimitros@mh-hannover.de; Rossi, Ranieri, E-mail: ranieri@unisi.it

2011-10-15

Both low-molecular-mass thiols (LMM-SH) and protein thiols (P-SH) can modulate the biological activity of S-nitrosothiols (RSNO) via S-transnitrosation reactions. It has been difficult to evaluate the entity of this effect in blood circulation by in vitro assays with isolated aorta rings so far, because media rich in proteins cannot be used due to the foaming as a consequence of the needed gas bubbling. We have modified the original apparatus for organ bioassay in order to minimize foaming and to increase analytical performance. By using this modified bioassay we investigated the vasodilatory potency of various endogenous RSNOs in the presence ofmore » physiologically relevant concentrations of albumin and LMM-SH. Our results show that the sulfhydryl group of the cysteine moiety of albumin and LMM-SH has a dramatic effect on the vasodilatory potency of RSNO. Considering the equilibrium constants for S-transnitrosation reactions and the concentration of P-SH and LMM-SH we measured in healthy humans (aged 18-85 years), we infer that the age-dependency of hematic levels of LMM-SH may have a considerable impact in RSNO-mediated vasodilation. S-Nitrosoproteins such as S-nitrosoalbumin may constitute a relatively silent and constant amount of circulating RSNO. On the other hand, LMM-SH may mediate and control the biological actions of S-nitrosoproteins via S-transnitrosation reactions, by forming more potent nitric oxide-releasing LMM-S-nitrosothiols. Lifestyle habits, status of health and individual age are proven factors that, in turn, may influence the concentration of these compounds. These aspects should be taken into consideration when testing the vasodilatory effects of RSNO in pre-clinical studies. - Highlights: > A modification of the organ chamber apparatus for aortic ring bioassays is proposed. > The new apparatus can work in the presence of albumin at physiological concentrations. > Potency of RSNOs was studied in the presence of albumin and low molecular mass -SH. > Plasma thiol levels decrease with age. > Potency of RSNOs varies in dependence of age and more in general of plasma thiol status.« less

Antimicrobial Cream Formulated with Supercritical Carbon Dioxide Extract of Tuberose Flowers Arrests Growth of Staphylococcus aureus.

PubMed

Ghosh, Probir Kumar; Bhattacharjee, Paramita; Das, Satadal

2016-01-01

Antimicrobial potency of herbal extracts is well known. The review of patents and research articles revealed that several herbal extracts have been employed in the formulation of topical products such as creams, exclusive of the cream reported in the present study. 0ur previous study has established antimicrobial potency of supercritical carbon dioxide extracts of tuberose flowers, better known for its sweet fragrance. The present work focuses on formulating a topical antimicrobial herbal cream with methyl eugenol (principal antimicrobial compound) rich - supercritical carbon dioxide extract of tuberose flowers, having good combination of phytochemical and antimicrobial potencies. Supercritical carbon dioxide parameters such as temperature, pressure and time were optimized using full factorial experimental design to obtain methyl eugenol-rich extracts. A cream was formulated using the extract having the best combination of phytochemical and antimicrobial potencies and was assayed further for in vitro antimicrobial potency; physiochemical and sensory properties. Two commercial antimicrobial cream samples were used as reference samples in the study. The extract obtained at 40°C, 10 MPa, 135 min at 1 L min-1 flow rate of gaseous C02 showed the best combination of phytochemical and antimicrobial potencies and was used for formulation of herbal creams. The cream formulated with 5% w/w of extract arrested growth of the common human skin pathogen Staphylococcus aureus and showed stable physiochemical properties and high sensory appeal for a year. The cream could be considered as a 'finished herbal product&' in compliance with the World Health 0rganization guidelines.

A FETAL RAT TESTIS ENDOCRINE AND GENOMIC "SIGNATURE"ACCURATELY PREDICTS THE PHTHALATE SYNDROME OF MALFORMATIONS.

EPA Science Inventory

ABSTRACT BODY: Phthalate esters (PE) vary greatly in their potency to induce malformations during sexual differentiation in the male rat. Since in vitro assay batteries are currently unable to generate useful information on the potential of chemicals within this class to disrupt ...

40 CFR 79.68 - Salmonella typhimurium reverse mutation assay.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Potency of Extracts of Diesel and Related Environmental Emissions: Study Design, Sample Generation... the present time, TA1535, TA1537, TA98, and TA100 are designated as tester strains. The fifth strain... this study shall be in accordance with good laboratory practice provisions under § 79.60. (1) Direct...

40 CFR 79.68 - Salmonella typhimurium reverse mutation assay.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Potency of Extracts of Diesel and Related Environmental Emissions: Study Design, Sample Generation... the present time, TA1535, TA1537, TA98, and TA100 are designated as tester strains. The fifth strain... this study shall be in accordance with good laboratory practice provisions under § 79.60. (1) Direct...

40 CFR 79.68 - Salmonella typhimurium reverse mutation assay.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Potency of Extracts of Diesel and Related Environmental Emissions: Study Design, Sample Generation... the present time, TA1535, TA1537, TA98, and TA100 are designated as tester strains. The fifth strain... this study shall be in accordance with good laboratory practice provisions under § 79.60. (1) Direct...

40 CFR 79.68 - Salmonella typhimurium reverse mutation assay.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Potency of Extracts of Diesel and Related Environmental Emissions: Study Design, Sample Generation... the present time, TA1535, TA1537, TA98, and TA100 are designated as tester strains. The fifth strain... this study shall be in accordance with good laboratory practice provisions under § 79.60. (1) Direct...

40 CFR 79.68 - Salmonella typhimurium reverse mutation assay.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Potency of Extracts of Diesel and Related Environmental Emissions: Study Design, Sample Generation... the present time, TA1535, TA1537, TA98, and TA100 are designated as tester strains. The fifth strain... this study shall be in accordance with good laboratory practice provisions under § 79.60. (1) Direct...

Evaluation of the performance of the reduced local lymph node assay for skin sensitization testing.

PubMed

Ezendam, Janine; Muller, Andre; Hakkert, Betty C; van Loveren, Henk

2013-06-01

The local lymph node assay (LLNA) is the preferred method for classification of sensitizers within REACH. To reduce the number of mice for the identification of sensitizers the reduced LLNA was proposed, which uses only the high dose group of the LLNA. To evaluate the performance of this method for classification, LLNA data from REACH registrations were used and classification based on all dose groups was compared to classification based on the high dose group. We confirmed previous examinations of the reduced LLNA showing that this method is less sensitive compared to the LLNA. The reduced LLNA misclassified 3.3% of the sensitizers identified in the LLNA and misclassification occurred in all potency classes and that there was no clear association with irritant properties. It is therefore not possible to predict beforehand which substances might be misclassified. Another limitation of the reduced LLNA is that skin sensitizing potency cannot be assessed. For these reasons, it is not recommended to use the reduced LLNA as a stand-alone assay for skin sensitization testing within REACH. In the future, the reduced LLNA might be of added value in a weight of evidence approach to confirm negative results obtained with non-animal approaches. Copyright © 2013 Elsevier Inc. All rights reserved.

DEVELOPMENT OF A COMPARATIVE POTENCY METHOD FOR CANCER RISK ASSESSMENT OF COMPLEX MIXTURES USING SHORT-TERM 'IN VIVO' AND 'IN VITRO' BIOASSAYS

EPA Science Inventory

A comparative potency method for cancer risk assessment has been developed based upon a constant relative potency hypothesis. This method was developed and tested using data from a battery of short-term mutagenesis bioassays, animal tumorigenicity data and human lung cancer risk ...

The discovery of a novel antagonist - Manduca sexta allatotropin analogue - as an insect midgut active ion transport inhibitor.

PubMed

Deng, Xi-le; Kai, Zhen-Peng; Chamberlin, Mary E; Horodyski, Frank M; Yang, Xin-Ling

2016-11-01

The midgut is an important site for both nutrient absorption and ionic regulation in lepidopteran larvae, major pests in agriculture. The larval lepidopteran midgut has become a potent insecticide target over the past few decades. Recent studies have shown that an insect neuropeptide, Manduca sexta allatotropin (Manse-AT), exhibits inhibition of active ion transport (AIT) across the larval midgut epithelium. The full characteristic of the AIT inhibition capacity of Manse-AT is essential to assay. In this study, AIT inhibition across the M. sexta midgut by Manse-AT and its analogues in a range of concentrations was assayed. The structure-activity relationship of Manse-AT was also studied by truncated and alanine-replacement strategies. Our results identified three residues, Thr4, Arg6 and Phe8, as the most important components for activity on the midgut. Replacement of Glu1, Met2 and Met3 reduced the potency of the analogues. The conservative substitution of Gly7 with alanine had little effect on the potency of the analogues. We demonstrated for the first time that Manse-AT (10-13) behaves as a potent antagonist in vitro on active ion transport across the epithelium of the posterior midgut in M. sexta. Structure-activity studies of Manse-AT are useful in developing lead compounds for the design and testing of synthetic antagonists, ultimately to develop potent and specific pest control strategies. Manse-AT (10-13) has been discovered as the first Manse-AT antagonist, with a significant effect and a short sequence compared with other insect neuropeptides. It may be a new potential pest control agent in the future. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Predicting QT prolongation in humans during early drug development using hERG inhibition and an anaesthetized guinea-pig model

PubMed Central

Yao, X; Anderson, D L; Ross, S A; Lang, D G; Desai, B Z; Cooper, D C; Wheelan, P; McIntyre, M S; Bergquist, M L; MacKenzie, K I; Becherer, J D; Hashim, M A

2008-01-01

Background and purpose: Drug-induced prolongation of the QT interval can lead to torsade de pointes, a life-threatening ventricular arrhythmia. Finding appropriate assays from among the plethora of options available to predict reliably this serious adverse effect in humans remains a challenging issue for the discovery and development of drugs. The purpose of the present study was to develop and verify a reliable and relatively simple approach for assessing, during preclinical development, the propensity of drugs to prolong the QT interval in humans. Experimental approach: Sixteen marketed drugs from various pharmacological classes with a known incidence—or lack thereof—of QT prolongation in humans were examined in hERG (human ether a-go-go-related gene) patch-clamp assay and an anaesthetized guinea-pig assay for QT prolongation using specific protocols. Drug concentrations in perfusates from hERG assays and plasma samples from guinea-pigs were determined using liquid chromatography-mass spectrometry. Key results: Various pharmacological agents that inhibit hERG currents prolong the QT interval in anaesthetized guinea-pigs in a manner similar to that seen in humans and at comparable drug exposures. Several compounds not associated with QT prolongation in humans failed to prolong the QT interval in this model. Conclusions and implications: Analysis of hERG inhibitory potency in conjunction with drug exposures and QT interval measurements in anaesthetized guinea-pigs can reliably predict, during preclinical drug development, the risk of human QT prolongation. A strategy is proposed for mitigating the risk of QT prolongation of new chemical entities during early lead optimization. PMID:18587422

A three-stage experimental strategy to evaluate and validate an interplate IC50 format.

PubMed

Rodrigues, Daniel J; Lyons, Richard; Laflin, Philip; Pointon, Wayne; Kammonen, Juha

2007-12-01

The serial dilution of compounds to establish potency against target enzymes or receptors can at times be a rate-limiting step in project progression. We have investigated the possibility of running 50% inhibitory concentration experiments in an interplate format, with dose ranges constructed across plates. The advantages associated with this format include a faster reformatting time for the compounds while also increasing the number of doses that can be potentially generated. These two factors, in particular, would lend themselves to a higher-throughput and more timely testing of compounds, while also maximizing chances to capture fully developed dose-response curves. The key objective from this work was to establish a strategy to assess the feasibility of an interplate format to ensure that the quality of data generated would be equivalent to historical formats used. A three-stage approach was adopted to assess and validate running an assay in an interplate format, compared to an intraplate format. Although the three-stage strategy was tested with two different assay formats, it would be necessary to investigate the feasibility for other assay types. The recommendation is that the three-stage experimental strategy defined here is used to assess feasibility of other assay formats used.

Influence of the ionic liquid 1-butyl-3-methylimidazolium bromide on amyloid fibrillogenesis in lysozyme: Evidence from photophysical and imaging studies.

PubMed

Basu, Anirban; Bhattacharya, Subhash Chandra; Kumar, Gopinatha Suresh

2018-02-01

Many proteins can abnormally fold to form pathological amyloid deposits/aggregates that are responsible for various degenerative disorders called amyloidosis. Here we have examined the anti-amyloidogenic potency of an ionic liquid, 1-butyl-3-methylimidazolium bromide, using lysozyme as a model system. Thioflavin T fluorescence assay demonstrated that the ionic liquid suppressed the formation of lysozyme fibrils significantly. This observation was further confirmed by the Congo red assay. Fluorescence microscopy, intrinsic fluorescence studies, nile red fluorescence assay, ANS binding assay and circular dichroism studies also testified diminishing of the fibrillogenesis in the presence of ionic liquid. Formation of amyloid fibrils was also characterized by α to β conformational transition. From far-UV circular dichroism studies it was observed that the β-sheet content of the lysozyme samples decreased in the presence of the ionic liquid which in turn implied that fibrillogenesis was supressed by the ionic liquid. Atomic force microscopy imaging unequivocally established that the ionic liquid attenuated fibrillogenesis in lysozyme. These results may be useful for the development of more effective therapeutics for amyloidosis. Copyright © 2017 Elsevier B.V. All rights reserved.

A microtitre-based method for measuring the haem polymerization inhibitory activity (HPIA) of antimalarial drugs.

PubMed

Basilico, N; Pagani, E; Monti, D; Olliaro, P; Taramelli, D

1998-07-01

The malaria parasite metabolizes haemoglobin and detoxifies the resulting haem by polymerizing it to form haemozoin (malaria pigment). A polymer identical to haemozoin, beta-haematin, can be obtained in vitro from haematin at acidic pH. Quinoline-containing anti-malarials (e.g. chloroquine) inhibit the formation of either polymer. Haem polymerization is an essential and unique pharmacological target. To identify molecules with haem polymerization inhibitory activity (HPIA) and quantify their potency, we developed a simple, inexpensive, quantitative in-vitro spectrophotometric microassay of haem polymerization. The assay uses 96-well U-bottomed polystyrene microplates and requires 24 h and a microplate reader. The relative amounts of polymerized and unpolymerized haematin are determined, based on solubility in DMSO, by measuring absorbance at 405 nm in the presence of test compounds as compared with untreated controls. The final product (a solid precipitate of polymerized haematin) was validated using infrared spectroscopy and the assay proved reproducible; in this assay, activity could be partly predicted based on the compound's chemical structure. Both water-soluble and water-insoluble compounds can be quantified by this method. Although the throughput of this assay is lower than that of radiometric methods, the assay is easier to set up and cheaper, and avoids the problems related to radioactive waste disposal.

Leveraging the contribution of thermodynamics in drug discovery with the help of fluorescence-based thermal shift assays.

PubMed

Hau, Jean Christophe; Fontana, Patrizia; Zimmermann, Catherine; De Pover, Alain; Erdmann, Dirk; Chène, Patrick

2011-06-01

The development of new drugs with better pharmacological and safety properties mandates the optimization of several parameters. Today, potency is often used as the sole biochemical parameter to identify and select new molecules. Surprisingly, thermodynamics, which is at the core of any interaction, is rarely used in drug discovery, even though it has been suggested that the selection of scaffolds according to thermodynamic criteria may be a valuable strategy. This poor integration of thermodynamics in drug discovery might be due to difficulties in implementing calorimetry experiments despite recent technological progress in this area. In this report, the authors show that fluorescence-based thermal shift assays could be used as prescreening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery.

Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15.

PubMed

Choi, Heejun; Yang, Zhilin; Weisshaar, James C

2015-01-20

Antibiotics target specific biochemical mechanisms in bacteria. In response to new drugs, pathogenic bacteria rapidly develop resistance. In contrast, antimicrobial peptides (AMPs) have retained broad spectrum antibacterial potency over millions of years. We present single-cell fluorescence assays that detect reactive oxygen species (ROS) in the Escherichia coli cytoplasm in real time. Within 30 s of permeabilization of the cytoplasmic membrane by the cationic AMP CM15 [combining residues 1-7 of cecropin A (from moth) with residues 2-9 of melittin (bee venom)], three fluorescence signals report oxidative stress in the cytoplasm, apparently involving O2 (-), H2O2, and •OH. Mechanistic studies indicate that active respiration is a prerequisite to the CM15-induced oxidative damage. In anaerobic conditions, signals from ROS are greatly diminished and the minimum inhibitory concentration increases 20-fold. Evidently the natural human AMP LL-37 also induces a burst of ROS. Oxidative stress may prove a significant bacteriostatic mechanism for a variety of cationic AMPs. If so, host organisms may use the local oxygen level to modulate AMP potency.

Quantitative determinations using portable Raman spectroscopy.

PubMed

Navin, Chelliah V; Tondepu, Chaitanya; Toth, Roxana; Lawson, Latevi S; Rodriguez, Jason D

2017-03-20

A portable Raman spectrometer was used to develop chemometric models to determine percent (%) drug release and potency for 500mg ciprofloxacin HCl tablets. Parallel dissolution and chromatographic experiments were conducted alongside Raman experiments to assess and compare the performance and capabilities of portable Raman instruments in determining critical drug attributes. All batches tested passed the 30min dissolution specification and the Raman model for drug release was able to essentially reproduce the dissolution profiles obtained by ultraviolet spectroscopy at 276nm for all five batches of the 500mg ciprofloxacin tablets. The five batches of 500mg ciprofloxacin tablets also passed the potency (assay) specification and the % label claim for the entire set of tablets run were nearly identical, 99.4±5.1 for the portable Raman method and 99.2±1.2 for the chromatographic method. The results indicate that portable Raman spectrometers can be used to perform quantitative analysis of critical product attributes of finished drug products. The findings of this study indicate that portable Raman may have applications in the areas of process analytical technology and rapid pharmaceutical surveillance. Published by Elsevier B.V.

Quality requirements for allergen extracts and allergoids for allergen immunotherapy.

PubMed

Zimmer, J; Bonertz, A; Vieths, S

2017-12-01

All allergen products for allergen immunotherapy currently marketed in the European Union are pharmaceutical preparations derived from allergen-containing source materials like pollens, mites and moulds. Especially this natural origin results in particular demands for the regulatory requirements governing allergen products. Furthermore, the development of regulatory requirements is complicated by the so far missing universal link between certain quality parameters, in particular biological potency, on the one hand and clinical efficacy on the other hand. As a consequence, each allergen product for specific immunotherapy has to be assessed individually for its quality, safety and efficacy. At the same time, biological potency of allergen products is most commonly determined using IgE inhibition assays based on human sera relative to product-specific in house references, ruling out full comparability of products from different manufacturers. This review article aims to summarize the current quality requirements for allergen products including the special requirements implemented for control of chemically modified allergen extracts (allergoids). Copyright © 2017 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.

Structure-Based Design and Optimization of Multitarget-Directed 2H-Chromen-2-one Derivatives as Potent Inhibitors of Monoamine Oxidase B and Cholinesterases.

PubMed

Farina, Roberta; Pisani, Leonardo; Catto, Marco; Nicolotti, Orazio; Gadaleta, Domenico; Denora, Nunzio; Soto-Otero, Ramon; Mendez-Alvarez, Estefania; Passos, Carolina S; Muncipinto, Giovanni; Altomare, Cosimo D; Nurisso, Alessandra; Carrupt, Pierre-Alain; Carotti, Angelo

2015-07-23

The multifactorial nature of Alzheimer's disease calls for the development of multitarget agents addressing key pathogenic processes. To this end, by following a docking-assisted hybridization strategy, a number of aminocoumarins were designed, prepared, and tested as monoamine oxidases (MAOs) and acetyl- and butyryl-cholinesterase (AChE and BChE) inhibitors. Highly flexible N-benzyl-N-alkyloxy coumarins 2-12 showed good inhibitory activities at MAO-B, AChE, and BChE but low selectivity. More rigid inhibitors, bearing meta- and para-xylyl linkers, displayed good inhibitory activities and high MAO-B selectivity. Compounds 21, 24, 37, and 39, the last two featuring an improved hydrophilic/lipophilic balance, exhibited excellent activity profiles with nanomolar inhibitory potency toward hMAO-B, high hMAO-B over hMAO-A selectivity and submicromolar potency at hAChE. Cell-based assays of BBB permeation, neurotoxicity, and neuroprotection supported the potential of compound 37 as a BBB-permeant neuroprotective agent against H2O2-induced oxidative stress with poor interaction as P-gp substrate and very low cytotoxicity.

Establishment of hepatitis A detection antibodies set BRR batch 3 for antigen content determination by ELISA.

PubMed

Morgeaux, S; Manniam, I; Variot, P; Daas, A; Costanzo, A

2015-01-01

The current batch of the European Pharmacopoeia (Ph. Eur.) Biological Reference Reagents (BRRs) used for the in vitro potency assay of hepatitis A vaccines (HAV) by ELISA (enzymelinked immunosorbent assay) was established in 2012 for use in conjunction with Ph. Eur. general chapter 2.7.14 Assay of hepatitis A vaccine. It is composed of a coating reagent and a set of detection antibodies. As stocks of the latter are running low, the European Directorate for the Quality of Medicines & HealthCare (EDQM) organised a collaborative study to qualify replacement batches. The candidate BRR antibodies (primary monoclonal antibody and labelled secondary antibody) were prepared under appropriate conditions from starting materials similar to those used for the current batches. The new batches of antibodies were tested alongside previous batches of BRRs to ensure continuity, and the results confirmed that they were suitable for use in the potency assay of hepatitis A vaccines by ELISA using the standard method referenced in Ph. Eur. general chapter 2.7.14 at the same final concentrations as the previous batches, i.e. 1:500 for the primary monoclonal antibody and 1:400 for the secondary conjugated antibody. The outcome of the study allowed their establishment by the Ph. Eur. Commission in March 2015 as anti-hepatitis A virus primary detection antibody BRR batch 3 and conjugated secondary detection antibody BRR batch 3 respectively. They are available from the EDQM as hepatitis A vaccine ELISA detection antibodies set BRR batch 3.

Comparative cytotoxic and genotoxic potential of 13 drinking water disinfection by-products using a microplate-based cytotoxicity assay and a developed SOS/umu assay.

PubMed

Zhang, Shao-Hui; Miao, Dong-Yue; Tan, Li; Liu, Ai-Lin; Lu, Wen-Qing

2016-01-01

The implications of disinfection by-products (DBPs) present in drinking water are of public health concern because of their potential mutagenic, carcinogenic and other toxic effects on humans. In this study, we selected 13 main DBPs found in drinking water to quantitatively analyse their cytotoxicity and genotoxicity using a microplate-based cytotoxicity assay and a developed SOS/umu assay in Salmonella typhimurium TA1535/pSK1002. With the developed SOS/umu test, eight DBPs: 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-fura3-chloro-4-(dichloromethyl)-5-hydroxy-2-[5H]-furanone (MX), dibromoacetonitrile (DBN), iodoacetic acid (IA), bromochloroacetonitrile (BCN), bromoacetic acid (BA), trichloroacetonitrile (TCN), dibromoacetic acid (DBA) and dichloroacetic acid (DCA) were significantly genotoxic to S. typhimurium. Three DBPs: chloroacetic acid (CA), trichloroacetic acid (TCA) and dichloroacetonitrile (DCN) were weakly genotoxic, whereas the remaining DBPs: chloroacetonitrile (CN) and chloral hydrate (CH) were negative. The rank order in decreasing genotoxicity was as follows: MX > DBN > IA > BCN > BA > TCN > DBA > DCA > CA, TCA, DCN > CN, CH. MX was approximately 370 000 times more genotoxic than DCA. In the microplate-based cytotoxicity assay, cytotoxic potencies of the 13 DBPs were compared and ranked in decreasing order as follows: MX > IA > DBN > BCN > BA > TCN > DCN > CA > DCA > DBA > CN > TCA > CH. MX was approximately 19 200 times more cytotoxic than CH. A statistically significant correlation was found between cytotoxicity and genotoxicity of the 13 DBPs in S. typhimurium. Results suggest that microplate-based cytotoxicity assay and the developed SOS/umu assay are feasible tools for analysing the cytotoxicity and genotoxicity of DBPs, particularly for comparing their toxic intensities quantitatively. © The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Synthesis and functional characterization of novel derivatives related to oxotremorine and oxotremorine-M.

PubMed

Dallanoce, C; Conti, P; De Amici, M; De Micheli, C; Barocelli, E; Chiavarini, M; Ballabeni, V; Bertoni, S; Impicciatore, M

1999-08-01

Two subseries of nonquaternized (5a-10a) and quaternized derivatives (5b-10b) related to oxotremorine and oxotremorine-M were synthesized and tested. The agonist potency at the muscarinic receptor subtypes of the new compounds was estimated in three classical in vitro functional assays: M1 rabbit vas deferens, M2 guinea pig left atrium and M3 guinea pig ileum. In addition, the occurrence of central muscarinic effects was evaluated as tremorigenic activity after intraperitoneal administration in mice. In in vitro tests a nonselective muscarinic activity was exhibited by all the derivatives with potencies values that, in some instances, surpassed those of the reference compounds (i.e. 8b). Functional selectivity was evidenced only for the oxotremorine-like derivative 9a, which behaved as a mixed M3-agonist/M1-antagonist (pD2 = 5.85; pA2 = 4.76, respectively). In in vivo tests non-quaternary compounds were able to evoke central muscarinic effects, with a potency order parallel to that observed in vitro.

Development and Optimization of a High-Throughput Assay To Measure Neutralizing Antibodies against Clostridium difficile Binary Toxin

PubMed Central

Xie, Jinfu; Horton, Melanie; Zorman, Julie; Antonello, Joseph M.; Zhang, Yuhua; Arnold, Beth A.; Secore, Susan; Xoconostle, Rachel; Miezeiewski, Matthew; Wang, Su; Price, Colleen E.; Thiriot, David; Goerke, Aaron; Gentile, Marie-Pierre; Skinner, Julie M.

2014-01-01

Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates. PMID:24623624

Screening of the MMV and GSK open access chemical boxes using a viability assay developed against the kinetoplastid Crithidia fasciculata.

PubMed

Kipandula, Wakisa; Young, Simon A; MacNeill, Stuart A; Smith, Terry K

2018-06-01

Diseases caused by the pathogenic kinetoplastids continue to incapacitate and kill hundreds of thousands of people annually throughout the tropics and sub-tropics. Unfortunately, in the countries where these neglected diseases occur, financial obstacles to drug discovery and technical limitations associated with biochemical studies impede the development of new, safe, easy to administer and effective drugs. Here we report the development and optimisation of a Crithidia fasciculata resazurin viability assay, which is subsequently used for screening and identification of anti-crithidial compounds in the MMV and GSK open access chemical boxes. The screening assay had an average Z' factor of 0.7 and tolerated a maximum dimethyl sulfoxide concentration of up to 0.5%. We identified from multiple chemical boxes two compound series exhibiting nanomolar potency against C. fasciculata, one centred around a 5-nitrofuran-2-yl scaffold, a well-known moiety in several existing anti-infectives, and another involving a 2-(pyridin-2-yl) pyrimidin-4-amine scaffold which seems to have pan-kinetoplastid activity. This work facilitates the future use of C. fasciculata as a non-pathogenic and inexpensive biological resource to identify mode of action/protein target(s) of potentially pan-trypanocidal potent compounds. This knowledge will aid in the development of new treatments for African sleeping sickness, Chagas disease and leishmaniasis. Copyright © 2018 Elsevier B.V. All rights reserved.

Small molecule antagonists of the urokinase (uPA): urokinase receptor (uPAR) interaction with high reported potencies show only weak effects in cell-based competition assays employing the native uPAR ligand.

PubMed

De Souza, Melissa; Matthews, Hayden; Lee, Jodi A; Ranson, Marie; Kelso, Michael J

2011-04-15

Binding of the urokinase-type plasminogen activator (uPA) to its cell-surface-bound receptor uPAR and upregulation of the plasminogen activation system (PAS) correlates with increased metastasis and poor prognosis in several tumour types. Disruptors of the uPA:uPAR interaction represent promising anti-tumour/metastasis agents and several approaches have been explored for this purpose, including the use of small molecule antagonists. Two highly potent non-peptidic antagonists 1 and 2 (IC(50)1=0.8 nM, IC(50)2=33 nM) from the patent literature were reportedly identified using competition assays employing radiolabelled uPAR-binding uPA fragments and appeared as useful pharmacological tools for studying the PAS. Before proceeding to such studies, confirmation was sought that 1 and 2 retained their potencies in physiologically relevant cell-based competition assays employing uPAR's native binding partner high molecular weight uPA (HMW-uPA). This study describes a new solution phase synthesis of 1, a mixed solid/solution phase synthesis of 2 and reports the activities of 1 and 2 in semi-quantitative competition flow cytometry assays and quantitative cell-based uPA activity assays that employed HMW-uPA as the competing ligand. The flow cytometry experiments revealed that high concentrations of 2 (10-100 μM) are required to compete with HMW-uPA for uPAR binding and that 1 shows no antagonist effects at 100 μM. The cell-based enzyme activity assays similarly revealed that 1 and 2 are poor inhibitors of cell surface-bound HMW-uPA activity (IC(50) >100 μM for 1 and 2). The report highlights the dangers of identifying false-positive lead uPAR antagonists from competition assays employing labelled competing ligands other than the native HMW-uPA. Copyright © 2011 Elsevier Ltd. All rights reserved.

Lyophilisation of influenza, rabies and Marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation -assay-based diagnostic kit.

PubMed

Mather, Stuart T; Wright, Edward; Scott, Simon D; Temperton, Nigel J

2014-12-15

Pseudotype viruses (PVs) are chimeric, replication-deficient virions that mimic wild-type virus entry mechanisms and can be safely employed in neutralisation assays, bypassing the need for high biosafety requirements and performing comparably to established serological assays. However, PV supernatant necessitates -80°C long-term storage and cold-chain maintenance during transport, which limits the scope of dissemination and application throughout resource-limited laboratories. We therefore investigated the effects of lyophilisation on influenza, rabies and Marburg PV stability, with a view to developing a pseudotype virus neutralisation assay (PVNA) based kit suitable for affordable global distribution. Infectivity of each PV was calculated after lyophilisation and immediate reconstitution, as well as subsequent to incubation of freeze-dried pellets at varying temperatures, humidities and timepoints. Integrity of glycoprotein structure following treatment was also assessed by employing lyophilised PVs in downstream PVNAs. In the presence of 0.5M sucrose-PBS cryoprotectant, each freeze-dried pseudotype was stably stored for 4 weeks at up to 37°C and could be neutralised to the same potency as unlyophilised PVs when employed in PVNAs. These results confirm the viability of a freeze-dried PVNA-based kit, which could significantly facilitate low-cost serology for a wide portfolio of emerging infectious viruses. Copyright © 2014 Elsevier B.V. All rights reserved.

Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches

PubMed Central

Ekland, Eric H.; Schneider, Jessica; Fidock, David A.

2011-01-01

Malarial parasites have evolved resistance to all previously used therapies, and recent evidence suggests emerging resistance to the first-line artemisinins. To identify antimalarials with novel mechanisms of action, we have developed a high-throughput screen targeting the apicoplast organelle of Plasmodium falciparum. Antibiotics known to interfere with this organelle, such as azithromycin, exhibit an unusual phenotype whereby the progeny of drug-treated parasites die. Our screen exploits this phenomenon by assaying for “delayed death” compounds that exhibit a higher potency after two cycles of intraerythrocytic development compared to one. We report a primary assay employing parasites with an integrated copy of a firefly luciferase reporter gene and a secondary flow cytometry-based assay using a nucleic acid stain paired with a mitochondrial vital dye. Screening of the U.S. National Institutes of Health Clinical Collection identified known and novel antimalarials including kitasamycin. This inexpensive macrolide, used for agricultural applications, exhibited an in vitro IC50 in the 50 nM range, comparable to the 30 nM activity of our control drug, azithromycin. Imaging and pharmacologic studies confirmed kitasamycin action against the apicoplast, and in vivo activity was observed in a murine malaria model. These assays provide the foundation for high-throughput campaigns to identify novel chemotypes for combination therapies to treat multidrug-resistant malaria.—Ekland, E. H., Schneider, J., Fidock, D. A. Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches. PMID:21746861

Development of an in vitro skin sensitization test based on ROS production in THP-1 cells.

PubMed

Saito, Kazutoshi; Miyazawa, Masaaki; Nukada, Yuko; Sakaguchi, Hitoshi; Nishiyama, Naohiro

2013-03-01

Recently, it has been reported that reactive oxygen species (ROS) produced by contact allergens can affect dendritic cell migration and contact hypersensitivity. The aim of the present study was to develop a new in vitro assay that could predict the skin sensitizing potential of chemicals by measuring ROS production in THP-1 (human monocytic leukemia cell line) cells. THP-1 cells were pre-loaded with a ROS sensitive fluorescent dye, 5-(and 6-)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), for 15min, then incubated with test chemicals for 30min. The fluorescence intensity was measured by flow cytometry. For the skin sensitizers, 25 out of 30 induced over a 2-fold ROS production at more than 90% of cell viability. In contrast, increases were only seen in 4 out of 20 non-sensitizers. The overall accuracy for the local lymph node assay (LLNA) was 82% for 50 chemicals tested. A correlation was found between the estimated concentration showing 2-fold ROS production in the ROS assay and the EC3 values (estimated concentration required to induce positive response) of the LLNA. These results indicated that the THP-1 cell-based ROS assay was a rapid and highly sensitive detection system able to predict skin sensitizing potentials and potency of chemicals. Copyright © 2012 Elsevier Ltd. All rights reserved.

76 FR 45254 - Report and Recommendations on the Usefulness and Limitations of the Murine Local Lymph Node Assay...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-28

... Contact Dermatitis in Humans AGENCY: Division of the National Toxicology Program (DNTP), National... categorizing the potency of substances with the potential to cause allergic contact dermatitis (ACD) as strong... Contact Dermatitis in Humans (NIH Publication No. 11-7709), describes ICCVAM's recommendations for using...

ASSESSMENT OF STANDARD REFERENCE COMPOUNDS FOR COMPARATIVE STUDIES USING THE SALMONELLA TYPHIMURIUM MUTAGENICITY ASSAY: I. WITHOUT EXOGENOUS ACTIVATION

EPA Science Inventory

Finney (1978) described a bioassay as an experiment for estimating the nature, constitution, or potency of a material by means of the eaction that follows its application to living matter. n this paper, two independent laboratories tested 10 known Salmonella mutagens in order to ...

Peroral bioassay of nucleopolyhedrosis viruses in larvae of western spruce budworm.

Treesearch

Mauro E. Martignoni; Paul J. Iwai

1981-01-01

The relative virulence of entomopathogenic viruses and the potency of virus preparations for control of destructive insects can be estimated reliably only by means of biological assay in the target species. A simple, yet sensitive peroral bioassay procedure is described for preparations of nucleopolyhedrosis viruses pathogenic for the western spruce budworm, ...

Substituted 2-benzothiazolamines as sodium flux inhibitors: quantitative structure-activity relationships and anticonvulsant activity.

PubMed

Hays, S J; Rice, M J; Ortwine, D F; Johnson, G; Schwarz, R D; Boyd, D K; Copeland, L F; Vartanian, M G; Boxer, P A

1994-10-01

Thirty-two aryl-substituted 2-benzothiazolamines have been tested for their ability to modulate sodium flux in rat cortical slices. A QSAR analysis, applied to these derivatives, showed a trend toward increasing potency as sodium flux inhibitors with increasing lipophilicity, decreasing size, and increasing electron withdrawal of the benzo ring substituents. Additionally, 4- or 5-substitution of the benzo ring was found to decrease potency. The combination of increased lipophilicity, small size, and electron withdrawal severely limited which groups were tolerated on the benzo ring, thus suggesting that the optimal substitution patterns have been prepared within this series. Nine of these compounds were potent inhibitors of veratridine-induced sodium flux (NaFl). These nine compounds also proved to be anticonvulsant in the maximal electroshock (MES) assay. Fourteen additional 2-benzothiazolamines demonstrated activity in the MES screen, yet exhibited no activity in the NaFl assay. These derivatives may be interacting at the sodium channel in a manner not discernible by the flux paradigm, or they may be acting by an alternative mechanism in vivo.

Isolation and Pharmacological Evaluation of Minor Cannabinoids from High-Potency Cannabis sativa.

PubMed

Radwan, Mohamed M; ElSohly, Mahmoud A; El-Alfy, Abir T; Ahmed, Safwat A; Slade, Desmond; Husni, Afeef S; Manly, Susan P; Wilson, Lisa; Seale, Suzanne; Cutler, Stephen J; Ross, Samir A

2015-06-26

Seven new naturally occurring hydroxylated cannabinoids (1-7), along with the known cannabiripsol (8), have been isolated from the aerial parts of high-potency Cannabis sativa. The structures of the new compounds were determined by 1D and 2D NMR spectroscopic analysis, GC-MS, and HRESIMS as 8α-hydroxy-Δ(9)-tetrahydrocannabinol (1), 8β-hydroxy-Δ(9)-tetrahydrocannabinol (2), 10α-hydroxy-Δ(8)-tetrahydrocannabinol (3), 10β-hydroxy-Δ(8)-tetrahydrocannabinol (4), 10α-hydroxy-Δ(9,11)-hexahydrocannabinol (5), 9β,10β-epoxyhexahydrocannabinol (6), and 11-acetoxy-Δ(9)-tetrahydrocannabinolic acid A (7). The binding affinity of isolated compounds 1-8, Δ(9)-tetrahydrocannabinol, and Δ(8)-tetrahydrocannabinol toward CB1 and CB2 receptors as well as their behavioral effects in a mouse tetrad assay were studied. The results indicated that compound 3, with the highest affinity to the CB1 receptors, exerted the most potent cannabimimetic-like actions in the tetrad assay, while compound 4 showed partial cannabimimetic actions. Compound 2, on the other hand, displayed a dose-dependent hypolocomotive effect only.

Translocation of arrestin induced by human A(3) adenosine receptor ligands in an engineered cell line: comparison with G protein-dependent pathways.

PubMed

Gao, Zhan-Guo; Jacobson, Kenneth A

2008-04-01

Structurally diverse ligands were studied in A(3) adenosine receptor (AR)-mediated beta-arrestin translocation in engineered CHO cells. The agonist potency and efficacy were similar, although not identical, to their G protein signaling. However, differences have also been found. MRS542, MRS1760, and other adenosine derivatives, A(3)AR antagonists in cyclic AMP assays, were partial agonists in beta-arrestin translocation, indicating possible biased agonism. The xanthine 7-riboside DBXRM, a full agonist, was only partially efficacious in beta-arrestin translocation. DBXRM was shown to induce a lesser extent of desensitization compared with IB-MECA. In kinetic studies, MRS3558, a potent and selective A(3)AR agonist, induced beta-arrestin translocation significantly faster than IB-MECA and Cl-IB-MECA. Non-nucleoside antagonists showed similar inhibitory potencies as previously reported. PTX pretreatment completely abolished ERK1/2 activation, but not arrestin translocation. Thus, lead candidates for biased agonists at the A(3)AR have been identified with this arrestin-translocation assay, which promises to be an effective tool for ligand screening.

Development of Special Biological Products

DTIC Science & Technology

1981-01-01

Rocky Mountain Spotted Fever (RMSF) 20. Continued B. Tissue Culture / ?Two production lots of FRhL-2 dnd three of MRC-5 were stabilized...104) was potency tested. J. Q Fever Vaccine Storage Stability Potency Testing Q fever vaccine (NDBR 105) was put on potency test. K. Rocky Mountain Spotted Fever (RMSF...Fever Vaccine Storage Stability Potency Testing Two lots of Q fever vaccine (NDBR 105) were put on potency test. K. Rocky Mountain Spotted Fever

Development, Optimization, and Validation of a Microplate Bioassay for Relative Potency Determination of Linezolid Using a Design Space Concept, and its Measurement Uncertainty.

PubMed

Saviano, Alessandro Morais; Francisco, Fabiane Lacerda; Ostronoff, Celina Silva; Lourenço, Felipe Rebello

2015-01-01

The aim of this study was to develop, optimize, and validate a microplate bioassay for relative potency determination of linezolid in pharmaceutical samples using quality-by-design and design space approaches. In addition, a procedure is described for estimating relative potency uncertainty based on microbiological response variability. The influence of culture media composition was studied using a factorial design and a central composite design was adopted to study the influence of inoculum proportion and triphenyltetrazolium chloride in microbial growth. The microplate bioassay was optimized regarding the responses of low, medium, and high doses of linezolid, negative and positive controls, and the slope, intercept, and correlation coefficient of dose-response curves. According to optimization results, design space ranges were established using: (a) low (1.0 μg/mL), medium (2.0 μg/mL), and high (4.0 μg/mL) doses of pharmaceutical samples and linezolid chemical reference substance; (b) Staphylococcus aureus ATCC 653 in an inoculum proportion of 10%; (c) antibiotic No. 3 culture medium pH 7.0±0.1; (d) 6 h incubation at 37.0±0.1ºC; and (e) addition of 50 μL of 0.5% (w/v) triphenyltetrazolium chloride solution. The microplate bioassay was linear (r2=0.992), specific, precise (repeatability RSD=2.3% and intermediate precision RSD=4.3%), accurate (mean recovery=101.4%), and robust. The overall measurement uncertainty was reasonable considering the increased variability inherent in microbiological response. Final uncertainty was comparable with those obtained with other microbiological assays, as well as chemical methods.

Inhibitors of Yellow Fever Virus replication based on 1,3,5-triphenyl-4,5-dihydropyrazole scaffold: Design, synthesis and antiviral evaluation.

PubMed

Fioravanti, Rossella; Desideri, Nicoletta; Carta, Antonio; Atzori, Elena Maria; Delogu, Ilenia; Collu, Gabriella; Loddo, Roberta

2017-12-01

By the antiviral screening of an in house library of pyrazoline compounds, 4-(3-(4-phenoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (5a) was identified as a promising hit compound for the development of anti- Yellow Fever Virus (YFV) agents. Structural optimization studies were focused on the development of 5a analogues which retain the potency as YFV inhibitors and show a reduced cytotoxicity. The synthesized 1-3,5-triphenyl-pyrazolines (4a-j, 5a-j, 6a-j) were evaluated in cell based assays for cytotoxicity and antiviral activity against representative viruses of two of the three genera of the Flaviviridae family, i.e.: Pestivirus (BVDV) and Flavivirus (YFV). These compounds were also tested against a large panel of different pathogenic RNA and DNA viruses. Most of the new 1-3,5-triphenyl-pyrazolines (4a-j, 5a-j, 6a-j) exhibited a specific activity against YFV, showing EC 50 values in the low micromolar range with almost a 10-fold improvement in potency compared to the reference inhibitor 6-azauridine. However, the selectivity indexes of the unsubstituted (4a-j) and the phenoxy (5a-j) analogues were generally modest due to the pronounced cytotoxicity against BHK-21 cells. Otherwise, the benzyloxy derivatives (6a-j) generally coupled high potency and selectivity. On the basis of both anti-YFV activity and selectivity index, pyrazolines 6a and 6b were chosen for time of addition experiments. The selected pyrazolines and the reference inhibitor 6-azauridine displayed maximal inhibition when added in the pretreatment or during the infection. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Design of potent substrate-analogue inhibitors of canine renin

NASA Technical Reports Server (NTRS)

Hui, K. Y.; Siragy, H. M.; Haber, E.

1992-01-01

Through a systematic study of structure-activity relationships, we designed potent renin inhibitors for use in dog models. In assays against dog plasma renin at neutral pH, we found that, as in previous studies of rat renin inhibitors, the structure at the P2 position appears to be important for potency. The substitution of Val for His at this position increases potency by one order of magnitude. At the P3 position, potency appears to depend on a hydrophobic side chain that does not necessarily have to be aromatic. Our results also support the approach of optimizing potency in a renin inhibitor by introducing a moiety that promotes aqueous solubility (an amino group) at the C-terminus of the substrate analogue. In the design of potent dog plasma renin inhibitors, the influence of the transition-state residue 4(S)-amino-3(S)-hydroxy-5-cyclohexylpentanoic acid (ACHPA)-commonly used as a substitute for the scissile-bond dipeptide to boost potency-is not obvious, and appears to be sequence dependent. The canine renin inhibitor Ac-paF-Pro-Phe-Val-statine-Leu-Phe-paF-NH2 (compound 15; IC50 of 1.7 nM against dog plasma renin at pH 7.4; statine, 4(S)-amino-3(S)-hydroxy-6-methylheptanoic acid; paF, para-aminophenylalanine) had a potent hypotensive effect when infused intravenously into conscious, sodium-depleted, normotensive dogs. Also, compound 15 concurrently inhibited plasma renin activity and had a profound diuretic effect.

Hazard assessment through hybrid in vitro / in silico approach: The case of zearalenone.

PubMed

Ehrlich, Veronika A; Dellafiora, Luca; Mollergues, Julie; Dall'Asta, Chiara; Serrant, Patrick; Marin-Kuan, Maricel; Lo Piparo, Elena; Schilter, Benoit; Cozzini, Pietro

2015-01-01

Within the framework of reduction, refinement and replacement of animal experiments, new approaches for identification and characterization of chemical hazards have been developed. Grouping and read across has been promoted as a most promising alternative approach. It uses existing toxicological information on a group of chemicals to make predictions on the toxicity of uncharacterized ones. In the present work, the feasibility of applying in vitro and in silico techniques to group chemicals for read across was studied using the food mycotoxin zearalenone (ZEN) and metabolites as a case study. ZEN and its reduced metabolites are known to act through activation of the estrogen receptor α (ERα). The ranking of their estrogenic potencies appeared highly conserved across test systems including binding, in vitro and in vivo assays. This data suggests that activation of ERα may play a role in the molecular initiating event (MIE) and be predictive of adverse effects and provides the rationale to model receptor-binding for hazard identification. The investigation of receptor-ligand interactions through docking simulation proved to accurately rank estrogenic potencies of ZEN and reduced metabolites, showing the suitability of the model to address estrogenic potency for this group of compounds. Therefore, the model was further applied to biologically uncharacterized, commercially unavailable, oxidized ZEN metabolites (6α-, 6β-, 8α-, 8β-, 13- and 15-OH-ZEN). Except for 15-OH-ZEN, the data indicate that in general, the oxidized metabolites would be considered a lower estrogenic concern than ZEN and reduced metabolites.

14-O-Methylmorphine: A Novel Selective Mu-Opioid Receptor Agonist with High Efficacy and Affinity.

PubMed

Zádor, Ferenc; Balogh, Mihály; Váradi, András; Zádori, Zoltán S; Király, Kornél; Szűcs, Edina; Varga, Bence; Lázár, Bernadette; Hosztafi, Sándor; Riba, Pál; Benyhe, Sándor; Fürst, Susanna; Al-Khrasani, Mahmoud

2017-11-05

14-O-methyl (14-O-Me) group in morphine-6-O-sulfate (M6SU) or oxymorphone has been reported to be essential for enhanced affinity, potency and antinociceptive effect of these opioids. Herein we report on the pharmacological properties (potency, affinity and efficacy) of the new compound, 14-O-methylmorphine (14-O-MeM) in in vitro. Additionally, we also investigated the antinociceptive effect of the novel compound, as well as its inhibitory action on gastrointestinal transit in in vivo. The potency and efficacy of test compound were measured by [ 35 S]GTPγS binding, isolated mouse vas deferens (MVD) and rat vas deferens (RVD) assays. The affinity of 14-O-MeM for opioid receptors was assessed by radioligand binding and MVD assays. The antinociceptive and gastrointestinal effects of the novel compound were evaluated in the rat tail-flick test and charcoal meal test, respectively. Morphine, DAMGO, Ile 5,6 deltorphin II, deltorphin II and U-69593 were used as reference compounds. 14-O-MeM showed higher efficacy (E max ) and potency (EC 50 ) than morphine in MVD, RVD or [ 35 S]GTPγS binding. In addition, 14-O-MeM compared to morphine showed higher affinity for μ-opioid receptor (MOR). In vivo, in rat tail-flick test 14-O-MeM proved to be stronger antinociceptive agent than morphine after peripheral or central administration. Additionally, both compounds inhibited the gastrointestinal peristalsis. However, when the antinociceptive and antitransit doses for each test compound are compared, 14-O-MeM proved to have slightly more favorable pharmacological profile. Our results affirm that 14-O-MeM, an opioid of high efficacy and affinity for MOR can be considered as a novel analgesic agent of potential clinical value. Copyright © 2017 Elsevier B.V. All rights reserved.

Pre-clinical development of a hydrogen peroxide-inactivated West Nile virus vaccine.

PubMed

Poore, Elizabeth A; Slifka, Dawn K; Raué, Hans-Peter; Thomas, Archana; Hammarlund, Erika; Quintel, Benjamin K; Torrey, Lindsay L; Slifka, Ariel M; Richner, Justin M; Dubois, Melissa E; Johnson, Lawrence P; Diamond, Michael S; Slifka, Mark K; Amanna, Ian J

2017-01-05

West Nile virus (WNV) is a mosquito-transmitted pathogen with a wide geographical range that can lead to long-term disability and death in some cases. Despite the public health risk posed by WNV, including an estimated 3 million infections in the United States alone, no vaccine is available for use in humans. Here, we present a scaled manufacturing approach for production of a hydrogen peroxide-inactivated whole virion WNV vaccine, termed HydroVax-001WNV. Vaccination resulted in robust virus-specific neutralizing antibody responses and protection against WNV-associated mortality in mice or viremia in rhesus macaques (RM). A GLP-compliant toxicology study performed in rats demonstrated an excellent safety profile with clinical findings limited to minor and transient irritation at the injection site. An in vitro relative potency (IVRP) assay was developed and shown to correlate with in vivo responses following forced degradation studies. Long-term in vivo potency comparisons between the intended storage condition (2-8°C) and a thermally stressed condition (40±2°C) demonstrated no loss in vaccine efficacy or protective immunity over a 6-month span of time. Together, the positive pre-clinical findings regarding immunogenicity, safety, and stability indicate that HydroVax-001WNV is a promising vaccine candidate. Copyright © 2016 Elsevier Ltd. All rights reserved.

The human skin/chick chorioallantoic membrane model accurately predicts the potency of cosmetic allergens.

PubMed

Slodownik, Dan; Grinberg, Igor; Spira, Ram M; Skornik, Yehuda; Goldstein, Ronald S

2009-04-01

The current standard method for predicting contact allergenicity is the murine local lymph node assay (LLNA). Public objection to the use of animals in testing of cosmetics makes the development of a system that does not use sentient animals highly desirable. The chorioallantoic membrane (CAM) of the chick egg has been extensively used for the growth of normal and transformed mammalian tissues. The CAM is not innervated, and embryos are sacrificed before the development of pain perception. The aim of this study was to determine whether the sensitization phase of contact dermatitis to known cosmetic allergens can be quantified using CAM-engrafted human skin and how these results compare with published EC3 data obtained with the LLNA. We studied six common molecules used in allergen testing and quantified migration of epidermal Langerhans cells (LC) as a measure of their allergic potency. All agents with known allergic potential induced statistically significant migration of LC. The data obtained correlated well with published data for these allergens generated using the LLNA test. The human-skin CAM model therefore has great potential as an inexpensive, non-radioactive, in vivo alternative to the LLNA, which does not require the use of sentient animals. In addition, this system has the advantage of testing the allergic response of human, rather than animal skin.

Evidence for Noncanonical Neurotransmitter Activation: Norepinephrine as a Dopamine D2-Like Receptor Agonist

PubMed Central

Sánchez-Soto, Marta; Bonifazi, Alessandro; Cai, Ning Sheng; Ellenberger, Michael P.; Newman, Amy Hauck

2016-01-01

The Gαi/o-coupled dopamine D2-like receptor family comprises three subtypes: the D2 receptor (D2R), with short and long isoform variants (D2SR and D2LR), D3 receptor (D3R), and D4 receptor (D4R), with several polymorphic variants. The common overlap of norepinephrine innervation and D2-like receptor expression patterns prompts the question of a possible noncanonical action by norepinephrine. In fact, previous studies have suggested that norepinephrine can functionally interact with D4R. To our knowledge, significant interactions between norepinephrine and D2R or D3R receptors have not been demonstrated. By using radioligand binding and bioluminescent resonance energy transfer (BRET) assays in transfected cells, the present study attempted a careful comparison between dopamine and norepinephrine in their possible activation of all D2-like receptors, including the two D2R isoforms and the most common D4R polymorphic variants. Functional BRET assays included activation of G proteins with all Gαi/o subunits, adenylyl cyclase inhibition, and β arrestin recruitment. Norepinephrine acted as a potent agonist for all D2-like receptor subtypes, with the general rank order of potency of D3R > D4R ≥ D2SR ≥ D2L. However, for both dopamine and norepinephrine, differences depended on the Gαi/o protein subunit involved. The most striking differences were observed with Gαi2, where the rank order of potencies for both dopamine and norepinephrine were D4R > D2SR = D2LR >> D3R. Furthermore the results do not support the existence of differences in the ability of dopamine and norepinephrine to activate different human D4R variants. The potency of norepinephrine for adrenergic α2A receptor was only about 20-fold higher compared with D3R and D4R across the three functional assays. PMID:26843180

Cytochrome P450 2C9-natural antiarthritic interactions: Evaluation of inhibition magnitude and prediction from in vitro data.

PubMed

Tan, Boon Hooi; Ahemad, Nafees; Pan, Yan; Palanisamy, Uma Devi; Othman, Iekhsan; Yiap, Beow Chin; Ong, Chin Eng

2018-04-01

Many dietary supplements are promoted to patients with osteoarthritis (OA) including the three naturally derived compounds, glucosamine, chondroitin and diacerein. Despite their wide spread use, research on interaction of these antiarthritic compounds with human hepatic cytochrome P450 (CYP) enzymes is limited. This study aimed to examine the modulatory effects of these compounds on CYP2C9, a major CYP isoform, using in vitro biochemical assay and in silico models. Utilizing valsartan hydroxylase assay as probe, all forms of glucosamine and chondroitin exhibited IC 50 values beyond 1000 μM, indicating very weak potential in inhibiting CYP2C9. In silico docking postulated no interaction with CYP2C9 for chondroitin and weak bonding for glucosamine. On the other hand, diacerein exhibited mixed-type inhibition with IC 50 value of 32.23 μM and K i value of 30.80 μM, indicating moderately weak inhibition. Diacerein's main metabolite, rhein, demonstrated the same mode of inhibition as diacerein but stronger potency, with IC 50 of 6.08 μM and K i of 1.16 μM. The docking of both compounds acquired lower CDOCKER interaction energy values, with interactions dominated by hydrogen and hydrophobic bondings. The ranking with respect to inhibition potency for the investigated compounds was generally the same in both in vitro enzyme assay and in silico modeling with order of potency being diacerein/rhein > various glucosamine/chondroitin forms. In vitro-in vivo extrapolation of inhibition kinetics (using 1 + [I]/K i ratio) demonstrated negligible potential of diacerein to cause interaction in vivo, whereas rhein was predicted to cause in vivo interaction, suggesting potential interaction risk with the CYP2C9 drug substrates. Copyright © 2018 John Wiley & Sons, Ltd.

Epoxy resin monomers with reduced skin sensitizing potency.

PubMed

O'Boyle, Niamh M; Niklasson, Ida B; Tehrani-Bagha, Ali R; Delaine, Tamara; Holmberg, Krister; Luthman, Kristina; Karlberg, Ann-Therese

2014-06-16

Epoxy resin monomers (ERMs), especially diglycidyl ethers of bisphenol A and F (DGEBA and DGEBF), are extensively used as building blocks for thermosetting polymers. However, they are known to commonly cause skin allergy. This research describes a number of alternative ERMs, designed with the aim of reducing the skin sensitizing potency while maintaining the ability to form thermosetting polymers. The compounds were designed, synthesized, and assessed for sensitizing potency using the in vivo murine local lymph node assay (LLNA). All six epoxy resin monomers had decreased sensitizing potencies compared to those of DGEBA and DGEBF. With respect to the LLNA EC3 value, the best of the alternative monomers had a value approximately 2.5 times higher than those of DGEBA and DGEBF. The diepoxides were reacted with triethylenetetramine, and the polymers formed were tested for technical applicability using thermogravimetric analysis and differential scanning calorimetry. Four out of the six alternative ERMs gave polymers with a thermal stability comparable to that obtained with DGEBA and DGEBF. The use of improved epoxy resin monomers with less skin sensitizing effects is a direct way to tackle the problem of contact allergy to epoxy resin systems, particularly in occupational settings, resulting in a reduction in the incidence of allergic contact dermatitis.

Comparative sensitizing potencies of fragrances, preservatives, and hair dyes.

PubMed

Lidén, Carola; Yazar, Kerem; Johansen, Jeanne D; Karlberg, Ann-Therese; Uter, Wolfgang; White, Ian R

2016-11-01

The local lymph node assay (LLNA) is used for assessing sensitizing potential in hazard identification and risk assessment for regulatory purposes. Sensitizing potency on the basis of the LLNA is categorized into extreme (EC3 value of ≤0.2%), strong (>0.2% to ≤2%), and moderate (>2%). To compare the sensitizing potencies of fragrance substances, preservatives, and hair dye substances, which are skin sensitizers that frequently come into contact with the skin of consumers and workers, LLNA results and EC3 values for 72 fragrance substances, 25 preservatives and 107 hair dye substances were obtained from two published compilations of LLNA data and opinions by the Scientific Committee on Consumer Safety and its predecessors. The median EC3 values of fragrances (n = 61), preservatives (n = 19) and hair dyes (n = 59) were 5.9%, 0.9%, and 1.3%, respectively. The majority of sensitizing preservatives and hair dyes are thus strong or extreme sensitizers (EC3 value of ≤2%), and fragrances are mostly moderate sensitizers. Although fragrances are typically moderate sensitizers, they are among the most frequent causes of contact allergy. This indicates that factors other than potency need to be addressed more rigorously in risk assessment and risk management. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Carbohydrate-Based Ice Recrystallization Inhibitors Increase Infectivity and Thermostability of Viral Vectors

NASA Astrophysics Data System (ADS)

Ghobadloo, Shahrokh M.; Balcerzak, Anna K.; Gargaun, Ana; Muharemagic, Darija; Mironov, Gleb G.; Capicciotti, Chantelle J.; Briard, Jennie G.; Ben, Robert N.; Berezovski, Maxim V.

2014-07-01

The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization. Viral potency after storage with all three conditions demonstrated that N-octyl-gluconamide (NOGlc) recovered the infectivity of shelf stored VV, 5.6 Log10 PFU mL-1 during 40 days, and HSV-1, 2.7 Log10 PFU mL-1 during 9 days. Carbon-linked antifreeze glycoprotein analogue ornithine-glycine-glycine-galactose (OGG-Gal) increases the recovery of VV and VSV more than 1 Log10 PFU mL-1 after 10 freeze-thaw cycles. In VSV, cryostorage with OGG-Gal maintains high infectivity and reduces temperature-induced aggregation of viral particles by 2 times that of the control. In total, OGG-Gal and NOGlc preserve virus potency during cryostorage. Remarkably, NOGlc has potential to eliminate the cold chain and permit room temperature storage of viral vectors.

Carbohydrate-Based Ice Recrystallization Inhibitors Increase Infectivity and Thermostability of Viral Vectors

PubMed Central

Ghobadloo, Shahrokh M.; Balcerzak, Anna K.; Gargaun, Ana; Muharemagic, Darija; Mironov, Gleb G.; Capicciotti, Chantelle J.; Briard, Jennie G.; Ben, Robert N.; Berezovski, Maxim V.

2014-01-01

The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization. Viral potency after storage with all three conditions demonstrated that N-octyl-gluconamide (NOGlc) recovered the infectivity of shelf stored VV, 5.6 Log10 PFU mL−1 during 40 days, and HSV-1, 2.7 Log10 PFU mL−1 during 9 days. Carbon-linked antifreeze glycoprotein analogue ornithine-glycine-glycine-galactose (OGG-Gal) increases the recovery of VV and VSV more than 1 Log10 PFU mL−1 after 10 freeze-thaw cycles. In VSV, cryostorage with OGG-Gal maintains high infectivity and reduces temperature-induced aggregation of viral particles by 2 times that of the control. In total, OGG-Gal and NOGlc preserve virus potency during cryostorage. Remarkably, NOGlc has potential to eliminate the cold chain and permit room temperature storage of viral vectors. PMID:25078058

Comparison between carcinogenicity and mutagenicity based on chemicals evaluated in the IARC monographs.

PubMed Central

Bartsch, H; Tomatis, L

1983-01-01

The qualitative relationship between carcinogenicity and mutagenicity (DNA-damaging activity), based on chemicals which are known to be or suspected of being carcinogenic to man and/or to experimental animals, is analyzed using 532 chemicals evaluated in Volumes 1-25 of the IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. About 40 compounds (industrial processes) were found to be either definitely or probably carcinogenic to man, and 130 chemicals have been adequately tested in rodents and most of them also in various short-term assays. For a comparison between the carcinogenicity of a chemical and its behavior in short-term tests, systems were selected that have a value for predicting carcinogenicity. These were divided into mutagenicity in (A) the S. typhimurium/microsome assay, (B) other submammalian systems and (C) cultured mammalian cells; (D) chromosomal abnormalities in mammalian cells; (E) DNA damage and repair; (F) cell transformation (or altered growth properties) in vitro. The following conclusions can be drawn. In the absence of studies in man, long-term animal tests are still today the only ones capable of providing evidence of the carcinogenic effect of a chemical. The development and application of an appropriate combination of short-term tests (despite current limitations) can significantly contribute to the prediction/confirmation of the carcinogenic effects of chemicals in animals/man. Confidence in positive tests results is increased when they are confirmed in multiple short-term tests using nonrepetitive end points and different activation systems. Assays to detect carcinogens which do not act via electrophiles (promoters) need to be developed. The results of a given short-term test should be interpreted in the context of other toxicological data. Increasing demand for quantitative carcinogenicity data requires further examination of whether or not there is a quantitative relationship between the potency of a carcinogen in experimental animals/man, and its genotoxic activity in short-term tests. At present, such a relationship is not sufficiently established for it to be used for the prediction of the carcinogenic potency of new compounds. PMID:6337827

Pharmacological and electrophysiological characterization of nine, single nucleotide polymorphisms of the hERG-encoded potassium channel

PubMed Central

Männikkö, R; Overend, G; Perrey, C; Gavaghan, CL; Valentin, J-P; Morten, J; Armstrong, M; Pollard, CE

2010-01-01

Background and purpose: Potencies of compounds blocking KV11.1 [human ether-ago-go-related gene (hERG)] are commonly assessed using cell lines expressing the Caucasian wild-type (WT) variant. Here we tested whether such potencies would be different for hERG single nucleotide polymorphisms (SNPs). Experimental approach: SNPs (R176W, R181Q, Del187-189, P347S, K897T, A915V, P917L, R1047L, A1116V) and a binding-site mutant (Y652A) were expressed in Tet-On CHO-K1 cells. Potencies [mean IC50; lower/upper 95% confidence limit (CL)] of 48 hERG blockers was estimated by automated electrophysiology [IonWorks™ HT (IW)]. In phase one, rapid potency comparison of each WT-SNP combination was made for each compound. In phase two, any compound-SNP combinations from phase one where the WT upper/lower CL did not overlap with those of the SNPs were re-examined. Electrophysiological WT and SNP parameters were determined using conventional electrophysiology. Key results: IW detected the expected sixfold potency decrease for propafenone in Y652A. In phase one, the WT lower/upper CL did not overlap with those of the SNPs for 77 compound-SNP combinations. In phase two, 62/77 cases no longer yielded IC50 values with non-overlapping CLs. For seven of the remaining 15 cases, there were non-overlapping CLs but in the opposite direction. For the eight compound-SNP combinations with non-overlapping CLs in the same direction as for phase 1, potencies were never more than twofold apart. The only statistically significant electrophysiological difference was the voltage dependence of activation of R1047L. Conclusion and implications: Potencies of hERG channel blockers defined using the Caucasian WT sequence, in this in vitro assay, were representative of potencies for common SNPs. This article is part of a themed section on QT safety. To view this issue visit http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2010 PMID:19673885

Development of at-line assay to monitor charge variants of MAbs during production.

PubMed

St Amand, M M; Ogunnaike, B A; Robinson, A S

2014-01-01

One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro-heterogeneity can result from post-translational modifications such as sialylation, galactosylation, C-terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on-line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired post-translational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on-line or at-line measurements of these attributes. In this work, we describe the development of an at-line assay to separate MAb charge variants in near real-time, which could ultimately be used to implement on-line quality control strategies for MAb production. The assay consists of a 2D-HPLC method with sequential in-line Protein A and WCX-10 HPLC column steps. To perform the 2D-HPLC assay at-line, the two columns steps were integrated into a single method using a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered to the 2D-HPLC for analysis. With this at-line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions. © 2013 American Institute of Chemical Engineers.

Development of an artificial neural network model for risk assessment of skin sensitization using human cell line activation test, direct peptide reactivity assay, KeratinoSens™ and in silico structure alert parameter.

PubMed

Hirota, Morihiko; Ashikaga, Takao; Kouzuki, Hirokazu

2018-04-01

It is important to predict the potential of cosmetic ingredients to cause skin sensitization, and in accordance with the European Union cosmetic directive for the replacement of animal tests, several in vitro tests based on the adverse outcome pathway have been developed for hazard identification, such as the direct peptide reactivity assay, KeratinoSens™ and the human cell line activation test. Here, we describe the development of an artificial neural network (ANN) prediction model for skin sensitization risk assessment based on the integrated testing strategy concept, using direct peptide reactivity assay, KeratinoSens™, human cell line activation test and an in silico or structure alert parameter. We first investigated the relationship between published murine local lymph node assay EC3 values, which represent skin sensitization potency, and in vitro test results using a panel of about 134 chemicals for which all the required data were available. Predictions based on ANN analysis using combinations of parameters from all three in vitro tests showed a good correlation with local lymph node assay EC3 values. However, when the ANN model was applied to a testing set of 28 chemicals that had not been included in the training set, predicted EC3s were overestimated for some chemicals. Incorporation of an additional in silico or structure alert descriptor (obtained with TIMES-M or Toxtree software) in the ANN model improved the results. Our findings suggest that the ANN model based on the integrated testing strategy concept could be useful for evaluating the skin sensitization potential. Copyright © 2017 John Wiley & Sons, Ltd.

Biarylmethoxy Nicotinamides As Novel and Specific Inhibitors of Mycobacterium tuberculosis.

PubMed

Kedari, Chaitanya Kumar; Roy Choudhury, Nilanjana; Sharma, Sreevalli; Kaur, Parvinder; Guptha, Supreeth; Panda, Manoranjan; Mukerjee, Kakoli; Ramachandran, Vasanthi; Bandodkar, Balachandra; Ramachandran, Sreekanth; Tantry, Subramanyam J

2014-05-08

A whole cell based screening effort on a focused library from corporate collection resulted in the identification of biarylmethoxy nicotinamides as novel inhibitors of M. tuberculosis (Mtu) H37Rv. The series exhibited tangible structure-activity relationships, and during hit to lead exploration, a cellular potency of 100 nM was achieved, which is an improvement of >200-fold from the starting point. The series is very specific to Mtu and noncytotoxic up to 250 μM as measured in the mammalian cell line THP-1 based cytotoxicity assay. This compound class retains its potency on several drug sensitive and single drug resistant clinical isolates, which indicate that the compounds could be acting through a novel mode of action.

Collaborative study for the establishment of the 4(th) International Standard for Streptomycin.

PubMed

Jorajuria, S; Raphalen, C; Dujardin, V; Daas, A

2015-01-01

An international collaborative study was organised to establish the 4(th) World Health Organization (WHO) International Standard (IS) for Streptomycin. Fourteen laboratories from different countries participated. Potencies of the candidate material were estimated by microbiological assays with sensitive micro-organisms. To ensure continuity between consecutive batches, the 3(rd) IS for Streptomycin was used as a reference. Based on the results of the study, the 4(th) IS for Streptomycin was adopted at the meeting of the WHO Expert Committee for Biological Standardization (ECBS) in 2015 with an assigned potency of 76 000 International Units (IU) per vial. The 4(th) IS for Streptomycin is available from the European Directorate for the Quality of Medicines & HealthCare (EDQM).

What level of estrogenic activity determined by in vitro assays in municipal waste waters can be considered as safe?

PubMed

Jarošová, Barbora; Bláha, Luděk; Giesy, John P; Hilscherová, Klára

2014-03-01

In vitro assays are broadly used tools to evaluate the estrogenic activity in Waste Water Treatment Plant (WWTP) effluents and their receiving rivers. Since potencies of individual estrogens to induce in vitro and in vivo responses can differ it is not possible to directly evaluate risks based on in vitro measures of estrogenic activity. Estrone, 17beta-estradiol, 17alfa-ethinylestradiol and to some extent, estriol have been shown to be responsible for the majority of in vitro estrogenic activity of municipal WWTP effluents. Therefore, in the present study safe concentrations of Estrogenic Equivalents (EEQs-SSE) in municipal WWTP effluents were derived based on simplified assumption that the steroid estrogens are responsible for all estrogenicity determined with particular in vitro assays. EEQs-SSEs were derived using the bioassay and testing protocol-specific in vitro potencies of steroid estrogens, in vivo predicted no effect concentration (PNECs) of these compounds, and their relative contributions to the overall estrogenicity detected in municipal WWTP effluents. EEQs-SSEs for 15 individual bioassays varied from 0.1 to 0.4ng EEQ/L. The EEQs-SSEs are supposed to be increased by use of location-specific dilution factors of WWTP effluents entering receiving rivers. They are applicable to municipal wastewater and rivers close to their discharges, but not to industrial waste waters. Copyright © 2013 Elsevier Ltd. All rights reserved.

Further experience with the local lymph node assay using standard radioactive and nonradioactive cell count measurements.

PubMed

Kolle, Susanne N; Basketter, David; Schrage, Arnhild; Gamer, Armin O; van Ravenzwaay, Bennard; Landsiedel, Robert

2012-08-01

In a previous study, the predictive capacity of a modified local lymph node assay (LLNA) based on cell counts, the LNCC, was demonstrated to be closely similar to that of the original assay. In addition, a range of substances, including some technical/commercial materials and a range of agrochemical formulations (n = 180) have also been assessed in both methods in parallel. The results in the LNCC and LLNA were generally consistent, with 86% yielding an identical classification outcome. Discordant results were associated with borderline data and were evenly distributed between the two methods. Potency information derived from each method also demonstrated good consistency (n = 101), with 93% of predictions being close. Skin irritation was observed only infrequently and was most commonly associated with positive results; it was not associated with the discordant results. Where different vehicles were used with the same test material, the effect on sensitizing activity was modest, consistent with historical data. Analysis of positive control data indicated that the LNCC and LLNA displayed similar levels of biological variation. When taken in combination with the previously published results on LLNA Performance Standard chemicals, it is concluded that the LNCC provides a viable non-radioactive alternative to the LLNA for the assessment of substances, including potency predictions, as well as for the evaluation of preparations. Copyright © 2012 John Wiley & Sons, Ltd.

ABBV-105, a selective and irreversible inhibitor of Bruton's Tyrosine Kinase (BTK), is efficacious in multiple preclinical models of inflammation.

PubMed

Goess, Christian; Harris, Christopher M; Murdock, Sara; McCarthy, Richard W; Sampson, Erik; Twomey, Rachel; Mathieu, Suzanne; Mario, Regina; Perham, Matthew; Goedken, Eric R; Long, Andrew J

2018-06-02

Bruton's Tyrosine Kinase (BTK) is a non-receptor tyrosine kinase required for intracellular signaling downstream of multiple immunoreceptors. We evaluated ABBV-105, a covalent BTK inhibitor, using in vitro and in vivo assays to determine potency, selectivity, and efficacy to validate the therapeutic potential of ABBV-105 in inflammatory disease. ABBV-105 potency and selectivity were evaluated in enzymatic and cellular assays. The impact of ABBV-105 on B cell function in vivo was assessed using mechanistic models of antibody production. Efficacy of ABBV-105 in chronic inflammatory disease was evaluated in animal models of arthritis and lupus. Measurement of BTK occupancy was employed as a target engagement biomarker. ABBV-105 irreversibly inhibits BTK, demonstrating superior kinome selectivity and is potent in B cell receptor, Fc receptor, and TLR-9-dependent cellular assays. Oral administration resulted in rapid clearance in plasma, but maintenance of BTK splenic occupancy. ABBV-105 inhibited antibody responses to thymus-independent and thymus-dependent antigens, paw swelling and bone destruction in rat collagen induced arthritis (CIA), and reduced disease in an IFNα-accelerated lupus nephritis model. BTK occupancy in disease models correlated with in vivo efficacy. ABBV-105, a selective BTK inhibitor, demonstrates compelling efficacy in pre-clinical mechanistic models of antibody production and in models of rheumatoid arthritis and lupus.

Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

PubMed

de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A

2013-12-31

Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the importance of new approaches for IFN potency determination. Copyright © 2013 Elsevier B.V. All rights reserved.

Transcriptome alterations in zebrafish embryos after exposure to environmental estrogens and anti-androgens can reveal endocrine disruption.

PubMed

Schiller, Viktoria; Wichmann, Arne; Kriehuber, Ralf; Schäfers, Christoph; Fischer, Rainer; Fenske, Martina

2013-12-01

Exposure to environmental chemicals known as endocrine disruptors (EDs) is in many cases associated with an unpredictable hazard for wildlife and human health. The identification of endocrine disruptive properties of chemicals certain to enter the aquatic environment relies on toxicity tests with fish, assessing adverse effects on reproduction and sexual development. The demand for quick, reliable ED assays favored the use of fish embryos as alternative test organisms. We investigated the application of a transcriptomics-based assay for estrogenic and anti-androgenic chemicals with zebrafish embryos. Two reference compounds, 17α-ethinylestradiol and flutamide, were tested to evaluate the effects on development and the transcriptome after 48h-exposures. Comparison of the transcriptome response with other estrogenic and anti-androgenic compounds (genistein, bisphenol A, methylparaben, linuron, prochloraz, propanil) showed commonalities and differences in regulated pathways, enabling us to classify the estrogenic and anti-androgenic potencies. This demonstrates that different mechanism of ED can be assessed already in fish embryos. Copyright © 2013 Elsevier Inc. All rights reserved.

Discovery of an Oxybenzylglycine Based Peroxisome Proliferator Activated Receptor [alpha] Selective Agonist 2-((3-((2-(4-Chlorophenyl)-5-methyloxazol-4-yl)methoxy)benzyl)(methoxycarbonyl)amino)acetic Acid (BMS-687453)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jun; Kennedy, Lawrence J.; Shi, Yan

2010-04-12

An 1,3-oxybenzylglycine based compound 2 (BMS-687453) was discovered to be a potent and selective peroxisome proliferator activated receptor (PPAR) {alpha} agonist, with an EC{sub 50} of 10 nM for human PPAR{alpha} and 410-fold selectivity vs human PPAR{gamma} in PPAR-GAL4 transactivation assays. Similar potencies and selectivity were also observed in the full length receptor co-transfection assays. Compound 2 has negligible cross-reactivity against a panel of human nuclear hormone receptors including PPAR{delta}. Compound 2 demonstrated an excellent pharmacological and safety profile in preclinical studies and thus was chosen as a development candidate for the treatment of atherosclerosis and dyslipidemia. The X-ray cocrystalmore » structures of the early lead compound 12 and compound 2 in complex with PPAR{alpha} ligand binding domain (LBD) were determined. The role of the crystal structure of compound 12 with PPAR{alpha} in the development of the SAR that ultimately resulted in the discovery of compound 2 is discussed.« less

DRP-1 is required for BH3 mimetic-mediated mitochondrial fragmentation and apoptosis

PubMed Central

Milani, Mateus; Byrne, Dominic P; Greaves, Georgia; Butterworth, Michael; Cohen, Gerald M; Eyers, Patrick A; Varadarajan, Shankar

2017-01-01

The concept of using BH3 mimetics as anticancer agents has been substantiated by the efficacy of selective drugs, such as Navitoclax and Venetoclax, in treating BCL-2-dependent haematological malignancies. However, most solid tumours depend on MCL-1 for survival, which is highly amplified in multiple cancers and a major factor determining chemoresistance. Most MCL-1 inhibitors that have been generated so far, while demonstrating early promise in vitro, fail to exhibit specificity and potency in a cellular context. To address the lack of standardised assays for benchmarking the in vitro binding of putative inhibitors before analysis of their cellular effects, we developed a rapid differential scanning fluorimetry (DSF)-based assay, and used it to screen a panel of BH3 mimetics. We next contrasted their binding signatures with their ability to induce apoptosis in a MCL-1 dependent cell line. Of all the MCL-1 inhibitors tested, only A-1210477 induced rapid, concentration-dependent apoptosis, which strongly correlated with a thermal protective effect on MCL-1 in the DSF assay. In cells that depend on both MCL-1 and BCL-XL, A-1210477 exhibited marked synergy with A-1331852, a BCL-XL specific inhibitor, to induce cell death. Despite this selectivity and potency, A-1210477 induced profound structural changes in the mitochondrial network in several cell lines that were not phenocopied following MCL-1 RNA interference or transcriptional repression, suggesting that A-1210477 induces mitochondrial fragmentation in an MCL-1-independent manner. However, A-1210477-induced mitochondrial fragmentation was dependent upon DRP-1, and silencing expression levels of DRP-1 diminished not just mitochondrial fragmentation but also BH3 mimetic-mediated apoptosis. These findings provide new insights into MCL-1 ligands, and the interplay between DRP-1 and the anti-apoptotic BCL-2 family members in the regulation of apoptosis. PMID:28079887

Rational screening of peroxisome proliferator-activated receptor-γ agonists from natural products: potential therapeutics for heart failure.

PubMed

Chen, Rui; Wan, Jing; Song, Jing; Qian, Yan; Liu, Yong; Gu, Shuiming

2017-12-01

Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. Activation of PPARγ pathway has been shown to enhance fatty acid oxidation, improve endothelial cell function, and decrease myocardial fibrosis in heart failure. Thus, the protein has been raised as an attractive target for heart failure therapy. This work attempted to discover new and potent PPARγ agonists from natural products using a synthetic strategy of computer virtual screening and transactivation reporter assay. A large library of structurally diverse, drug-like natural products was compiled, from which those with unsatisfactory pharmacokinetic profile and/or structurally redundant compounds were excluded. The binding mode of remaining candidates to PPARγ ligand-binding domain (LBD) was computationally modelled using molecular docking and their relative binding potency was ranked by an empirical scoring scheme. Consequently, eight commercially available hits with top scores were selected and their biological activity was determined using a cell-based reporter-gene assay. Four natural product compounds, namely ZINC13408172, ZINC4292805, ZINC44179 and ZINC901461, were identified to have high or moderate agonistic potency against human PPARγ with EC 50 values of 0.084, 2.1, 0.35 and 5.6 μM, respectively, which are comparable to or even better than that of the approved PPARγ full agonists pioglitazone (EC 50  =   0.16 μM) and rosiglitazone (EC 50  =   0.034 μM). Hydrophobic interactions and van der Waals contacts are the primary chemical forces to stabilize the complex architecture of PPARγ LBD domain with these agonist ligands, while few hydrogen bonds, salt bridges and/or π-π stacking at the complex interfaces confer selectivity and specificity for the domain-agonist recognition. The integrated in vitro-in silico screening strategy can be successfully applied to rational discovery of biologically active compounds. The newly identified natural products with PPARγ agonistic potency are considered as promising lead scaffolds to develop novel chemical therapeutics for heart failure.

An Assessment of the Model of Concentration Addition for Predicting the Estrogenic Activity of Chemical Mixtures in Wastewater Treatment Works Effluents

PubMed Central

Thorpe, Karen L.; Gross-Sorokin, Melanie; Johnson, Ian; Brighty, Geoff; Tyler, Charles R.

2006-01-01

The effects of simple mixtures of chemicals, with similar mechanisms of action, can be predicted using the concentration addition model (CA). The ability of this model to predict the estrogenic effects of more complex mixtures such as effluent discharges, however, has yet to be established. Effluents from 43 U.K. wastewater treatment works were analyzed for the presence of the principal estrogenic chemical contaminants, estradiol, estrone, ethinylestradiol, and nonylphenol. The measured concentrations were used to predict the estrogenic activity of each effluent, employing the model of CA, based on the relative potencies of the individual chemicals in an in vitro recombinant yeast estrogen screen (rYES) and a short-term (14-day) in vivo rainbow trout vitellogenin induction assay. Based on the measured concentrations of the four chemicals in the effluents and their relative potencies in each assay, the calculated in vitro and in vivo responses compared well and ranged between 3.5 and 87 ng/L of estradiol equivalents (E2 EQ) for the different effluents. In the rYES, however, the measured E2 EQ concentrations in the effluents ranged between 0.65 and 43 ng E2 EQ/L, and they varied against those predicted by the CA model. Deviations in the estimation of the estrogenic potency of the effluents by the CA model, compared with the measured responses in the rYES, are likely to have resulted from inaccuracies associated with the measurement of the chemicals in the extracts derived from the complex effluents. Such deviations could also result as a consequence of interactions between chemicals present in the extracts that disrupted the activation of the estrogen response elements in the rYES. E2 EQ concentrations derived from the vitellogenic response in fathead minnows exposed to a series of effluent dilutions were highly comparable with the E2 EQ concentrations derived from assessments of the estrogenic potency of these dilutions in the rYES. Together these data support the use of bioassays for determining the estrogenic potency of WwTW effluents, and they highlight the associated problems for modeling approaches that are reliant on measured concentrations of estrogenic chemicals. PMID:16818252

A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests

PubMed Central

2011-01-01

Background Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative, human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power. Results We have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation, using 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an area under the ROC curve of 0.98. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict sensitizing potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization. Conclusions A gene signature predicting sensitization, using a human cell line in vitro, has been identified. This simple and robust cell-based assay has the potential to completely replace or drastically reduce the utilization of test systems based on experimental animals. Being based on human biology, the assay is proposed to be more accurate for predicting sensitization in humans, than the traditional animal-based tests. PMID:21824406

Proarrhythmia liability assessment and the comprehensive in vitro Proarrhythmia Assay (CiPA): An industry survey on current practice.

PubMed

Authier, Simon; Pugsley, Michael K; Koerner, John E; Fermini, Bernard; Redfern, William S; Valentin, Jean-Pierre; Vargas, Hugo M; Leishman, Derek J; Correll, Krystle; Curtis, Michael J

2017-07-01

The Safety Pharmacology Society (SPS) has conducted a survey of its membership to identify industry practices related to testing considered in the Comprehensive In vitro Proarrhythmia Assay (CiPA). Survey topics included nonclinical approaches to address proarrhythmia issues, conduct of in silico studies, in vitro ion channel testing methods used, drugs used as positive controls during the conduct of cardiac ion channel studies, types of arrhythmias observed in non-clinical studies and use of the anticipated CiPA ion channel assay. In silico studies were used to evaluate effects on ventricular action potentials by only 15% of responders. In vitro assays were used mostly to assess QT prolongation (95%), cardiac Ca 2+ and Na + channel blockade (82%) and QT shortening or QRS prolongation (53%). For de-risking of candidate drugs for proarrhythmia, those assays most relevant to CiPA including cell lines stably expressing ion channels used to determine potency of drug block were most frequently used (89%) and human stem cell-derived or induced pluripotent stem cell cardiomyocytes (46%). Those in vivo assays related to general proarrhythmia derisking include ECG recording using implanted telemetry technology (88%), jacketed external telemetry (62%) and anesthetized animal models (53%). While the CiPA initiative was supported by 92% of responders, there may be some disconnect between current practice and future expectations, as explained. Proarrhythmia liability assessment in drug development presently includes study types consistent with CiPA. It is anticipated that CiPA will develop into a workable solution to the concern that proarrhythmia liability testing remains suboptimal. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Structure-based lead optimization to improve antiviral potency and ADMET properties of phenyl-1H-pyrrole-carboxamide entry inhibitors targeted to HIV-1 gp120.

PubMed

Curreli, Francesca; Belov, Dmitry S; Kwon, Young Do; Ramesh, Ranjith; Furimsky, Anna M; O'Loughlin, Kathleen; Byrge, Patricia C; Iyer, Lalitha V; Mirsalis, Jon C; Kurkin, Alexander V; Altieri, Andrea; Debnath, Asim K

2018-05-12

We are continuing our concerted effort to optimize our first lead entry antagonist, NBD-11021, which targets the Phe43 cavity of the HIV-1 envelope glycoprotein gp120, to improve antiviral potency and ADMET properties. In this report, we present a structure-based approach that helped us to generate working hypotheses to modify further a recently reported advanced lead entry antagonist, NBD-14107, which showed significant improvement in antiviral potency when tested in a single-cycle assay against a large panel of Env-pseudotyped viruses. We report here the synthesis of twenty-nine new compounds and evaluation of their antiviral activity in a single-cycle and multi-cycle assay to derive a comprehensive structure-activity relationship (SAR). We have selected three inhibitors with the high selectivity index for testing against a large panel of 55 Env-pseudotyped viruses representing a diverse set of clinical isolates of different subtypes. The antiviral activity of one of these potent inhibitors, 55 (NBD-14189), against some clinical isolates was as low as 63 nM. We determined the sensitivity of CD4-binding site mutated-pseudoviruses to these inhibitors to confirm that they target HIV-1 gp120. Furthermore, we assessed their ADMET properties and compared them to the clinical candidate attachment inhibitor, BMS-626529. The ADMET data indicate that some of these new inhibitors have comparable ADMET properties to BMS-626529 and can be optimized further to potential clinical candidates. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Developmental Effects of the ToxCast™ Phase I and Phase II Chemicals in Caenorhabditis elegans and Corresponding Responses in Zebrafish, Rats, and Rabbits

PubMed Central

Boyd, Windy A.; Smith, Marjolein V.; Co, Caroll A.; Pirone, Jason R.; Rice, Julie R.; Shockley, Keith R.; Freedman, Jonathan H.

2015-01-01

Background: Modern toxicology is shifting from an observational to a mechanistic science. As part of this shift, high-throughput toxicity assays are being developed using alternative, nonmammalian species to prioritize chemicals and develop prediction models of human toxicity. Methods: The nematode Caenorhabditis elegans (C. elegans) was used to screen the U.S. Environmental Protection Agency’s (EPA’s) ToxCast™ Phase I and Phase II libraries, which contain 292 and 676 chemicals, respectively, for chemicals leading to decreased larval development and growth. Chemical toxicity was evaluated using three parameters: a biologically defined effect size threshold, half-maximal activity concentration (AC50), and lowest effective concentration (LEC). Results: Across both the Phase I and Phase II libraries, 62% of the chemicals were classified as active ≤ 200 μM in the C. elegans assay. Chemical activities and potencies in C. elegans were compared with those from two zebrafish embryonic development toxicity studies and developmental toxicity data for rats and rabbits. Concordance of chemical activity was higher between C. elegans and one zebrafish assay across Phase I chemicals (79%) than with a second zebrafish assay (59%). Using C. elegans or zebrafish to predict rat or rabbit developmental toxicity resulted in balanced accuracies (the average value of the sensitivity and specificity for an assay) ranging from 45% to 53%, slightly lower than the concordance between rat and rabbit (58%). Conclusions: Here, we present an assay that quantitatively and reliably describes the effects of chemical toxicants on C. elegans growth and development. We found significant overlap in the activity of chemicals in the ToxCast™ libraries between C. elegans and zebrafish developmental screens. Incorporating C. elegans toxicological assays as part of a battery of in vitro and in vivo assays provides additional information for the development of models to predict a chemical’s potential toxicity to humans. Citation: Boyd WA, Smith MV, Co CA, Pirone JR, Rice JR, Shockley KR, Freedman JH. 2016. Developmental effects of the ToxCast™ Phase I and II chemicals in Caenorhabditis elegans and corresponding responses in zebrafish, rats, and rabbits. Environ Health Perspect 124:586–593; http://dx.doi.org/10.1289/ehp.1409645 PMID:26496690

Isolation and characterization of α-conotoxin LsIA with potent activity at nicotinic acetylcholine receptors.

PubMed

Inserra, Marco C; Kompella, Shiva N; Vetter, Irina; Brust, Andreas; Daly, Norelle L; Cuny, Hartmut; Craik, David J; Alewood, Paul F; Adams, David J; Lewis, Richard J

2013-09-15

A new α-conotoxin LsIA was isolated from the crude venom of Conus limpusi using assay-guided RP-HPLC fractionation. Synthetic LsIA was a potent antagonist of α3β2, α3α5β2 and α7 nAChRs, with half-maximal inhibitory concentrations of 10, 31 and 10 nM, respectively. The structure of LsIA determined by NMR spectroscopy comprised a characteristic disulfide bond-stabilized α-helical structure and disordered N-terminal region. Potency reductions of up to 9-fold were observed for N-terminally truncated analogues of LsIA at α7 and α3β2 nAChRs, whereas C-terminal carboxylation enhanced potency 3-fold at α3β2 nAChRs but reduced potency 3-fold at α7 nAChRs. This study gives further insight into α-conotoxin pharmacology and the molecular basis of nAChR selectivity, highlighting the influence of N-terminal residues and C-terminal amidation on conotoxin pharmacology. Copyright © 2013. Published by Elsevier Inc.

Neuromedin N: high affinity interaction with brain neurotensin receptors and rapid inactivation by brain synaptic peptidases.

PubMed

Checler, F; Vincent, J P; Kitabgi, P

1986-07-31

Neuromedin N (NN) is a novel neurotensin (NT)-like hexapeptide recently isolated from porcine spinal cord. NN competitively inhibited the binding of monoiodinated [Trp11]NT to rat brain synaptic membranes with a 19-fold lower potency than NT. In the presence of 1 mM 1,10-phenanthroline or 10 microM bestatin, the potency of NN relative to NT was increased about 5-fold. NN was readily degraded by rat brain synaptic membranes, and NN-(2-6) was the major degradation product. NN-(2-6) did not bind to NT receptors at concentrations up to 1 microM whether or not peptidase inhibitors were present in the binding assay. The rate of degradation by synaptic membranes was nearly 2.5 times higher for NN than for NT. NN degradation by membranes was totally prevented by 1,10-phenanthroline and markedly inhibited by bestatin. The presence of NN in the central nervous system, its high potency to interact with brain NT receptors and its rapid inactivation by brain synaptic peptidases make it a potential neurotransmitter candidate acting at the NT receptor.

Conditions that Stabilize Membrane Domains Also Antagonize n-Alcohol Anesthesia

NASA Astrophysics Data System (ADS)

Machta, Benjamin B.; Gray, Ellyn; Nouri, Mariam; McCarthy, Nicola L. C.; Gray, Erin M.; Miller, Ann L.; Brooks, Nicholas J.; Veatch, Sarah L.

2016-08-01

Diverse molecules induce general anesthesia with potency strongly correlated both with their hydrophobicity and their effects on certain ion channels. We recently observed that several n-alcohol anesthetics inhibit heterogeneity in plasma membrane derived vesicles by lowering the critical temperature ($T_c$) for phase separation. Here we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n-alcohols and effects on $T_c$. First we show that hexadecanol acts oppositely to n-alcohol anesthetics on membrane mixing and antagonizes ethanol induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described `intoxication reversers' raise $T_c$ and counter ethanol's effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that hydrostatic pressure, long known to reverse anesthesia, also raises $T_c$ in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together, these results demonstrate that $\\Delta T_c$ predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.

Development of a QPatch automated electrophysiology assay for identifying KCa3.1 inhibitors and activators.

PubMed

Jenkins, David Paul; Yu, Weifeng; Brown, Brandon M; Løjkner, Lars Damgaard; Wulff, Heike

2013-01-01

The intermediate-conductance Ca(2+)-activated K(+) channel KCa3.1 (also known as KCNN4, IK1, or the Gárdos channel) plays an important role in the activation of T and B cells, mast cells, macrophages, and microglia by regulating membrane potential, cellular volume, and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders, whereas KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca(2+) sensitivity of KCa3.1 was studied using varying intracellular Ca(2+) concentrations. A free Ca(2+) concentration of 1 μM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca(2+) concentration (∼200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay.

Biological profiling and dose-response modeling tools ...

EPA Pesticide Factsheets

Through its ToxCast project, the U.S. EPA has developed a battery of in vitro high throughput screening (HTS) assays designed to assess the potential toxicity of environmental chemicals. At present, over 1800 chemicals have been tested in up to 600 assays, yielding a large number of concentration-response data sets. Standard processing of these data sets involves finding a best fitting mathematical model and set of model parameters that specify this model. The model parameters include quantities such as the half-maximal activity concentration (or “AC50”) that have biological significance and can be used to inform the efficacy or potency of a given chemical with respect to a given assay. All of this data is processed and stored in an online-accessible database and website: http://actor.epa.gov/dashboard2. Results from these in vitro assays are used in a multitude of ways. New pathways and targets can be identified and incorporated into new or existing adverse outcome pathways (AOPs). Pharmacokinetic models such as those implemented EPA’s HTTK R package can be used to translate an in vitro concentration into an in vivo dose; i.e., one can predict the oral equivalent dose that might be expected to activate a specific biological pathway. Such predicted values can then be compared with estimated actual human exposures prioritize chemicals for further testing.Any quantitative examination should be accompanied by estimation of uncertainty. We are developing met

Human beta-endorphin: synthesis and biological activity of analogs with substitutions in positions 2, 9, 18, 27 and 31.

PubMed

Yeung, H W; Yamashiro, D; Tseng, L F; Chang, W C; Li, C H

1981-02-01

Four analogs of the opioid peptide human beta-endorphin (Bh-EP) have been synthesized: [D-Lys9, Phe27, Gly31]-beta h-EP, [D-PHe18,Phe27,Gly31]-beta h-EP, [D-Thr2,D-Lys9,Phe27,Gly31]-beta h-EP, and [D-Thr2,D-Phe18,Phe27,Gly31]-beta h-EP. All are practically indistinguishable from beta h-EP in the guinea pig ileum assay. All show diminished analgesic potency in the mouse tail-flick assay.

A study of antioxidant potential of Perilladehyde

NASA Astrophysics Data System (ADS)

Malu, T. J.; Banerjee, Nitesh; Singh, Avinash Kumar; Kannadasan, S.; Ethiraj, K. R.

2017-11-01

The use of plants as food, medicine is credited to a biological property of their secondary metabolites. These naturally occurring secondary metabolites are found to have great importance in controlling the formation of free radicles. These antioxidants are capable to catch the free radicles present in the body and maintain its balance. Antioxidant activity and potency of Perillaldehyde using various in vitro biochemical assays were studied. The assay involves various levels of antioxidant action such as free radical scavenging activity through DPPH, reducing power determination, nitric oxide scavenging ability, metal chelation power, scavenging of hydrogen peroxide, membrane stabilizing activity, and lipid peroxidation study.

Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease

PubMed Central

Zheng, Wei; Padia, Janak; Urban, Daniel J.; Jadhav, Ajit; Goker-Alpan, Ozlem; Simeonov, Anton; Goldin, Ehud; Auld, Douglas; LaMarca, Mary E.; Inglese, James; Austin, Christopher P.; Sidransky, Ellen

2007-01-01

Gaucher disease is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene. Missense mutations result in reduced enzyme activity that may be due to misfolding, raising the possibility of small-molecule chaperone correction of the defect. Screening large compound libraries by quantitative high-throughput screening (qHTS) provides comprehensive information on the potency, efficacy, and structure–activity relationships (SAR) of active compounds directly from the primary screen, facilitating identification of leads for medicinal chemistry optimization. We used qHTS to rapidly identify three structural series of potent, selective, nonsugar glucocerebrosidase inhibitors. The three structural classes had excellent potencies and efficacies and, importantly, high selectivity against closely related hydrolases. Preliminary SAR data were used to select compounds with high activity in both enzyme and cell-based assays. Compounds from two of these structural series increased N370S mutant glucocerebrosidase activity by 40–90% in patient cell lines and enhanced lysosomal colocalization, indicating chaperone activity. These small molecules have potential as leads for chaperone therapy for Gaucher disease, and this paradigm promises to accelerate the development of leads for other rare genetic disorders. PMID:17670938

Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15

PubMed Central

Yang, Zhilin; Weisshaar, James C.

2015-01-01

Antibiotics target specific biochemical mechanisms in bacteria. In response to new drugs, pathogenic bacteria rapidly develop resistance. In contrast, antimicrobial peptides (AMPs) have retained broad spectrum antibacterial potency over millions of years. We present single-cell fluorescence assays that detect reactive oxygen species (ROS) in the Escherichia coli cytoplasm in real time. Within 30 s of permeabilization of the cytoplasmic membrane by the cationic AMP CM15 [combining residues 1–7 of cecropin A (from moth) with residues 2–9 of melittin (bee venom)], three fluorescence signals report oxidative stress in the cytoplasm, apparently involving O2−, H2O2, and •OH. Mechanistic studies indicate that active respiration is a prerequisite to the CM15-induced oxidative damage. In anaerobic conditions, signals from ROS are greatly diminished and the minimum inhibitory concentration increases 20-fold. Evidently the natural human AMP LL-37 also induces a burst of ROS. Oxidative stress may prove a significant bacteriostatic mechanism for a variety of cationic AMPs. If so, host organisms may use the local oxygen level to modulate AMP potency. PMID:25561551

Cryopreserved or Fresh Mesenchymal Stromal Cells: Only a Matter of Taste or Key to Unleash the Full Clinical Potential of MSC Therapy?

PubMed

Moll, Guido; Geißler, Sven; Catar, Rusan; Ignatowicz, Lech; Hoogduijn, Martin J; Strunk, Dirk; Bieback, Karen; Ringdén, Olle

2016-01-01

Mesenchymal stromal cells (MSCs) harbor great therapeutic potential for numerous diseases. From early clinical trials, success and failure analysis, bench-to-bedside and back-to-bench approaches, there has been a great gain in knowledge, still leaving a number of questions to be answered regarding optimal manufacturing and quality of MSCs for clinical application. For treatment of many acute indications, cryobanking may remain a prerequisite, but great uncertainty exists considering the therapeutic value of freshly thawed (thawed) and continuously cultured (fresh) MSCs. The field has seen an explosion of new literature lately, outlining the relevance of the topic. MSCs appear to have compromised immunomodulatory activity directly after thawing for clinical application. This may provide a possible explanation for failure of early clinical trials. It is not clear if and how quickly MSCs recover their full therapeutic activity, and if the "cryo stun effect" is relevant for clinical success. Here, we will share our latest insights into the relevance of these observations for clinical practice that will be discussed in the context of the published literature. We argue that the differences of fresh and thawed MSCs are limited but significant. A key issue in evaluating potency differences is the time point of analysis after thawing. To date, prospective double-blinded randomized clinical studies to evaluate potency of both products are lacking, although recent progress was made with preclinical assessment. We suggest refocusing therapeutic MSC development on potency and safety assays with close resemblance of the clinical reality.

Umbilical cord tissue-derived mesenchymal stromal cells maintain immunomodulatory and angiogenic potencies after cryopreservation and subsequent thawing.

PubMed

Bárcia, Rita N; Santos, Jorge M; Teixeira, Mariana; Filipe, Mariana; Pereira, Ana Rita S; Ministro, Augusto; Água-Doce, Ana; Carvalheiro, Manuela; Gaspar, Maria Manuela; Miranda, Joana P; Graça, Luis; Simões, Sandra; Santos, Susana Constantino Rosa; Cruz, Pedro; Cruz, Helder

2017-03-01

The effect of cryopreservation on mesenchymal stromal cell (MSC) therapeutic properties has become highly controversial. However, data thus far have indiscriminately involved the assessment of different types of MSCs with distinct production processes. This study assumed that MSC-based products are affected differently depending on the tissue source and manufacturing process and analyzed the effect of cryopreservation on a specific population of umbilical cord tissue-derived MSCs (UC-MSCs), UCX ® . Cell phenotype was assessed by flow cytometry through the evaluation of the expression of relevant surface markers such as CD14, CD19, CD31, CD34, CD44, CD45, CD90, CD105, CD146, CD200, CD273, CD274 and HLA-DR. Immunomodulatory activity was analyzed in vitro through the ability to inhibit activated T cells and in vivo by the ability to reverse the signs of inflammation in an adjuvant-induced arthritis (AIA) model. Angiogenic potential was evaluated in vitro using a human umbilical vein endothelial cell-based angiogenesis assay, and in vivo using a mouse model for hindlimb ischemia. Phenotype and immunomodulatory and angiogenic potencies of this specific UC-MSC population were not impaired by cryopreservation and subsequent thawing, both in vitro and in vivo. This study suggests that potency impairment related to cryopreservation in a given tissue source can be avoided by the production process. The results have positive implications for the development of advanced-therapy medicinal products. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Structure-Activity Relationship Studies Using Natural and Synthetic Okadaic Acid/Dinophysistoxin Toxins.

PubMed

Twiner, Michael J; Doucette, Gregory J; Pang, Yucheng; Fang, Chao; Forsyth, Craig J; Miles, Christopher O

2016-11-04

Okadaic acid (OA) and the closely related dinophysistoxins (DTXs) are algal toxins that accumulate in shellfish and are known serine/threonine protein phosphatase (ser/thr PP) inhibitors. Phosphatases are important modulators of enzyme activity and cell signaling pathways. However, the interactions between the OA/DTX toxins and phosphatases are not fully understood. This study sought to identify phosphatase targets and characterize their structure-activity relationships (SAR) with these algal toxins using a combination of phosphatase activity and cytotoxicity assays. Preliminary screening of 21 human and yeast phosphatases indicated that only three ser/thr PPs (PP2a, PP1, PP5) were inhibited by physiologically saturating concentrations of DTX2 (200 nM). SAR studies employed naturally-isolated OA, DTX1, and DTX2, which vary in degree and/or position of methylation, in addition to synthetic 2- epi -DTX2. OA/DTX analogs induced cytotoxicity and inhibited PP activity with a relatively conserved order of potency: OA = DTX1 ≥ DTX2 > 2- epi -DTX. The PPs were also differentially inhibited with sensitivities of PP2a > PP5 > PP1. These findings demonstrate that small variations in OA/DTX toxin structures, particularly at the head region (i.e., C1/C2), result in significant changes in toxicological potency, whereas changes in methylation at C31 and C35 (tail region) only mildly affect potency. In addition to this being the first study to extensively test OA/DTX analogs' activities towards PP5, these data will be helpful for accurately determining toxic equivalence factors (TEFs), facilitating molecular modeling efforts, and developing highly selective phosphatase inhibitors.

ScreenBEAM: a novel meta-analysis algorithm for functional genomics screens via Bayesian hierarchical modeling | Office of Cancer Genomics

Cancer.gov

Functional genomics (FG) screens, using RNAi or CRISPR technology, have become a standard tool for systematic, genome-wide loss-of-function studies for therapeutic target discovery. As in many large-scale assays, however, off-target effects, variable reagents' potency and experimental noise must be accounted for appropriately control for false positives.

Multivalency of Sonic hedgehog conjugated to linear polymer chains modulates protein potency.

PubMed

Wall, Samuel T; Saha, Krishanu; Ashton, Randolph S; Kam, Kimberly R; Schaffer, David V; Healy, Kevin E

2008-04-01

A potently active multivalent form of the protein Sonic hedgehog (Shh) was produced by bioconjugation of a modified recombinant form of Shh to the linear polymers poly(acrylic acid) (pAAc) and hyaluronic acid (HyA) via a two-step reaction exploiting carboimiide and maleimide chemistry. Efficiency of the conjugation was approximately 75% even at stoichiometric ratios of 30 Shh molecules per linear HyA chain (i.e., 30:1 Shh/HyA). Bioactivity of the conjugates was tested via a cellular assay across a range of stoichiometric ratios of Shh molecules to HyA linear chains, which was varied from 0.6:1 Shh/HyA to 22:1 Shh/HyA. Results indicate that low conjugation ratios decrease Shh bioactivity and high ratios increase this activity beyond the potency of monomeric Shh, with approximately equal activity between monomeric soluble Shh and conjugated Shh at 7:1 Shh/HyA. In addition, high-ratio constructs increased angiogenesis determined by the in vivo chick chorioallantoic membrane (CAM) assay. These results are captured by a kinetic model of multiple interactions between the Shh/HyA conjugates and cell surface receptors resulting in higher cell signaling at lower bulk Shh concentrations.

Compound Selectivity and Target Residence Time of Kinase Inhibitors Studied with Surface Plasmon Resonance.

PubMed

Willemsen-Seegers, Nicole; Uitdehaag, Joost C M; Prinsen, Martine B W; de Vetter, Judith R F; de Man, Jos; Sawa, Masaaki; Kawase, Yusuke; Buijsman, Rogier C; Zaman, Guido J R

2017-02-17

Target residence time (τ) has been suggested to be a better predictor of the biological activity of kinase inhibitors than inhibitory potency (IC 50 ) in enzyme assays. Surface plasmon resonance binding assays for 46 human protein and lipid kinases were developed. The association and dissociation constants of 80 kinase inhibitor interactions were determined. τ and equilibrium affinity constants (K D ) were calculated to determine kinetic selectivity. Comparison of τ and K D or IC 50 values revealed a strikingly different view on the selectivity of several kinase inhibitors, including the multi-kinase inhibitor ponatinib, which was tested on 10 different kinases. In addition, known pan-Aurora inhibitors resided much longer on Aurora B than on Aurora A, despite having comparable affinity for Aurora A and B. Furthermore, the γ/δ-selective PI3K inhibitor duvelisib and the δ-selective drug idelalisib had similar 20-fold selectivity for δ- over γ-isoform but duvelisib resided much longer on both targets. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular docking study, synthesis and biological evaluation of Schiff bases as Hsp90 inhibitors.

PubMed

Dutta Gupta, Sayan; Snigdha, D; Mazaira, Gisela I; Galigniana, Mario D; Subrahmanyam, C V S; Gowrishankar, N L; Raghavendra, N M

2014-04-01

Heat shock protein 90 (Hsp90) is an emerging attractive target for the discovery of novel cancer therapeutic agents. Docking methods are powerful in silico tools for lead generation and optimization. In our mission to rationally develop novel effective small molecules against Hsp90, we predicted the potency of our designed compounds by Sybyl surflex Geom X docking method. The results of the above studies revealed that Schiff bases derived from 2,4-dihydroxy benzaldehyde/5-chloro-2,4-dihydroxy benzaldehyde demonstrated effective binding with the protein. Subsequently, a few of them were synthesized (1-10) and characterized by IR, (1)HNMR and mass spectral analysis. The synthesized molecules were evaluated for their potential to suppress Hsp90 ATPase activity by Malachite green assay. The anticancer studies were performed by 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method. The software generated results was in satisfactory agreement with the evaluated biological activity. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Prediction of intracellular exposure bridges the gap between target- and cell-based drug discovery

PubMed Central

Gordon, Laurie J.; Wayne, Gareth J.; Almqvist, Helena; Axelsson, Hanna; Seashore-Ludlow, Brinton; Treyer, Andrea; Lundbäck, Thomas; West, Andy; Hann, Michael M.; Artursson, Per

2017-01-01

Inadequate target exposure is a major cause of high attrition in drug discovery. Here, we show that a label-free method for quantifying the intracellular bioavailability (Fic) of drug molecules predicts drug access to intracellular targets and hence, pharmacological effect. We determined Fic in multiple cellular assays and cell types representing different targets from a number of therapeutic areas, including cancer, inflammation, and dementia. Both cytosolic targets and targets localized in subcellular compartments were investigated. Fic gives insights on membrane-permeable compounds in terms of cellular potency and intracellular target engagement, compared with biochemical potency measurements alone. Knowledge of the amount of drug that is locally available to bind intracellular targets provides a powerful tool for compound selection in early drug discovery. PMID:28701380

Collaborative study for the establishment of the 2(nd) International Standard for Bleomycin Complex A2/B2.

PubMed

Jorajuria, S; Raphalen, C; Dujardin, V; Daas, A

2015-01-01

Organization (WHO) International Standard (IS) for bleomycin complex A2/B2. Eight laboratories from different countries participated. Potencies of the candidate material were estimated by microbiological assays with sensitive micro-organisms. To ensure continuity between consecutive batches, the 1(st) IS for bleomycin complex A2/B2 was used as a reference. Based on the results of the study, the 2(nd) IS for bleomycin complex A2/B2 was adopted at the meeting of the WHO Expert Committee for Biological Standardization (ECBS) in 2014 with an assigned potency of 12 500 International Units (IU) per vial. The 2(nd) IS for bleomycin complex A2/B2 is available from the European Directorate for the Quality of Medicines & HealthCare (EDQM).

Discovery of Highly Potent Tyrosinase Inhibitor, T1, with Significant Anti-Melanogenesis Ability by zebrafish in vivo Assay and Computational Molecular Modeling

NASA Astrophysics Data System (ADS)

Chen, Wang-Chuan; Tseng, Tien-Sheng; Hsiao, Nai-Wan; Lin, Yun-Lian; Wen, Zhi-Hong; Tsai, Chin-Chuan; Lee, Yu-Ching; Lin, Hui-Hsiung; Tsai, Keng-Chang

2015-01-01

Tyrosinase is involved in melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders that can be treated with depigmenting agents. A natural product T1, bis(4-hydroxybenzyl)sulfide, isolated from the Chinese herbal plant, Gastrodia elata, is a strong competitive inhibitor against mushroom tyrosinase (IC50 = 0.53 μM, Ki = 58 +/- 6 nM), outperforms than kojic acid. The cell viability and melanin quantification assay demonstrate that 50 μM of T1 apparently attenuates 20% melanin content of human normal melanocytes without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that T1 effectively reduces melanogenesis with no adverse side effects. The acute oral toxicity study evidently confirms that T1 molecule is free of discernable cytotoxicity in mice. Furthermore, the molecular modeling demonstrates that the sulfur atom of T1 coordinating with the copper ions in the active site of tyrosinase is essential for mushroom tyrosinase inhibition and the ability of diminishing the human melanin synthesis. These results evident that T1 isolated from Gastrodia elata is a promising candidate in developing pharmacological and cosmetic agents of great potency in skin-whitening.

RELATIVE POTENCY OF FUNGAL EXTRACTS IN INDUCING ALLERGIC ASTHMA-LIKE RESPONSES IN BALB/C MICE

EPA Science Inventory

Indoor mold has been associated with the development of allergic asthma. However, relative potency of molds in the induction of allergic asthma is not clear. In this study, we tested the relative potency of fungal extracts (Metarizium anisophilae [MACA], Stachybotrys ...