Jessica Whritenour

Drug-induced Skin Lesions in Cynomolgus Macaques Treated with Metabotropic Glutamate Receptor 5 (mGluR5) Negative Allosteric Modulators.

Three orally administered metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators caused skin lesions consistent with delayed type-IV hypersensitivity in cynomolgus macaques in 2- and 12-week toxicity studies. Several monkeys developed macroscopic skin lesions in multiple locations after 8 to 9 days of dosing; the most prominent effects involved the genital region of males and generalized erythema occurred in both sexes. Microscopic lesions occurred in both clinically affected and unaffected areas and were characterized by lymphocytic interface inflammation, subepidermal bullae, and individual keratinocyte vacuolation/necrosis. In the 12-week study, clinical effects in 2 animals resolved with continued dosing, whereas in others the inflammatory process progressed with 1 female exhibiting systemic lymphocytic inflammation in multiple tissues. The inflammatory infiltrate consisted of CD3 and CD4 positive T lymphocytes with minimal CD68 positive macrophages and only rare CD8 positive T lymphocytes. A subset of animals given a dosing holiday was subsequently rechallenged with similar lesions developing but with a more rapid clinical onset. These skin lesions were consistent with type-IV delayed hypersensitivity with some features comparable to bullous drug eruptions in humans. A relationship between these findings and the intended mode of action for these compounds could not be ruled out, given the occurrence across different chemotypes.

Development and partial validation of a mouse model for predicting drug hypersensitivity reactions

Abstract Animal models that can be used to predict the allergenic potential of drug candidates have not been adequately optimized, validated, or characterized. While initial validation data from an inter-laboratory study of the mouse lymph node proliferation assay (LNPA) appeared promising, no additional investigations in this model have been reported. The objectives of this study were to use positive and negative control drugs to further optimize and validate the LNPA utilizing a non-radioactive endpoint and determine the sensitivity, specificity, and predictivity of the model. Drugs associated with hypersensitivity reactions in the literature were chosen to test in the model in addition to drugs with few or no reports of hypersensitivity. Mice received a subcutaneous injection of drug or vehicle into the scruff of the neck once daily for a period of 3 days. On Day 6, draining lymph nodes were harvested, single cell suspensions prepared, and total cell numbers determined for each animal by flow cytometry. A stimulation index was calculated by dividing the mean total cell number for the drug-treated group by the mean total cell number for the vehicle-treated animals. Based on statistical analysis of the data, animals with a total cell number ≥2.5× the mean of the vehicle group were classified as ‘responders’. Based on data generated to date with 12 positive control and six negative control drugs, the model had a sensitivity of 75%, a specificity of 74%, and a relatively good predictive value (measured by the Receiver Operating Characteristic AUC of 0.80). The data here suggest that this model may be a useful tool for identifying drug candidates with the potential to produce allergic responses in the clinic. Future studies will investigate the mechanism(s) for the lymph node responses in order to develop additional endpoints that may increase the sensitivity and specificity of the model.

Development of a modified lymphocyte transformation test for diagnosing drug-induced liver injury associated with an adaptive immune response

Abstract Drug-induced liver injury (DILI) is a growing problem. Diagnostic methods to differentiate DILI caused by an adaptive immune response from liver injury of other causes or to identify the responsible drug in patients receiving multiple drugs, herbals and/or dietary supplements (polypharmacy) have not yet been established. The lymphocyte transformation test (LTT) has been proposed as a diagnostic method to determine if a subject with an apparent hypersensitivity reaction has become sensitized to a specific drug. In this test, peripheral blood mononuclear cells (PBMC) collected from a subject are incubated with drug(s) suspected of causing the reaction. Cell proliferation, measured by the incorporation of [3H]-thymidine into new DNA, is considered evidence of a drug-specific immune response. The objectives of the current studies were to: (1) develop and optimize a modified version of the LTT (mLTT) and (2) investigate the feasibility of using the mLTT for diagnosing DILI associated with an adaptive immune response and identifying the responsible drug. PBMC collected from donors with a history of drug hypersensitivity reactions to specific drugs (manifested as skin rash) were used as positive controls for assay optimization. Following optimization, samples collected from 24 subjects enrolled in the U.S. Drug-Induced Liver Injury Network (DILIN) were tested in the mLTT. Using cytokine and granzyme B production as the primary endpoints to demonstrate lymphocyte sensitization to a specific drug, most samples from the DILIN subjects failed to respond. However, robust positive mLTT responses were observed for two of four samples from three DILIN subjects with hepatitis due to isoniazid (INH). We conclude that the mLTT, as performed here on frozen and thawed PBMC, is not a reliable test for diagnosing DILI caused by all drugs, but that it may be useful for confirming the role of the adaptive immune response in DILI ascribed to INH.

Mechanisms of Skin Toxicity Associated with Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators

Cutaneous reactions represent one of the most common adverse drug effects observed in clinical trials leading to substantial compound attrition. Three negative allosteric modulators (NAMs) of metabotropic glutamate receptors (mGluRs), which represent an important target for neurological diseases, developed by Pfizer, were recently failed in preclinical development due to delayed type IV skin hypersensitivity observed in non-human primates (NHPs). Here we employed large-scale phenotypic profiling in standardized panels of human primary cell/co-culture systems to characterize the skin toxicity mechanism(s) of mGluR5 NAMs from two different series. Investigation of a database of chemicals tested in these systems and transcriptional profiling suggested that the mechanism of toxicity may involve modulation of nuclear receptor targets RAR/RXR, and/or VDR with AhR antagonism. The studies reported here demonstrate how phenotypic profiling of preclinical drug candidates using human primary cells can provide insights into the mechanisms of toxicity and inform early drug discovery and development campaigns.

Development of a screening assay to evaluate the potential of drugs to cause immune-mediated hypersensitivity reactions.

Abstract Evidence suggests that bio-activation of drugs to generate chemically reactive metabolites (RM) that act as haptens to form immunogenic protein conjugates may be an important cause of immune-mediated drug hypersensitivity reactions (IDHR). Although many drugs that form RMs raise concerns about producing IDHR, standard non-clinical testing methods are rarely able to identify compounds with the potential to produce IDHR in humans. The objective of this study was to develop a predictive assay for IDHR that involves: (1) the use of an in vitro drug-metabolizing system to generate the RM that is captured by GSH, (2) conjugating the RM-GSH conjugate to mouse serum albumin (MSA) by using a chemical cross-linker, (3) immunization of mice with RM-GSH-MSA adducts, and (4) ex vivo challenge with RM-GSH-MSA adduct and measurement of lymphocyte proliferation to determine if the RM is immunogenic. The predictivity of the assay was evaluated by using drugs that produce RM and have been strongly, weakly, or not associated with IDHRs in the clinic. While this method requires additional validation with more drugs, the results demonstrate the feasibility of identifying drugs strongly associated with IDHR and the utility of the assay for rank ordering drugs with respect to their potential to cause IDHR.

Identification of MHC Haplotypes Associated with Drug-induced Hypersensitivity Reactions in Cynomolgus Monkeys

Drug-induced hypersensitivity reactions can significantly impact drug development and use. Studies to understand risk factors for drug-induced hypersensitivity reactions have identified genetic association with specific human leukocyte antigen (HLA) alleles. Interestingly, drug-induced hypersensitivity reactions can occur in nonhuman primates; however, association between drug-induced hypersensitivity reactions and major histocompatibility complex (MHC) alleles has not been described. In this study, tissue samples were collected from 62 cynomolgus monkeys from preclinical studies in which 9 animals had evidence of drug-induced hypersensitivity reactions. Microsatellite analysis was used to determine MHC haplotypes for each animal. A total of 7 haplotypes and recombinant MHC haplotypes were observed, with distribution frequency comparable to known MHC I allele frequency in cynomolgus monkeys. Genetic association analysis identified alleles from the M3 haplotype of the MHC I B region (B*011:01, B*075:01, B*079:01, B*070:02, B*098:05, and B*165:01) to be significantly associated (χ2 test for trend, p < 0.05) with occurrence of drug-induced hypersensitivity reactions. Sequence similarity from alignment of alleles in the M3 haplotype B region and HLA alleles associated with drug-induced hypersensitivity reactions in humans was 86% to 93%. These data demonstrate that MHC alleles in cynomolgus monkeys are associated with drug-induced hypersensitivity reactions, similar to HLA alleles in humans.

Characterization of the draining lymph node response in the mouse drug allergy model: A model for drug hypersensitivity reactions

Abstract The mouse drug allergy model (MDAM) was developed as a tool to predict the potential of systemically administered drugs to produce hypersensitivity reactions (HR). Drugs associated with HR in the clinic produce a marked increase in the cellularity of the draining lymph nodes (DLN) in the MDAM. The objective of this study was to characterize the changes in the DLN following exposure to drugs associated with HR and to investigate whether lymphocyte migration and/or proliferation play a role in the response. These endpoints were also investigated in the local lymph node assay (LLNA) to determine whether responses between the two assays occur via similar mechanisms. Results demonstrated that total numbers of T- and B-cells were proportionally increased in the DLN of mice treated with positive control drugs (i.e. abacavir, amoxicillin, ofloxacin, and sulfamethoxazole) compared to animals administered the vehicle or negative control drugs (metformin and cimetidine). In contrast, a significant increase in the B-cell population of the DLN was observed for 2,4-dinitrofluorobenzene (DNFB) following the LLNA protocol. Down-regulation of CD62L and up-regulation of CCR7 were observed for T-cells from the DLN of the positive control treated mice in the MDAM, but not with DNFB in the LLNA. A mild increase in T-cell proliferation was observed in the MDAM with positive control drugs, while DNFB in the LLNA induced proliferation within the B-cell population only. Anti-CD40L antibody administration inhibited MDAM responses to positive control drugs, but did not affect DNFB-induced increases in total cell number in the LLNA. These results suggest that the increased cellularity of the DLN in the MDAM may be the result of drug-induced alterations in lymphocyte migration and/or effects on lymphocyte proliferation. Moreover, it appears that different mechanisms may be involved in driving the MDAM and LLNA responses.

Discovery and Characterization of (R)-6-Neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894), an Alkyne-Lacking Metabotropic Glutamate Receptor 5 Negative Allosteric Modulator Profiled in both Rat and Nonhuman Primates

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu5 negative allosteric modulator (NAM) 7. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu5 NAMs. Increasing the sp3 character of high-throughput screening hit 40 afforded a novel morpholinopyrimidone mGlu5 NAM series. Its prototype, (R)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894, 8), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound 8 did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that 8 did not form any reactive metabolites. However, 8 caused the identical microscopic skin lesions in NHPs found with 7, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is required to confirm this and determine clinical relevance.

Chapter 17:Immunotoxicity Testing

The field of immunotoxicology has been growing rapidly and a wide variety of immunotoxicity testing methods is now available. This chapter first discusses approaches to developing immunotoxicity testing strategies and provides a general overview of the types of assays currently available and how they are used. These assays are classified either according to the type of immune response being measured, the experimental design, or the number of immune cell types involved. The chapter then focuses on in vitro and ex vivo assays used during drug development that have been developed more recently, rather than on the more established immunotoxicity assays (e.g., in vivo T-dependent antibody response). These assays address concerns regarding hypersensitivity reactions, immunoenhancement and immunosuppression. Assays for hypersensitivity include measuring human mast-cell function and activation of the complement cascade, while those for immunoenhancement include the cytokine release assay. Immunosuppression can be measured using assays that use co-cultures of different types of immune cells, such as the human lymphocyte activation assay, three-dimensional culture systems, and by assays that measure T-cell function. Several of the assays presented are in vitro assays that use human immune cells to enhance the translatability to human risk. In addition, assays that can be added to standard toxicology studies are discussed.