Considerations when deriving compound-specific limits for extractables and leachables from pharmaceutical products: Four case studies.

Leachables from pharmaceutical container closure systems are a subset of impurities that present in drug products and may pose a risk to patients or compromise product quality. Extractable studies can identify potential leachables, and extractables and leachables (E&Ls) should be evaluated during development of the impurity control strategy. Currently, there is a lack of specific regulatory guidance on how to risk assess E&Ls; this may lead to inconsistency across the industry. This manuscript is a cross-industry Extractables and Leachables Safety Information Exchange (ELSIE) consortium collaboration and follow-up to Broschard et al. (2016), which aims to provide further clarity and detail on the conduct of E&L risk assessments. Where sufficient data are available, a health-based exposure limit termed Permitted Daily Exposure (PDE) may be calculated and to exemplify this, case studies of four common E&Ls are described herein, namely bisphenol-A, butylated hydroxytoluene, Irgafos® 168, and Irganox® 1010. Relevant discussion points are further explored, including the value of extractable data, how to perform route-to-route extrapolations and considerations around degradation products. By presenting PDEs for common E&L substances, the aim is to encourage consistency and harmony in approaches for deriving compound-specific limits.

Skin sensitization in silico protocol.

The assessment of skin sensitization has evolved over the past few years to include in vitro assessments of key events along the adverse outcome pathway and opportunistically capitalize on the strengths of in silico methods to support a weight of evidence assessment without conducting a test in animals. While in silico methods vary greatly in their purpose and format; there is a need to standardize the underlying principles on which such models are developed and to make transparent the implications for the uncertainty in the overall assessment. In this contribution, the relationship between skin sensitization relevant effects, mechanisms, and endpoints are built into a hazard assessment framework. Based on the relevance of the mechanisms and effects as well as the strengths and limitations of the experimental systems used to identify them, rules and principles are defined for deriving skin sensitization in silico assessments. Further, the assignments of reliability and confidence scores that reflect the overall strength of the assessment are discussed. This skin sensitization protocol supports the implementation and acceptance of in silico approaches for the prediction of skin sensitization.

Managing emerging mutagenicity risks: Late stage mutagenic impurity control within the atovaquone second generation synthesis.

The mutagenic-impurity control strategy for a second generation manufacturing route to the non-mutagenic antipneumocystic agent atovaquone (2-((1R,4R)-4-(4-chlorophenyl)cyclohexyl)-3-hydroxynaphthalene-1,4-dione) 1 is described. Preliminary assessment highlighted multiple materials of concern which were largely discharged either through returning a negative bacterial mutagenicity assay or through confidence that the impurity would be purged during the downstream processing from when it was first introduced. Additional genotoxicity testing highlighted two materials of concern where initial assessment suggested that testing for these impurities at trace levels within the drug substance would be required. Following a thorough review of process purging detail, spiking and purging experimentation, and an understanding of the process parameters to which they were exposed an ICH M7 Option 4 approach could be justified for their control. The development of two 1H NMR spectroscopy methods for measurement of these impurities is also described as well as a proposed summary table for describing the underlying rationale for ICH M7 control rationales to regulators. This manuscript demonstrates that process purging of potential mutagenic impurities can be realised even when they are introduced in the later stages of a process and highlights the importance of scientific understanding rather than relying on a stage-counting approach.

Evaluation of a statistics-based Ames mutagenicity QSAR model and interpretation of the results obtained.

The relative wealth of bacterial mutagenicity data available in the public literature means that in silico quantitative/qualitative structure activity relationship (QSAR) systems can readily be built for this endpoint. A good means of evaluating the performance of such systems is to use private unpublished data sets, which generally represent a more distinct chemical space than publicly available test sets and, as a result, provide a greater challenge to the model. However, raw performance metrics should not be the only factor considered when judging this type of software since expert interpretation of the results obtained may allow for further improvements in predictivity. Enough information should be provided by a QSAR to allow the user to make general, scientifically-based arguments in order to assess and overrule predictions when necessary. With all this in mind, we sought to validate the performance of the statistics-based in vitro bacterial mutagenicity prediction system Sarah Nexus (version 1.1) against private test data sets supplied by nine different pharmaceutical companies. The results of these evaluations were then analysed in order to identify findings presented by the model which would be useful for the user to take into consideration when interpreting the results and making their final decision about the mutagenic potential of a given compound.

It's difficult, but important, to make negative predictions.

At the confluence of predictive and regulatory toxicologies, negative predictions may be the thin green line that prevents populations from being exposed to harm. Here, two novel approaches to making confident and robust negative in silico predictions for mutagenicity (as defined by the Ames test) have been evaluated. Analyses of 12 data sets containing >13,000 compounds, showed that negative predictivity is high (∼90%) for the best approach and features that either reduce the accuracy or certainty of negative predictions are identified as misclassified or unclassified respectively. However, negative predictivity remains high (and in excess of the prevalence of non-mutagens) even in the presence of these features, indicating that they are not flags for mutagenicity.

Evaluation of aromatic amines with different purities and different solvent vehicles in the Ames test.

Of all the in vitro mutagenicity assays, the Ames test displays the best correlation with rodent carcinogenicity and therefore carries significant weight with the food and drug regulatory bodies. Aromatic amines (AA) are ubiquitous structural groups in food and drug molecules despite the well-documented mutagenic and carcinogenic propensity for many representatives. Furthermore, recent regulatory guidelines (that is ICH M7) requires the hazard assessment of actual and potential impurities by two complementary (Q)SAR prediction methodologies if no carcinogenicity or bacterial mutagenicity data is available. One methodology should be expert-rule-based and the second should be statistics-based. Having encountered numerous reports of contradictory Ames results for members of this chemotype, we undertook systematic Ames tests on a diverse set of 14 AAs of differing purities in different solvents, and as free bases and their salts. The aim of this work was to investigate the reliability of the Ames test for this chemotype leading to the creation of a reference set of AAs for use by medicinal chemists and in silico modelling. Contrary to previous experience, which led to the investigations reported in this publication, the anticipated transformation from an Ames-positive to an Ames-negative after purification only occurred for one compound. Furthermore, this result proved inconclusive after testing as the HCl salt in DMSO and in water. The anticipated change in class from mutagen to non-mutagen, did not occur and this can be read as evidence for the reliability of the Ames test for AAs.

In vitro assays for induction of drug metabolism.

Hepatic microsomal cytochrome P450 (CYP) forms have a major role in the metabolism of drugs and other chemicals. Primary hepatocyte cultures from humans and experimental animals are a valuable in vitro system for studying the effects of chemicals on CYP forms. This chapter describes methods to evaluate CYP form induction in human and rat hepatocytes cultured in a 96-well plate format. The use of a 96-well plate format permits studies to be performed with relatively small numbers of hepatocytes and obviates the need to harvest cells and prepare subcellular fractions prior to the assay of enzyme activities. The induction of CYP1A and CYP3A forms in human and rat hepatocytes can be determined by measurement of 7-ethoxyresorufin O-deethylase and testosterone 6beta-hydroxylase activities, respectively, whereas 7-benzyloxy-4-trifluoromethylcoumarin (BFC) O-debenzylase can be employed to assess both CYP1A and CYP2B form induction in rat hepatocytes. An assay for determining the protein content of hepatocytes cultured in a 96-well plate format is also described.

Effect of Pyrethrins on cytochrome P450 forms in cultured rat and human hepatocytes.

High doses of Pyrethrins produce liver and thyroid gland tumours in rats by modes of action involving the induction of hepatic xenobiotic metabolising enzymes. The aim of this study was to compare the effects of Pyrethrins with those of the rat liver and thyroid tumour promoter sodium Phenobarbital on some cytochrome P450 (CYP) forms in cultured rat and human hepatocytes. The treatment of female Sprague-Dawley rat and human (both male and female) hepatocytes for 72 h with 0-1000 microM Pyrethrins and 0-1000 microM Phenobarbital did not result in any marked cytotoxicity. In rat hepatocytes both Pyrethrins and Phenobarbital produced an induction of 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase activity (a CYP1A/2B form marker) and CYP2B1 and CYP2B1/2 mRNA levels. Pyrethrins and Phenobarbital also induced CYP3A-dependent testosterone 6beta-hydroxylase activity in rat hepatocytes. In human hepatocytes Pyrethrins and Phenobarbital induced both testosterone 6beta-hydroxylase activity and CYP3A4 mRNA levels and also increased CYP2B6 mRNA levels. The effects of Pyrethrins and Phenobarbital were concentration-dependent and exhibited a threshold. These results demonstrate that the effects of Pyrethrins on CYP forms in cultured rat and human hepatocytes are qualitatively similar to those of Phenobarbital. Pyrethrins induce CYP2B and CYP3A forms in cultured rat hepatocytes and can induce CYP3A and CYP2B forms in human hepatocytes. While CYP form induction by Pyrethrins, Phenobarbital and related compounds can be associated with liver and thyroid gland tumour formation in rodents, epidemiological data for Phenobarbital suggests that such effects do not occur in humans.

Discovery of a First-in-Class Gut-Restricted RET Kinase Inhibitor as a Clinical Candidate for the Treatment of IBS.

Abdominal pain and abnormal bowel habits represent major symptoms for irritable bowel syndrome (IBS) patients that are not adequately managed. Although the etiology of IBS is not completely understood, many of the functions of the gastrointestinal (GI) tract are regulated by the enteric nervous system (ENS). Inflammation or stress-induced expression of growth factors or cytokines may lead to hyperinnervation of visceral afferent neurons in GI tract and contribute to the pathophysiology of IBS. Rearranged during transfection (RET) is a neuronal growth factor receptor tyrosine kinase critical for the development of the ENS as exemplified by Hirschsprung patients who carry RET loss-of-function mutations and lack normal colonic innervation leading to colonic obstruction. Similarly, RET signaling in the adult ENS maintains neuronal function by contributing to synaptic formation, signal transmission, and neuronal plasticity. Inhibition of RET in the ENS represents a novel therapeutic strategy for the normalization of neuronal function and the symptoms of IBS patients. Herein, we describe our screening effort and subsequent structure-activity relationships (SARs) in optimizing potency, selectivity, and mutagenicity of the series, which led to the discovery of a first-in-class, gut-restricted RET kinase inhibitor, 2-(4-(4-ethoxy-6-oxo-1,6-dihydropyridin-3-yl)-2-fluorophenyl)-N-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)acetamide (15, GSK3179106), as a clinical candidate for the treatment of IBS. GSK3179106 is a potent, selective, and gut-restricted pyridone hinge binder small molecule RET kinase inhibitor with a RET IC50 of 0.3 nM and is efficacious in vivo.

International Pig-a gene mutation assay trial (stage III): results with N-methyl-N-nitrosourea.

N-methyl-N-nitrosourea (MNU) was evaluated in the in vivo Pig-a mutation assay as part of an International Collaborative Trial to investigate laboratory reproducibility, 28-day study integration, and comparative analysis with micronucleus (MN), comet, and clinical pathology endpoints. Male Sprague Dawley rats were treated for 28 days with doses of 0, 2.5, 5, and 10 mg MNU/kg/day in two independent laboratories, GlaxoSmithKline (GSK) and Bristol Myers Squibb (BMS). Additional studies investigated the low-dose region (<2.5 mg/kg/day). Reticulocytes were evaluated for Pig-a phenotypic mutation, CD59-negative reticulocytes/erythrocytes (RETs(CD592-)/ RBCs(CD592-)) on Days 1, 4, 15, 29, 43, and 57, and for micronucleated reticulocytes (MN-RETs) on Days 4 and 29. Comet analysis was conducted for liver and whole blood, and hematology and clinical chemistry was investigated. Dose-dependent increases in the frequency of RETs(CD592-) and RBCs(CD592-) were observed by Day 15 or 29, respectively. Dose-dependent increases were observed in %MN-RET on Days 4 and 29, and in mean %tail intensity in liver and in blood. Hematology/clinical chemistry data demonstrated bone marrow toxicity. Data comparison between GSK and BMS indicated a high degree of concordance with the Pig-a mutation assay results, consistent with previous observations with MNU and N-ethyl-N-nitrosourea. These data confirm that complementary genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that can provide information on gene mutation, chromosome damage, and DNA strand breaks in a single repeat dose rodent study. Collectively, this would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints.

International Pig-a gene mutation assay trial: evaluation of transferability across 14 laboratories.

A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBC(CD59-)) and CD59-negative reticulocytes (RET(CD59-)). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3). Each laboratory studied 0, 20, and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RET(CD59-) , and frequency of RBC(CD59-) . The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose-related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87-0.99). Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories.

Structure based drug design: development of potent and selective factor IXa (FIXa) inhibitors.

On the basis of our understanding on the binding interactions of the benzothiophene template within the FIXa active site by X-ray crystallography and molecular modeling studies, we developed our SAR strategy by targeting the 4-position of the template to access the S1 beta and S2-S4 sites. A number of highly selective and potent factor Xa (FXa) and FIXa inhibitors were identified by simple switch of functional groups with conformational changes toward the S2-S4 sites.