Mode of action and human relevance assessment of male CD-1 mouse renal adenocarcinoma associated with lifetime exposure to empagliflozin.

Inhibition of sodium-glucose cotransporter-2 (SGLT2) has been shown to be a safe and efficacious approach to support managing Type 2 diabetes. In the 2-year carcinogenicity study with the SGLT2 inhibitor empagliflozin in CD-1 mice, an increased incidence of renal tubular adenomas and carcinomas was identified in the male high-dose group but was not observed in female mice. An integrated review of available nonclinical data was conducted to establish a mode-of-action hypothesis for male mouse-specific tumorigenesis. Five key events were identified through systematic analysis to form the proposed mode-of-action: (1) Background kidney pathology in CD-1 mice sensitizes the strain to (2) pharmacology-related diuretic effects associated with SGLT2 inhib ition. (3) In male mice, metabolic demand increases with the formation of a sex- and species-specific empagliflozin metabolite. These features converge to (4) deplete oxidative stress handling reserve, driving (5) constitutive cellular proliferation in male CD-1 mice. The proposed mode of action requires all five key events for empagliflozin to present a carcinogenicity risk in the CD-1 mouse. Considering that empagliflozin is not genotoxic in the standard battery of genotoxicity tests, and not all five key events are present in the context of female mice, rats, or humans, nor for other osmotic diuretics or other SGLT2 inhibitors, the observed male mouse renal tumors are not considered relevant to humans.

Histone Methyltransferase G9a Regulates Expression of Nuclear Receptors and Cytochrome P450 Enzymes in HepaRG Cells at Basal Level and in Fatty Acid Induced Steatosis.

Obesity and nonalcoholic fatty liver disease (NAFLD) affect expression and function of cytochrome P450 genes (P450s). The increased expression of inflammatory cytokines is a major driver of the downregulation of P450 expression in NAFLD. Decrease in P450 expression could potentially lead to drug-drug interaction, inefficient pharmacological effect of a drug, or hepatotoxicity. An epigenetic modifier, histone 3 lysine 9 methyl transferase enzyme (G9a), known to increase histone 3 lysine 9 methylation, is downregulated in diet-induced obesity animal models. In a liver-specific G9a knockout animal model, expression of P450s was downregulated. Currently, the role of G9a in regulation of P450s in steatosis is unknown. Our hypothesis is that in steatosis G9a plays a role in downregulation of P450 expression. In this study, we used HepaRG cells to induce steatosis using a combination of free fatty acids oleic acid and palmitic acid. The G9a was knocked down and overexpressed using small interfering RNA and adenovirus mediated approaches, respectively. Knockdown and overexpression of G9a in the absence of steatosis decreased and increased expression of nuclear receptors constitutive androstane receptor (CAR), pregnane X receptor, small heterodimer partner, and CYP2B6, 2E1, 2C8, 2C9, and 3A4, respectively. In steatotic conditions, overexpression of G9a prevented fatty acid mediated decreased expression of CAR, CYP2C19, 2C8, 7A1, and 3A4. Our current study suggests that G9a might serve as a key regulator of P450 expression at both the basal level and in early steatotic conditions. Single nucleotide polymorphism of G9a leading to loss/gain of function could lead to the poor metabolizer or ultrarapid metabolizer phenotypes. SIGNIFICANCE STATEMENT: The current study demonstrates that histone modification enzyme G9a is involved in the regulation of expression of nuclear receptors constitutive androstane receptor, pregnane X receptor, and small heterodimer partner as well as drug-metabolizing cytochrome P450s (P450s) at basal conditions and in fatty acid induced cellular model of steatosis. Histone 3 lysine 9 methylation should be considered together with histone 3 lysine 4 and histone 3 lysine 27 methylation as the epigenetic mechanisms controlling gene expression of P450s.

Pathogenesis of Renal Injury and Gene Expression Changes in the Male CD-1 Mouse Associated with Exposure to Empagliflozin.

An increased incidence of renal tubular adenomas and carcinomas was identified in the 2-year CD-1 mouse carcinogenicity study with empagliflozin (sodium-glucose transporter 2 inhibitor) in high dose (1,000 mg/kg/day) male mice. A 13-week mouse renal investigative pathogenesis study was conducted with empagliflozin to evaluate dose dependency and temporal onset of nonneoplastic degenerative/regenerative renal tubular and molecular (genes, pathways) changes which precede neoplasia. Male and female CD-1 mice were given daily oral doses of 0, 100, 300, or 1,000 mg/kg/day (corresponding carcinogenicity study dose levels) for 1, 2, 4, 8, or 13 weeks. The maximum expected pharmacology with secondary osmotic diuresis was observed by week 1 at ≥100 mg/kg/day in both genders. Histopathologic kidney changes were first detected after 4 weeks of dosing in the male 1,000 mg/kg/day dose group, with progressive increases in the incidence and/or number of findings in this dose group so that they were more readily detected during weeks 8 and 13. Changes detected starting on week 4 consisted of minimal single-cell necrosis and minimal increases in mitotic figures. These changes persisted at an increased incidence at weeks 8 and 13 and were accompanied by minimal to mild tubular epithelial karyomegaly, minimal proximal convoluted tubular epithelial cell hyperplasia, and a corresponding increase in Ki-67-positive nuclei in epithelial cells of the proximal convoluted tubules. There were no corresponding changes in serum chemistry or urinalysis parameters indicative of any physiologically meaningful effect on renal function and thus these findings were not considered to be adverse. Similar changes were not identified in lower-dose groups in males nor were they present in females of any dose group. RNA-sequencing analysis revealed male mouse-specific changes in kidney over 13 weeks of dosing at 1,000 mg/kg/day. Treatment-related changes included genes and pathways related to p53-regulated cell cycle and proliferation, transforming growth factor ß, oxidative stress, and renal injury and the number of genes with significant expression change dramatically increased at week 13. These treatment-related changes in genes and pathways were predominant in high-dose males and complemented the observed temporal renal tubular changes. Overall, these mouse investigative study results support the role of early empagliflozin-related degenerative/regenerative changes only observed in high-dose male CD-1 mice as a key contributing feature to a nongenotoxic mode of renal tumor pathogenesis.

A Predominant Oxidative Renal Metabolite of Empagliflozin in Male Mice Is Cytotoxic in Mouse Renal Tubular Cells but not Genotoxic.

In a previously reported CD-1 mouse 2-year carcinogenicity study with the sodium glucose cotransporter-2 inhibitor empagliflozin, an increased incidence of renal tubular adenomas and carcinomas was identified only in the male high-dose group. Follow-up investigative studies have shown that the renal tumors in male high-dose mice were preceded by a number of renal degenerative/regenerative findings. Prior cross-species in vitro metabolism studies using microsomes identified an oxidative metabolite (M466/2) predominantly formed in the male mouse kidney and which spontaneously degrades to a metabolite (M380/1) and reactive 4-OH crotonaldehyde (CTA). In order to further evaluate potential modes of action for empagliflozin-associated male mouse renal tumors, we report here a series of in vitro investigative toxicology studies conducted to evaluate the cytotoxic and genotoxic potential of empagliflozin and M466/2. To assess the cytotoxic potential of empagliflozin and M466/2, a primary mouse renal tubular epithelial (mRTE) cell model was used. In mRTE cells, M466/2-derived in vitro 4-OH CTA exposure was cytotoxic, while empagliflozin was not cytotoxic or mitogenic. Empagliflozin and M466/2 were not genotoxic, supporting an indirect mode of action for empagliflozin-associated male mouse renal tumorigenesis. In conclusion, these in vitro data show that M466/2-derived 4-OH CTA exposure is associated with cytotoxicity in renal tubule cells and may be involved in promoting compound-related in vivo renal metabolic stress and chronic low-level renal injury, in turn supporting a nongenotoxic mode of tumor pathogenesis specific to the male mouse.

A simple orchidometric method for the preliminary assessment of maturity status in male cynomolgus monkeys (Macaca fascicularis) used for nonclinical safety studies.

INTRODUCTION: The identification and use of mature male non-human primates in nonclinical toxicology studies could be important for evaluating candidate drugs for which the profile of toxicity may differ depending on sexual maturity. This investigation sought to establish operational criteria to complement the current standard of histological evaluation for defining sexual maturity in male cynomolgus monkeys (Macaca fascicularis) used for toxicology studies, and to identify a practical non-invasive measure to select mature males for study. METHOD: Retrospectively, the relationships between body weight, testicular weight and testis histology were established in control males (n=126) used in previous toxicology studies. Prospectively, testicular volumes were measured in-life by orchidometry using comparative scrotal palpation (n=23 males used for study), then compared to testicular weights measured at necropsy. RESULTS: Consistent with previous literature, a weak relationship was observed between body weight and testicular weight. There was, however, a very good relationship between testicular weight and histological maturation level, which was based upon microscopic examination of testes, epididymides and prostates. Orchidometric measurement of testicular volume was found to be a reasonable predictor of testicular weight and served to rapidly select sexually mature males for study, and a total testicular volume (left and right combined) of >20 ml correlated with the histological appearance of maturity. CONCLUSION: Based upon this preliminary exploratory study, the initial simple measurement of testicular volume by orchidometry may provide a non-invasive alternative approach for assessing the sexual maturity of male cynomolgus monkeys in research colonies or during toxicology studies that will require more thorough validation.

Bioluminescent Salmonella reverse mutation assay: a screen for detecting mutagenicity with high throughput attributes.

Here, we describe the development and evaluation of a novel bioluminescent high-throughput Salmonella reverse mutation assay applicable to the screening of large numbers of small molecules. The bioluminescent Salmonella assay utilizes genetically engineered standard Salmonella tester strains TA98 and TA100 expressing the lux(CDABE) operon from Xenorhabdus luminescence. In principle, the assay employs bioluminescence as a sensor of changes in bacterial metabolism associated with starvation or energy depletion effectively identifying colonies of histidine-independent revertant cells in a high-throughput fashion. The assay provides highly concordant data with the outcome in the standard Salmonella plate incorporation reverse mutation assay. Since the results of the standard Salmonella plate assay are required by various regulatory agencies for approval of new drugs, the bioluminescent Salmonella assay can be effectively used for prioritization of compounds in pharmaceutical drug discovery as well as the evaluation of environmental and industrial chemicals. Because of its high throughput attributes, the assay permits effective, fast and economical screening of a large series of structural analogs enabling the investigation of structure-activity relationships.

Genetic toxicity assessment: employing the best science for human safety evaluation Part VII: Why not start with a single test: a transformational alternative to genotoxicity hazard and risk assessment.

A transformational alternative for genotoxicity hazard and risk assessment is proposed to the current standard regulatory test battery. In principle, the proposed approach consists of a single in vitro test system with high genomic sequence homology to humans that addresses the relevant principal genetic lesions assessed in the current test battery. The single test system also possesses higher throughput attributes to permit the screening of large numbers of compounds and allow for an initial differentiation of genotoxic mechanisms (i.e., direct vs. indirect mechanisms) by how the hazard end point is measured. To differentiate compounds showing positive results, toxicogenomic analysis can be conducted to evaluate genotoxic mechanisms and further support risk assessment. Lastly, the results from the single test system can be followed up with a complementary in vivo assessment to establish mechanistic relevance at potential target tissues. Here, we propose the in vitro (yeast) DNA deletion (DEL) recombination assay as a single test alternative to the current genotoxicity test battery with a mechanistic follow up toxicogenomic analysis of genotoxic stress response as one approach that requires broader evaluation and validation. In this assay, intrachromosomal recombination events between a repeated DNA sequence lead to DNA deletions, which have been shown to be inducible by a variety of carcinogens including those both negative and positive in the standard Salmonella Ames assay. It is hoped that the general framework outlined along with this specific example will provoke broader interest to propose other potential test systems.

An evaluation of the sensitivity of the Ames assay to discern low-level mutagenic impurities.

Low level impurities often reside in active pharmaceutical ingredients (API). Some of these impurities are potentially genotoxic since reactive intermediates are used in the synthetic route for the production of API. Routine mutagenicity testing is conducted in support of clinical trials with the intent to identify genotoxic hazards associated with API. Depending on the amount of impurity present in the API tested, the potency of the impurities and the relative sensitivity of the Ames assay, it is possible that mutagenicity associated with the presence of genotoxic impurities could also be detected while testing API. Therefore, we evaluated published data and generated new information to understand the sensitivity of the Ames assay. Based on a literature survey of approximately 450 mutagens, it was estimated that 85% of mutagens are detected at concentrations of 250 microg/plate or less. Based on this estimate, most mutagens should be detected in an Ames assay testing API concentrations up to 5000 microg/plate if present at a 5% or greater concentration. Data from experiments where several direct and indirect-acting mutagens were spiked into representative API further support the literature-based evaluation. Some limitations of this approach, including toxicity of API and competing metabolism are discussed.

Strategy for genotoxicity testing--metabolic considerations.

The report from the 2002 International Workshop on Genotoxicity Tests (IWGT) Strategy Expert Group emphasized metabolic considerations as an important area to address in developing a common strategy for genotoxicity testing. A working group convened at the 2005 4th IWGT to discuss this area further and propose practical strategy recommendations. To propose a strategy, the working group reviewed: (1) the current status and deficiencies, including examples of carcinogens "missed" in genotoxicity testing, established shortcomings of the standard in vitro induced S9 activation system and drug metabolite case examples; (2) the current status of possible remedies, including alternative S9 sources, other external metabolism systems or genetically engineered test systems; (3) any existing positions or guidance. The working group established consensus principles to guide strategy development. Thus, a human metabolite of interest should be represented in genotoxicity and carcinogenicity testing, including evaluation of alternative genotoxicity in vitro metabolic activation or test systems, and the selection of a carcinogenicity test species showing appropriate biotransformation. Appropriate action triggers need to be defined based on the extent of human exposure, considering any structural knowledge of the metabolite, and when genotoxicity is observed upon in vitro testing in the presence of metabolic activation. These triggers also need to be considered in defining the timing of human pharmaceutical ADME assessments. The working group proposed two strategies to consider; a more proactive approach, which emphasizes early metabolism predictions to drive appropriate hazard assessment; and a retroactive approach to manage safety risks of a unique or "major" metabolite once identified and quantitated from human clinical ADME studies. In both strategies, the assessment of the genotoxic potential of a metabolite could include the use of an alternative or optimized in vitro metabolic activation system, or direct testing of an isolated or synthesized metabolite. The working group also identified specific areas where more data or experiences need to be gained to reach consensus. These included defining a discrete exposure action trigger for safety assessment and when direct testing of a metabolite of interest is warranted versus the use of an alternative in vitro activation system, a universal recommendation for the timing of human ADME studies for drug candidates and the positioning of metabolite structural knowledge (through in silico systems, literature, expert analysis) in supporting metabolite safety qualification. Lastly, the working group outlined future considerations for refining the initially proposed strategies. These included the need for further evaluation of the current in vitro genotoxicity testing protocols that can potentially perturb or reduce the level of metabolic activity (potential alterations in metabolism associated with both the use of some solvents to solubilize test chemicals and testing to the guidance limit dose), and proposing broader evaluations of alternative metabolic activation sources or engineered test systems to further challenge the suitability of (or replace) the current induced liver S9 activation source.

Informed toxicity assessment in drug discovery: systems-based toxicology.

Technological advances in the biological, chemical and in silico sciences have transformed many scientific disciplines, including toxicology. A vast new palate of toxicity testing tools is now available to investigators, enabling the generation of enormous amounts of data using only small amounts of test sample and at relatively low cost. In addition to these tools, the pharmaceutical industry has an urgent need for toxicity testing earlier in the process, based on the recognition that safety issues are the single largest cause of drug candidate attrition from development portfolios and the marketplace. However, along with the opportunity provided by new testing tools comes the dilemma of deciding which tools to use and, equally as important, when and why to use them. It may well be unwise to apply a new toxicity test or screening system simply because one can, as both false positive and false negative outcomes can quickly negate the value of a toxicity test system and may even have a net negative impact on drug discovery productivity. This can be true even of test systems that are considered to be 'validated' in the traditional sense. How then is an investigator or drug discovery organization to decide which of the new tools to use, and when to use them? Proposed herein is a strategy for identifying high-value toxicity testing systems and strategies based on program knowledge and informed decision-making. The decision to apply a certain toxicity testing system in this strategy is informed by knowledge of the pharmacological target, the chemical features of molecules active at the pharmacological target, and existing public domain or institutional learning. This 'fit-for-purpose' approach limits non-targeted or 'uninformed' toxicity screening to only those few test systems with high specificity, strong outcome concordance and molecular relevance to frequently encountered toxicity risks (eg, genotoxicity). Additional toxicity testing and screening is then conducted to address specific known or potential toxicity risks, based on existing knowledge of the target pharmacology and secondary pharmacology or chemical attributes with known or suspect risk, and by active 'interrogation' of both the target and active chemical moieties during the drug discovery process. This model for toxicity testing decision-making is illustrated by two case studies from recent experience.

The application of structure-based assessment to support safety and chemistry diligence to manage genotoxic impurities in active pharmaceutical ingredients during drug development.

Starting materials and intermediates used to synthesize pharmaceuticals are reactive in nature and may be present as impurities in the active pharmaceutical ingredient (API) used for preclinical safety studies and clinical trials. Furthermore, starting materials and intermediates may be known or suspected mutagens and/or carcinogens. Therefore, during drug development due diligence need be applied from two perspectives (1) to understand potential mutagenic and carcinogenic risks associated with compounds used for synthesis and (2) to understand the capability of synthetic processes to control genotoxic impurities in the API. Recently, a task force comprised of experts from pharmaceutical industry proposed guidance, with recommendations for classification, testing, qualification and assessing risk of genotoxic impurities. In our experience the proposed structure-based classification, has differentiated 75% of starting materials and intermediates as mutagenic and non-mutagenic with high concordance (92%) when compared with Ames results. Structure-based assessment has been used to identify genotoxic hazards, and prompted evaluation of fate of genotoxic impurities in API. These two assessments (safety and chemistry) culminate in identification of genotoxic impurities known or suspected to exceed acceptable levels in API, thereby triggering actions needed to assure appropriate control and measurement methods are in place. Hypothetical case studies are presented demonstrating this multi-disciplinary approach.