Magnitude of Urine Albumin and KIM-1 Changes Can be Used to Differentiate Glomerular Injury From Tubular Injury in Rats.

Emerging urinary kidney safety biomarkers have been evaluated in recent years and have been shown to be superior to the serum parameters blood urea nitrogen (BUN) and creatinine (sCr) for monitoring kidney injury in the proximal tubule. However, their potential application in differentiating the location of the initial kidney injury (eg, glomerulus vs tubule) has not been fully explored. Here, we assessed the performance of two algorithms that were constructed using either an empirical or a mathematical model to predict the site of kidney injury using a data set consisting of 22 rat kidney toxicity studies with known urine biomarker and histopathologic outcomes. Two kidney safety biomarkers used in both models, kidney injury molecule 1 (KIM-1) and albumin (ALB), were the best performers to differentiate glomerular injury from tubular injury. The performance of algorithms using these two biomarkers against the gold standard of kidney histopathologic examination showed high sensitivity in differentiating the location of the kidney damage to either the glomerulus or the proximal tubules. These data support the exploration of such an approach for use in clinical settings, leveraging urinary biomarker data to aid in the diagnosis of either glomerular or tubular injury where histopathologic assessments are not conducted.

Early-Onset albuminuria and Associated Renal Pathology in Leucine-Rich Repeat Kinase 2 Knockout Rats.

Activating mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are associated with Parkinson disease (PD), prompting development of LRRK2 inhibitors as potential treatment for PD. However, kidney safety concerns have surfaced from LRRK2 knockout (KO) mice and rats and from repeat-dose studies in rodents administered LRRK2 inhibitors. To support drug development of this therapeutic target, we conducted a study of 26 weeks' duration in 2-month-old wild-type and LRRK2 KO Long-Evans Hooded rats to systematically examine the performance of urinary safety biomarkers and to characterize the nature of the morphological changes in the kidneys by light microscopy and by ultrastructural evaluation. Our data reveal the time course of early-onset albuminuria at 3 and 4 months in LRRK2 KO female and male rats, respectively. The increases in urine albumin were not accompanied by concurrent increases in serum creatinine, blood urea nitrogen, or renal safety biomarkers such as kidney injury molecule 1 or clusterin, although morphological alterations in both glomerular and tubular structure were identified by light and transmission electron microscopy at 8 months of age. Diet optimization with controlled food intake attenuated the progression of albuminuria and associated renal changes.

Kidney Injury Monitoring in Tobramycin-Treated Rhesus Monkeys: Supplementing Urinary Kidney Biomarkers With Kidney Biopsy Gene Expression Profiling.

Kidney biopsies are used sparingly to diagnose kidney injury in the clinic. Here we have conducted a small exploratory study to directly compare the low-grade kidney injury monitoring performance of serum safety biomarkers, novel urine safety biomarkers, microscopic histopathology and targeted gene expression alterations in kidney biopsy specimens in rhesus monkeys treated with tobramycin. Targeted gene expression increases were observed in the kidney biopsy samples and whole kidney sections for kidney injury molecule 1 (KIM-1), clusterin (CLU), osteopontin (OPN) messenger RNA transcripts. In addition, increases of the urinary kidney safety protein biomarkers including KIM-1, CLU, OPN were also observed. These increases in gene expression and urinary protein end point were in concordance with the eventual low-grade kidney lesions seen in terminal tissue sections. In contrast, conventional serum biomarkers blood urea nitrogen and serum creatinine were not as sensitive in monitoring kidney injury. Although these data do not support routinely adding kidney biopsies to regular toxicology studies, they provide evidence on the value and limitations of incorporating gene expression profiling on kidney biopsy specimens, further underscore the value of urinary kidney safety biomarkers for improved low-grade kidney injury monitoring, and open the door for future definitive studies.

Survey of tumorigenic sensitivity in 6-month rasH2-Tg mice studies compared with 2-year rodent assays.

The pharmacokinetic endpoint of a 25-fold increase in human exposure is one of the specified criteria for high-dose selection for 2-year carcinogenicity studies in rodents according to ICH S1C(R2). However, this criterion is not universally accepted for 6-month carcinogenicity tests in rasH2-Tg mice. To evaluate an appropriate multiple for rasH2-Tg mice, we evaluated data for 53 compounds across five categories of rasH2-Tg mouse-positive [(1) genotoxic and (2) non-genotoxic] carcinogens and rasH2-Tg mouse-negative [(3) non-genotoxic carcinogens with clear or uncertain human relevance; (4) non-genotoxic rodent-specific carcinogens; and (5) non-carcinogens], and surveyed their tumorigenic activities and high doses in rasH2-Tg mice and 2-year rodent models. Our survey indicated that area under the curve (AUC) margins (AMs) or body surface area-adjusted dose ratios (DRs) of tumorigenesis in rasH2-Tg mice to the maximum recommended human dose (MRHD) were 0.05- to 5.2-fold in 6 category (1) compounds with small differences between models and 0.2- to 47-fold in 7 category (2) including three 2-year rat study-negative compounds. Among all 53 compounds, including 40 compounds of the rasH2-Tg mouse-negative category (3), (4), and (5), no histopathologic risk factors for rodent neoplasia were induced only at doses above 50-fold AM or DR in rasH2-Tg mice except for two compounds, which induced hyperplasia and had no relationship with the tumors observed in the rasH2-Tg mouse or 2-year rodent studies. From the results of these surveys, we confirmed that exceeding a high dose level of 50-fold AM in rasH2-Tg mouse carcinogenicity studies does not appear to be of value.

A Two-Tiered In Vitro Approach to De-Risk Drug Candidates for Potential Bile Salt Export Pump Inhibition Liabilities in Drug Discovery.

Hepatocellular accumulation of bile salts by inhibition of bile salt export pump (BSEP/ABCB11) may result in cholestasis and is one proposed mechanism of drug-induced liver injury (DILI). To understand the relationship between BSEP inhibition and DILI, we evaluated 64 DILI-positive and 57 DILI-negative compounds in BSEP, multidrug resistance protein (MRP) 2, MRP3, and MRP4 vesicular inhibition assays. An empirical cutoff (5 µM) for BSEP inhibition was established based on a relationship between BSEP IC50 values and the calculated maximal unbound concentration at the inlet of the human liver (fu*Iin,max, assay specificity = 98%). Including inhibition of MRP2-4 did not increase DILI predictivity. To further understand the potential to inhibit bile salt transport, a selected subset of 30 compounds were tested for inhibition of taurocholate (TCA) transport in a long-term human hepatocyte micropatterned co-culture (MPCC) system. The resulting IC50 for TCA in vitro biliary clearance and biliary excretion index (BEI) in MPCCs were compared with the compound's fu*Iin,max to assess potential risk for bile salt transport perturbation. The data show high specificity (89%). Nine out of 15 compounds showed an IC50 value in the BSEP vesicular assay of <5µM, but the BEI IC50 was more than 10-fold the fu*Iin,max, suggesting that inhibition of BSEP in vivo is unlikely. The data indicate that although BSEP inhibition measured in membrane vesicles correlates with DILI risk, that measurement of this assay activity is insufficient. A two-tiered strategy incorporating MPCCs is presented to reduce BSEP inhibition potential and improve DILI risk. SIGNIFICANCE STATEMENT: This work describes a two-tiered in vitro approach to de-risk compounds for potential bile salt export pump inhibition liabilities in drug discovery utilizing membrane vesicles and a long-term human hepatocyte micropatterned co-culture system. Cutoffs to maximize specificity were established based on in vitro data from a set of 121 DILI-positive and -negative compounds and associated calculated maximal unbound concentration at the inlet of the human liver based on the highest clinical dose.

Evaluation of 10 Urinary Biomarkers for Renal Safety With 5 Nephrotoxicants in Nonhuman Primates.

To date, there has been very little published data evaluating the performance of novel urinary kidney biomarkers in nonhuman primates (NHPs). To assess the biomarker performance and characterize the corresponding histomorphologic patterns of tubular renal injury in the NHP, several studies were conducted using mechanistically diverse nephrotoxicants including cefpirome, cisplatin, naproxen, cyclosporine, and a combination of gentamicin with everninomicin. An evaluation of 10 urinary biomarkers (albumin, clusterin, cystatin C, kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, liver-type fatty acid-binding protein, N-acetyl-ß-D-glucosaminidase, osteopontin, retinol binding protein 4 and total protein) was performed on urine collected from these studies. Each of these 5 treatments resulted in kidney proximal tubule injury of various severities. Histomorphologic features observed following treatment were generally consistent with analogous drug-induced changes in humans described in the literature. Most of the analyzed biomarkers were able to detect the injury earlier and with greater sensitivity than blood urea nitrogen and serum creatinine. Across all studies, KIM-1 and clusterin showed the highest overall performance. Differences in the patterns of biomarker responsiveness were noted among certain studies that may be informing tubular injury severity and recovery potential, underlying histopathologic processes, and prognosis. These findings demonstrate the utility of urinary kidney translational safety biomarkers in NHPs and provide additional supporting evidence for translating these biomarkers for use in clinical trial settings to further ensure patient safety.

Tg.rasH2 Mouse Model for Assessing Carcinogenic Potential of Pharmaceuticals: Industry Survey of Current Practices.

The Tg.rasH2 mouse was developed as an alternative model to the traditional 2-year mouse bioassay for pharmaceutical carcinogenicity testing. This model has found extensive use in support of pharmaceutical drug development over the last few decades. It has the potential to improve quality and timeliness, reduce animal usage, and in some instances allow expedient decision-making regarding the human carcinogenicity potential of a drug candidate. Despite the increased use of the Tg.rasH2 model, there has been no systematic survey of current practices in the design, interpretation of results from the bioassay, and global health authority perspectives. Therefore, the aim of this work was to poll the pharmaceutical industry on study design practices used in the dose range finding and definitive 6-month studies and on results relative to the ongoing negotiations to revise The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S1 Guidance. Twenty-two member companies of International Consortium for Innovation and Quality in Pharmaceutical Development DruSafe Leadership Group participated in the survey, sharing experiences from studies conducted with 55 test compounds between 2010 and 2018. The survey results provide very useful insights into study design and interpretation. Importantly, the results identified several key opportunities for reducing animal use and increasing the value of testing for potential human carcinogenicity using this model. Recommended changes to study designs that would reduce animal usage include eliminating the requirement to include positive control groups in every study, use of nontransgenic wild-type littermates in the dose range finding study, and use of microsampling to reduce or eliminate satellite groups for toxicokinetics.

Can Galactose Be Converted to Glucose in HepG2 Cells? Improving the in Vitro Mitochondrial Toxicity Assay for the Assessment of Drug Induced Liver Injury.

Human hepatocellular carcinoma cells, HepG2, are often used for drug mediated mitochondrial toxicity assessments. Glucose in HepG2 culture media is replaced by galactose to reveal drug-induced mitochondrial toxicity as a marked shift of drug IC50 values for the reduction of cellular ATP. It has been postulated that galactose sensitizes HepG2 mitochondria by the additional ATP consumption demand in the Leloir pathway. However, our NMR metabolomics analysis of HepG2 cells and culture media showed very limited galactose metabolism. To clarify the role of galactose in HepG2 cellular metabolism, U-13C6-galactose or U-13C6-glucose was added to HepG2 culture media to help specifically track the metabolism of those two sugars. Conversion to U-13C3-lactate was hardly detected when HepG2 cells were incubated with U-13C6-galactose, while an abundance of U-13C3-lactate was produced when HepG2 cells were incubated with U-13C6-glucose. In the absence of glucose, HepG2 cells increased glutamine consumption as a bioenergetics source. The requirement of additional glutamine almost matched the amount of glucose needed to maintain a similar level of cellular ATP in HepG2 cells. This improved understanding of galactose and glutamine metabolism in HepG2 cells helped optimize the ATP-based mitochondrial toxicity assay. The modified assay showed 96% sensitivity and 97% specificity in correctly discriminating compounds known to cause mitochondrial toxicity from those with prior evidence of not being mitochondrial toxicants. The greatest significance of the modified assay was its improved sensitivity in detecting the inhibition of mitochondrial fatty acid ß-oxidation (FAO) when glutamine was withheld. Use of this improved assay for an empirical prediction of the likely contribution of mitochondrial toxicity to human DILI (drug induced liver injury) was attempted. According to testing of 65 DILI positive compounds representing numerous mechanisms of DILI together with 55 DILI negative compounds, the overall prediction of mitochondrial mechanism-related DILI showed 25% sensitivity and 95% specificity.

Adverse outcome pathway-driven identification of rat liver tumorigens in short-term assays.

Chemicals induce liver cancer in rodents through well characterized adverse outcome pathways (AOPs). We hypothesized that measurement of molecular initiating events (MIEs) and downstream key events (KEs) in liver cancer AOPs in short-term assays will allow early identification of chemicals and their associated doses that are likely to be tumorigenic in the liver in two-year bioassays. We tested this hypothesis using the rat in vivo TG-GATES study data to measure MIEs (genotoxicity, cytotoxicity, AhR, CAR, ER, PPARα) and associated KEs (oxidative stress, cell proliferation, liver to body weights) across 77 chemicals that could be linked to doses with previously established effects on rat liver tumor induction. Gene expression biomarkers for MIEs generally considered to be rodent specific and human irrelevant (CAR, PPARα) and for MIEs that would be considered of greater risk at human relevant exposures (ER, AhR) were built using microarray comparisons from the livers of rats treated with prototypical activators of the receptors. The genotoxicity biomarker, also a potentially human relevant MIE, was comprised of 7 p53-responsive genes known to be induced upon DNA damage. The ability of the biomarkers to accurately predict MIE activation ranged from 91% to 98%. The Toxicological Priority Index (ToxPi) was used to rank chemicals based on their ability to activate MIEs/KEs. Chemicals administered at tumorigenic doses clearly gave the highest ranked scores. Our AOP-directed approach could be used in short term assays to identify chemicals and their doses that would be predicted to cause liver tumors in rats.

Performance Assessment of New Urinary Translational Safety Biomarkers of Drug-induced Renal Tubular Injury in Tenofovir-treated Cynomolgus Monkeys and Beagle Dogs.

Newer urinary protein kidney safety biomarkers can outperform the conventional kidney functional biomarkers blood urea nitrogen (BUN) and serum creatinine (SCr) in rats. However, there is far less experience with the relative performance of these biomarkers in dogs and nonhuman primates. Here, we report urine protein biomarker performance in tenofovir-treated cynomolgus monkeys and beagle dogs. Tenofovir intravenous daily dosing in monkeys for 2 or 4 weeks at 30 mg/kg/day resulted in minimal to moderate tubular degeneration and regeneration, and tenofovir disoproxil fumarate oral dosing in dogs for 10 days at 45 mg/kg/day resulted in mild to marked tubular degeneration, necrosis, and regeneration. Among biomarkers tested, kidney injury molecule 1 (Kim-1) and clusterin (CLU) clearly outperformed BUN and SCr and were the most reliable in detecting the onset and progression of tenofovir-induced tubular injury. Cystatin C, retinol binding protein 4, ß2-microglobulin, neutrophil gelatinase-associated lipocalin, albumin, and total protein also performed better than BUN and SCr and added value when considered together with Kim-1 and CLU. These findings demonstrate the promising utility of these urinary safety biomarkers in monkeys and dogs and support their further evaluation in human to improve early detection of renal tubular injury.

Vascular Imaging of Matrix Metalloproteinase Activity as an Informative Preclinical Biomarker of Drug-induced Vascular Injury.

Lack of biomarkers specific to and either predictive or diagnostic of drug-induced vascular injury (DIVI) continues to be a major obstacle during drug development. Biomarkers derived from physiologic responses to vessel injury, such as inflammation and vascular remodeling, could make good candidates; however, they characteristically lack specificity for vasculature. We evaluated whether vascular remodeling-associated protease activity, as well as changes to vessel permeability resulting from DIVI, could be visualized ex vivo in affected vessels, thereby allowing for visual monitoring of the pathology to address specificity. We found that visualization of matrix metalloproteinase activation accompanied by increased vascular leakage in the mesentery of rats treated with agents known to induce vascular injury correlated well with incidence and severity of histopathological findings and associated inflammation as well as with circulating levels of tissue inhibitors of metalloproteinase 1 and neutrophil gelatinase-associated lipocalin. The weight of evidence approach reported here shows promise as a composite DIVI preclinical tool by means of complementing noninvasive monitoring of circulating biomarkers of inflammation with direct imaging of affected vasculature and thus lending specificity to its interpretation. These findings are supportive of a potential strategy that relies on translational imaging tools in conjunction with circulating biomarker data for high-specificity monitoring of VI both preclinically and clinically.

Role of chronic toxicology studies in revealing new toxicities.

Chronic (>3 months) preclinical toxicology studies are conducted to support the safe conduct of clinical trials exceeding 3 months in duration. We have conducted a review of 32 chronic toxicology studies in non-rodents (22 studies in dogs and 10 in non-human primates) and 27 chronic toxicology studies in rats dosed with Merck compounds to determine the frequency at which additional target organ toxicities are observed in chronic toxicology studies as compared to subchronic studies of 3 months in duration. Our review shows that majority of the findings are observed in the subchronic studies since additional target organs were not observed in 24 chronic non rodent studies and in 21 chronic rodent studies. However, 6 studies in non rodents and 6 studies in rodents yielded new findings that were not seen in studies of 3-month or shorter duration. For 3 compounds the new safety findings did contribute to termination of clinical development plans. Although the incidence of compound termination associated with chronic toxicology study observations is low (∼10%), the observations made in these studies can be important for evaluating human safety risk.

Regulatory Forum Commentary* Counterpoint: Dose Selection for Tg.rasH2 Mouse Carcinogenicity Studies.

High-dose selection for 6-month carcinogenicity studies of pharmaceutical candidates in Tg.rasH2-transgenic mice currently primarily relies on (1) estimation of a maximum tolerated dose (MTD) from the results of a 1-month range-finding study, (2) determination of the maximum dose administrable to the animals (maximum feasible dose [MFD]), (3) demonstration of a plateau in systemic exposure, and (4) use of a limit dose of 1,500 mg/kg/day for products with human daily doses not exceeding 500 mg. Eleven 6-month Tg.rasH2 carcinogenicity studies and their corresponding 1-month range-finding studies conducted at Merck were reviewed. High doses were set by estimation of the MTD in 6, by plateau of exposure in 3, and by MFD in 2 cases. For 4 of 6 studies where MTD was used for high-dose selection, the 1-month study accurately predicted the 6-month study tolerability whereas in the remaining 2 studies the high doses showed poorer tolerability than expected. The use of 3 or more drug-treated dose levels proved useful to ensure that a study would successfully and unambiguously demonstrate that a drug candidate was adequately evaluated for carcinogenicity at a minimally toxic high dose level, especially when the high dose may be found to exceed the MTD.

Regulatory Forum commentary: alternative mouse models for future cancer risk assessment.

International regulatory and pharmaceutical industry scientists are discussing revision of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S1 guidance on rodent carcinogenicity assessment of small molecule pharmaceuticals. A weight-of-evidence approach is proposed to determine the need for rodent carcinogenicity studies. For compounds with high human cancer risk, the product may be labeled appropriately without conducting rodent carcinogenicity studies. For compounds with minimal cancer risk, only a 6-month transgenic mouse study (rasH2 mouse or p53+/- mouse) or a 2-year mouse study would be needed. If rodent carcinogenicity testing may add significant value to cancer risk assessment, a 2-year rat study and either a 6-month transgenic mouse or a 2-year mouse study is appropriate. In many cases, therefore, one rodent carcinogenicity study could be sufficient. The rasH2 model predicts neoplastic findings relevant to human cancer risk assessment as well as 2-year rodent models, produces fewer irrelevant neoplastic outcomes, and often will be preferable to a 2-year rodent study. Before revising ICH S1 guidance, a prospective evaluation will be conducted to test the proposed weight-of-evidence approach. This evaluation offers an opportunity for a secondary analysis comparing the value of alternative mouse models and 2-year rodent studies in the proposed ICH S1 weight-of-evidence approach for human cancer risk assessment.

ICH S1 prospective evaluation study and weight of evidence assessments: commentary from industry representatives.

Industry representatives on the ICH S1B(R1) Expert Working Group (EWG) worked closely with colleagues from the Drug Regulatory Authorities to develop an addendum to the ICH S1B guideline on carcinogenicity studies that allows for a weight-of-evidence (WoE) carcinogenicity assessment in some cases, rather than conducting a 2-year rat carcinogenicity study. A subgroup of the EWG composed of regulators have published in this issue a detailed analysis of the Prospective Evaluation Study (PES) conducted under the auspices of the ICH S1B(R1) EWG. Based on the experience gained through the Prospective Evaluation Study (PES) process, industry members of the EWG have prepared the following commentary to aid sponsors in assessing the standard WoE factors, considering how novel investigative approaches may be used to support a WoE assessment, and preparing appropriate documentation of the WoE assessment for presentation to regulatory authorities. The commentary also reviews some of the implementation challenges sponsors must consider in developing a carcinogenicity assessment strategy. Finally, case examples drawn from previously marketed products are provided as a supplement to this commentary to provide additional examples of how WoE criteria may be applied. The information and opinions expressed in this commentary are aimed at increasing the quality of WoE assessments to ensure the successful implementation of this approach.

A mechanistic biomarker investigation of fialuridine hepatotoxicity using the chimeric TK-NOG Hu-liver mouse model and in vitro micropatterned hepatocyte cocultures.

Fialuridine (FIAU) is a nucleoside-based drug that caused liver failure and deaths in a human clinical trial that were not predicted by nonclinical safety studies. A recent report concluded that a TK-NOG humanized liver (hu-liver) mouse model detected human-specific FIAU liver toxicity, and broader use of that model could improve drug safety testing. We further evaluated this model at similar dose levels to assess FIAU sensitivity and potential mechanistic biomarkers. Although we were unable to reproduce the marked acute liver toxicity with two separate studies (including one with a "sensitized" donor), we identified molecular biomarkers reflecting the early stages of FIAU mitochondrial toxicity, which were not seen with its stereoisomer (FIRU). Dose dependent FIAU-induced changes in hu-liver mice included more pronounced reductions in mitochondrial to nuclear DNA (mtDNA/nucDNA) ratios in human hepatocytes compared to mouse hepatocytes and kidneys of the same animals. FIAU treatment also triggered a p53 transcriptional response and opposing changes in transcripts of nuclear- and mitochondrial-encoded mitochondrial proteins. The time dependent accumulation of FIAU into mtDNA is consistent with the ≥9-week latency of liver toxicity observed for FIAU in the clinic. Similar changes were observed in an in vitro micro-patterned hepatocyte coculture system. In addition, FIAU-dependent mtDNA/nucDNA ratio and transcriptional alterations, especially reductions in mitochondrially encoded transcripts, were seen in livers of non-engrafted TK-NOG and CD-1 mice dosed for a shorter period. Conclusion: These mechanistic biomarker findings can be leveraged in an in vitro model and in a more routine preclinical model (CD-1 mice) to identify nucleosides with such a FIAU-like mitochondrial toxicity mechanistic liability potential. Further optimization of the TK-NOG hu-liver mouse model is necessary before broader adoption for drug safety testing.

Inhibin B response to testicular toxicants hexachlorophene, ethane dimethane sulfonate, di-(n-butyl)-phthalate, nitrofurazone, DL-ethionine, 17-alpha ethinylestradiol, 2,5-hexanedione, or carbendazim following short-term dosing in male rats.

BACKGROUND: Inhibin B is a heterodimer glycoprotein that downregulates follicle-stimulating hormone and is produced predominantly by Sertoli cells. The potential correlation between changes in plasma Inhibin B and Sertoli cell toxicity was evaluated in male rats administered testicular toxicants in eight studies. Inhibin B fluctuations over 24 hr were also measured. METHODS: Adult rats were administered one of eight testicular toxicants for 1 to 29 days. The toxicants were DL-ethionine, dibutyl phthalate, nitrofurazone, 2,5-hexanedione, 17-alpha ethinylestradiol, ethane dimethane sulfonate, hexachlorophene, and carbendazim. In a separate study plasma was collected throughout a 24-hr period via an automatic blood sampler. RESULTS: Histomorphologic testicular findings included seminiferous tubule degeneration, round and elongate spermatid degeneration/necrosis, seminiferous tubule vacuolation, aspermatogenesis, and interstitial cell degeneration. There was a varying response of plasma Inhibin B levels to seminiferous tubule toxicity, with three studies showing high correlation, three studies with a response only at a certain time or dose, and two studies with no Inhibin B changes. In a receiver operating characteristics exclusion model analysis, where treated samples without histopathology were excluded, Inhibin B showed a sensitivity of 70% at 90% specificity in studies targeting seminiferous tubule toxicity. CONCLUSION: Decreases in Inhibin B correlated with Sertoli cell toxicity in the majority of studies evaluated, demonstrating the value of Inhibin B as a potential biomarker of testicular toxicity. There was no correlation between decreases in Inhibin B and interstitial cell degeneration. In addition, a pattern of Inhibin B secretion could not be identified over 24 hr.

Medical applications of microarray technologies: a regulatory science perspective.

The potential medical applications of microarrays have generated much excitement, and some skepticism, within the biomedical community. Some researchers have suggested that within the decade microarrays will be routinely used in the selection, assessment, and quality control of the best drugs for pharmaceutical development, as well as for disease diagnosis and for monitoring desired and adverse outcomes of therapeutic interventions. Realizing this potential will be a challenge for the whole scientific community, as breakthroughs that show great promise at the bench often fail to meet the requirements of clinicians and regulatory scientists. The development of a cooperative framework among regulators, product sponsors, and technology experts will be essential for realizing the revolutionary promise that microarrays hold for drug development, regulatory science, medical practice and public health.

Using human genetics to improve safety assessment of therapeutics.

Human genetics research has discovered thousands of proteins associated with complex and rare diseases. Genome-wide association studies (GWAS) and studies of Mendelian disease have resulted in an increased understanding of the role of gene function and regulation in human conditions. Although the application of human genetics has been explored primarily as a method to identify potential drug targets and support their relevance to disease in humans, there is increasing interest in using genetic data to identify potential safety liabilities of modulating a given target. Human genetic variants can be used as a model to anticipate the effect of lifelong modulation of therapeutic targets and identify the potential risk for on-target adverse events. This approach is particularly useful for non-clinical safety evaluation of novel therapeutics that lack pharmacologically relevant animal models and can contribute to the intrinsic safety profile of a drug target. This Review illustrates applications of human genetics to safety studies during drug discovery and development, including assessing the potential for on- and off-target associated adverse events, carcinogenicity risk assessment, and guiding translational safety study designs and monitoring strategies. A summary of available human genetic resources and recommended best practices is provided. The challenges and future perspectives of translating human genetic information to identify risks for potential drug effects in preclinical and clinical development are discussed.

Universal Accessible Biomarkers of Drug-Induced Tissue Injury and Systemic Inflammation in Rat: Performance Assessment of TIMP-1, A2M, AGP, NGAL, and Albumin.

The ability to monitor for general drug-induced tissue injury (DITI) or systemic inflammation in any tissue using blood-based accessible biomarkers would provide a valuable tool in early exploratory animal studies to understand potential drug liabilities. Here we describe the evaluation of 4 biomarkers of tissue remodeling and inflammation (α2-macroglobulin [A2M], α1-acid glycoprotein [AGP], neutrophil gelatinase-associated lipocalin [NGAL], and tissue inhibitor of metalloproteinases [TIMP-1]) as well as the traditional serum parameter albumin as potential blood-based biomarkers of DITI and systemic inflammatory response (SIR). Biomarker performance was assessed in 51 short-term rat in vivo studies with various end-organ toxicities or SIR and receiver operating characteristic curves were generated to compare relative performances. All 4 biomarkers performed well in their ability to detect DITI and SIR with an area under the curve (AUC) of 0.82-0.78, however TIMP-1 achieved the best sensitivity (at 95% specificity) of 61%; AGP, NGAL, and A2M sensitivity was 51%-52%. AUC for albumin was 0.72 with sensitivity of 39%. A2M was the best performer in studies with only SIR (AUC 0.91). In the subset of studies with drug-induced vascular injury, TIMP-1 performed best with an AUC of 0.96. Poor performance of all tested biomarkers was observed in samples with CNS toxicity. In summary, TIMP-1, A2M, AGP, and NGAL demonstrated performance as sensitive accessible biomarkers of DITI and SIR, supporting their potential application as universal accessible tissue toxicity biomarkers to quickly identify dose levels associated with drug-induced injury in early exploratory rat safety and other studies.