RESUMO
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.
Assuntos
Estudo de Associação Genômica Ampla , Genética Humana , Animais , HumanosRESUMO
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.
Assuntos
Injúria Renal Aguda , alfa 2-Macroglobulinas Associadas à Gravidez , Albuminas , Animais , Biomarcadores , Feminino , Inflamação , Lipocalina-2 , Orosomucoide/metabolismo , Gravidez , Curva ROC , Ratos , Inibidor Tecidual de Metaloproteinase-1RESUMO
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.
Assuntos
Testes de Carcinogenicidade/métodos , Carcinógenos/toxicidade , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Indústria Farmacêutica/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Animais , Bioensaio , Genes ras , Camundongos Transgênicos , Projetos de Pesquisa , Inquéritos e QuestionáriosRESUMO
The robust transcriptional plasticity of liver mediated through xenobiotic receptors underlies its ability to respond rapidly and effectively to diverse chemical stressors. Thus, drug-induced gene expression changes in liver serve not only as biomarkers of liver injury, but also as mechanistic sentinels of adaptation in metabolism, detoxification, and tissue protection from chemicals. Modern RNA sequencing methods offer an unmatched opportunity to quantitatively monitor these processes in parallel and to contextualize the spectrum of dose-dependent stress, adaptation, protection, and injury responses induced in liver by drug treatments. Using this approach, we profiled the transcriptional changes in rat liver following daily oral administration of 120 different compounds, many of which are known to be associated with clinical risk for drug-induced liver injury by diverse mechanisms. Clustering, correlation, and linear modeling analyses were used to identify and optimize coexpressed gene signatures modulated by drug treatment. Here, we specifically focused on prioritizing 9 key signatures for their pragmatic utility for routine monitoring in initial rat tolerability studies just prior to entering drug development. These signatures are associated with 5 canonical xenobiotic nuclear receptors (AHR, CAR, PXR, PPARα, ER), 3 mediators of reactive metabolite-mediated stress responses (NRF2, NRF1, P53), and 1 liver response following activation of the innate immune response. Comparing paradigm chemical inducers of each receptor to the other compounds surveyed enabled us to identify sets of optimized gene expression panels and associated scoring algorithms proposed as quantitative mechanistic biomarkers with high sensitivity, specificity, and quantitative accuracy. These findings were further qualified using public datasets, Open TG-GATEs and DrugMatrix, and internal development compounds. With broader collaboration and additional qualification, the quantitative toxicogenomic framework described here could inform candidate selection prior to committing to drug development, as well as complement and provide a deeper understanding of the conventional toxicology study endpoints used later in drug development.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Desenvolvimento de Medicamentos , Fígado/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Transcriptoma , Xenobióticos/toxicidade , Animais , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Fígado/metabolismo , Fígado/patologia , Masculino , Ratos Sprague-Dawley , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Medição de Risco , Transdução de Sinais , Testes de Toxicidade , Toxicogenética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Galactose/metabolismo , Glucose/metabolismo , Mitocôndrias Hepáticas/metabolismo , Amiodarona/farmacologia , Benzobromarona/farmacologia , Células Hep G2 , Humanos , Metabolômica , Mitocôndrias Hepáticas/efeitos dos fármacos , Piperazinas/farmacologia , Triazóis/farmacologia , Troglitazona/farmacologia , Células Tumorais CultivadasRESUMO
The kidney's role as a major route of metabolism and clearance of xenobiotics and its ability to concentrate the glomerular filtrate make it particularly vulnerable to drug-induced toxicity. Improving kidney safety is an active area of research and there is a need in early stages of drug development for strategies and model systems to reliably identify nephrotoxic compounds and sufficiently characterize mechanisms to support drug pipeline decision making. In later stages of drug development the value of sensitive translational biomarkers to monitor kidney toxicity across species in nonclinical and clinical settings is gaining realization. Various tools and strategies for kidney safety assessment have emerged over the past decade; however, there is currently no clear consensus on best practices for their use across different phases of drug development. Here, we provide perspective on the scope of this problem in drug development, and an overview of progress in the field of kidney safety including several informative case examples of kidney toxicity de-risking scenarios encountered in the pharmaceutical industry. The results of a survey of pharmaceutical companies conducted through the Innovation and Quality Drug Safety consortium provides additional insight into recent experiences with compound attrition and different de-risking approaches across the industry.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Desenvolvimento de Medicamentos , Insuficiência Renal Crônica/induzido quimicamente , Ensaios Clínicos como Assunto , Humanos , Modelos Animais , Medição de RiscoRESUMO
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.
Assuntos
Injúria Renal Aguda/urina , Biomarcadores/urina , Túbulos Renais/efeitos dos fármacos , Tenofovir/toxicidade , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Administração Oral , Animais , Biomarcadores/sangue , Cães , Feminino , Injeções Intravenosas , Túbulos Renais/patologia , Macaca fascicularis , Masculino , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
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 (â¼10%), the observations made in these studies can be important for evaluating human safety risk.
Assuntos
Testes de Toxicidade Crônica/métodos , Testes de Toxicidade Subcrônica/métodos , Animais , Bases de Dados Factuais , Relação Dose-Resposta a Droga , Indústria Farmacêutica , Humanos , Modelos Animais , Reprodutibilidade dos Testes , Medição de Risco , Especificidade da Espécie , Fatores de TempoRESUMO
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.
Assuntos
Testes de Carcinogenicidade/normas , Modelos Animais de Doenças , Neoplasias/tratamento farmacológico , Medição de Risco , Animais , Avaliação Pré-Clínica de Medicamentos , Indústria Farmacêutica , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Camundongos , Camundongos TransgênicosRESUMO
One promise of new translational safety biomarkers (TSBs) is their ability to demonstrate that toxicities in animal studies are monitorable at an early stage, such that human relevance of potential adverse effects of drugs can be safely and definitively evaluated in clinical trials. Another is that they would provide an earlier, more definitive and deeper insight to patient prognosis compared with conventional biomarkers. Recent experience with regulatory authorities indicates that resource demands for new TSB qualifications under the current framework are daunting and the rate of their expansion will be slow, particularly in light of mounting financial pressures on the pharmaceutical industry. Sponsors face a dilemma over engaging in safety biomarker qualification consortia. While it is clear new TSBs could be considered catalysts to drug development and that patient health, business and scientific benefits, described here using examples, should outweigh qualification costs, concerns exist that early ambiguities in biomarker interpretations at the introduction of such new TSBs might hinder drug development.
Assuntos
Biomarcadores Farmacológicos/metabolismo , Animais , Ensaios Clínicos como Assunto/economia , Ensaios Clínicos como Assunto/métodos , Avaliação Pré-Clínica de Medicamentos/economia , Indústria Farmacêutica/economia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Regulamentação Governamental , Humanos , Preparações Farmacêuticas/economia , RatosAssuntos
Biomarcadores , Pesquisa Biomédica , Descoberta de Drogas/normas , Preparações Farmacêuticas , Indústria Farmacêutica , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Europa (Continente) , Humanos , Rim/efeitos dos fármacos , Rim/lesões , Preparações Farmacêuticas/normas , Estados Unidos , United States Food and Drug Administration , UniversidadesRESUMO
International guidelines allow for use of a short-term cancer bioassay (twenty-six weeks) in transgenic mice as a substitute for one of the two required long-term rodent bioassays in the preclinical safety evaluation of pharmaceuticals. The two models that have gained the widest acceptance by sponsors and regulatory authorities are the CB6F1-RasH2 mouse hemizygous for a human H-ras transgene and the B6.129N5-Trp53 mouse heterozygous for a p53 null allele. The p53(+/-) model is of particular value for compounds with residual concern that genotoxic activity may contribute to tumorigenesis. The rasH2 model is an appropriate alternative without regard to evidence of genotoxic potential. Since results from a short-term bioassay can be obtained relatively early in drug development, there is the potential for more timely assessment of cancer risk for individuals in long-term clinical trials. Use of these models in preclinical safety evaluation also significantly reduces animal use, time, and manpower. Preliminary findings indicate that prediction of two-year rat bioassay outcomes based on data from chronic rat toxicity studies, together with early assessment of carcinogenic potential in short-term transgenic models, may have the potential to increase the timeliness and efficiency of strategies for the identification of human carcinogenic hazards.
Assuntos
Testes de Carcinogenicidade/métodos , Indústria Farmacêutica , Camundongos Transgênicos , Animais , Modelos Animais de Doenças , Descoberta de Drogas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Camundongos , Ratos , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Species differences in drug metabolism present two challenges that may confound the nonclinical safety assessment of candidate drugs. The first challenge is encountered when metabolites are formed uniquely or disproportionately in humans. Another challenge is understanding the human relevance of toxicities associated with metabolites formed uniquely or disproportionately in a nonclinical species. One potential approach to minimize the impact of metabolite related challenges is to consider genetically engineered mouse models that express human P450 enzymes. Human P450 expressing mouse models may have the ability to generate major human metabolites and eliminate or reduce the formation of mouse specific metabolites. Prior to determining the utility of any particular model, it is important to qualify by characterizing protein expression, establishing whether the model generates an in vivo metabolite profile more closely related to that of humans than the wild-type mouse, verifying genetic stability, and evaluating animal health. When compared to the current strategy for handling metabolite challenges (i.e., direct administration of metabolite), identifying an appropriate human P450 expressing model could provide a number of benefits. Such benefits include improved scientific relevance of the evaluation, decreased resource needs, and a possible reduction in the number of animals used. These benefits may ultimately improve the quality and speed by which promising new drug candidates are developed and delivered to patients.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Camundongos Transgênicos , Modelos Animais , Preparações Farmacêuticas/metabolismo , Testes de Toxicidade/métodos , Administração Oral , Animais , Custos e Análise de Custo , Sistema Enzimático do Citocromo P-450/genética , Cães , Avaliação Pré-Clínica de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Guias como Assunto , Humanos , Legislação de Medicamentos , Camundongos , Camundongos Knockout , Ratos , Medição de Risco , Testes de Toxicidade/economiaRESUMO
Determining the carcinogenic potential of materials to which humans have significant exposure is an important, complex and imperfect exercise. Not only are the methods for such determinations protracted, expensive and utilize large numbers of animals, extrapolation of data from such studies to human risk is imprecise. Toxicologists have long recognized these shortcomings but the 2-year chronic rodent study has remained the gold standard. Recent developments in the field of molecular oncology and development of methods to insert or inactivate specific genes in animals have provided the tools with which to develop the next generation of carcinogenicity assays. With improved understanding of oncogene activation and tumor suppressor gene inactivation a number of animal models have been developed to dramatically reduce latency for chemically induced cancers. This has led to the development of shorter carcinogenicity assays. Also, because the spontaneous tumor frequencies in these animals are low during the in-life portion of the study, and studies are terminated well before the health complications of advanced aging are observed, it has been possible to reduce the group sizes and reduce animal usage. FDA's adoption of ICH S1B in 1997, (ICH, 1997) "Testing for the Carcinogenicity of Pharmaceuticals," opened the door for the use of such transgenic models in regulatory toxicology. This presentation reviews the current state of the science and its application to regulatory issues.
Assuntos
Testes de Carcinogenicidade/métodos , Carcinógenos/toxicidade , Neoplasias Experimentais/induzido quimicamente , Medição de Risco/tendências , Toxicologia/métodos , Animais , Modelos Animais de Doenças , Regulamentação Governamental , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genética , Estados Unidos , United States Food and Drug AdministrationRESUMO
Microarrays allow for the simultaneous measurement of changes in the levels of thousands of messenger RNAs within a single experiment. As such, the potential for the application of transcription profiling to preclinical safety assessment and mechanism-based risk assessment is profound. However, several practical and technical challenges remain. Among these are nomenclature issues, platform-specific data formats, and the lack of uniform analysis methods and tools. Experiments were designed to address biological, technical, and methodological variability, to evaluate different approaches to data analysis, and to understand the application of the technology to other profiling methodologies and to mechanism-based risk assessment. These goals were addressed using experimental information derived from analysis of the biological response to three mechanistically distinct nephrotoxins: cisplatin, gentamicin, and puromycin aminonucleoside. In spite of the technical challenges, the transcription profiling data yielded mechanistically and topographically valuable information. The analyses detailed in the articles from the Nephrotoxicity Working Group of the International Life Sciences Institute Health and Environmental Sciences Institute suggest at least equal sensitivity of microarray technology compared to traditional end points. Additionally, microarray analysis of these prototypical nephrotoxicants provided an opportunity for the development of candidate bridging biomarkers of nephrotoxicity. The potential future extension of these applications for risk assessment is also discussed.
Assuntos
Perfilação da Expressão Gênica , Rim/efeitos dos fármacos , Rim/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Antibacterianos/toxicidade , Antimetabólitos Antineoplásicos/toxicidade , Cisplatino/toxicidade , Relação Dose-Resposta a Droga , Gentamicinas/toxicidade , Masculino , Puromicina/toxicidade , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Medição de RiscoRESUMO
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.