The role of early in vivo toxicity testing in drug discovery toxicology

The cost impact of late-stage failures of drug candidates has motivated the pharmaceutical industry to develop, validate, and implement a more proactive testing paradigm, including an emphasis on conducting predictive in vitro and in vivo studies earlier. The goal of drug discovery toxicology is not to reduce or eliminate attrition, as is often mis-stated as such, but rather to reprioritize efforts to shift attrition of future failing molecules upstream in discovery. This shift in attrition requires additional studies and investment earlier in the candidate evaluation process in order to avoid spending resources on molecules with soon-to-be-discovered development-limiting liabilities. While in silico and in vitro models will continually be developed and refined, in vivo preclinical safety models remain the gold standard for assessing human risk. For in vivo testing to influence early discovery effectively, it must: i) require low amounts of compound; ii) provide rapid results to drive decision-making and medicinal chemistry efforts; and iii) be flexible and provide results relevant to the development plan tailored to each target, drug class, and/or indication.     

The role of mitochondrial and plasma membrane nucleoside transporters in drug toxicity

Many anticancer and antiviral drugs are nucleoside analogues, which interfere with nucleotide metabolism and DNA replication to produce pharmacological effects. Clinical efficacy and toxicity of nucleoside drugs are closely associated with nucleoside transporters because they mediate the transport of nucleoside drugs across biological membranes. Two families of human nucleoside transporters (equilibrative nucleoside transporters and concentrative nucleoside transporters) have been extensively studied for several decades. They are widely distributed, from the plasma membrane to membranes of organelles such as mitochondria, and the distribution differs in different tissues. In addition, they have different specificities to nucleoside drugs. The characteristics of equilibrative and concentrative nucleoside transporters affect the therapeutic outcomes achieved with anticancer and antiviral nucleoside drugs. In this review, an overview of the role of mitochondrial and plasma membrane nucleoside transporters in nucleoside drug toxicity is provided. Rational design and therapeutic application of nucleoside analogues are also discussed.     

The role of early in vivo toxicity testing in drug discovery toxicology

The cost impact of late-stage failures of drug candidates has motivated the pharmaceutical industry to develop, validate, and implement a more proactive testing paradigm, including an emphasis on conducting predictive in vitro and in vivo studies earlier. The goal of drug discovery toxicology is not to reduce or eliminate attrition, as is often mis-stated as such, but rather to reprioritize efforts to shift attrition of future failing molecules upstream in discovery. This shift in attrition requires additional studies and investment earlier in the candidate evaluation process in order to avoid spending resources on molecules with soon-to-be-discovered development-limiting liabilities. While in silico and in vitro models will continually be developed and refined, in vivo preclinical safety models remain the gold standard for assessing human risk. For in vivo testing to influence early discovery effectively, it must: i) require low amounts of compound; ii) provide rapid results to drive decision-making and medicinal chemistry efforts; and iii) be flexible and provide results relevant to the development plan tailored to each target, drug class, and/or indication.     

The significance of chirality in drug design and development

Proteins are often enantioselective towards their binding partners. When designing small molecules to interact with these targets, one should consider stereoselectivity. As considerations for exploring structure space evolve, chirality is increasingly important. Binding affinity for a chiral drug can differ for diastereomers and between enantiomers. For the virtual screening and computational design stage of drug development, this problem can be compounded by incomplete stereochemical information in structure libraries leading to a "coin toss" as to whether or not the "ideal" chiral structure is present. Creating every stereoisomer for each chiral compound in a structure library leads to an exponential increase in the number of structures resulting in potentially unmanageable file sizes and screening times. Therefore, only key chiral structures, enantiomeric pairs based on relative stereochemistry need be included, and lead to a compromise between exploration of chemical space and maintaining manageable libraries. In clinical environments, enantiomers of chiral drugs can have reduced, no, or even deleterious effects. This underscores the need to avoid mixtures of compounds and focus on chiral synthesis. Governmental regulations emphasizing the need to monitor chirality in drug development have increased. The United States Food and Drug Administration issued guidelines and policies in 1992 concerning the development of chiral compounds. These guidelines require that absolute stereochemistry be known for compounds with chiral centers and that this information should be established early in drug development in order that the analysis can be considered valid. From exploration of structure space to governmental regulations it is clear that the question of chirality in drug design is of vital importance.     

Adequate immunotoxicity testing in drug development

Modulation of the immune system can lead to either immunostimulation or immunosuppression and can be either intended or unintended. While many effects on the immune system's components can be found as a result of a drug treatment or chemical exposure, true immunotoxicity occurs when such treatment results in adverse effects or defects in the immune response. Regulatory expectations to evaluate potential adverse effects of pharmaceuticals warrants a need for reliable and readily standardized methods. Moreover, criteria to classify a drug as an "immunotoxicant" need to be established. Examples of studies using a modified approach to measure T-cell-dependent antibody responses (the rat KLH model) and interpretation of the results in the context of immunotoxicity evaluation are discussed in this paper.     

The PIT method: an automated in vitro technique for drug toxicity testing

An automated in vitro technique for drug toxicity testing is described. Human tumor cells were cultured for 2 days in 96-well microtiter plates before the addition of serial dilutions of drugs. At day 5 the cultures were terminated by the addition of a solution containing propidium iodide, ink and triton X-100 (PIT). Triton X-100 lysed all cells, which were subsequently stained by the DNA specific fluorescing propidium iodide. The ink effectively quenched all background fluorescence. The plates were read on a computer controlled automated microscope with a photomultiplier. The results showed a linear relationship between cell number and fluorescence intensity. Reproducible dose-response curves were obtained for 6 drugs tested. A computer program calculated ID₅₀ values, making use of adequate growth and no-growth controls.     

线粒体毒性评价及其在创新药物安全性评价中的意义

线粒体是细胞内能量和活性氧自由基(ROS)的主要来源,在病理条件下对细胞的存活与死亡具有十分重要的调控作用。线粒体是药物毒性作用的重要靶标。一些抗病毒药物、抗肿瘤药物和抗生素等可显著诱导肝脏和心脏等靶器官线粒体损伤。药物诱导的线粒体损伤可能涉及多条途径和多种机制。近年来研究表明,线粒体毒性可能是多种已上市药物被迫撤市或受到美国FDA"黑框"警告以及候选药物研发失败的重要原因。因此,在创新药物研发过程中,开展线粒体毒性评价具有十分重要的意义。     

Food and drug administration guidelines for reproductive toxicity testing

The Food and Drug Administration generally requires reproductive toxicity testing of all new drugs to be used by pregnant women or women or men of reproductive potential. These requirements may vary among the centers within the FDA. Reproductive and developmental toxicity is usually tested in one or two animal species and is divided into three segments to represent treatment throughout the reproductive process. The FDA monitors adverse drug effects on human reproduction through postmarketing surveillance.     

Bringing primary cells to mainstream drug development and drug testing

Although cell-based screening is already an essential tool in drug discovery, the cell models currently available are fast becoming inadequate. The use of transformed cells as models in almost every step of the discovery pipeline needs to be substituted with more relevant, disease-oriented models, and the use of patient-derived primary cells should logically become the next best strategy. In the past the use of such cells has been restricted by their scarcity and difficulty in manipulation and general handling; however, recent advances in isolation and growth, as well as assay miniaturization, transfection efficiency and assay sensitivity, have enabled their use in the mainstream of drug discovery. This review explores some of these enabling technologies, as well as some of the most critical uses of primary cells that may dramatically alter the landscape of drug discovery and drug testing.     

The industry view on long-term toxicology testing in drug development of human pharmaceuticals

The approach to chronic toxicity testing over the past decade is reviewed and assessed in the light of developing ICH guidelines. The 1990's have seen a general acceptance that studies with a maximal duration of 6 months in rodents are all that is required for adequate safety assessment of developmental pharmaceutical agents. However, controversy has arisen concerning the most appropriate duration for chronic toxicity testing in non-rodents. Initial suggestions that 6 months duration was sufficient have been countered by findings noted in 12-month studies that were not seen in shorter-term studies. Retrospective analysis of available data eventually lead to a subsequent ICH recommendation that studies of 9 months duration would be now acceptable. However, until recently the FDA position on this recommendation was unclear and an analysis of industry practices since the ICH recommendation was made in 1997 has shown that the 9-month guideline is not widely applied. Recent clarification by the FDA will probably result in a continued but limited use of this alternative. An industry view on the future of chronic toxicology testing in rodents and non-rodents is presented.     

The heterozygous Sod2(+/-) mouse: modeling the mitochondrial role in drug toxicity

Mitochondria have been increasingly implicated in being a crucial subcellular target and amplifying oxidative injury induced by many drugs. Among the major cytoprotective antioxidants is the mitochondrial matrix protein, superoxide dismutase-2 (SOD2). Genetic modification of the expression of SOD2 by transgenic techniques or gene silencing has generated a number of distinct animal models with SOD2 deficiency including the heterozygous Sod2(+/-) knockout mouse model. These mice display a discreet underlying mitochondrial stress but are otherwise phenotypically normal and thus model a variety of clinically silent mitochondrial abnormalities. The model has found application in oxidative stress and age-related research, but it is only recently that it has been successfully used to study mechanisms of idiosyncratic drug-induced liver injury.     

Mechanisms of drug toxicity and relevance to pharmaceutical development

Toxicity has been estimated to be responsible for the attrition of approximately one-third of drug candidates and is a major contributor to the high cost of drug development, particularly when not recognized until late in clinical trials or post-marketing. The causes of drug toxicity can be classified in several ways and include mechanism-based (on-target) toxicity, immune hypersensitivity, off-target toxicity, and bioactivation/covalent modification. In addition, idiosyncratic responses are rare but can be one of the most problematic issues; several hypotheses for these have been advanced. Although covalent binding of drugs to proteins was described almost 40 years ago, the significance to toxicity has been difficult to establish; recent literature in this field is considered. The development of more useful biomarkers and short-term assays for rapid screening of drug toxicity early in the drug discovery/development process is a major goal, and some progress has been made using "omics" approaches.     

转运体在药物排泄中的作用及在新药研发中的意义

本文介绍了药物转运体在药物排泄过程中的作用,探讨了其在新药研发和临床应用中的可能性。通过对药物转运体功能的了解和利用,可以开发出对某些器官有靶向性的药物,或避免药物分布到某些器官中,从而提高药物的疗效,降低其毒副作用;也可以通过对转运体介导的药物相互作用及肝肠循环的研究,指导临床更加安全有效的用药。在药物研发的初始阶段,就开始重视其药动学特性,这一观念近年来已被很多人所接受。对药物转运体的深入认识和利用,建立高通量的药物转运体筛选体系,对于加速新药研发的进程将具有极其重要的意义。     

Pluripotent human stem cells as novel tools in drug discovery and toxicity testing

Improved technologies are urgently needed to develop effective and safe new drugs in a cost-efficient manner. Cell-based assays have many advantages in drug research, particularly because these assays can be adapted in a high-throughput format. In addition, technological advances in the areas of instrumentation and automation are providing expanding opportunities for high-content analyses. However, in cell-based research, none of these systems is particularly useful unless the cells that are being evaluated are clinically relevant. Pluripotent human stem cells are expected to revolutionize the accessibility to a variety of human cell types. The possibility to propagate pluripotent human stem cells and to subsequently differentiate these cells into desired target cell types will provide a stable supply of cells for a range of applications in drug discovery and toxicity testing. This feature discusses some of the research opportunities for pluripotent human stem cells.     

The C. elegans model in toxicity testing

Caenorhabditis elegans is a small nematode that can be maintained at low cost and handled using standard in vitro techniques. Unlike toxicity testing using cell cultures, C. elegans toxicity assays provide data from a whole animal with intact and metabolically active digestive, reproductive, endocrine, sensory and neuromuscular systems. Toxicity ranking screens in C. elegans have repeatedly been shown to be as predictive of rat LD₅₀ ranking as mouse LD₅₀ ranking. Additionally, many instances of conservation of mode of toxic action have been noted between C. elegans and mammals. These consistent correlations make the case for inclusion of C. elegans assays in early safety testing and as one component in tiered or integrated toxicity testing strategies, but do not indicate that nematodes alone can replace data from mammals for hazard evaluation. As with cell cultures, good C. elegans culture practice (GCeCP) is essential for reliable results. This article reviews C. elegans use in various toxicity assays, the C. elegans model's strengths and limitations for use in predictive toxicology, and GCeCP. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Journal of Applied Toxicology published by John Wiley & Sons Ltd.     

The challenge of predicting drug toxicity in silico

Poor pharmacokinetics, side effects and compound toxicity are frequent causes of late-stage failures in drug development. A safe in silico identification of adverse effects triggered by drugs and chemicals would be highly desirable as it not only bears economical potential but also spawns a variety of ecological benefits: sustainable resource management, reduction of animal models and possibly less risky clinical trials. In computer-aided drug discovery, both existing and hypothetical compounds may be studied; the methods are fast, reproducible, and typically based on human bioregulators, making the question of transferability obsolete. In the recent past, our laboratory contributed towards the development of in silico concepts (--> multi-dimensional QSAR) and validated a series of "virtual test kits" based on the oestrogen, androgen, thyroid, and aryl hydrocarbon receptor (endocrine disruption, receptor-mediated toxicity) as well as on the enzyme cytochrome P450 3A4 (metabolic transformations, drug-drug interactions). The test kits are based on the three-dimensional structure of their target protein (i.e. ER(alphabeta), AR, TR(alphabeta), CYP450) or a surrogate thereof (AhR) and were trained using a representative selection of 362 substances. Subsequent evaluation of 107 compounds different therefrom showed that binding affinities are predicted close to experimental uncertainty. These results suggest that our approach is suited for the in silico identification of adverse effects triggered by drugs and chemicals and encouraged us to compile an Internet Database for the virtual screening of drugs and chemicals for toxic effects.