Preclinical toxicology studies are required for every therapeutic development program as these studies answer fundamental questions on the local and systemic effects of the test drug on the patient. Typically, tox studies are performed in small and large animal models and use defined endpoints. The guidance issued by the FDA has clearly stated the minimum requirements for preclinical toxicology studies include PK/PD profiling, acute toxicity studies in two species (rats and dogs are the most commonly used) and short-term toxicity studies to evaluate continued and potentially delayed onset adverse effects1. Traditional toxicology studies have been more observational and record the ADME characteristics, biodistribution and PK/PD profiling along with optimal dose ranges that have acceptable off target effects. However, there is an increasing shift towards a more active investigational toxicology approach that can be either prospective or retrospective2. Prospective investigative toxicology, as the name suggests, is performed during the discovery stage to quickly identify promising drug assets that have low toxicity and can move forward into efficacy evaluation. This approach supports the concept of “fail early and fail fast” so that assets with unacceptably high levels of toxicity are removed early from the development pipeline, thus saving significant time and downstream costs. These prospective studies are typically performed in translational in vitro models that range from simple 2D cell culture models and 3D organoids to highly complex microphysiological systems (MPS) such as organ-chips3. The retrospective approach is focused on understanding the mechanism of action of adverse effects identified in in vivo animal models or clinical trial patients. These studies can use multiomics-based approaches to review global changes in gene and protein expression profiles in response to drug exposure combined with ADME, histopathology and PK/PD data. The retrospective analysis is very useful to design next-generation therapies that can bypass the signaling triggers that cause off-target effects.

Prospective investigative toxicology studies are recently gaining traction due to the interest in responsible animal use and regulatory willingness to accept data generated in in vitro and ex vivo models. The FDA Modernization Act in the US and the activities by European medical agencies to promote animal-free testing has accelerated the development of complex in vitro model systems to predict toxic effects. It is important to note that in vitro model development has moved at different paces depending on the organ. For example, lung MPS model development was very rapid in response to the COVID-19 pandemic, while liver and kidney MPS development are moving at a slower pace in part due to tissue complexity. The availability of high-quality input materials impacts the pace of development – for example, researchers are dependent on hepatocellular carcinoma (HepG2) cells or primary hepatocytes to test therapies for drug induced liver injury (DILI), which is a critical tox readout. These models are not fully representative of the in vivo state and, in the case of primary hepatocytes, supply and quality continue to be issues. The development of reliable, high-quality iPSC-derived hepatocytes has been a challenge but as reprogramming technologies continue to improve, it is likely that this challenge will be solved. Another example is the development of translational complex kidney models. Simple 2D overexpression models have been used for several years to study drug-drug interactions (DDI) but the recapitulation of kidney glomeruli in vitro is a complex issue. Nephrons, the functional units of the kidney, consists of over 20 cell types that are arranged in a complex structure4 but MPS platforms typically use 2 cell types – epithelial cells and endothelial cells. Micro-physiological systems (MPS) for kidney cell culture were first reported in 2013 with the development of a kidney chip5 that showed expression of uptake and efflux transporters, resulting in accurate and reproducible responses to known transporter inhibitors such as cimetidine. Bioprinting is another technology that is being investigated to develop a 3D model of the kidney for the use in investigative toxicology studies4.

It is clear that the development of complex in vitro models for investigative toxicology is on an accelerated pace. As the development of input materials and culture systems continue to improve and evolve, the combination of biology and engineering will result in human in vitro systems that recapitulate the in vivo state to better predict off target effects.







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