Novel Alternative Methods RFI

The American Physiological Society (APS) appreciates the opportunity to comment on the Request for Information on Catalyzing the Development and Use of Novel Alternative Methods to Advance Biomedical Research (NOT-OD-23-140). APS represents a multidisciplinary community of nearly 10,000 scientists and educators focused on understanding the function of living organisms. APS strongly agrees with recent statements from NIH recognizing that animal models are critically important to biomedical research (1). APS also supports the principles of the “3Rs” (replacement, reduction, and refinement) and the development and use of alternative approaches, including novel alternative methods (NAMs), where they are scientifically justified and appropriate. With recent rapid advancement of microfluidic devices, researchers have access to novel in vitro tools including organ-on-a-chip and body-on-a-chip systems which allow more complex modeling of biological systems. However, despite the sophistication of these technologies, they do not recapitulate the total complexity of a living animal and should be viewed as supplemental to animal models as tools to understand life, health, and disease.

Microphysiological systems such as organ-on-a-chip are of particular interest to the pharmaceutical industry. As preclinical models they offer potential as high-throughput drug screening platforms and for accurate prediction of pharmacokinetic parameters, promoting a “fail early, fail fast” approach that could significantly lower the cost of drug development (2). The technology offers several advantages over traditional cell culture models. Microfluidic devices enable the study of cell and tissue function in the presence of physiologically relevant factors such as mechanical stretching and perfusion, which are difficult to model in other in vitro systems. By using fluidic coupling of multiple chambers lined by different organ cell types, a body-on-a-chip system was demonstrated to predict the pharmacokinetics and pharmacodynamics of cisplatin and nicotine (3). A number of other case studies have been summarized in a report from a workshop with the FDA and representatives of the pharmaceutical industry (4).

Although these successes underscore the promise of NAMs, there are several important limitations to these technologies as currently implemented. While they can be much more complex than traditional cell culture, microphysiological systems are reductive models, and are unable to replicate the entire physiology of an organ. Additionally, data derived from NAMs are generally limited to parameters that can be easily observed and measured, such as the concentration of a metabolite or the rate of cell death. It is difficult to design a system capable of measuring a range of physiological responses, including tissue and organ morphology and function, and impossible to measure neuropsychological responses such as behavioral changes or pain. This limits the utility of NAMs as tools for drug discovery. Because of these drawbacks, researchers will continue to rely on the currently available tools for drug development, including animal models.

Ultimately, the clinical value of these technologies depends on their ability to predict toxicological or therapeutic results in human trials. As the technologies are still in development, there is a lack of validating data to verify their predictive power. NIH investment should support the validation and assessment of these technologies alongside their development. NIH should also clarify for investigators that translational and preclinical studies should not rely on data from unvalidated models when established models are available. Finally, NIH should continue to invest in research using established models while the appropriate role of NAMs in the drug development process remains unclear.

1. Jorgenson L. 8 Dec. 2022. Catalyzing Research with Novel Alternative Methods [Online]. National Institutes of Health. https://osp.od.nih.gov/catalyzing-research-with-novel-alternative-methods/. [22 July 2023].
2. Ewart L, et al. Performance assessment and economic analysis of a human Liver-Chip for predictive toxicology. Commun. Med. 2, 154, 2022. https://doi.org/10.1038/s43856-022-00209-1
3. Herland A, et al. Quantitative prediction of human pharmacokinetic responses to drugs via fluidically coupled vascularized organ chips. Nat. Biomed. Eng. 4(4):421-436, 2020. https://doi.org/10.1038/s41551-019-0498-9
4. Baran SW, et al. Perspectives on the evaluation and adoption of complex in vitro models in drug development: Workshop with the FDA and the pharmaceutical industry (IQ MPS Affiliate). ALTEX-Altern. Anim. Ex. 39(2):297-314, 2022. https://doi.org/10.14573/altex.2112203

Translatability of preclinical models is a persistent challenge in biomedical research and one of the leading causes for the high cost of drug and therapy development. NAMs such as organ-on-a-chip and multi-organ chips have the potential to become powerful tools for drug efficacy and toxicology testing, but they face several unique challenges that must be addressed to maximize their predictive power. To account for these challenges and make the best use of available resources for fundamental and translational research, APS makes the following recommendations:

• NIH should support the characterization of the cells used in NAMs, including the development of new immortalized organotypic cell lines and cell culture methodologies that improve the consistency and replicability of cell behavior.
• NIH should prioritize independent validation of NAMs before they can be considered as physiologically relevant models of function or disease.
• NIH should emphasize the supplementary role of NAMs to established methods used in academic research, including the use of animal models.

As more microphysiological model systems reach the market, reproducibility will be a significant concern. Run-to-run reproducibility is highly dependent on the source of cells used. Systems which rely on the use of primary cells may face challenges with consistent sourcing of cells, as well as the susceptibility of primary cells to differentiation. Induced pluripotent stem cells (iPS cells) may be used to provide improved consistency between experiments, but they can be limited in their ability to exhibit the fully mature differentiated phenotypes of cells found in a living organism. Neither iPS cells nor primary cells are easy to scale for high-throughput applications, so while they are not considered ideal as physiologically relevant models, established cell lines are sometimes used. However, many of the most widely used cell lines poorly represent organotypic cell function in a human, and improper stewardship of many cell lines has resulted in contamination with viruses, mycoplasma, and other cell lines. The most appropriate types of cells will need to be determined for each use case, and validation standards will need to account for any potential variability due to the types of cells used. NIH should direct effort toward the characterization of cells used in microphysiological models and improved reproducibility in cell behavior.

Before NAMs are adopted as reliable tools in the drug development pipeline, they will require rigorous demonstration of their performance. Because most academic studies only use a small number of chips, often with unique device configurations, validation will need to involve large-scale studies using standardized protocols. NIH should work with manufacturers to ensure that validation studies are carried out by independent third-party entities to avoid conflicts of interest and minimize experimental bias. NIH should also consider NAMs derived from animal cells as a tool for technology development and validation. Animal chips can support validation through assessing concordance of results based on parallel in vivo studies or extensive existing pharmacology data. Showing strong agreement of results between animal and human NAMs can help to demonstrate clinical relevance.

Because of the tremendous potential economic benefit of integrating NAMs into drug development workflows, the pharmaceutical industry will almost certainly be a leader in exploring potential applications. This will likely occur in a stepwise manner as researchers identify applications that provide superior results to existing models. To maximize the research value of NIH investments in NAMs, APS provides the following recommendations:

• NIH should prioritize the development and validation of NAMs for use in fields that lack models that are relevant to human health, such as liver toxicity studies and certain infectious diseases.
• NIH should improve the reproducibility of results from NAMs by supporting training of personnel, standardization of experimental procedures, and facilitating sharing of data, methods, and reagents.
• NIH should support fundamental research at academic institutions to characterize cellular and molecular physiology and to support the development and validation of NAMs.

There is particular value in the development of NAMs for use in fields of study that do not already have access to physiologically relevant in vitro or animal models, or where such models are poor representatives of human function. Examples include modeling of liver toxicity, infectious diseases with narrow host ranges, and genetic diseases that are not recapitulated by animal models. Each context of use may have unique chip design criteria depending on the disease or condition studied and the number of measurable outputs desired. However, it remains critical to validate these models for each use case. NIH should continue to engage with the research community to identify gaps in the availability of models and focus investments to address those gaps.

Reproducibility will be a significant challenge as NAMs become more widely utilized, and NIH should take steps to minimize undesired variance across labs. Important steps include coordination of personnel training and standardization of methods and procedures. Collaborative initiatives such as postdoc exchanges can help personnel understand best practices for performing experiments. Additionally, efforts to stimulate sharing of data, materials and reagents, and detailed experimental protocols will be essential to controlling the variability that can occur due to small differences in techniques.

NIH should also consider the role of academic researchers in the development and improvement of these new platforms. The efforts of academic researchers to characterize human cellular and molecular physiology have been indispensable to the development of NAMs. Fundamental research to identify the mechanisms of function and disease in biological systems can enable better, more physiologically relevant models to be developed, and improve our ability to validate these models. NIH should provide funding opportunities to enable researchers to contribute to the development and characterization of NAMs. However, continued funding of research using established methods remains crucial to further our understanding of biological systems. The importance of fundamental research to the development of new models underscores the value in continued investment in a variety of research tools and methods, including animal models, cell culture, and organoids, and emphasizes the role of NAMs as supplemental to existing methods.

APS appreciates the efforts of the Working Group to engage with stakeholders to understand the needs of the research community. As these technologies continue to mature, they will likely enable significant progress in basic and translational biomedical research and may provide a humane alternative to animal models in some contexts. Coordination and collaboration across federal agencies, particularly FDA, will help to maximize their potential. We look forward to continued engagement with the Working Group as they move toward final recommendations.