End-to-end automation of organ-on-a-chip culture and drug testing: Enhancing scalability and expandability.

New approach methodologies in non-clinical safety assessment of vaccines

Traditional animal-based preclinical models, including xenografts and genetically engineered mice, have been used for assessing pharmacodynamics, toxicity, efficacy, and safety for decades. Despite their limited ability to mimic human tumor heterogeneity, immune interactions, and microenvironmental complexity, over 90% of oncology candidates that succeed in animal studies fail in clinical trials. The New Approach Methodologies (NAMs), which include patient-derived organoids, organ-on-chip platforms, and AI-driven computational models, provide human-relevant solutions that can improve predictive validity, mechanistic insight, and ethics. Through these technologies, it will be possible to replicate tumor biology specific to patients, to support co-clinical trial designs, and to facilitate biomarker discovery while reducing animal testing. Several recent regulatory reforms, including the Food and Drug Administration (FDA) Modernization Act 2.0 and the European Medicines Agency’s NAM qualification framework, have established clear pathways for the integration of validated NAMs into preclinical drug evaluation. Critically evaluating the scientific rationale, comparative performance, and regulatory context of key NAM platforms in oncology, this review highlights opportunities for synergistic integration, technical refinement, and global harmonization in order to accelerate the development of clinically effective cancer therapeutics based on preclinical findings.

The U.S. Food and Drug Administration (FDA), Center for Drug Evaluation and Research (CDER), and Office of New Drugs (OND) has continuously encouraged the submission of nonclinical tests utilizing new approach methodologies (NAMs) and sponsor engagement with regulators to optimize NAM utility in supporting the safety and efficacy of new drugs. Previously, we published an FDA/CDER perspective on nonclinical testing strategies, discussed the opportunities and challenges of using NAMs to replace, reduce, and refine animal testing in drug development, and reported gaps and challenges underserved by existing nonclinical testing approaches that CDER Pharmacology/Toxicology reviewers face. Here, we demonstrate how FDA/CDER has historically incorporated NAMs into standard nonclinical assessments, describing how specific tests became validated and internationally accepted alternatives to animal testing for regulatory decision-making. We also provide a CDER/OND Pharmacology/Toxicology reviewer perspective on NAMs submitted to support new drug development, in an effort to provide insight into our experience with NAMs submitted for CDER-regulated products. Furthermore, we provide a CDER/OND Pharmacology/Toxicology reviewer perspective on the future of NAM incorporation into nonclinical development programs for new drugs as scientific technology continues to evolve. Ultimately, we hope that by sharing the FDA/CDER/OND experience with NAMs thus far and providing considerations for refining NAM submissions, we will (1) illustrate our scientific approach to evaluating NAM submissions, (2) reiterate FDA/CDER's steadfast commitment to the 3Rs, and (3) foster confidence in our continued efforts to encourage nonclinical test NAM submissions for regulatory decision-making, while maintaining our mission to protect public health and patients from unintended harm.

Background/Objectives: The rapid expansion of New Approach Methodologies (NAMs) is transforming oral biopharmaceutics by offering mechanistically rich, human-relevant tools that can reduce reliance on animal testing while improving translational confidence. Regulatory agencies, including the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), are increasingly open to NAM-generated evidence, provided that methods are fit-for-purpose and scientifically justified. This review synthesizes current advances and evaluates how NAMs can be integrated across drug-development stages to enhance the prediction of oral absorption, formulation performance, and regulatory decision-making. Methods: A comprehensive literature review was conducted across classical and emerging methodologies, including in vitro permeability and solubility models, organoids, organ-on-a-chip (OoC) systems, machine learning frameworks, and mechanistic approaches such as the physiologically based pharmacokinetic (PBPK) and biopharmaceutics (PBBM) models. Emphasis was placed on physiological relevance, predictive performance, validation status, and regulatory applicability. Results: Classical tools remain essential for the Biopharmaceutics Classification System (BCS)-based biowaivers and risk-based assessments, yet they often lack physiological fidelity. NAMs provide enhanced representation of intestinal architecture, hydrodynamics, transporter activity, and metabolism. Organoids and microphysiological systems generate high-quality permeability and metabolic data, while computational NAMs enable scalable prediction of ADME properties and formulation behavior. When integrated into PBPK/PBBM models, these methods have great potential in predicting in vivo performance in humans. Evidence demonstrates that NAMs can refine, reduce, and, in specific contexts, replace animal studies without compromising scientific rigor. Conclusions: NAMs complement, rather than displace, classical biopharmaceutic tools, enabling a more mechanistic, human-centered, and ethically responsible framework for drug development. Their effective implementation will depend on continued validation, standardization, and regulatory harmonization as the field transitions toward fully NAM-supported biopharmaceutical assessment.

Animal models are an essential part of preclinical research, serving to protect patients through safety and efficacy studies before human trials begin. Prior to the passage of the Food and Drug Administration (FDA) Modernization Act 2.0 in 2022, animal studies were generally required by regulators in preclinical safety evaluations. This Act amended the Federal Food, Drug, and Cosmetic Act to clarify that while animal testing had been the default standard, it is no longer a mandatory requirement for regulatory filings involving drugs and biological products1. Additionally, the Act explicitly specifies in vitro, in silico, and in chemico testing as alternatives to assess pre-clinical safety and efficacy. The FDA and others refer to these alternative platforms as New Approach Methodologies (NAMs) with an emphasis on the 3Rs of replacing, reducing and refining the use of animal testing2. This shift opens new pathways for developing and evaluating precision therapies in cardiovascular disease particularly if human-derived, genetically accurate models can outperform animal studies in predictive power and translatability.

Despite unprecedented technological progress, most drug candidates continue to fail in clinical trials, reflecting a persistent gap between preclinical models and human biology. New approach methodologies (NAMs), by spanning human-derived cellular systems, microphysiological platforms, and artificial intelligence, offer a paradigm shift in human-centric drug development and biomedical research. Recent regulatory reforms, such as the US Food and Drug Administration (FDA) Modernization Act 3.0, have begun to position NAMs as a complement to or replacement for animal testing. This Review synthesizes emerging biological and computational NAMs and examines how their integration is reshaping drug development. We also discuss regulatory and ethical frameworks enabling this transition and outline a roadmap for embedding human-based science in a predictive, efficient, and ethically grounded infrastructure of human-centered drug development.

Introduction: ushering in an era of humanrelevant drug evaluationOn April 10, 2025, the U.S. Food and Drug Administration (FDA) issued an announcement announcing that it will gradually eliminate animal testing requirements in the development of monoclonal antibodies (mAbs) and other drugs.The main contents of the new policy include (1) policy background and overall objectives; (2) new approach methodologies (NAMs) roadmap; (3) key alternative technologies; (4) regulatory incentives and implementation arrangements; and (5) cooperation mechanisms and follow-up plans.The initiative aims to reduce animal testing in preclinical safety studies using scientifically validated NAMs, including cell-based tests, organoids, organ-on-achips (OoCs), microphysiological systems (MPS), in silico tools and computational models, and real-world evidence (RWE) [1] .The 2025 "Roadmap to Reduce Animal Testing in Preclinical Safety Studies" adopts a phased approach, initially focusing on mAbs, and is expected to significantly reduce or replace animal studies within 3-5 years [2] .Animal model-based testing of mAbs has been particularly challenging, as species-specific targets and immunogenicity limit the predictive power of animal models.This underscores the need for NAMs in different therapeutic domains [3] .This article supports the FDA's policy shift as a scientifically necessary, ethically imperative, and technologically enabled evolution.It represents a move towards a drug development paradigm centered on human biology, promising accelerated development timelines, reduced costs, enhanced safety prediction, and ultimately, a "win-win for public health and ethics" [1] .For the Cell Organoid community, this transition highlights the critical role that advanced in vitro models, particularly organoid technologies, will play in shaping the future of medicine.

New approach methodologies for drug discovery.

Alternatives to animal testing to assess MASH drugs and hepatotoxicity.

The transition away from animal experimentation is gaining momentum across scientific disciplines, driven by technological advancements, ethical imperatives and policy support. At the forefront of this movement is the development of New Approach Methodologies (NAMs). Rather than serving as mere substitutes for animal testing, NAMs represent a paradigm shift. Their growing acceptance has been reinforced by recent policy advancements, such as the US Food and Drug Administration Modernization Act 2.0 and the European Chemicals Agency's commitment to transitioning toward an animal-free regulatory system, both of which promote the adoption of NAMs. However, the term NAMs has become increasingly ambiguous, leading to confusion and potential misuse. To address this, we propose a unified definition: NAMs are species-specific methodologies, not including the use of living animals. NAMs prioritize the use of the target species in some form or another, eliminating the need for interspecies extrapolation. If the target species is human, this simple definition implies that working with experimental animals as a model never qualifies as a NAM. This distinction positions NAMs as a fundamentally different approach to the 3Rs framework of Replacement, Reduction and Refinement, and practical examples are discussed. Critically, this definition extends beyond toxicology and regulatory science, encompassing basic, applied and translational research. By leveraging cutting-edge technologies, including organoids, organ-on-chip, microfluidics, 3D bioprinting, bioinformatics, artificial intelligence and machine learning, NAMs provide novel biological insights that differ from those obtained through animal models while enhancing species-relevant data accuracy. Although challenges remain, particularly in their validation and widespread adoption, continued innovation, policy support and interdisciplinary collaboration will be key to unlocking their full potential, fostering more sustainable, humane and human-relevant approaches in biomedical research.

New Approach Methodologies (NAMs), including organoids, microphysiological systems, and computer modeling, are gaining increased popularity for toxicological testing and even mechanistic research. With the use of human cells, the primary objectives of NAMs are to develop more human-relevant test systems and to reduce, and ultimately eliminate, animal experiments. There are many advantages of using NAMs for biological research. For example, NAMs can be used to test the dose- and time-dependent toxicity of numerous chemicals and mixtures in a cost-effective way and reduce animal use. Although these are worthwhile goals when considering the big picture, the problems lie in the details. First, in vivo insight is needed to build and refine NAMs, including computer modeling. Second, primary human cells are difficult to obtain reliably and in sufficient quantities; substitutes such as immortalized cell lines or induced pluripotent stem cells have the advantage of being more robust and available in unlimited numbers, but their basal and stress-induced gene expression profiles are quite different compared with primary cells. Third, critical aspects such as metabolic competency, the presence of various cell types in an organ, spatial aspects, oxygen gradients, and the role of inflammatory cells are very difficult to replicate in vitro. Therefore, in vivo experiments are necessary to verify results obtained with NAMs. Importantly, the results of both NAMs and the in vivo animal experiments need to be translatable to human disease processes. The advantages and limitations of NAMs are being discussed using the challenges of investigating mechanisms of drug hepatotoxicity as an example.

“Black box” 101: How the Food and Drug Administration evaluates, communicates, and manages drug benefit/risk

Organ-on-chip (OOC) microphysiological systems have emerged as a promising alternative to traditional in vitro and animal models for drug development. These advanced platforms recreate the complex microenvironment and physiological functions of human organs, enabling more accurate predictions of drug efficacy and toxicity. The complex tissue-tissue interfaces, biochemical gradients, and mechanical cues found in vivo can be simulated effectively by OOC systems providing a powerful way for preclinical drug screening and testing. The integration of OOC systems into early phase clinical trials has the potential to revolutionize drug development by bridging the gap between preclinical studies and human clinical outcomes. This approach allows for the evaluation of drug candidates in a more physiologically relevant context, taking into account factors such as organ-specific responses, inter-individual variability, and disease-specific conditions. The incorporation of patient-derived cells and the development of multi-organ platforms further enhance the predictive power of OOC systems, enabling personalized medicine approaches and the assessment of systemic effects. However, challenges such as standardization, validation, scalability, and regulatory acceptance need to be addressed to fully realize the potential of OOC systems in clinical settings. As the knowledge on OOC systems advances, their integration into early phase clinical trials is expected to streamline the drug development process, reduce the reliance on animal testing, and accelerate the translation of basic research into safe and effective therapies for patients.

Drug-induced kidney injury (DIKI) is a frequently reported adverse event, associated with acute kidney injury, chronic kidney disease, and end-stage renal failure. Prospective cohort studies on acute injuries suggest a frequency of around 14%-26% in adult populations and a significant concern in pediatrics with a frequency of 16% being attributed to a drug. In drug discovery and development, renal injury accounts for 8 and 9% of preclinical and clinical failures, respectively, impacting multiple therapeutic areas. Currently, the standard biomarkers for identifying DIKI are serum creatinine and blood urea nitrogen. However, both markers lack the sensitivity and specificity to detect nephrotoxicity prior to a significant loss of renal function. Consequently, there is a pressing need for the development of alternative methods to reliably predict drug-induced kidney injury (DIKI) in early drug discovery. In this article, we discuss various aspects of DIKI and how it is assessed in preclinical models and in the clinical setting, including the challenges posed by translating animal data to humans. We then examine the urinary biomarkers accepted by both the US Food and Drug Administration (FDA) and the European Medicines Agency for monitoring DIKI in preclinical studies and on a case-by-case basis in clinical trials. We also review new approach methodologies (NAMs) and how they may assist in developing novel biomarkers for DIKI that can be used earlier in drug discovery and development.

In their editorial entitled “Pediatric Research and Scholarship,” Tobin et al. (1) question whether the Food and Drug Administration (FDA) has taken an acceptably proactive role in efforts to encourage high quality clinical trials of drugs in the pediatric patient population when those studies are performed by individual academic investigators. In particular, they question the obligation of academic investigators to abide by federal regulations when conducting small clinical trials with drugs not yet approved for the pediatric population. In response, we will review the role of the FDA in clinical research conducted by individual academic investigators, summarize recent progress in pediatric drug development, and offer suggestions to academic investigators who wish to perform these types of drug trials. FDA: THE INVESTIGATIONAL NEW DRUG (IND) APPLICATION Most investigational use of drugs is subject to the IND regulations. While these clearly apply to unapproved new drugs, in certain circumstances, federal regulations require that a protocol utilizing an approved drug product must be reviewed by the FDA before a clinical drug trial may proceed. The FDA Web site offers guidance to investigators to help them determine when their proposed study will require FDA review (http://www.fda.gov/cder/guidance/phase1.pdf). When FDA review of a protocol is required, the investigator must submit an IND application. FDA review is always required when the intent of an investigation is to change a product’s label, such as to permit the product’s use in a new, potentially more vulnerable population. For example, the novel use of a product in pediatric patients would require a pharmaceutical company to perform all clinical research under an IND. However, even in the setting of a small-scale academic investigation, an IND would likely be required in a vulnerable population such as pediatric patients, whether or not there is common off-label use of this drug in the pediatric population. If an investigator is uncertain whether an IND application is necessary, he or she should contact the FDA for advice prior to initiating subject recruitment. When an IND application is required, the FDA review process can benefit the investigator and reduce the risk to subjects participating in the clinical study. For example, if there is preliminary evidence that a particular drug is associated with organ toxicity in an animal model of growth and development, the FDA may be able to advise the IND applicant how to safely monitor patients, and provide this information without compromising proprietary work by other investigators. Furthermore, because the FDA is often aware of trials that have failed, the agency can advise an IND applicant when the risk of planned research exceeds the putative benefit. Academic investigators who are not expert in conducting clinical drug research can also receive assistance in developing an appropriate trial design based on FDA experience in the evaluation of previously completed trials (2–4). In contradistinction to statements by Tobin et al. (1), the FDA clearly recognizes the utility of alternative designs to placebo-controlled trials (CFR 314.126) when appropriate. The review of an IND application is conducted by a team consisting of chemists, nonclinical pharmacologists/toxicologists, clinical pharmacologists, statisticians, and physicians, concluding with a determination of whether or not the trial is considered safe to proceed. This process is typically completed within 30 days. When a clinical trial that required an IND review has been conducted without FDA approval, the FDA Division of Scientific Investigations may perform an evaluation at the research site to assess the potential risk that was incurred by subjects during that study. In such cases, the FDA evaluation of the trial and any subsequent regulatory action do not depend on whether the trial data have been or will be published in the medical literature. The magnitude of the risk to research subjects is incorporated into the assessment and can result in a legal action when appropriate. PROGRESS IN PEDIATRIC DRUG DEVELOPMENT Most medications are initially studied and approved in the adult population, which is appropriate given the unknowns of safety and efficacy during initial development. However, this means that children are often treated off-label with these medications prior to the development of well-controlled clinical trials in the pediatric population. Consequently, physicians rely on extrapolating adult experience and on anecdotal information to determine safety and effectiveness of many medications for the treatment of pediatric patients. Congress has enacted several laws intended to directly promote drug development for the pediatric population. These measures have increased the amount of information on the safe and efficacious use of drugs for pediatric patients. The first law, passed in 1997, was the Food and Drug Administration Modernization Act (FDAMA). FDAMA offered pharmaceutical companies a 6-month period of marketing exclusivity if they performed studies in pediatric patients in response to a Written Request (WR) issued by the FDA. Marketing exclusivity incentives attach to a period of existing patent protection or exclusivity and were effective in prompting industry to conduct needed pediatric trials for drugs with existing patent protection or exclusivity. This program did not provide an incentive for the study of off-patent, mostly generic, drugs. The Best Pharmaceuticals for Children Act (BPCA) was signed into law on January 4, 2002, shortly after the pediatric exclusivity provision of FDAMA expired on January 1, 2002. The BPCA reauthorizes the exclusivity incentive enacted originally in FDAMA. Further, section 409I(a)(2) of the BPCA provides a process for the study of off-patent drugs (approved drugs that have no remaining patent protection or exclusivity). This law directs the National Institute of Health (NIH), in consultation with the FDA and experts in pediatric research, to develop and prioritize a list of “off-patent” drugs for which pediatric studies are most urgently needed. The list was originally published in January 2003 and is updated at least annually. WRs for pediatric studies will be issued by the FDA for drugs on the priority list requiring studies to develop adequate pediatric labeling. In turn, the NIH will issue contracts for the studies. Once the trials are performed and the results are analyzed, the data become publicly available for dissemination and incorporation into the label (http://www.fda.gov/cder/pediatric/70FR3937.txt). Since the inception of FDAMA and BPCA, over 450 proposed pediatric study requests from industrial sponsors have been received and more than 300 pediatric WRs have been issued. This has resulted in revisions to more than 100 drug labels to include new pediatric information. The Pediatric Rule, which became effective on April 1, 1999, required that manufacturers of certain new and marketed drugs and biological products conduct studies to provide adequate labeling for the use of these products in children. A District of Columbia federal district court invalidated the Pediatric Rule on October 17, 2002. In December 2003, Congress enacted the Pediatric Research Equity Act (PREA), which requires (retroactively to April 1, 1999) all applications for new active ingredients, new dosage forms, new indications, new routes of administration, and new dosing regimens to contain an assessment of the safety and effectiveness of the product in pediatric patients. PREA is complimentary to BPCA and allows the FDA to obtain pediatric information when applications are submitted. This requirement may be waived or deferred depending on existing labeling, public health benefit, and usefulness of the drug in different pediatric populations. With passage of the BPCA and the PREA, there have been gains in the information available in labeling for the appropriate use of medications in children. While much has been achieved, there are still many drugs that need to be studied for the pediatric population. The anesthesia community can become involved by alerting the leadership of their specialty societies regarding drugs when they feel additional information would benefit the pediatric population. Academic investigators can compete for NIH funding to support pediatric clinical trials. SUGGESTIONS FOR FURTHER RESEARCH IN PEDIATRIC DRUG DEVELOPMENT The editorial by Tobin et al. (1) suggests that a consensus statement by children’s advocacy groups, increased commitment by pharmaceutical companies, and additional resources at FDA are needed to improve pediatric drug development. We concur that all stakeholders should work together to find new ways to advance the development of pediatric drug development. The FDA continues to be active in organizing an increased commitment to this vital area of research. For example, the Newborn Drug Development Initiative: Improving Neonatal Therapeutics workshop, sponsored by the NIH and FDA, included summary recommendations for pain control by members of the academic anesthesia community (5,6). Full-length articles on procedural pain, sedation and analgesia, perioperative pain, and study designs resulting from this effort have also been published (7–10). High-caliber academic investigations, when conducted under an approved IND application, can continue to make valuable contributions to improve the spectrum and quality of therapeutics available to pediatric patients.