Many scientific breakthroughs have depended on animal research, yet the ethical concerns surrounding the use of animals in experimentation have long prompted discussions about humane treatment and responsible scientific practice. First articulated by Russell and Burch, the 3Rs Principles of Replacement, Reduction, and Refinement have gained widespread recognition as basic guidelines for animal research. Over time, the 3Rs have transcended the research community, influencing policy decisions, animal welfare advocacy and public perception of animal experimentation. Despite their broad acceptance, interpretations of the 3Rs vary substantially, shaping statutory frameworks at various levels, with both technical and practical impacts.

The kinetics of passive transport of ketoprofen and metformin, as model substances for high and low permeability, respectively, across the artificial membrane under the influence of the pH of donor solution was investigated. There was an upward trend in the apparent permeation coefficient (Papp) of ketoprofen with the decrease in pH to a value close to pKa. At the pH value below pKa the permeation coefficient had lower value, due to the higher retention of ketoprofen in the artificial membrane. Metformin is a low permeable compound, and the highest permeation values were recorded at pH 7.4. Two dissociation constants determine that metformin at physiological pH exists as a hydrophilic cationic molecule, i.e. predominantly in ionized form. At pH values below 2.8, metformin mainly exists in diprotonated form, and it was, thus, very poorly permeable. The highest retention, i.e. affinity of both ketoprofen and metformin to the membrane, was at the lowest pH values, which is explained by different mechanisms. At higher pH values of the donor compartment, the substances showed significantly less affinity to the membrane. The obtained values of apparent permeation coefficients at studied pH values showed a good correlation with the obtained experimental values by other in vitro methods.

The antidiabetic drug gliclazide is partly metabolized by CYP2C19, the main enzyme involved in omeprazole metabolism. The aim of the study was to explore the interaction between omeprazole and gliclazide in relation to CYP2C19 phenotype using physiologically based pharmacokinetic (PBPK) modeling approach. Developed PBPK models were verified using in vivo pharmacokinetic profiles obtained from a clinical trial on omeprazole-gliclazide interaction in healthy volunteers, CYP2C19 normal/rapid/ultrarapid metabolizers (NM/RM/UM). In addition, the association of omeprazole cotreatment with gliclazide-induced hypoglycemia was explored in 267 patients with type 2 diabetes (T2D) from the GoDARTS cohort, Scotland. The PBPK simulations predicted 1.4–1.6-fold higher gliclazide area under the curve (AUC) after 5-day treatment with 20 mg omeprazole in all CYP2C19 phenotype groups except in poor metabolizers. The predicted gliclazide AUC increased 2.1 and 2.5-fold in intermediate metabolizers, and 2.6- and 3.8-fold in NM/RM/UM group, after simulated 20-day dosing with 40 mg omeprazole once and twice daily, respectively. The predicted results were corroborated by findings in patients with T2D which demonstrated 3.3-fold higher odds of severe gliclazide-induced hypoglycemia in NM/RM/UM patients concomitantly treated with omeprazole. Our results indicate that omeprazole may increase exposure to gliclazide and thus increase the risk of gliclazide-associated hypoglycemia in the majority of patients.

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.