Poor Translatability of Biomedical Research Using Animals - A Narrative Review.

The failure rate for the translation of drugs from animal testing to human treatments remains at over 92%, where it has been for the past few decades. The majority of these failures are due to unexpected toxicity - that is, safety issues revealed in human trials that were not apparent in animal tests - or lack of efficacy. However, the use of more innovative tools, such as organs-on-chips, in the preclinical pipeline for drug testing, has revealed that these tools are more able to predict unexpected safety events prior to clinical trials and so can be used for this, as well as for efficacy testing. Here, we review several disease areas, and consider how the use of animal models has failed to offer effective new treatments. We also make some suggestions as to how the more human-relevant new approach methodologies might be applied to address this.

Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings.

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.

Recent efforts to elucidate the scientific validity of animal-based drug tests by the pharmaceutical industry, pro-testing lobby groups, and animal welfare organisations.

BACKGROUND: Even after several decades of human drug development, there remains an absence of published, substantial, comprehensive data to validate the use of animals in preclinical drug testing, and to point to their predictive nature with regard to human safety/toxicity and efficacy. Two recent papers, authored by pharmaceutical industry scientists, added to the few substantive publications that exist. In this brief article, we discuss both these papers, as well as our own series of three papers on the subject, and also various views and criticisms of lobby groups that advocate the animal testing of new drugs. MAIN TEXT: We argue that there still remains no published evidence to support the current regulatory paradigm of animal testing in supporting safe entry to clinical trials. In fact, the data in these recent studies, as well as in our own studies, support the contention that tests on rodents, dogs and monkeys provide next to no evidential weight to the probability of there being a lack of human toxicity, when there is no apparent toxicity in the animals. CONCLUSION: Based on these data, and in particular on this finding, it must be concluded that animal drug tests are therefore not fit for their stated purpose. At the very least, it is now incumbent on-and we very much encourage-the pharmaceutical industry and its regulators to commission, conduct and/or facilitate further independent studies involving the use of substantial proprietary data.

Advances in neuroscience imply that harmful experiments in dogs are unethical.

Functional MRI (fMRI) of fully awake and unrestrained dog 'volunteers' has been proven an effective tool to understand the neural circuitry and functioning of the canine brain. Although every dog owner would vouch that dogs are perceptive, cognitive, intuitive and capable of positive emotions/empathy, as indeed substantiated by ethological studies for some time, neurological investigations now corroborate this. These studies show that there exists a striking similarity between dogs and humans in the functioning of the caudate nucleus (associated with pleasure and emotion), and dogs experience positive emotions, empathic-like responses and demonstrate human bonding which, some scientists claim, may be at least comparable with human children. There exists an area analogous to the 'voice area' in the canine brain, enabling dogs to comprehend and respond to emotional cues/valence in human voices, and evidence of a region in the temporal cortex of dogs involved in the processing of faces, as also observed in humans and monkeys. We therefore contend that using dogs in invasive and/or harmful research, and toxicity testing, cannot be ethically justifiable.

Does the stress of laboratory life and experimentation on animals adversely affect research data? A critical review.

Recurrent acute and/or chronic stress can affect all vertebrate species, and can have serious consequences. It is increasingly and widely appreciated that laboratory animals experience significant and repeated stress, which is unavoidable and is caused by many aspects of laboratory life, such as captivity, transport, noise, handling, restraint and other procedures, as well as the experimental procedures applied to them. Such stress is difficult to mitigate, and lack of significant desensitisation/habituation can result in considerable psychological and physiological welfare problems, which are mediated by the activation of various neuroendocrine networks that have numerous and pervasive effects. Psychological damage can be reflected in stereotypical behaviours, including repetitive pacing and circling, and even self-harm. Physical consequences include adverse effects on immune function, inflammatory responses, metabolism, and disease susceptibility and progression. Further, some of these effects are epigenetic, and are therefore potentially transgenerational: the biology of animals whose parents/grandparents were wild-caught and/or have experienced chronic stress in laboratories could be altered, as compared to free-living individuals. It is argued that these effects must have consequences for the reliability of experimental data and their extrapolation to humans, and this may not be recognised sufficiently among those who use animals in experiments.

Non-human primates in neuroscience research: The case against its scientific necessity.

Public opposition to non-human primate (NHP) experiments is significant, yet those who defend them cite minimal harm to NHPs and substantial human benefit. Here we review these claims of benefit, specifically in neuroscience, and show that: a) there is a default assumption of their human relevance and benefit, rather than robust evidence; b) their human relevance and essential contribution and necessity are wholly overstated; c) the contribution and capacity of non-animal investigative methods are greatly understated; and d) confounding issues, such as species differences and the effects of stress and anaesthesia, are usually overlooked. This is the case in NHP research generally, but here we specifically focus on the development and interpretation of functional magnetic resonance imaging (fMRI), deep brain stimulation (DBS), the understanding of neural oscillations and memory, and investigation of the neural control of movement and of vision/binocular rivalry. The increasing power of human-specific methods, including advances in fMRI and invasive techniques such as electrocorticography and single-unit recordings, is discussed. These methods serve to render NHP approaches redundant. We conclude that the defence of NHP use is groundless, and that neuroscience would be more relevant and successful for humans, if it were conducted with a direct human focus. We have confidence in opposing NHP neuroscience, both on scientific as well as on ethical grounds.

Predicting human drug toxicity and safety via animal tests: can any one species predict drug toxicity in any other, and do monkeys help?

Animals are still widely used in drug development and safety tests, despite evidence for their lack of predictive value. In this regard, we recently showed, by producing Likelihood Ratios (LRs) for an extensive data set of over 3,000 drugs with both animal and human data, that the absence of toxicity in animals provides little or virtually no evidential weight that adverse drug reactions will also be absent in humans. While our analyses suggest that the presence of toxicity in one species may sometimes add evidential weight for risk of toxicity in another, the LRs are extremely inconsistent, varying substantially for different classes of drugs. Here, we present further data from analyses of other species pairs, including non-human primates (NHPs), which support our previous conclusions, and also show in particular that test results inferring an absence of toxicity in one species provide no evidential weight with regard to toxicity in any other species, even when data from NHPs and humans are compared. Our results for species including humans, NHPs, dogs, mice, rabbits, and rats, have major implications for the value of animal tests in predicting human toxicity, and demand that human-focused alternative methods are adopted in their place as a matter of urgency.

Monkey-based research on human disease: the implications of genetic differences.

Assertions that the use of monkeys to investigate human diseases is valid scientifically are frequently based on a reported 90-93% genetic similarity between the species. Critical analyses of the relevance of monkey studies to human biology, however, indicate that this genetic similarity does not result in sufficient physiological similarity for monkeys to constitute good models for research, and that monkey data do not translate well to progress in clinical practice for humans. Salient examples include the failure of new drugs in clinical trials, the highly different infectivity and pathology of SIV/HIV, and poor extrapolation of research on Alzheimer's disease, Parkinson's disease and stroke. The major molecular differences underlying these inter-species phenotypic disparities have been revealed by comparative genomics and molecular biology - there are key differences in all aspects of gene expression and protein function, from chromosome and chromatin structure to post-translational modification. The collective effects of these differences are striking, extensive and widespread, and they show that the superficial similarity between human and monkey genetic sequences is of little benefit for biomedical research. The extrapolation of biomedical data from monkeys to humans is therefore highly unreliable, and the use of monkeys must be considered of questionable value, particularly given the breadth and potential of alternative methods of enquiry that are currently available to scientists.

An analysis of the use of animal models in predicting human toxicology and drug safety.

Animal use continues to be central to preclinical drug development, in spite of a lack of its demonstrable validity. The current nadir of new drug approvals and the drying-up of pipelines may be a direct consequence of this. To estimate the evidential weight given by animal data to the probability that a new drug may be toxic to humans, we have calculated Likelihood Ratios (LRs) for an extensive data set of 2,366 drugs, for which both animal and human data are available, including tissue-level effects and MedDRA Level 1-4 biomedical observations. This was done for three preclinical species (rat, mouse and rabbit), to augment our previously-published analysis of canine data. In common with our dog analysis, the resulting LRs show: a) that the absence of toxicity in the animal provides little or virtually no evidential weight that adverse drug reactions (ADRs) will also be absent in humans; and b) that, while the presence of toxicity in these species can add considerable evidential weight for human risk, the LRs are extremely inconsistent, varying by over two orders of magnitude for different classes of compounds and their effects. Therefore, our results for these additional preclinical species have important implications for their use in predicting human toxicity, and suggest that alternative methods are urgently required.

An analysis of the use of dogs in predicting human toxicology and drug safety.

Dogs remain the main non-rodent species in preclinical drug development. Despite the current dearth of new drug approvals and meagre pipelines, this continues, with little supportive evidence of its value or necessity. To estimate the evidential weight provided by canine data to the probability that a new drug may be toxic to humans, we have calculated Likelihood Ratios (LRs) for an extensive dataset of 2,366 drugs with both animal and human data, including tissue-level effects and Medical Dictionary for Regulatory Activities (MedDRA) Level 1-4 biomedical observations. The resulting LRs show that the absence of toxicity in dogs provides virtually no evidence that adverse drug reactions (ADRs) will also be absent in humans. While the LRs suggest that the presence of toxic effects in dogs can provide considerable evidential weight for a risk of potential ADRs in humans, this is highly inconsistent, varying by over two orders of magnitude for different classes of compounds and their effects. Our results therefore have important implications for the value of the dog in predicting human toxicity, and suggest that alternative methods are urgently required.

Modernizing Medical Research to Benefit People and Animals.

In the context of widespread public and political concern around the use of animals in research, we sought to examine the scientific, ethical and economic arguments around the replacement of animals with New Approach Methodologies (NAMs) and to situate this within a regulatory context. We also analyzed the extent to which animal replacement aligns with British public and policymakers' priorities and explored global progress towards this outcome. The global context is especially relevant given the international nature of regulatory guidance on the safety testing of new medicines. We used a range of evidence to analyze this area, including scientific papers; expert economic analysis; public opinion polls and the Hansard of the UK Parliament. We found evidence indicating that replacing animals with NAMs would benefit animal welfare, public health and the economy. The majority of the British public is in favor of efforts to replace animals and focusing on this area would help to support the British Government's current policy priorities. We believe that this evidence underlines the need for strong action from policymakers to accelerate the transition from animal experiments to NAMs. The specific measure we suggest is to introduce a new ministerial position to coordinate and accelerate the replacement of animals with NAMs.

The future of Parkinson's disease research: A new paradigm of human-specific investigation is necessary and possible.

Parkinson's disease (PD) is a complex neurodegenerative condition with a multifactorial origin. To date, approaches to drug discovery for PD have resulted in symptomatic therapies for the motor manifestations and signs associated with neurodegeneration but have failed to identify preventive or curative therapies. This failure mainly originates from the persistence of major gaps in our understanding of the specific molecular basis of PD initiation and progression. New approach methodologies (NAMs) hold the potential to advance PD research while facilitating a move away from ani-mal-based research. We report a workshop involving NAM experts in the field of PD and neurodegenerative diseases, who discussed and identified a scientific strategy for successful, human-specific PD research. We outline some of the most important human-specific NAMs, along with their main potentials and limitations, and suggest possible ways to overcome the latter. Key recommendations to advance PD research include integrating NAMs while accounting for multiple levels of complexity, from molecular to population level; learning from recent advances in Alzheimer's disease research; increasing the sharing of data; promoting innovative pilot studies on disease pathogenesis; and accessing philanthropic funding to enable studies using novel approaches. Collaborative efforts between different stakeholders, including researchers, clinicians, and funding agencies, are urgently needed to create a scientific roadmap and support a paradigm change towards effective, human-specific research for neurodegenerative diseases without animals, as is already happening in the field of toxicology.

Lessons from chimpanzee-based research on human disease: the implications of genetic differences.

Assertions that the use of chimpanzees to investigate human diseases is valid scientifically are frequently based on a reported 98-99% genetic similarity between the species. Critical analyses of the relevance of chimpanzee studies to human biology, however, indicate that this genetic similarity does not result in sufficient physiological similarity for the chimpanzee to constitute a good model for research, and furthermore, that chimpanzee data do not translate well to progress in clinical practice for humans. Leading examples include the minimal citations of chimpanzee research that is relevant to human medicine, the highly different pathology of HIV/AIDS and hepatitis C virus infection in the two species, the lack of correlation in the efficacy of vaccines and treatments between chimpanzees and humans, and the fact that chimpanzees are not useful for research on human cancer. The major molecular differences underlying these inter-species phenotypic disparities have been revealed by comparative genomics and molecular biology - there are key differences in all aspects of gene expression and protein function, from chromosome and chromatin structure to post-translational modification. The collective effects of these differences are striking, extensive and widespread, and they show that the superficial similarity between human and chimpanzee genetic sequences is of little consequence for biomedical research. The extrapolation of biomedical data from the chimpanzee to the human is therefore highly unreliable, and the use of the chimpanzee must be considered of little value, particularly given the breadth and potential of alternative methods of enquiry that are currently available to science.

An assessment of the use of chimpanzees in hepatitis C research past, present and future: 2. Alternative replacement methods.

The use of chimpanzees in hepatitis C virus (HCV) research was examined in the report associated with this paper (1: Validity of the chimpanzee model), in which it was concluded that claims of past necessity of chimpanzee use were exaggerated, and that claims of current and future indispensability were unjustifiable. Furthermore, given the serious scientific and ethical issues surrounding chimpanzee experimentation, it was proposed that it must now be considered redundant--particularly in light of the demonstrable contribution of alternative methods to past and current scientific progress, and the future promise that these methods hold. This paper builds on this evidence, by examining the development of alternative approaches to the investigation of HCV, and by reviewing examples of how these methods have contributed, and are continuing to contribute substantially, to progress in this field. It augments the argument against chimpanzee use by demonstrating the comprehensive nature of these methods and the valuable data they deliver. The entire life-cycle of HCV can now be investigated in a human (and much more relevant) context, without recourse to chimpanzee use. This also includes the testing of new therapies and vaccines. Consequently, there is no sound argument against the changes in public policy that propose a move away from chimpanzee use in US laboratories.

An assessment of the use of chimpanzees in hepatitis C research past, present and future: 1. Validity of the chimpanzee model.

The USA is the only significant user of chimpanzees in biomedical research in the world, since many countries have banned or limited the practice due to substantial ethical, economic and scientific concerns. Advocates of chimpanzee use cite hepatitis C research as a major reason for its necessity and continuation, in spite of supporting evidence that is scant and often anecdotal. This paper examines the scientific and ethical issues surrounding chimpanzee hepatitis C research, and concludes that claims of the necessity of chimpanzees in historical and future hepatitis C research are exaggerated and unjustifiable, respectively. The chimpanzee model has several major scientific, ethical, economic and practical caveats. It has made a relatively negligible contribution to knowledge of, and tangible progress against, the hepatitis C virus compared to non-chimpanzee research, and must be considered scientifically redundant, given the array of alternative methods of inquiry now available. The continuation of chimpanzee use in hepatitis C research adversely affects scientific progress, as well as chimpanzees and humans in need of treatment. Unfounded claims of its necessity should not discourage changes in public policy regarding the use of chimpanzees in US laboratories.