Lecture Outline. Darwin and Animal Welfare

Transcription

1 The Future of Animal Research in the Context of the 3Rs (Replacement, Reduction & Refinement) Joanne Zurlo, PhD Johns Hopkins Center for Alternatives to Animal Testing Lecture Outline Brief history of animals in research Laboratory animal protection and legislation in the US and the Three Rs Advances in replacement and reduction toxicity testing and drug development Advances in refinement Final thoughts A Short History Lesson A few key names: Rene Descartes 17th century (animals are automata) Jeremy Bentham 18th century ( The question is not, can they reason? Nor can they talk? But, can they suffer?) Claude Bernard 19th century (dubbed by Pasteur as Physiology itself) Darwin and Animal Welfare Both animal rightists and scientists embraced Darwin as their own in a period of spirited discussion regarding the use of animals in research leading up to the passage of the 1876 Cruelty to Animals Act in England In truth, Darwin was very much a centrist, but certainly conflicted as evidenced by some of his quotes Darwin Quotes on Animals "You ask about my opinion on vivisection. I quite agree that it is justifiable for real investigations on physiology; but not for mere damnable and detestable curiosity. It is a subject which makes me sick with horror, so I will not say another word about it, else I shall not sleep to night. From all that I have heard, however, I fear that in some parts of Europe little regard is paid to the sufferings of animals, and if this be the case, I should be glad to hear of legislation against inhumanity in any such country. Letter to Prof. Frithiof Holmgren (1881) Letter to Professor R. Lankester (1871) 1

2 On the other hand, I know that physiology cannot possibly progress except by means of experiments on living animals, and I feel the deepest conviction that he who retards the progress of physiology commits a crime against mankind. Letter to Prof. Frithiof Holmgren (1881) The difference in mind between man and the higher animals, great as it is, certainly is one of degree and not of kind. The Descent of Man (1871) In his book The Expressions of the Emotions in Man and Animals (1872), Darwin asserted that facial and bodily expressions reveal inner feelings, e.g. pain, fear, pleasure, in both humans and animals Since Darwin s time, advances made in ethology have shown that some animals are capable of: Self awareness Facial recognition Empathy and grief Tool making and usage Cooperativity Altruism Language Aggression Laboratory Animal Protection and Legislation in the US U.S. ANIMAL PROTECTION LEGISLATION 1966 Laboratory Animal Welfare Act focused on dealers to prevent pet theft (included dogs, cats, nonhuman primates, guinea pigs & rabbits) 1970 amendment to LAWA expanded to include all warm blooded animals used in research (except farm animals) 1971 USDA excluded rats, mice and birds from LAWA U.S. ANIMAL PROTECTION LEGISLATION (cont d) Animal Welfare Act (1985) amendment of LAWA: Created Institutional Animal Care and Use Committees (IACUCs) Mandated minimization of pain and distress Specified exercise requirements for dogs Established guidelines to maintain psychological well being of nonhuman primates 2

3 Public Health Service Policy on Humane Care and Use of Laboratory Animals Applies to all institutions with PHS funding Protects ALL vertebrate animals Institutions must comply with guidelines set forth in the Guide for the Care and Use of Laboratory Animals (NRC) Each assured facility must have an IACUC and report whether they are accredited by AAALAC International Association for Assessment and Accreditation of Laboratory Animal Care International Voluntary accreditation for laboratory animal care programs ( Uses the Guide for the Care and Use of Laboratory Animals (the Guide) as the basis for accreditation (along with other reference documents) Conducts announced site visits every three years Recent Changes to NIH Policy Random source dogs and cats for use in NIHfunded research may no longer be obtained from Class B dealers (based on recommendations of the ILAR report) Most use of chimpanzees for biomedical research is unnecessary and any proposed future research must meet strict criteria (based on recommendations from IOM report) Guiding principles for the use of animals in research the 3Rs Replacement Reduction Refinement WMS Russell and R Burch (1959) Principles of Humane Experimental Technique by now it is widely recognized that [the most humane] possible treatment of experimental animals, far from being an obstacle, is actually a prerequisite for successful animal experiments. W.M.S. Russell and R. Burch The Principles of Humane Experimental Technique, 1959 Replacement considerations Living systems, e.g., organ, tissue, or cell culture techniques (in vitro), invertebrate models, microorganisms, and if necessary less sentient animal species Non living systems (chemical, physical) Computer models or simulations (in silico) 17 3

4 Reduction considerations: Rational selection of group size including pilot studies and power analysis Careful experimental design and appropriate statistical analysis Maximizing use of animals Correct choice of model Optimal animal husbandry (monitoring for infections, avoiding overbreeding) Refinement Considerations Pain Recognition and alleviation Distress Minimization (prevention), recognition and alleviation Humane Endpoints Environmental enrichment 20 Toxicology Advances in Replacement and Reduction Toxicity Testing Informally The science of poisons Paracelsus the dose makes the poison More formally Toxicology is the study of the adverse effects of chemical, physical, or biological agents on people, animals, and the environment. Toxicity Testing Systematic approach to assessing adverse effects of a drug, chemical or other agent on human health Historically done in animals to measure a variety of endpoints Why test? A Brief History of Toxicity Testing Federal Food, Drug and Cosmetic Act (FDCA) 1938 Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) 1947 Toxic Substances Control Act (TSCA) 1976 Endocrine Disruptor Screening Program

5 Toxic Substances Control Act (TSCA) 1976 Control of new and existing industrial chemicals not regulated by other legislation No specific toxicity testing was required by EPA Chemicals in commerce at the time TSCA was passed (60,000) could remain in commerce unless EPA could show that a chemical posed a hazard For new chemicals, companies must bring data to EPA to help EPA assess potential adverse effects Many thousands of chemicals remain untested (e.g.,4 methylcyclohexanemethanol spill in WV Traditional Toxicity Testing Paradigm Relies on administering high doses of chemicals and drugs to animals, then results are extrapolated for low dose exposures Animals are typically inbred strains, and the results are extrapolated to heterogeneous human populations Results from animal studies do not always reflect results in humans Vision of the Committee A 2007 report by the National Research Council of the National Academies requested by the EPA To effect a paradigm shift in the approach to toxicity testing that would: Provide broad coverage of chemicals, chemical mixtures, outcomes and life stages Reduce the cost and time of testing Use fewer animals and cause minimal suffering in the animals used Develop a more robust scientific basis for assessing health effects of environmental agents Option I In Vivo Options for Future Toxicity Testing Strategies Option II Tiered In Vivo Option III In Vitro/In Vivo Animal biology Animal biology Primarily human biology High doses High doses Broad range of doses Low throughput Improved throughput High and medium throughput Option IV In vitro Primarily human biology Broad range of doses High throughput Expensive Less expensive Less expensive Less expensive Time consuming Less time consuming Less time consuming Relative large number of animals Fewer animals Substantially fewer animals Apical endpoints Apical endpoints Perturbations of toxicity pathways Some in silico and in vitro screens In silico screens possible Less time consuming Virtually no animals Perturbations of toxicity pathways In silico screens A New Paradigm: Activation of Toxicity Pathways Biologic Inputs (Courtesy of Mel Andersen) Exposure Tissue Dose Biologic Interaction Perturbation Adaptive Stress Responses Early Cellular Changes Low Dose Higher Dose Higher yet Normal Biologic Function Cell Injury Morbidity and Mortality 5

6 Toxicity Pathways Toxicity Pathway: A cellular response pathway that, when sufficiently perturbed, is expected to result in an adverse health effect. Progress in Implementation NTP Vision and Roadmap Memorandum of Understanding US EPA (National Center for Computational Toxicology) NIH/NIEHS (National Toxicology Program) NIH (National Center for Advancing Translational Sciences) US FDA Center for Drug Evaluation & Regulation Agency Commitment With an advanced field of An atmosphere of departure in regulatory science, new toxicology tools, including functional genomics, proteomics, New technologies metabolomics, from highthroughput (bio-screening, and biotech and )informatics systems revolution biology, we can Mapping of pathways of toxicity (PoT) replace current toxicology assays with tests that incorporate the mechanistic underpinnings of disease and of underlying toxic side effects. M.A. Hamburg, FDA 2011 We propose a shift from primarily in vivo animal studies to in vitro assays, in vivo assays with lower organisms, and computational modeling for toxicity assessments F. Collins, NIH, 2008 Uses automated chemical screening technologies, called high throughput screening assays, to expose living cells or isolated proteins to chemicals. Phase I: 309 data rich chemicals (primarily pesticides) having over 30 years of traditional animal studies valued at $2B; 500 assays Phase II: screened over 2000 chemicals from a broad range of sources (e.g., industrial and consumer products, food additives, failed drugs) using 700 assays for over 300 toxicity pathways Now being used for Endocrine Disruptor Screening 34 ToxCast Program (EPA) ToxCast Program (EPA) Emerging Technologies Omics technologies genomics, transcriptomics, metabolomics, proteomics Organ/Human on a Chip building a whole human, organ by organ Project supported by FDA, NIH, DARPA and DTRA Desire to achieve better predictability of new drugs for humans (cf. FDA animal rule for medical countermeasures) 35 6

7 Lung on a Chip (Wyss Institute for Biologically Inspired Engineering at Harvard University) Liver Heart Kidney Bone Marrow GI Brain Other Organs on Chips Published by AAAS Published by AAAS Future of Testing HUMAN on a chip????? achieve integration of all organ systems to create a virtual human Scientific and Animal Welfare Innovations in Drug Development and Safety Assessment Some Advances In vitro methods used early in drug development to identify potentially toxic agents leads to significant reduction in animal use In vitro assays to identify biomarkers for toxicity reduces numbers of animals needed Use of chemical structure analysis and information in databases to help select potential new drugs with less toxicity Advances (cont d) Use of stem cell cultures to assess cardiotoxicity, and less so for hepatotoxicity and neurotoxicity Use of human and animal hepatocyte cocultures to assess metabolism and liver toxicity Mechanism based investigative toxicology to identify critical organ systems and potential intervention systems 7

8 Advances in Refinement Specific Refinement Issues for Laboratory Animals Meeting biological needs Confinement in cages little freedom of movement Few guidelines for enrichment are laboratory animals normal? Caregivers not proficient in animal behavior of laboratory animals Abstract Mus musculus enjoys pride of place at the center of contemporary biomedical research. Despite being the current model system of choice for in vivo mechanistic analysis, mice have clear limitations. The literature is littered with examples of therapeutic approaches that showed promise in mouse models but failed in clinical trials. More generally, mice often provide poor mimics of the human diseases being modeled. Available data suggest that the cold stress to which laboratory mice are ubiquitously subjected profoundly affects mouse physiology in ways that impair the modeling of human homeostasis and disease. Experimental attention to this key, albeit largely ignored, environmental variable is likely to have a broad transformative effect on biomedical research. From Medical Unit blog: ABCnews.com From C.L. Karp (2012) Unstressing intemperate models: How cold stress undermines mouse modeling. J Exp Med 209, Environmental Enrichment Providing novel or complex stimuli to encourage species specific behaviors in a laboratory setting, and/or to avoid distress and stereotypical behaviors resulting from boredom or fear Enrichment group housing considerations Is the species social or solitary in the wild? Normal complex socialization in the wild, e.g. rhesus macaques different for males and females 48 8

9 Enrichment group housing benefits and costs Benefits include support of species specific behavior, social buffering, increased resistance to disease, increased immune response Costs include increased aggression and wounding, food competition, infant mortality, increased variability (dominance rank in NHP groups), separation effects, e.g., depression Other Types of Enrichment Nonhuman Primates Arboreal component in cage Visual barriers Food enrichment Novel foods Puzzle feeders Foraging opportunities Manipulanda (toys, tools) Visual (TV, computer games) Audio (music) Other Types of Enrichment Dogs Human interaction*** Handling grooming Visual access to conspecifics Toys Audio (music) Access to outdoors Considerations for Rodents Cage conditions Rats prefer opaque cages when humans are present (Cloutier & Newberry, 2010) and like hiding shelters Mice like to burrow and build nests (nest building also helps mice to maintain body temperature Lighting Noise Positive Reinforcement Training (PRT) Used to habituate laboratory animals to routine procedures Much in the literature on nonhuman primates Other animals can be trained as well Provides a positive experience + reward for the animals Results in significantly less stress Can eliminate stereotypic behavior The Future of Refinement Linked to advances in animal behavior research Necessary to understand normal behavior to meet animals needs Further incorporation of positive reinforcement training to enhance animal welfare 54 9

10 Final Thoughts Will there be an end to animal use for invasive research? Not if but when When is difficult to predict: depends on scientific advances, funding for research, commitment of agencies Until that time, we must employ the principles of the 3Rs Thank you! Questions? 10