Joint submission
by
The Dr Hadwen Trust for Humane Research
and
The Humane Society International
to
The Public Consultation on the SCHER working mandate and the call for scientific information on the use of non-human primates in experiments
June 2008
Contact:
Dr Gill Langley, Science Director, The Dr. Hadwen Trust for Humane Research e-mail: [email protected]
Emily McIvor, EU Director, The Humane Society International e-mail: [email protected]
Dr Hadwen Trust, June 2008 1 1. Introduction
This is a joint submission by the Dr Hadwen Trust for Humane Research, England, and the Humane Society International, in response to:
-- the public consultation on a working mandate for the Scientific Committee on Health and Environmental Risks (SCHER) to issue an expert scientific opinion on the ‘need’ for non-human primates in biomedical experiments, and
-- the call for information on the use of primates in biomedical experiments and the possibility of replacing their use.
The Dr Hadwen Trust for Humane Research is the UK's leading medical research charity funding exclusively non-animal research techniques to replace animal experiments, benefiting people and animals. The Dr Hadwen Trust has 38 years’ experience of funding high-quality, peer-reviewed and innovative research aimed at both advancing medical progress and replacing procedures on animals. Much of our research has had a significant impact in replacing experiments on primates.
The Dr Hadwen Trust is founded on anti-vivisection principles and is opposed, on ethical grounds, to all experiments on animals. We believe that excellence in medical research can and should be pursued without animal experiments.
The Humane Society International (HSI) is the international arm of the Humane Society of the United States (HSUS), the largest animal protection organisation in the world. The HSI works to reduce suffering and to create meaningful change for animals by advocating for greater protection of animals through legislation, investigating cruelty, educating the public and conducting hands-on programmes.
The HSI addresses issues such as inhumane conditions affecting companion and farm animals, the illegal trade in wildlife, threats to endangered species, slaughter of marine mammals and the use of animals in research and testing.
2. The Working Mandate
2a. We strongly support the priority given by DG Environment to replacing experiments on primates with alternative methods.
We urge that these methods should be 21st-century, advanced non-animal techniques. These hold the most promise for improving the quality of scientific research and testing whilst also addressing the concerns of European Union (EU) citizens about all animal experiments, not only those conducted on primates. The joint report by our two organisations, launched at a meeting in the European Parliament in May 2008, provides powerful arguments for putting the replacement of all animal experiments at the heart of the revision of Directive 86/609/EEC 1.
2b. It is important that, in the development of its opinion, SCHER acknowledges and reports on the lack of independent, systematic evidence supporting the value of experiments on non-human primates. The Working Mandate currently contains the phrase:
“[the use of primates] is considered essential in several research programs such as on immune based diseases (e.g. multiple sclerosis), neuro-degenerative disorders (Parkinson [sic], Alzheimer [sic], etc), infectious diseases (HIV, Malaria, TB, Hepatitis, SARS, etc.) and other serious diseases.”
Dr Hadwen Trust, June 2008 2 However, that statement reflects little more than an opinion. We argue in this submission that there is insufficient scientific evidence to support that contention. It should not be repeated by SCHER unless and until the committee finds and presents objective, transparent evidence in support of this claim.
2c. In reviewing, prioritising and discussing the replacement of primate experiments, it is essential that SCHER reviews each type of research study or category of test on a specific case-by-case basis. It is impossible to generalise and it would not be scientific to attempt to do so.
3. Our position on primate experiments
Our two organisations are ethically opposed to all experiments on sentient animals, believing that it is impossible to breed, supply, transport, house, handle and conduct experiments on such animals without causing them harm. Harming unconsenting individuals who will not benefit from an experiment, in the hopes of achieving scientific or medical progress for others, cannot be morally justified.
EU legislation is founded on the understanding that other animals can experience “pain, distress, suffering and lasting harm”. The advanced cognitive skills of primates add significantly to the case against using them in experiments, and their complex behavioural and social needs make it impossible to capture them, breed them, transport them to and/or keep them in laboratories, without compromising their physical and psychological health2.
Several EU member states have imposed specific rules and legislative requirements concerning the use of great apes in experiments. Austria and the Netherlands have adopted legislation that explicitly bans on the use of great apes, and Sweden bans all harmful great ape experiments. Britain has agreed that no great ape experiments will be authorised, and other non-EU countries, such as New Zealand and Australia have also adopted legislation to impose stringent restrictions. In Switzerland, the Swiss Commission on Animal Experiments and the Swiss Ethics Committee on Non-Human Biotechnology have proposed that great ape experiments should be prohibited.
The results of DG Environment’s 2006 expert questionnaire 3 revealed strong support for a ban on the use of all great apes, on the grounds of the ethical aspects and the positive impact on animal welfare, protection and biodiversity.
In Britain experiments on great apes were administratively prohibited 10 years ago due to their moral standing. There has never been a need to rescind that ban.
The experts’ questionnaire results also showed that many people felt a ban on wild-caught primates would have a negative impact on specific biomedical research programmes; and would have cost implications for user establishments. However, those comments were made without specific, credible evidence for the value of such experiments (see section 5 of this submission). The absence of such evidence seriously weakens the import of the comments.
We believe there are equally compelling moral arguments for ending the use of all primates in research and testing and we urge SCHER to make this recommendation.
If primate experiments are not prohibited throughout the EU, it is absolutely essential that the following policies are implemented in order to afford them the maximum possible protection from suffering and distress:
• An end to the use wild-caught primates. In Britain, wild-caught primates have not been used in experiments for many years.
Dr Hadwen Trust, June 2008 3 • All uses of primates including in basic biological and medical research, those killed for their tissues, and those used in the production and maintenance of genetically modified strains, must meet stringent animal protection requirements and be specifically authorised through EU legislation. • Authorisation of all proposals to use primates must be dependent on ethical review, successful inspection reports, and satisfactory training and infrastructure in the laboratories concerned. • Ethical review must include evidence of mandatory application of replacement, reduction and refinement techniques, as well as scrutiny of the justification given for requesting authorisation to use primates. • All experiments involving primates should also be subject to compulsory retrospective ethical review, in which the intended purpose of the experiment is compared with the actual outcome. These experiments thus receive additional scrutiny in the interest of improving research and ensuring that justifications given for using primates are regularly reviewed, according to a transparent assessment of evidence as it becomes available. • Immediate suspension of experiments failing to follow ethical review or authorisation constraints on their purpose or conduct, or those entailing more severe suffering than authorised. • The breeding, euthanasia and anaesthesia of animals must be strictly controlled so that minimising animal suffering and distress is the key priority. • A complete ban on experiments that may cause severe or prolonged pain, suffering or distress. • The Commission must collect and publish annual information from member states as detailed statistics of animal use, especially primates, including for the production and maintenance of genetically modified animals and the killing of animals used solely for their tissues. • EU and member state structures are needed to mandate the development and use of replacement, reduction and refinement (Three Rs) techniques to address all scientific uses of animals, especially primates. • The EU and member states should commission, fund and conduct research into the Three Rs, and provide training in use of these techniques. • An open-access EU database on Three Rs approaches, especially on the replacement of primate experiments, should be established. • Establish open-access databases of negative and unpublished primate studies to improve research opportunities, save animals and money and prevent duplication. • Setting targets for decreasing primate use through the application of replacement techniques. • Regular EU and member state reporting on the development and use of Three Rs techniques.
4. Primate experiments in Europe
Member states of the European Union (EU) collectively used approximately 10,451 (10,392 in EU15) primates (including prosimians) in 2005 4, representing an increase overall of 43% from EU15 since 1999 5. In 2005, France (3,789) and Britain (3,115) accounted for the largest numbers of primates, with Germany (2,086) a distant third. No experiments on great apes were reported in the EU in 2002 or 2005.
67% of primates used in the EU in 2005 were for toxicological (safety) purposes. These tests include:
● short-term toxicity tests (which may be lethal). ● sub-acute toxicity tests with repeat dosing, that last for 14-28 days. ● sub-chronic and chronic toxicity tests with repeat dosing, that last 90 days or more. ● toxicokinetics tests to assess the absorption, distribution, metabolism and excretion of medicines.
Dr Hadwen Trust, June 2008 4 ● safety pharmacology to characterise the side effects of new medicines. ● assessing new medicines for toxic effects on the immune system, and ● ‘other’ (unspecified) studies of medicines, and medical and dental devices.
Most medicines safety tests on primates are conducted to provide data to regulatory authorities.
The second most common use of primates in the EU is for fundamental biological research which used 1,456 animals.
“Fundamental biological research” includes fundamental research without immediate practical applications, such as basic studies of the function of the brain including research into thought, learning and memory (cognition), vision, hearing, taste and movement.
This category also includes basic medical research into human illnesses such as Parkinson’s disease, Alzheimer’s disease, AIDS, multiple sclerosis, epilepsy, schizophrenia, reproduction and fertility. Some primate ‘models’ of human diseases are used to research and develop new medicines including vaccines.
The third largest category is research, development, production and quality control of products and devices for human medicine, dentistry and veterinary medicine which used 981 primates in 2005. Production and quality control of medicines, mainly vaccines used 416 primates, with the routine testing of batches of polio vaccine probably accounted for much of this.
Forty-two primates were used in education and training, 16 to diagnose disease and 536 in unspecified studies.
5. Judging the ‘need’ for primate experiments: where is the scientific evidence?
Those who claim there is a need for primate experiments say that such experiments have been essential for past medical breakthroughs, and will continue to be necessary for medical progress.
However, these are simplistic and generalised claims based mainly on long-standing assumptions and perceptions rather than on hard scientific evidence or critical analysis, and are thus insufficient for EU policy-making. Policy development must be based on strong scientific evidence.
A recent article in the Journal of the Royal Society of Medicine challenged the scientific community for making unsubstantiated and unjustified claims about the value of animal experiments 6.
Claims that medical breakthroughs in the past have relied on primate research should only be considered by SCHER if there is clear supporting evidence on a specific case-by- case basis. This evidence should be objective, transparent and evidence-based, for example by means of Cochrane-style reviews showing whether primate studies were of a high quality and translated successfully to the human situation.
Such evidence is virtually unavailable. What evidence there is about the quality, necessity and validity of primate experiments suggests that most have not proved effective in advancing human medical progress.
For example, a 2007 review of chimpanzee experiments found they had made a minimal contribution to the advancement of biomedical knowledge 7. The author selected 95 chimpanzee experiments (from a total of 749 published worldwide between 1995 and
Dr Hadwen Trust, June 2008 5 2004). 49.5% (47/95) of the chimpanzee experiments were not subsequently cited. 14.7% (14/95) of the experiments were cited by 27 papers describing well-developed methods for tackling human diseases.
However, detailed examination of those medical papers revealed that in vitro studies, human clinical and epidemiological studies, molecular assays and methods, and genomic studies contributed most to the development of those effective methods. The author found that no chimpanzee study out of the 95 reviewed made an essential contribution (or even, in most cases, a significant contribution of any kind) to the development of the medical method described.
This is an astonishing finding. If experiments on great apes have made such a poor contribution to medical progress, experiments on other primates are hardly likely to do better.
In the absence of transparent, objective systematic reviews of the value of primate experiments, SCHER should carefully look at the concerns increasingly being raised about the validity of primate use in specific cases. Annex 1 of our submission includes case studies relating to neuroprotective drugs and stroke; the British example of TGN1412; and the development of GDNF for Parkinson’s disease.
In Switzerland, a request by scientists to conduct research into human depression using marmosets was referred to the Swiss Commission on Animal Experiments and the Swiss Ethics Committee on Non-Human Biotechnology.
The two committees produced a report 8 expressing their concern about marmoset suffering, but they also considered the scientific validity of the proposed marmoset ‘model’ of human depression. Unanimously, they “...questioned the relevance of the marmoset animal model to provide any meaningful findings for research into depression.”
Their final, also unanimous, recommendations were that the marmoset experiments should not be permitted; that the development of alternatives in depression research must be encouraged; and that experiments on great apes should be explicitly prohibited.
Despite their similarities to humans, there are many reasons why experiments on primates cannot be reliably extrapolated to humans.
5a. Other primates do not naturally suffer human illnesses such as Alzheimer’s disease, Parkinson’s disease, stroke or AIDS. Artificially inducing in other primates some pre-selected signs and symptoms that superficially resemble human diseases cannot sufficiently replicate the actual human disease being studied.
For example, surgically blocking an artery in the brain of a marmoset does not replicate the complexity of a human stroke, and applying the toxin MPTP to the brain of a macaque does not create a reliable surrogate of Parkinson’s disease.
Because disease symptoms are artificially induced in primates, by chemical poisoning or physical interference or other damage, it is very rare for primate research to shed any light on the underlying causes of human illness.
5b. Primates used in disease research are young and otherwise healthy animals. In contrast, human neurological diseases are usually found in middle-aged or elderly patients with complicating co-morbidities (e.g. atherosclerosis preceding a stroke). Neither do primates used in research have the full spectrum of symptoms which develop during the natural progression of a disease 9.
5c. Species differences between different primates, and between primates and humans, frequently occur: in anatomy (body structures), metabolism (e.g. drug handling),
Dr Hadwen Trust, June 2008 6 physiology (system functions), biochemistry or pharmacology (e.g. cellular receptors, drug effects). Even subtle molecular differences can have a significant effect on the validity of results for extrapolation from primates to humans, including in neurological and immunological research.
This was acknowledged in the conclusions of the 2002 report of the Scientific Committee on Animal Health and Animal Welfare, which re-stated that there is a great variation between the different species in the taxonomic order of primates 10 .
Our contention that objective, scientific evidence of the value of primate experiments is lacking was supported by Dr Vicky Robinson, chief executive of the British government’s National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), who said:
“The Weatherall Report made the scientific case for primate use in some areas of research, but it was not a comprehensive or systematic review. We are pleased that the UK's major funders of primate research are taking seriously their responsibility to retrospectively assess the value of the work they have funded. Such an assessment should weigh the outcomes from the research against the actual harms caused to the animals involved and the availability of alternative approaches.
“There is growing recognition in UK science and industry that looking critically at research using animals can benefit the scientific outcomes as well as the animals, and also a greater willingness to think innovatively about where the use of primates can be reduced. The Government and the funders should be seeking to develop a national strategy that is not just about the continued use of primates, but which has the clear aim of replacing, refining and reducing that use wherever possible.”
As well as the use of primates in basic medical research, the assessment of medicines for safety and efficacy involves tests on primates. These primate tests are not specifically required by regulatory authorities and can be misleading. For example, several reviews of adverse drug effects in dogs, monkeys and humans reveal, perhaps surprisingly, that the dog may be a better predictor of toxicity in humans than monkeys 11 . Many drugs that tested safe and effective in primates have proved ineffective and/or unsafe in patients (see Annex 2) including numerous experimental treatments for Parkinson’s disease 12 .
In fact, the US Food and Drug Administration reported that 92% of new drugs which pass extensive preclinical tests (which always include tests on primates) have failed to reach the market, either because of safety or efficacy problems 13 . That is itself an appalling indictment of the value of primate studies, which are clearly not fit for purpose.
6. Replacing primate experiments with non-animal methods
An important part of SCHER’s working mandate is to assess the availability of alternative methods to replace primate experiments. Replacing animal experiments benefits from a multi-disciplinary approach, combining techniques such as molecular, cell and tissue, post-mortem tissue, clinical, human volunteer, epidemiological and computer methods.
Here we discuss replacement possibilities in two main areas: medicines testing and neurological research.
6a. Development and testing of medicines
Most primates are used in the development and testing of new medicines, but alternative routes for drug development without primate studies are becoming increasingly realistic.
Dr Hadwen Trust, June 2008 7 The knowledge and databases in the archives of the world’s drug regulatory agencies, such as the FDA, EMEA and MHRA, are huge repositories of in vitro and primate data that are linked with actual human outcomes data. These data should be exploited as resources for the development and validation of non-animal (including non-primate) methods of drug testing.
To illustrate what could already be achieved without using animal tests, here is a hypothetical model for drug development and testing based on existing technologies:
1. Use in vitro human-based molecular (e.g. receptor binding), cell and tissue studies, to identify novel drug candidates. 2. Apply computer methods such as (Q)SAR models and three-dimensional computer modelling of drug molecules to confirm desired pharmaceutical activity and screen for toxicity. 3. Assess drug metabolism in vitro using human liver cells in culture. 4. Conduct in vitro mutagenesis assays to assess safety for early human dosing. 5. Conduct early, healthy volunteer microdosing (phase 0) studies, to assess pharmacokinetics and/or pharmacodynamics Footnote 1 & reference 14 . 6. Apply in vitro , cell-based assays to predict acute and longer term toxicity (including use of the outcomes of EU-funded research, such as ACuteTox and Predictomics). 7. Computer simulations such as physiologically-based pharmacokinetic (PBPK) and other modelling methods to scale up doses, treatment schedules, route of delivery etc. from all available molecular, in vitro and human microdosing data. 8. Careful dose escalation phase I trials should be considered in volunteer patients (rather than healthy volunteers) as they stand to benefit from any possible therapeutic effect. 9. Monitor drug efficacy in volunteers using state-of-the-art imaging (e.g. dopamine levels, brain cell loss, etc). 10. Monitor pharmacokinetics of the drug. 11. Carefully monitor physiology of volunteers and analyse body fluids to assess any early signs of toxicity.
Primates are used to test the immunogenicity of medicines and vaccines, and it is predicted that there may be an increase in demand for primate studies of protein-based medicines.
Many of these medicines are recombinant human or ‘humanised’ proteins that can cause unwanted but highly species-specific effects. Animal tests seldom predict accurately the likelihood of immunological reactions in people and even tests in primates are unreliable: “...animal studies, even those conducted in non-human primates, have limited predictive power for immunogenicity in humans...” 15 .
The experience of the drug company TeGenero with TGN1412 is a striking recent example (see Annex 1), where a monoclonal antibody almost caused the deaths of six healthy human volunteers, because tests in animals – including non-human primates – using extremely high doses of TGN1412 failed to predict the massive immune reaction that occurred in humans.
1 Human microdose studies rely on ultra-sensitive analytical methods such as positron emission tomography (PET) and accelerator mass spectrometry (AMS), to measure extremely small – and hence very safe – doses of medicines in volunteers. AMS can quantify drug and metabolite concentrations in the low picogram and femtogram range. Such sensitivity makes it possible to conduct human pharmacokinetic and metabolism studies using sub-toxic and sub-pharmacological doses of experimental drugs, very early in the development process. Early human microdose studies will enhance the selection of optimal drug candidates and reduce the likelihood of later failure, by early elimination of drug candidates with poor human pharmacokinetics or metabolism. Thus many ultimately useless animal tests, including primate studies, would be pre-empted and the likely success of phase I trials would be improved.
Dr Hadwen Trust, June 2008 8
More predictive assays for protein-based medicines are required and a better solution may lie in combining human-oriented computational and in vitro techniques. Computational methods that predict protein interactions with T-cells, for example on the basis of MHC/peptide binding motifs, have been developed by several research groups 16 . These aid candidate protein selection, minimising the need for subsequent tests.
This virtual screening would be followed by in vitro assays. These include MHC/peptide binding studies, peptide presentation assays and T-cell recognition assays. The latter usually use primed T-cells from immunised mice or from exposed human donors, which limits their applicability for completely novel proteins. Human dendritic and T-cells from donated blood samples can now be primed in vitro to detect immunogenic protein sequences 17 .
In a recent development, the US company VaxDesign has produced a highly relevant in vitro human model called MIMIC, to study and assess immune system reactions related to medicines, vaccines and chemicals 18 .
MIMIC is a two-stage microscale model of the human immune system, set in the wells of cell culture plates. Human T-cells, B-cells and monocytes are extracted from blood donations and applied to wells containing a layer of human endothelial cells above a layer of collagen. The blood cells migrate through the endothelial cells into the collagen matrix and spontaneously differentiate into dendritic cells. These reverse migrate into the supernatant and are activated by a challenge antigen.
The antigen-presenting dendritic cells are removed and added to the lymph node model, in a second cell culture plate. The immune response is characterised in terms of cytokines, antibodies and activated lymphocytes, to create a full in vitro model used for automated high-throughput testing.
VaxDesign says of the MIMIC technology:
“We developed MIMIC to address inherent flaws with current animal testing models, namely that animals can never offer completely predictive results of a new vaccine or therapy because of basic physiological differences between humans and animals. We created the MIMIC technology not only as an alternative to animal testing, but one that is more predictive of human responses.”
The International AIDS Vaccine Initiative recently awarded VaxDesign its Innovation Fund Award because of the progress MIMIC represents over animal studies in developing a vaccine for HIV. MIMIC is faster, more translatable to humans and can be used to test responses from genetically diverse populations before a vaccine enters humans in vivo .
6b. Neurological research
In medical research, primates are most widely used as neurological ‘models’ of human disorders such as Parkinson’s disease, multiple sclerosis, chronic pain, epilepsy and strokes; and to study cognitive, sensory, motor and other functions.
These experiments aim to understand the structures, connections and functions of the human brain, but for decades more was known about the brains of monkeys than humans and findings from the primate research remains of unproven relevance to patients suffering from neurological conditions. Many of these primate studies involve electrophysiology, using intracellular or field electrodes and/or lesioning techniques; while in others, monkeys are brain damaged, sometimes in long-term experiments.
The advent of safe brain imaging technologies has revolutionised understanding of neurological conditions, by making it possible to study, in vivo , the brains of human
Dr Hadwen Trust, June 2008 9 volunteers – healthy subjects and patients with serious conditions that urgently need researching.
Non-invasive imaging with human volunteers yields highly relevant information on human brain structure and function at the cell-population and systems levels, especially when used multi-modally. Safe and ethical human imaging studies have already replaced some primate research and much more progress is achievable.
Dynamic magnetic resonance imaging
Dynamic magnetic resonance with contrast enhancers and magnetisation transfer imaging provide information on the permeability of the human blood-brain barrier, which is disrupted in various diseases. Studies of disease progression in patients have already provided a better understanding of the nature of multiple sclerosis lesions, for example, than experiments on animals 19 .
Magnetic resonance spectroscopy non-invasively measures the distribution and concentration of neuronal and glial cell markers, relating these to human brain function in health and disease. Magnetic resonance imaging reveals cellular changes characteristic of disease states and their progression, which are simply unavailable from animal studies that depend on artificially created, surrogate forms of human illnesses.
Diffusion tensor MRI (DT-MRI)
Diffusion tensor magnetic resonance imaging (DT-MRI) provides high-resolution images showing the structure and architecture of deep white matter: the pathways that connect areas of the brain together.
Until recently, more was known about brain connectivity in monkeys than in humans, through invasive and usually terminal experiments (such as electrode studies and tract- tracing). Apart from the ethical issues of primate research, species differences can complicate interpretation of such data 20 . For example, before DT-MRI, detailed information about circuitry between motor areas and the striatum was available only from primate experiments, but these circuits have now been mapped directly in human volunteers 21 .
The value of tract-tracing primate models in studying brain networks is limited by:
1. Potential differences in connectivity patterns between species. 2. The restricted number of tracer injection sites that can be studied in each animal. 3. Technical difficulties in obtaining accurate, quantitative connectivity values from tracer experiments, and 4. The poor fidelity of animal models of human brain disorders such as stroke, schizophrenia and chronic pain.
In contrast to tracer experiments in animals, DT-MRI offer quantitative human data including connectivity information on the whole human brain, obtained in a single scan , in patient populations of interest.
DT-MRI can’t, at least yet, provide the high level of detail and accuracy possible in invasive animal studies. But as technology advances, improvements in spatial resolution and the development of more sophisticated fibre-tracing imaging methods, will allow more detailed mapping of the human brain and combined structure/function analyses of very small regions, in a single human individual.
DT-MRI is already being applied to research into ageing and cognition, neuropsychiatric disorders (e.g. schizophrenia 22 ), chronic pain, HIV infection and multiple sclerosis 23 . Because of its non-invasive nature, it can replace certain primate experiments with safe, highly relevant human volunteer studies, investigating relationships between brain
Dr Hadwen Trust, June 2008 10 structure, function, genes and behaviour 24 . It can be validated without animal experiments, e.g. by comparison with post-mortem human brain measurements, with existing imaging data, and with other functional data from the same volunteers 25 .
Transcranial magnetic stimulation (TMS)
Transcranial magnetic stimulation (TMS) is used to create momentary and reversible virtual ‘lesions’ in volunteers, to study the location of functions in the brain. TMS research with human volunteers has already replaced some primate experiments.
In early TMS studies funded by the Dr Hadwen Trust, the functions of two different parts of the parietal lobe of the brain were compared in volunteers. It was believed the two areas were separately critical for applying concentration to what one sees or does. The finding was a clear dissociation of visual and motor concentration abilities – an important result with respect to parietal lobe function 26 .
Then the role of one of these human brain regions in learning was examined with TMS 27 with striking results. These two early TMS studies in human volunteers had taken the research further than it could have gone in monkeys: the first exploited a difference between left and right cerebral hemispheres in humans that does not exist in monkeys, and the second required re-testing after training which isn’t possible when a brain region has been permanently removed or damaged, as it would be in primate experiments.
The Dr Hadwen Trust research provided proof of concept for TMS studies at a time when there was only one TMS system in the whole of the UK. TMS is now widely accepted as a technique that can replace some primate experiments. The British Royal Society, for example, has made clear that 28 :
“This ability to use human subjects in lesion experiments now obviates the need for some lesion experiments in non-human primates... TMS also has very high temporal resolution and cortical functions can be disrupted within time windows as small as 5 milliseconds, thus allowing a temporal dimension to lesion analysis that is difficult to achieve in monkeys with other methods.”
Human studies with TMS have advantages over primate experiments: TMS has transient effects, so that learning and plasticity can be studied; it enables research into temporal functions and systems-level activities, which electrode experiments do not; and it eliminates species difference problems.
Magnetoencephalography (MEG)
MEG generates high-resolution functional maps of the human cortex, with a temporal resolution of a few milliseconds and spatial discrimination of around two millimetres.
Ground-breaking human volunteer research with MEG was undertaken at Aston University in England in the 1990s, funded by the Dr Hadwen Trust. It showed that accurate cortical function data from humans can be obtained with MEG, and it laid the foundations for subsequent MEG studies of human brain functional architecture 29 . Consequently, Aston later received a Wellcome/HEFCE infrastructure grant of £1.5 million, which was used to commission the UK’s first whole-head MEG; and it is now an internationally recognised neuroimaging centre.
Using Synthetic Aperture Magnetometry with human volunteers, researchers have demonstrated gamma oscillations in human visual cortex, indicating that MEG does provide a direct and reliable representation of underlying neuronal activity 30 .
Dr Hadwen Trust, June 2008 11 Cortical representation maps with high levels of resolution have been produced using MEG and EEG, with an accuracy (approaching one cubic millimetre of brain tissue) close to that achieved by highly invasive microelectrode recordings in primates.
Combining MEG and magnetic resonance spectroscopy may provide models of human neurochemical measures linked to anatomically pinpointed electrical behaviour, in the cerebral cortex of human volunteers. This pharmaco-imaging approach would have wide applications to understanding normal brain function, treatment of neurological disorders and the targeted design of new CNS drugs. Modelling these parameters in volunteers would generate results more relevant to humans, as well as replacing some invasive animal experiments.
Multi-disciplinary approaches
Safe brain-imaging methods in volunteers can be combined with cell and tissue based studies, post-mortem tissue studies, molecular and genetic epidemiology and computer simulations and models, to replace primate experiments.
We describe here two case studies, in pain research and Parkinson’s disease research.
Pain research: Pain research experts from academia and industry recently reported on strategies and challenges in replacing animal experiments in pain research with ethically conducted studies of human patients and healthy volunteers, in combination with in vitro methods. Their report 31 considers how a range of neuroimaging techniques, singly and combined, can address important features of human pain conditions. In addition, microdialysis in human subjects; genome-wide association research, twin studies and other epidemiological approaches; and in vitro cell and tissue research, offer complementary replacement potential in this field, but also in many others.
Parkinson’s disease research: In this research, marmosets and macaques are injected with neurotoxins, such as 6-hydroxydopamine and MPTP, to create brain damage which, in limited respects, resembles that seen in Parkinson's disease32 . However this does not accurately recreate Parkinson's disease in animals and the causes and progression of PD in humans remain largely unknown 33 .
Primate 'models' of Parkinson's disease have many serious limitations which include:
1. The rapid appearance of symptoms in primates, yet the causes of the human illness are unknown and symptoms are slow to develop 2. Compensatory mechanisms in undamaged brain regions are likely to be different in lesioned monkeys compared to human patients with Parkinson’s disease 3. If toxic injections stop, primates show partial but variable recovery (with variation between species). However, humans always show a progressive worsening of the symptoms over time 4. Parkinson’s disease usually affects older people with co-morbidities, but the monkeys used in research are young and otherwise healthy 5. Lewy bodies are either never seen, or only very infrequently seen, in primates; but in patients these cellular inclusions are the classic hallmark of the illness 6. MPTP-treated primates show sporadic limb tremor but in patients tremor is marked and sustained. The cognitive patterns of impairment also differ between primates and patients 34 7. In primates, specific dopamine-containing brain cells are damaged in one part of the brain. In Parkinson’s disease, damage is more widespread and involves other neurotransmitter systems, in addition to dopamine.
Combining a range of non-animal but human-relevant approaches offers a different, more species-specific route to researching Parkinson’s disease. Positron emission tomography (PET) imaging has been used to measure levels of dopaminergic activity in the brains of
Dr Hadwen Trust, June 2008 12 Parkinson's patients, shedding light on the pathophysiology of the condition, and permitting direct study of disease progression at a biochemical level 35 .
Volunteer studies have provided evidence that direct dopamine agonists (mimics) can inhibit the release of endogenous dopamine, possibly by the activation of presynaptic dopamine receptors 36 . Functional MRI has been applied to patients to study impaired connectivity between frontal cortical regions of the brain underlying the movement disorders of Parkinson’s disease 37 .
Some researchers have studied in cell culture the role of the protein synuclein 38 (found in Lewy bodies in the brains of Parkinson's patients). Cell cultures have also been used to study oxidative stress and microglial activation as factors in Parkinson's disease.
Human post-mortem tissue studies have provided evidence of biochemical damage underlying the progression of Parkinson’s disease 39 . Molecular epidemiology of human populations is elucidating the genetic and environmental factors which interact to cause Parkinson's disease. A molecular genetic approach has identified three genes and two or more additional genetic locations in the rarer familial forms of Parkinson's disease 40 .
7. Summary and recommendations
We believe that the use of all non-human primates in laboratories should be prohibited: on ethical grounds, reflecting the strength of public concern; and on scientific grounds, because advanced non-animal methods offer superior scientific outcomes.
If SCHER is considering recommending that certain primate experiments continue, then it could only do so on the basis of objective, scientific evidence that such experiments are effective, reliable and predictive of human situations. Since the value of primate research to human medical progress has not been subjected to independent and systematic scrutiny, SCHER is not in a position to justify the ‘necessity’ of primate experiments.
Independent and expert systematic analyses should be undertaken of historical primate studies of all kinds, to assess their relevance to humans. These must be published and used to inform the process of ending primate experiments.
Some primate experiments can and should be replaced now by non-animal methods, and SCHER should carefully review these areas and make appropriate recommendations.
Other uses of primates need urgent review, on a case-by-case basis, and prioritisation for research to enable them to be replaced quickly, maximising the implementation of advanced non-animal techniques. SCHER should produce a targeted and timetabled strategy to achieve the replacement of all primate experiments, and should recommend that the necessary political will and funding resources are urgently brought to bear.
The conceptual shifts required in the primate-using community should be facilitated to advance different modes of research and to implement non-animal methods as replacements. Equally, researchers and toxicologists should be made aware that an excessive reliance on and confidence in the value of animal studies, can itself lead to serious unexpected outcomes. This has been demonstrated by the examples of TGN1412, and by the unexpected life-threatening psychological effects in humans of drugs such as Seroxat and Roaccutane.
If SCHER does not recommend a prohibition of all primate experiments, then the level of protection given to primates must be greatly enhanced and the use of primates should be phased out as part of an overarching EU strategy to replace all animal experiments. The scientific validity of primate experiments must be constantly re-assessed on the basis of new evidence obtained through retrospective ethical reviews and from all other sources.
Dr Hadwen Trust, June 2008 13 Transparency and accountability should be improved through detailed and comprehensive reporting and enforcement measures.
Dr Hadwen Trust, June 2008 14 Annex 1
Specific case studies where primate experiments have failed to predict human consequences
Case study 1 – Neuroprotective drugs for stroke
NXY-059 was hailed as a likely breakthrough in the treatment of patients who suffer strokes. AstraZeneca predicted in 2002 that, by carefully following guidelines on the conduct of animal tests for such drugs, NXY-059 had increased chances of being successful in clinical trials 41 .
This drug was probably tested more extensively in primates that any preceding stroke drug, being assessed in marmosets with deliberately induced ‘strokes’ which involved substantial suffering. However a series of clinical trials finally demonstrated in 2006 that the drug was completely ineffective in patients.
This is the 114th drug of this kind to have succeeded in animal tests but failed in patients. An editorial in The Lancet 42 advised: “Translation of positive results obtained in the laboratory into the clinic has been exceptionally elusive, and the stroke [research] community needs to think long and hard about whether these animal models are financially and ethically viable”.
Case study 2 – Parkinson’s disease and GDNF
GDNF, a nerve growth factor, was considered a potential treatment for Parkinson’s disease and 1996 saw the first report of a monkey study.
However, a clinical trial of GDNF in a Parkinson’s disease patient had been started before the primate test results were available 43 . Then a phase I clinical trial, started in 2000, showed benefits for patients without side effects, and a larger trial followed. But this was stopped in 2004, because longer-term tests in monkeys had revealed side effects (cerebellar damage) 44 . Patients sued the drug company for withholding a treatment that they found beneficial, on the basis solely of tests on monkeys. The side effects they developed have not been found in Parkinson’s disease patients.
This case study illustrates a number of points. Species differences in brain size meant that two methods of delivering GDNF to the brain which were successful in marmosets, were not both effective in patients 45 . Monkey studies are often claimed to be important for ‘scaling up’ from the rodent brain to the larger human brain for predicting human doses, routes of delivery, injection sites and administration protocols. Clearly, the primate studies failed in this case. Moreover, the clinical trials of GDNF began before primate data were available, which begs the question: how ‘necessary’ were the primate tests?
Case study 3 – TGN1412
Six volunteers were nearly killed in 2006 while participating in a clinical trial of the humanised monoclonal antibody TGN1412 — a protein designed to interact very specifically with human immune cells in the bloodstream. Rats and primates endured deliberate inflammation of their joints in tests of the drug. Toxicity tests used cynomolgus and rhesus monkeys (as well as rats and rabbits), but revealed no serious side effects.
TGN1412 was intended to activate regulatory immune cells, which would then ‘damp down’ the immune system overall. Instead, and despite studies in two species of primates, the drug unexpectedly massively stimulated the volunteers’ immune systems, causing organ failure. Before the human trial, the drug company claimed that binding sites for TGN1412 in rhesus and cynomolgus monkeys are identical to those in humans. However,
Dr Hadwen Trust, June 2008 15 other experts had reported differences of up to 4% between rhesus and human binding sites. This means the strength of the drug's effect could be substantially different in monkeys and humans, and the monkey tests were insufficient to assure safe predictions for humans.
The expert group set up by the government to investigate the disaster reported that later studies, using a different test-tube method, showed a clear contrast in the responses of monkey and human cells 46 . If the test had been conducted before the clinical trial, the species difference would have been apparent and the disaster might have been averted.
Dr Hadwen Trust, June 2008 16 Annex 2
Many drugs which were tested in primates (as well as in other animals) caused unexpected and sometimes fatal effects in humans.
A short list includes:
TGN1412 (T-cell stimulator; caused massive immune reaction in volunteers)
Amrinone (heart drug; caused haemorrhage in humans)
Fenclofenac (anti-inflammatory; caused jaundice in humans)
Vioxx (pain-killer; raised risk of heart attack & stroke in humans)
Fialuridine (anti-viral; caused liver failure in humans)
Carbenoxalone (for ulcers; caused heart failure in patients)
5FU (cancer; was metabolised differently in humans)
Benoxaprofen (for arthritis; killed some patients)
Flosequinan (for heart failure; increased deaths in patients)
Nomifensine (anti-depressant; caused liver damage in humans)
Losartan (for high blood pressure; metabolic differences caused side effects)
Methoxyflurane (anaesthetic; caused kidney failure in humans)
Indinavir (anti-AIDS drug; metabolic differences between humans and primates)
Flosint (for arthritis; lethal in humans)
Isoprenaline (anti-asthmatic; unsafe dose for humans).
Dr Hadwen Trust, June 2008 17 Annex 3
Advanced Non-Animal Techniques Include:
• Gene-hunting tools to find and understand the role of disease-causing genes in people;
• Cell and molecular tests for the safety of medicines and chemicals;
• Biosensors that synergise cell research with microelectronics, to study drug metabolism, toxicity and disease biomarkers;
• Ultra-sensitive analytical techniques such accelerator mass spectrometry allow safe, ethical, microdose studies of drugs without animal testing;
• Advanced microscopic techniques for imaging and analysing human cell functions in health and disease;
• High-powered computer models that realistically simulate the human body and its component systems and organs, and their reactions to medicines;
• Studies of post-mortem tissues bequeathed by patients for research into human illnesses;
• Tissue engineering that re-creates three-dimensional human tissues in the test-tube, for disease research, drug development and safety testing;
• Computer predictions of medicinal effects based on the structures of molecules;
• High-technology, safe imaging of the human brain to understand neurological disorders and drug effects on the brain;
• Molecular methods to study disease using human cells in the test-tube.
Dr Hadwen Trust, June 2008 18 Contact details
Dr Gill Langley Dr Hadwen Trust, 84A Tilehouse Street, Hitchin, Herts. SG5 2DY, England. Tel: +00 44 (0)1462 436819 Email: [email protected] Web site: www.drhadwentrust.org Science web site: www.scienceroom.org
Emily McIvor Humane Society International www.hsi.org Tel: + 00 44 (0)7812 354144 Email: [email protected]
Dr Hadwen Trust, June 2008 19 References
1 Dr Hadwen Trust and Humane Society International (2008). Towards a European Science Without Animal Experiments: Opportunities for the replacement of animal experiments provided through the revision of Directive 86/609/|EEC. Publ. DHT/HSI, May 2008. 2 Langley G (2006). Next of Kin: A Report on the Use of Primates in Experiments. London: BUAV. www.buav.org 3 Results of the expert questionnaire on the revision of Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes. 16 June - 18 August 2006. http://ec.europa.eu/environment/chemicals/lab_animals/questionnaire2.htm 4 The figures are approximate partly because France provided statistics for 2004 instead of 2005. 5 Annex to the Report on the Statistics on the Number of Animals used for Experimental and other Scientific Purposes in the Member States of the European Union in the year 1999. 6 Matthews RAJ (2008). Medical progress depends on animal models - doesn't it? Journal of the Royal Society of Medicine, 101:95-98. 7 Knight, A (2007). The Poor Contribution of Chimpanzee Experiments to Biomedical Progress. Journal of Applied Animal Welfare Science, 10: 281–308. 8 Swiss Committee on Animal Experiments/Swiss Ethics Committee on Non-Human Biotechnology (2006). Research on Primates — An Ethical Evaluation. Available in English via: www.ekah.ch 9 Hackam DG (2007). Translating animal research into clinical benefit. Br. Med. J. 334:163-164. 10 The welfare of non-human primates used in research. Report of the Scientific Committee on Animal Health and Animal Welfare. Adopted on 17 December 2002. http://ec.europa.eu/food/fs/sc/scah/out83_en.pdf 11 Greaves P et al (2004). First dose of potential new medicines to humans: how animals help. Nature Rev. Drug Discov. 3:226-236. 12 Linazasoro G (2004). Recent failures of new potential symptomatic treatments for Parkinson’s disease: Causes and solutions. Movement Dis. 19:743-754. 13 US FDA (2004). Report on Challenge and Opportunity on the Critical Path to New Medical Products, March 2004. www.fda.gov/oc/initiatives/criticalpath/whitepaper.html 14 Combes RD et al (2003). Early microdose studies in human volunteers can minimise animal testing: Proceedings of a workshop organised by Volunteers in Research and Testing. Eur. J. Pharm. Sci. 19:1-11. 15 Bugelski PJ & Treacy G (2004). Predictive power of preclinical studies in animals for the immunogenicity of recombinant therapeutic proteins in humans. Curr. Opin. Mol. Ther. 6:10-16. 16 Koren E, Zuckerman LA & Mire-Sluis AR (2002). Immune responses to therapeutic proteins in humans — clinical significance, assessment and prediction. Curr. Pharm. Biotechnol. 3:349-360. 17 Stickler MM, Estell DA & Harding FA (2000). CD4+ T-cell epitope determination using unexposed human donor peripheral blood mononuclear cells. J. Immunother. 23:654-660. 18 See the VaxDesign website, http://www.vaxdesign.com/advanced-immunotherapy-testing/index.php 19 Langley G et al (2000). Volunteer studies replacing animal experiments in brain research: Report and recommendations of a Volunteers in Research and Testing Workshop. ATLA 28:315-331. 20 Yamamoto T et al (2004). Cerebellar activation of cortical motor regions: Comparisons across mammals. Prog. Brain Res. 143:309-317. 21 Lehericy S et al (2004). 3-D diffusion tensor axonal tracking shows distinct SMA and pre-SMA projections to the human striatum. Cereb. Cortex 14:1302-1309. 22 Szeszko PR et al (2005). White matter abnormalities in first-episode schizophrenia or schizoaffective disorder: a diffusion tensor imaging study. Am. J. Psychiatr. 162:602-605. 23 Schmierer K, Wheeler-Kingshott CA, Boulby PA, Scaravilli F, Altmann DR, Barker GJ, Tofts PS & Miller DH (2007). Diffusion tensor imaging of post mortem multiple sclerosis brain. Neuroimage 35:467-477. 24 Tomassini V et al (2007). Diffusion-weighted imaging tractography-based parcellation of the human lateral premotor cortex identifies dorsal and ventral subregions with anatomical and functional specializations. J Neurosci 27:10259-10269. 25 Johansen-Berg H et al (2005). Functional-anatomical validation and individual variation of diffusion tractography-based segmentation of the human thalamus. Cereb. Cortex 15:31-39. 26 Rushworth MFS, Ellison A & Walsh V (2001). Complementary localization and lateralization of orienting and motor attention. Nature Neuroscience 4:656-661. 27 Walsh V & Pascual-Leone A (2003). Magnetic stimulation and cognition. MIT Press. 28 Royal Society (2004). The use of non-human animals in research: a guide for scientists. 29 Fylan F et al (1997). Magnetoencephalographic investigation of human cortical area V1 using color stimuli. NeuroImage 6:47-57; and Singh KD et al (2002). Task-related changes in cortical synchronisation are spatially coincident with the hemodynamic response. NeuroImage 16:103-114. 30 Hall SD, Holliday IE, Hillebrand A et al (2005). The missing link: analogous human and primate cortical gamma oscillations. Neuroimage 26:13-17. 31 Langley CK, Aziz Q, Bountra C, Gordon N, Hawkins P, Jones A, Langley G, Nurmikko T & Tracey I (2008). Volunteer studies in pain research – Opportunities and challenges to replace animal experiments. The Report and Recommendations of a Focus on Alternatives Workshop. http://dx.doi.org/10.1016/j.neuroimage.2008.05.030 32 Hurley MJ et al (2005). Immunoautoradiographic analysis of NMDA receptor subunits and associated postsynaptic density proteins in the brain of dyskinetic MPTP-treated common marmosets. Eur. J. Neurosci. 21:3240-3250.
Dr Hadwen Trust, June 2008 20
33 Calne DB (2003). Parkinson's Disease over the last 100 years. In: Parkinson's Disease: Advances in Neurology 91:1-8. 34 Collins P et al (2000). The effect of dopamine depletion from the caudate nucleus of the common marmoset (Callithrix jacchus) on tests of prefrontal cognitive function. Behav. Neurosci. 114:3-17. 35 Leenders KL & Oertel WH (2001). Parkinson's disease: clinical signs and symptoms, neural mechanisms, positron emission tomography, and therapeutic interventions. Neural Plast. 8:99-110. 36 de la Fuente-Fernandez R et al (2001). Apomorphine-induced changes in synaptic dopamine levels: positron emission tomography evidence for presynaptic inhibition. J. Cerebr. Blood Flow Metab. 21:1151- 1159. 37 Rowe J et al (2002). Attention to action in Parkinson's disease: impaired effective connectivity among frontal cortical regions. Brain 125:276-289. 38 Tofaris GK et al (2001). alpha-synuclein metabolism and aggregation is linked to ubiquitin-independent degradation by the proteasome. FEBS Lett. 509:22-26. 39 Dawson TM & Dawson VL (2003). Molecular pathways of neurodegeneration in Parkinson's Disease. Science 302:819-822. 40 Shastry BS (2001). Parkinson's disease: etiology, pathogenesis and future of gene therapy. Neurosci. Res. 41:5-12. 41 Green R (2002). Why do neuroprotective drugs that are so promising in animals fail in the clinic? An industry perspective. Clin. Exp. Pharmacol. Physiol. 29:1010-1034. 42 Editorial (2006). Neuroprotection: The end of an era? Lancet 368:1548. 43 Brundin P (2002). GDNF treatment in Parkinson’s disease: time for controlled clinical trials? Brain 125:2149- 2151. 44 Anon. (2005). Patient choice in clinical trials. Lancet 365:1984. 45 Gill SS et al (2003). Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson’s disease. Nat. Med. 9:589-595. 46 Expert Scientific Group on Phase I Clinical Trials (2006). Final report. 30 November 2006. www.dh.gov.uk
Dr Hadwen Trust, June 2008 21