GLOBAL CAMPAIGN TO CLOSE DOWN HUNTINGDON LIFE SCIENCES
Alternatives to animal testing

Besides saving countless animal lives, alternatives to animal tests are efficient and reliable. At the start of the 21st Century, non-animal techniques have become the cutting edge of medical research. Forward-thinking companies are exploring modern alternatives. For example, Pharmagene Laboratories, based in Royston, England, is the first company to use only human tissues and sophisticated computer technologies in the process of drug development and testing. With tools from molecular biology, biochemistry, and analytical pharmacology, Pharmagene conducts extensive studies of human genes and how drugs affect those genes or the proteins they make. While some companies have used animal tissues for this purpose, Pharmagene scientists believe that the discovery process is much more efficient with human tissues. “If you have information on human genes, what’s the point of going back to animals?” says Pharmagene cofounder Gordon Baxter.[1]

CELL CULTURE
It is possible to obtain human cells and tissues from biopsies, post-mortems, placentas, or as waste from surgery, and grow them in the laboratory.

Drug testing can use cell culture to great advantage, and many forward thinking scientists use cell culture in tests that traditionally have used animals – like screening drugs for a positive effect or the potential to do damage. In 1996 a team based at Uppsala, Sweden, compared animal test data, human experience and the results of cell culture tests for a range of chemicals. Their aim was to discover whether animals or cell culture were better predictors of what happens in humans. The cell culture results were found to be significantly more accurate.[2] Since then further advances have been made, such as the use of lasers in cell culture tests, and 3D tissue structures, making cell cultures superior by a greater margin.

Skin tests on animals cannot be justified given the existence of the EpiDerm test which uses human skin cells and is accepted as accurate, and Epipack which uses sheets of cloned human skin cells. The Human Keratinocyte Bioassay enables a computer to measure damage to the epithelial cells, which cover the skin and eyes. Corrositex detects skin damage using a membrane and a chemical detection fluid, and gives results in 4 hours – compared with 4 weeks for animal tests.[3] The MatTek EpiOcular test has been using human cells since 1985 to evaluate eye irritancy, and is one of many that do the job.[4] Incredibly, animals are still used in these sorts of tests.

Ensuring safety for pregnant mothers is a popular use of animals. The Embryonic Cell Test (EST) is a highly accurate test which has been available since 2001, and a medical journal review recently claimed that it alone was more valuable than all animal tests combined in this area.[5] The Micromass (MM) test is also invaluable, and is proven particularly effective for chemicals causing specific forms of damage to the growing embryo.[6]

By comparison, animal tests are extremely inaccurate, and rarely more accurate that pure guesswork. Over 97% of substances predicted as dangerous to pregnant humans are entirely safe[7]. Substances claimed to be dangerous in the animal lab include oxygen, several vitamins, and many safe fruit and vegetable extracts. For substances that are dangerous to humans, animals are rarely more effective than could be expected by chance.[8] 70% of dangerous drugs are safe in pregnant monkeys.[9]

The American National Cancer Institute (NCI) now favours cell culture over animals in drug screening and can “screen up to 20,000 compounds per year for potential anticancer activity” using “60 different human tumor cell lines, representing leukaemia, melanoma, and cancers of the lung, colon, brain, ovary, breast, prostate and kidney.”[10]

The alternative is to use animals, which is not effective. A textbook explains: "despite 25 years of intensive research and positive results in animal models, not a single anti-tumour drug emerged from this work." [11]

It is also important that human cells, rather than animal cells, are used for medical research, to avoid the problem of relating results from one species to another. To encourage the use of human tissue the Dr Hadwen Trust has helped establish the Human Tissue Bank at Leicester. The Dr Hadwen Trust has funded research using human cells and tissues to replace animal experiments, into Alzheimer's disease, cancer, rheumatism, cataracts, allergies, meningitis, and more.

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COMPUTERS
An area where animal use is particularly popular yet also especially flawed in predicting effective drugs and identifying dangerous ones – yet HLS are still using this outdated, inaccurate method. Computers have revolutionised this area, as their ability to handle millions of interactions simultaneously enables them to model physical conditions.

Among various models of the human heart is the one developed by Denis Noble at Oxford University. It beats, develops illnesses and reacts to drugs. Drug companies have been using it since 2001 to predict drug reactions and eliminate dangerous drugs.[12] It can replay reactions, show them in slow motion, and be subjected to extremes that animals and patients can’t.

Other computer packages predict drug effects – one specialises in those in babies and children, an area animal tests have shown their failure with dramatic results for the children involved.[13] The goal of developing an entire virtual human is being achieved already, with organs and their interactions being simulated accurately along with reactions to drugs.[14]

The sort of technology scientists are watching with great interest is microdosing. Patients are given 1% of a test drug while their body is scanned using Accelerator Mass Spectrometry. This shows where the drug is and monitors it’s activity and effects. Evaluation of microdosing has shown it to be surprisingly accurate, even on drugs than have unusual, unexpected characteristics.[15]

It’s even been proven to work at lower test levels. Tests using one millionth (0.0001%) of therapeutic doses still enabled evaluation of drug concentrations in blood, saliva, urine, DNA and white blood cells. An expert explained "we can say with confidence that between 30 min and 45 minutes after dosing, 0.09% of the oral dose resided within the white blood cells in the blood. We were also able to show uptake of AZT into the genetic material of these cells, which is ultimately how antivirals like AZT inhibit viral replication. Such data could not have been obtained by any other method".[16] By comparison, animals are known to metabolise medicines along different routes in the body[17]. The majority of dangerous reactions are missed in animal tests[18], and most dangerous reactions predicted at that stage never happen in humans[19].

Part of the problem with animals studies is that straight away there’s a complex animal with millions of interactions that are too complex to unravel. Now it’s increasingly obvious that we need to understand what’s happening with individual cells, and even within individual cells. Proteomics is the study of how proteins are arranged in individual cells, and already this area has enabled advances: for example working out how to enable cancer dugs to interact with target cells.[20]

Now projects have started to catalogue protein activity in cells to understand what happens to cause illnesses. An expert explains: “Proteins are central to our understanding of cellular function and disease processes and without a concerted effort in proteomics the fruits of genomics will go unrealised. The necessity of proteomics cannot be avoided”[21]

No animal liver is similar to a human liver, which is a major problem because this organ is central to the way a drug is handled in the body. But now human liver has been grown in the lab, and can be used to test drugs. A report said that the discovery “eliminates the need for animal experiments for drug testing.”[22]

With this wealth of scientific methodology available, there clearly isn’t a need for animal testing. Ironically, HLS’ vehement defence of their animal testing practices could prove their undoing. As the industry moves on, the new methods are those that will enable commercial survival. The Chairman of Charles River, largest lab animal supplier in the world, was asked about the company’s diversification which has reduced the animal trade from 80% to 40% of business activity in just five years. “I don’t want to sit here and say ‘Hey, there goes our animal business’ ” he explained, while explaining a new non animal test that was far superior to the old animal methods.[23] HLS’ obstinacy and refusal to embrace the new methods ignores the revolution which is leaving animal tests as scientifically undesirable and enabling scientists to gain a previously impossible understanding of medical science. This technology could also play a part in hastening the demise of HLS.

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MICROORGANISMS
Tests with simple microorganisms, such as bacteria and yeasts, are being used as early indicators of chemicals likely to be harmful, and are frequently faster, cheaper and more humane than animal tests. Bacteria can be genetically manipulated to manufacture useful products previously obtained from animals, such as human insulin and monoclonal antibodies.

The Trust's research into diabetes successfully used a microscopic organism called Hydra, as an alternative to diabetic animals. Whilst another Trust researcher has developed a test-tube method of growing the microbes responsible for causing sleeping sickness, a fatal tropical illness, replacing the mice normally used for research into this disease.

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MOLECULAR METHODS
Technological advances are resulting in new and improved molecular methods for analysing and identifying new compounds and medicines. The Trust has provided analytical equipment to researchers selecting new anti-cancer and anti-malaria drugs, based on their molecular interaction with DNA, as an alternative to selecting drugs by animal tests.

Research at the molecular level is being used to understand the biochemistry and genetics underlying various illnesses, and leading to better treatments. A Trust researcher is using newly devised technology to rapidly analyse DNA from patients all over Europe and identify genes that predispose individuals to fibrosing lung disease. This approach is an alternative to modelling the illness in animals such as genetically modified mice.

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POPULATION RESEARCH
Studying the diseases in human populations, and the effects of lifestyle, diet and occupation, has already revealed a great deal about cancer, heart disease, osteoporosis , and birth defects. Such information is vital to improving human health and providing clues to the causes of illnesses. The Trust is funding part of a large population study into how fetal and infant growth influences the development of heart disease in later life, as an alternative to experiments on pregnant animals.

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VOLUNTEER STUDIES
One of the best ways to conduct medical research is by studying the whole human being. New scanning and imaging techniques are making it increasingly possible to conduct safe and ethical studies of human volunteers, where previously animals had been used.

Trust projects use a variety of sophisticated imaging techniques to non-invasively investigate the intact human body. These include using a MEG scanner to study epileptic patients; investigating pain in patients with fMRI; and developing a novel technique, TMS, to study the function of the human brain in healthy volunteers.

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PRACTICAL EXAMPLES
The Dr Hadwen Trust is the UK's leading medical research charity funding exclusively non-animal techniques to replace animal experiments, benefiting humans and animals. By looking at some of their current research projects we can see how alternatives can be developed and practically applied in real life research scenarios to the benefit of humans and animals. Click here for a full list of projects.

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CYSTIC FIBROSIS

The Problem
Most cystic fibrosis (CF) patient illness and death is due to persistent lung infections caused by bacteria, the most important of which is Pseudomonas aeruginosa. Once infection has been established these bacteria are never eradicated. During “exacerbations” patients are particularly ill and require hospitalisation and intravenous antibiotic therapy. At present doctors have very poor information upon which to base their choice of antibiotics, since isolates of bacteria from CF sputum samples show an astonishing variation in their antibiotic susceptibilities and in many other characteristics.

Animal Experiments to be Replaced
Current modeling bacterial populations and testing of antibiotics is conducted on rats with chronic respiratory infections and mouse models of CF, usually genetically engineered knock out mice who are infected with P.aeruginosa. Each experiment uses hundreds of animals and results may not be directly applicable to human patients.

The Alternative
This study will monitor changes in bacterial populations in CF patient sputum to improve our understanding of what happens during exacerbations and periods of stability. The project will also test the usefulness of an artificial sputum medium (ASM) as an alternative to animals for studying the behaviour of bacterial populations in response to challenge with antibiotics. These methods could help doctors to make better informed choices of antibiotic treatments and directly benefit CF patients.

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BRAIN RESEARCH

The Problem
Non-invasive brain imaging methods can now be used to study the human brain and identify which areas of the brain ‘light up’ or become active during mental tasks. However important questions still remain as to how different areas of the brain interact and what each area contributes when carrying out tasks. To date, these kinds of questions have been answered using monkeys. The Dr Hadwen Trust funded early work that showed how a new research tool called transcranial magnetic stimulation (TMS) could be used to temporarily disrupt areas of the brain in volunteers. This project will develop TMS to the next stage by applying it to investigate how different areas of the brain interact.

Animal Experiments to be Replaced
Brain research experiments on monkeys often involve subjecting animals to long periods of training and testing. They undergo surgery to expose an area of the brain and a device is fixed to the skull for applying recording and stimulating electrodes. They may also have regions of the brain damaged and the monkeys are usually killed at the end of the experiments.

The Alternative
Instead of studying monkeys, this project will use dual-site TMS to shed light on the interaction of two areas of the human brain known to be involved in visual attention. The research will increase our understanding of how the human brain works and provide an alternative to animal experiments. It will also have also implications for the treatment of psychiatric conditions involving attention deficit, such as schizophrenia, and the consequences of brain damage.

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ASTHMA (1)

The Problem
Asthma is a serious worldwide problem affecting over 300 million people and its prevalence among children is increasing. Abnormal mucus production is a major feature of asthma, and other respiratory conditions such as cystic fibrosis and COPD (chronic pulmonary obstructive disease). Mucus is not easily cleared from the lungs and can block the airways leading to suffocation. Understanding how airway cells control the production of mucus and finding ways to switch off over-production of mucus could be crucial to finding new treatments.

Animal Experiments to be Replaced
Many laboratories use animals in asthma research usually injecting them with allergens to induce inflammation of the lungs and produce asthma-like symptoms, although no animal study exactly replicates human asthma. Mice are usually used, but other species have included rats, guinea pigs, dogs and cats. In particular, there are differences in the number and distribution of mucus-secreting cells between species, as well as other significant anatomical and immunological differences.

The Alternative
The research project will use cells collected from asthmatic patients to create a three-dimensional cell culture model of mucus production. This new model will represent the human condition and will be used to investigate how mucus production is controlled and to find ways to switch off over-production of mucus. Importantly, this cell culture model will provide an alternative to animal studies in this area, and could also be
adapted to study other serious respiratory conditions.

ASTHMA (2)

The Problem
Asthma rates are soaring worldwide, especially in children, although the reasons for this remain unclear. Asthma affects an estimated eight million people in the UK, that’s one in 13 adults and one in eight children. Animals are widely used in asthma research, but important species differences between the lungs of humans, rats, mice and other animals, mean that there are marked variations between findings in animal experiments and humans with asthma.

Animal Experiments to be Replaced
Rats, mice, rabbits, guinea pigs and increasingly genetically engineered mice are used in asthma research. Animals are subjected to repeated and distressing treatments, including multiple injections in the abdomen. Asthma-like symptoms are induced by sensitising the animals’ lungs, producing inflamed airways and difficultly in breathing.

The Alternative
Our asthma project at King’s College London is investigating changes that occur in the airways of asthmatics, instead of studying animals with induced asthma-like symptoms. The latest imaging and genetic techniques are being applied to biopsy samples of airway smooth muscle cells taken from volunteers with and without asthma. This project will establish the use of these human cells in culture as a research tool to replace animal experiments.

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MULTIPLE SCLEROSIS

The Problem
Multiple sclerosis (MS) is a devastating disease that affects around 2.5 million people and for which there is no cure. It causes a range of symptoms, including muscle weakness, loss of co-ordination, problems with speech and vision, severe fatigue, pain and depression.

Animal Experiments to be Replaced
For decades much research into MS has involved inducing an experimental condition called autoimmune encephalomyelitis (EAE) in rodents, monkeys, rabbits and guinea-pigs as a ‘model’ of human MS. Animals suffer inflammation and damage to the nervous system resulting in paralysis, in experiments that can cause distress and suffering. More recently EAE has been studied in genetically modified mice, either ‘humanized’ by the addition of human genes or with genes ‘knocked out’.

Despite more than 10,000 published experiments on animals with EAE, the human disease MS remains poorly understood, treatments are very limited, and a cure remains elusive. More advanced non-animal approaches to studying MS are urgently needed.

The Alternative
The Dr Hadwen Trust is funding a one-year pilot study to investigate the potential of applying a new molecular technique to MS research to replace animal studies.

Hallmark damage to the nervous system seen in MS is believed to be caused by the patient’s own immune system attacking and damaging the nerves. Patients’ immune cells can be obtained from blood samples and studied in culture.

In our project, a new molecular technique called RNA knockdown will be applied to immune cells from MS patients. Particular genes in the immune cells will be turned-off to see which ones are contributing to the immune responses that underlie MS. This approach will replace experiments on knockout mice with induced EAE, which are currently used to investigate contribution of immune system genes to MS.

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3D MODELS OF SKIN CANCER

The Problem
Basal cell carcinoma (BCC), a type of skin cancer associated with sun exposure and ageing, is the commonest human cancer. Diagnosis and treatment places a large burden on our health services. BCC is usually easy to treat if caught early, but it can become dangerous if left untreated. Treatment may involve complicated surgery that can leave unsightly scarring.

Animal Experiments to be Replaced
At present there are no cell culture models of this type of cancer and so research is often carried out on mice genetically modified to develop tumours. In one recent experiment, genetically modified (knockout) mice were subjected to irradiation to induce large aggressive tumours on their undersides. Sometimes human cells may be implanted into mice with deficient immune systems to model the disease. A single experiment may use as many as 400 mice.

The Alternative
The Dr Hadwen Trust is funding researchers at Queen Mary’s School of Medicine and Dentistry who are creating the first cell culture model of BCC to replace the use of mice in research. They are attempting to incorporate human BCC cells into complex three-dimensional cell culture models of human skin. Their aim is to create realistic laboratory models of this common form of human skin cancer that can replace widespread experiments on mice. The research will also help us to understand the development of BCC and why some forms are more aggressive than others, and could shed light on other types of skin cancer too.

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AIDS-RELATED PNEUMONIA

The Problem
Pneumocystis jirovecii is an infectious fungus that grows in the lungs of immunocompromised patients and causes pneumonia, particularly in AIDS patients. At present it is not possible to culture this pathogen in the test-tube, and so much research has instead focused on rats and mice infected with a related, but different fungus.

Animal Experiments to be Replaced
A fungus that causes a form of pneumonia in rodents is grown in the lungs of animals whose immune systems have been damaged, either with chemicals or by genetic mutation. Rats and mice are used as living incubators in which to grow the fungus, which is inoculated into their lungs. Animals are likely to suffer breathing difficulties as the disease progresses. Once they develop pneumonia they become seriously ill and are killed.

The Alternative
A Dr Hadwen Trust project at University College London is devising the first-ever test-tube method for culturing the human pathogen, to replace experiments on infected rodents with purposely damaged immune systems. The project is investigating both short- and long-term culture methods, using donated samples of human lungs cells from infected patients. The fruits of this project could revolutionise research in this field, which for so long has focused on the wrong species.

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BRAIN TUMOUR INVASION

The Problem
Brain tumours are one of the most difficult forms of cancer to treat and they are becoming more common. Tumours in the brain are particularly resistant to drug treatment and radiotherapy, and new approaches to treatment are urgently needed.

Animal Experiments to be Replaced
Much research into brain tumour therapies involves experiments on rats and mice. Animals either have brain tumours chemically induced, or pieces of human brain tumour are surgically implanted in their brains. Each experiment typically uses around 100 rodents, who are all killed for autopsy. In America, researchers have studied experimental brain tumours in dogs. Many differences between human and animal brain tumours make animal experiments ‘grossly inadequate’. Artificially induced animal brain tumours can be ‘cured’ but the human disease has a very poor outlook.

The Alternative
Dr Hadwen Trust-funded researchers at Portsmouth University are creating a three-dimensional culture model of human brain tumour invasion. Human brain cells are ethically obtained from patients undergoing surgery. Normal brain cells are grown in the lab alongside balls of tumour cells (spheroids) to produce a model of brain tumour invasion. The very latest microscope and live-cell imaging techniques are being used to study the model and to investigate potential anti-tumour therapies, instead of experiments in rats or mice.

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COMPUTER MODELLING OF VIRTUAL ORGANS

The Problem
Animal tests are notoriously bad at predicting the safety and effectiveness of new drugs. According to a recent report by the American drug regulatory body, 92% of drugs that pass animal tests subsequently fail in human trials. Clearly, more accurate non-animal tests are needed. Computer modelling holds enormous potential to replace animal experiments in medical research and testing.

Animal Experiments to be Replaced
More than half a million animals are used in UK pharmaceutical research and testing. These include monkeys, dogs, cats, sheep, pigs, rabbits, ferrets, guinea pigs, mice, rats, birds, and more.

The Alternative
Dr Hadwen Trust funding is supporting the construction of computer models of the human heart, uterus and spinal cord at Leeds University, using data acquired with the very latest imaging technology, diffusion tensor imaging (DTI). The computer models will be used to conduct virtual experiments on human organs and will have a wide range of applications. For example, to screen new heart drugs (anti-arrhythmics) in place of experiments on dogs, rodents, rabbits, pigs, goats, guinea-pigs and cats. To study spinal cord pathways and new nerve injury therapies, instead of spinal cord injury experiments on rats and mice. And for research into labour and premature labour, replacing experiments on pregnant sheep and guinea-pigs.

Prof Holden’s laboratory is a member of BioSim, a European Network of Excellence, which brings together research groups working in this area. This will help to ensure that new computer simulations are readily distributed and adopted by other researchers.

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TISSUE ENGINEERING OF HUMAN LIVER

The Problem
The liver is a large organ that plays a vital role in the body’s metabolism, and it continues to be the focus of much animal experimentation. In the development of new medicines, effects on the liver are always an important consideration, and routine tests are conducted in rats, monkeys and dogs. However, species differences mean that results from animal tests cannot reliably predict how humans will respond.

Animal Experiments to be Replaced
Serious liver infections, such as hepatitis viruses, are investigated in infected ground squirrels, woodchucks, monkeys, and genetically modified mice. Chimpanzees experimentally infected with hepatitis C virus continue to be studied in Japan and the USA. Again, species differences make the findings from such animal experiments of dubious relevance to human patients with liver diseases.

The Alternative
This project is using the very latest tissue engineering techniques to culture human liver cells on 3D micro-scaffolds, to create realistic cell culture models for the study of liver diseases, such as hepatitis, and for drug research and testing. Developing advanced human liver tissue cultures will help to replace the routine use of animals in these areas of research.

The aim is to establish the next generation of long-lived, exclusively human liver cultures that will maintain liver functions in the test-tube. Realistic and functional test-tube models of human liver would be invaluable for replacing animal research into liver diseases and for screening and testing new medicines. These improved in vitro models of human liver could have important implications for both human health and animal replacement.

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References:

[1] “Pioneers Cut Out Animal Experiments,” New Scientist, 31 Aug. 1996.
[2] Clemedson C, McFarlane-Abdulla E, Andersson M, et al. MEIC Evaluation of Acute Systemic Toxicity. ATLA 1996;24:273-311
[3] www.mbresearch.com 31/12/2006
[4] www.mbresearch.com 31/12/2006
[5] Biogenic Amines Vol. 19, No. 2, pp. 97–145 (2005)
[6] Biogenic Amines Vol. 19, No. 2, pp. 97–145 (2005)
[7] Lewis, R. J., Sr. (1989). Sax’s Dangerous Properties of Industrial Materials. 7th edn. John Wiley, New York. Wilson, J. G. (1977). Current status of teratology. General principles and mechanisms derived from animal studies, in: Handbook of Teratology, pp. 1–47. Plenum Press, New York
[8] Biogenic Amines Vol. 19, No. 2, pp. 97–145 (2005)
[9] Developmental Toxicology: Mechanisms and Risk JA McLachlan, RM Pratt, C L Markert (Eds) 1987 p313
[10] http://dtp.nco.nih.gov/branches/btb/ivclsp.html
[11] JCW Salen, Animal Models-Principles and Problems in Handbook of Laboratory Animal Science 1994
[12] Christine Soares ‘Virtually Human’ New Scientist 16 June 2001
[13] ‘New Technology Detects Risk of Drugs to Heart Sooner’ Yorkshire Today 16th January 2006
[14] Business Guardian, Tuesday March 7th 2006
[15] http://www.drugresearcher.com/news/ng.asp?n=65500-xceleron-microdosing-adme-pk-pharmacokinetics
[16] http://www.emediawire.com/releases/2005/10/emw300761.htm
Vitalea Science Pioneers "Accelerator Technology" for HIV-Drug Testing: Results from First Microdose Study of the Antiretroviral AZT Released

[17] Parke/Smith (eds) Drug Metabolism from Microbe to man, quoted Page "Viv. Unv." p45
[18] Clin Pharmacol Ther 1962; pp665-672
[19] AP Fletcher in Proc R Soc med, 1978;71, 693-8
[20] John Hopkins Medical institutions Press Release Aug 6th 2002 “Structure of key receptor unlocked; Related proteins will fall like dominoes”
[21] www.xensei.com/users/chi/2001/hpr/hpr_pressrelease.htm
[22] This is London. 31 October 2006.
Animal Aid press release: Another nail in the coffin of animal research, Tuesday, October 31, 2006.
[23] Boston Globe, 27th Feb 2002

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