Ethics of Research involving animals
Use of animals in current pharmaceutical research and development - continuation I
8.10 The molecules that are studied in stages 1 and 2 are screened against animals, animal tissues and cloned human receptors. The numbers of animals involved are small, probably less than ten percent of the total number used in pharmaceutical research.15 Animal tissues are used for some in vitro tests, but cloned human receptors are preferred as these are more selective. GM mice are most commonly used to assess the importance of a drug target by examining the effects of deleting genes responsible for the synthesis of proteins such as receptors or other potential drug targets. The way in which the welfare of these animals is affected depends on the precise nature of the genetic modification that has been applied. Phenotypic effects may range from a lack of detectable changes to stunted growth and developmental abnormalities, and early death (paragraphs 4.57–4.58). Assessments need to be on a case by case basis as it is difficult to make generalisations.
Vaccines
8.11 Advances in genomic research have had a significant impact on the use of animals in the vaccine discovery process, often reducing the number involved or, leading to the replacement of animals such as primates with genetically modified mice.16 Bacterial and viral genomes have been sequenced and potential vaccine targets are tested in high-throughput screening. The main difference in comparison to drug development is that the potential medical product under test is usually not an inorganic chemical molecule, but a biological product such as a fragment of a virus. Mapping of the human genome has also allowed the discovery of biological products that may eventually protect from, or even treat, diseases such as cancer.17
Stages 3 and 4: the characterisation of promising candidate medicines
8.12 In stages 3 and 4 the pharmacological properties of potential medicines are characterised more fully. These techniques combine use of non-animal approaches such as computer studies and analysis, chemistry and cell culture, with animal-based techniques such as advanced surgery, behavioural analysis, imaging such as MRI, and tissue and body fluid analysis (see paragraphs 4.53–4.56). New technologies such as telemetry now allow much more information to be obtained from each animal. For example, data from multiple measurements of physiological parameters such as heart rate or levels of neurotransmitters can be combined. With regard to welfare, post-operative pain can be controlled by pain relieving medicines, but sometimes they may interfere with experiments on pain and may not be given (see Box 8.3). The choice of pain relieving medicine can therefore be critical. Occasionally, distress can also be caused by devices used in telemetry (see paragraph 4.56).18
Stage 3: identification of ‘leads’
8.13 Potential drug compounds (‘hits’) that have been identified by means of high-throughput screening are further examined in this stage, commonly using more complex cell cultures or assays based on animal or human tissue. The number of compounds entering this phase is usually in the hundreds. Through ‘hit-to-lead chemistry’, these hits are converted into a significantly lower number of compounds known as ‘leads’. Lead compounds are chemicals that influence the target in a way that indicates that they have high potential to be developed into effective treatments.
Stage 4: lead optimisation
8.14 Lead compounds are further refined by synthetic chemical modification, leading to the identification of a subset of the compounds that fulfil the requirements for clinical usefulness.19 Animal and non-animal techniques are used to test for attributes such as absorption, duration of action and delivery to the target. The results determine whether the lead compounds have the potential for subsequent testing in human trials, and therefore the qualities to become candidates for medicines.
Use of animals
8.15 Most of the animals used by the pharmaceutical industry are involved in stages 3 and 4, comprising up to 80% percent of the total. Some techniques, such as methods for administering a medicine and measuring the level in blood, are generic for all types of research and testing (see paragraphs 4.31–4.59), but specific animal models of disease are used in particular areas of research. For example, one model may be used to identify targets for compounds to treat acute tissue damage after a stroke, whereas another may seek to find targets relevant to long-term recovery from a stroke (see Box 8.2). As we have said, an animal need not share all properties of humans to be an effective model. It is sufficient for the model to be similar in relevant aspects of the disease being studied (see paragraph 4.10).
8.16 The involvement of GM animals, usually mice, during stages 3 and 4, is becoming increasingly common. They are generally used either to determine if a gene is important as a target (target validation) or, once its importance is known, as a much more specific animal model of a disease.20 Some tests of bioavailability (the degree or rate at which a medicine or other substance is absorbed or becomes available at the intended site in the body after administration), drug disposition and pharmacogenetic models21 may also be used in a more limited way at this stage.
| Box 8.1: The characterisation of promising candidate medicines (stages 3 and 4): example of animal research undertaken during of the development process of a new medicine Jin, Q, Nie H, McCleland BW et al. (2004) Discovery of potent and orally bioavailable N,N’-diarylurea antagonists for the CXCR2 chemokine receptor Bioorg Med Chem Lett 14:4375-8.* The aim of this research was to test the ability of a series of compounds to bind to the CXCR2 chemokine receptor (thus blocking its function). CXCR chemokines are signalling molecules that play an important role in transporting neutrophils (a type of white blood cell) to sites of inflammation in disease processes involved in arthritis, asthma and reperfusion injury (where the body’s attempt to restore blood flow to an injury causes damage by oxidation). A non-animal in vitro assay was used to identify compounds which may bind to the CXCR receptor. Six compounds were identified and their affinity for the CXCR2 receptor, as well as their effect in a living body, was investigated. The degree of binding to the CXCR2 receptor was then assessed in cell lines originally derived from the kidneys of Chinese hamsters. In a further test, the compounds were injected into groups of three rats. This was first done intravenously and then, in a later experiment, injected into the peritoneal cavity. This experimental format is designed to both reduce the number of animals used and experimental variation. At various intervals after administration of the compounds, blood samples were taken from the lateral tail vein of the rats. Further in vitro studies using components of rat and human liver cells were carried out to investigate the way that the liver metabolises these compounds. These cells were obtained from euthanised rats and from human tissue which had been removed during surgery. The research yielded a new class of CXCR2 compounds that are potent and effective in binding and blocking CXCR2 receptor function. * This is an example of animal research that has been carried out in the UK and published in a peer-reviewed journal. Details relate to this specific example and should not be taken to represent a ‘typical’ animal experiment. It is important to note that individually published experiments usually form one part of a continuing area of research, and the significance of the results may therefore be difficult to interpret. |
| Box 8.2: The characterisation of promising candidate medicines (stages 3 and 4): example of animal research undertaken during the development process of a new medicine Irving EA, Vinson M, Rosin C et al. (2005) Identification of neuroprotective properties of anti-MAG antibody: a novel approach for the treatment of stroke? J Cereb Blood Flow Metab 25: 98–107.* It had been previously hypothesised that a protein called myelin-associated glycoprotein (MAG) was a contributing factor to the lack of regeneration of the CNS after injury, such as stroke. This research project demonstrated that the antibody specific to this protein, anti-MAG, possessed the ability to neutralise the inhibitory effect of MAG on neurons following an induced stroke and, in addition, protected certain CNS cells from cell death in vitro. Rats given the antibody improved in their motor function ability after the stroke compared with control animals, measured by their ability to walk along a cylindrical beam. The authors concluded that the data indicated potential for the use of the antibody as a therapeutic agent for the treatment of stroke. Under anaesthesia, small tubes were inserted into the brains of rats to enable the induction of a stroke. Two weeks later the rats were anaesthetised and a stroke was induced by causing a transient blockage of an artery in the brain for 90 minutes. Rats that displayed circling stereotypic behaviour one hour following the surgical procedure were judged to be suitable models and therefore only these rats were included in the study. During the following week, the rats were administered with the test antibody at 1, 24 and 72 hours after the stroke either into the brain or intravenously. They were then euthanised. |
8.17 Information about research carried out during stages 3 and 4 is often provided through oral communications and posters at scientific meetings, and is later reported in scientific publications.22 Many thousands of such posters and publications are published annually by industry. More recently, the Home Office has begun to make available abstracts of licensed research (see Box 13.4), which are likely to include many types of experiment undertaken to identify and optimise pharmaceutical leads. We consider issues relating to publication of research in more detail in Chapter 15 (see paragraph 15.35).
Footnotes15 The statistics collected by the Home Office do not include these data and companies vary in how they implement the various stages, making this figure difficult to estimate.
16 See The Associate Parliamentary Group for Animal Welfare (2005) The Use of Animals in Vaccine Testing for Humans, p21,
available at: http://apgaw.org/userimages/Vaccinetesting.pdf. Accessed on: 26 Apr 2005; see also paragraph 6.35.
17 For example, see Berthet FX, Coche T and Vinals C (2001) Applied genome research in the field of human vaccines
J Biotechnol 85: 213–26.
18 Morton DB, Hawkins P, Bevan R et al. (2003) Seventh report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement: Refinements in telemetry procedures Lab Anim 37: 261–99.
19 Physico-chemical, pharmacokinetic and toxicological properties are important criteria in assessing potential clinical
usefulness.
20 Wellcome Trust (2003) Transgenic mice, available at: http://www.wellcome.ac.uk/en/genome/technologies/hg17b012.html
Accessed on: 26 Apr 2005.
21 See MacGregor JT (2003) The future of regulatory toxicology: impact of the biotechnology revolution Toxicol Sci 75: 236–48.
22 See PubMed, a service of the US National Library of Medicine, which includes over 15 million citations for biomedical articles
dating back to the 1950s, available at: http://www.ncbi.nlm.nih.gov/pubmed. Accessed on: 26 Apr 2005.