Animal experimentation in snake venom research and in vitro alternatives

Toxicon 42 (2003) 115–133 www.elsevier.com/locate/toxicon

Review

Animal experimentation in snake venom research and in vitro alternatives Paula G. Sells* Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK Received 30 January 2003; accepted 23 April 2003

Abstract Current experimental techniques used in snake venom research (with and without the use of animals) are reviewed. The emphasis is on the reduction of the use of animals in the development of antivenoms for the clinical treatment of snakebite. Diagnostic and research techniques for the major pathologies of envenoming are described and those using animals are contrasted with non-sentient methods where possible. In particular, LD50 and ED50 assays using animals (in vivo) and fertilised eggs (in vivo, non-sentient) are compared as well as in vitro procedures (ELISA and haemolytic test) for ED50 estimations. The social context of antivenom production, supply and demand is outlined together with the consequent tension between the benefits derived and the increase in opposition to experiments on animals. Stringent regulations governing the use of animals, limited research funds and public pressure all focus the need for progress towards non-animal, or non-sentient, research methods. Some achievements are noted but success is hampered by lack of detailed knowledge of the many constituents of venom which have to be assessed as a whole rather than individually. The only way to evaluate the net pathological effect of venom is to use a living system, usually a rodent, and similarly, the efficacy of antivenoms is also measured in vivo. The pre-clinical testing of antivenoms in animals is therefore a legal requirement in many countries and is strictly monitored by government authorities. New technologies applied to the characterisation of individual venom proteins should enable novel in vitro assays to be designed thus reducing the number of animals required. In the meantime, the principles of Reduce, Refine and Replace relating to animals in research are increasingly endorsed by those working in the field and the many agencies regulating ethical and research policy. q 2003 Elsevier Ltd. All rights reserved. Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Estimation of venom lethality (LD50 testing) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. In vivo rodent test for LD50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. In vivo egg test for LD50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Estimation of antivenom preclinical efficacy (ED50 testing) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. In vivo rodent test for ED50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. In vivo egg test for ED50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. In vitro ELISA test for ED50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. In vitro haemolytic test for ED50. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. General comments on LD50 and ED50 tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Estimation of haemorrhagic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * Tel.: þ44-1745-710114; fax: þ44-151-705-3371. E-mail address: [email protected] (P.G. Sells). 0041-0101/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0041-0101(03)00125-9

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5.1. In vivo rodent test for haemorrhagic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. In vivo egg test (a) for haemorrhagic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. In vivo egg test (b) for haemorrhagic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. In vitro ELISA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Gelatin-degradation ELISA for haemorrhagic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. In vitro gelatin zymography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Advantages and disadvantages of the egg embryo assay system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. General points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Estimation of coagulant activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. In vivo rodent test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. In vitro clotting test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Estimation of neurotoxic activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. In vivo rodent estimation of neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. In vitro chick biventer cervicis muscle-nerve preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. In vitro mouse hemidiaphragm phrenic nerve preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4. In vitro frog rectus abdominis muscle preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5. In vitro guinea pig isolated ileum preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6. In vitro estimation of neurotoxicity using cultured cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7. In vitro estimation of neurotoxicity and its neutralisation by ELISA . . . . . . . . . . . . . . . . . . . . . . 9. Estimation of myotoxic activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. In vivo rodent estimation of myotoxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. In vitro measurement of myotoxicity on cultured cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Estimation of necrotising activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1. In vivo rodent estimation of necrosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2. In vitro estimation of necrosis on cultured cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3. In vitro assays for phospholipase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Validation of new procedures and trends in animal use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Keywords: Snake venom; animal experimentation; in vitro

1. Introduction Early pioneering work in medicine, surgery and anaesthetics depended on animal work which continues to be an integral part of medical and veterinary research today. Among the earliest published animal studies in snake venom research are those on the effect of viper venom on cats, dogs and rabbits (Geoffroy and Hunault, 1737; Fontana, 1781). The first demonstration of specific immunity, induced in pigeons treated with pygmy rattlesnake (Sistrurus catenatus tergeminus) venom, is attributed to Sewall (1887), while Calmette (1896) and other workers established that neutralising antivenoms could be developed in rabbits and guinea pigs (reviewed by Boquet (1979)). This led to the combined use of snakes (providing venom) and a variety of immunised mammals (providing antibodies) which culminated in the production of protective antivenoms for use in human snake bite therapy.

A number of potential drugs have been developed from snake venom. For example, captopril (Ondetti et al., 1977) a component isolated from the venom of the pit viper (Jararaca), was the first orally active inhibitor of angiotensin-converting enzyme (Rocha e Silva et al., 1949). The drug was approved by the FDA (US Food and Drug Administration) in 1981 and is used in the treatment of high blood pressure, renal disease in diabetics and heart failure. More recently, captopril and similar related antihypertensive drugs have been synthesised. Ancrod (Arvin or Aggrastat), a fibrinogen-depleting protease derived from the venom of the Malayan pit viper (Calloselasma rhodostoma), reduces disability associated with acute ischaemic stroke and has been used in the treatment of patients undergoing coronary by-pass surgery (Lathan and Staggers, 1996). Examples of other venom-derived reagents include Atroxase (Tu, 1996), an anticoagulation enzyme from the venom of the Western diamondback rattlesnake (Crotalus atrox) and Cobra Venom Factor (CVF), an

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anticomplementary venom protein originally isolated from the venom of the Indian cobra, Naja naja (Ballow and Cochrane, 1969). CVF is now genetically engineered (Andra et al., 2000) and is of potential use for the depletion of complement in patients with autoimmune disorders. All such medicines are tested at some stage of their development in animals before being licensed for clinical use. Pharmaceutical companies continue to focus on novel venom-derived peptides which may have potential medical benefit; for example, the use of venom disintegrins as antiangiogenesis agents in cancer studies (Zhou et al., 2000) and fibrolase for the degradation of blood clots (Bolger et al., 2001). In addition, venoms are also powerful probes for physiologists; they bind to specific vertebrate and invertebrate epitopes thus helping to elucidate the structure and function of receptors; for example, the acetylcholine receptor in neurobiology (Changeux et al., 1970; Raftery et al., 1980) and platelet receptors in haematology (Markland, 1998; Andrews et al., 2001). Physiological research requires targets (usually proteins) which function as receptors, immune system epitopes or enzymes. In vitro assays are more useful for the investigation of venom fractions with specific modes of action than are whole animal models. An in vitro assay can simulate a specific physiological process but the requirement for whole animal assays is based upon the need to measure the total in vivo pathological effect of a venom and the capacity of an antivenom to neutralise that net effect. Snake venom pharmacology and antivenom activity is not yet sufficiently understood to permit precise in vitro assays to be designed as an alternative. Therefore, preclinical testing of antivenoms in animals is currently a legal requirement governed by the Medicines Control Agency (MCA) in the UK, the European Agency for the Evaluation of Medicinal Products (EMEA) in Europe and the FDA in the USA. Antivenom research to improve the treatment of snakebite uses the ‘notorious’ lethality assays (usually in mice) which include the Minimum Lethal Dose of venom (LD50) to quantify the lethality of a venom and the Minimum Effective Dose of antivenom (ED50) which estimates the in vivo neutralising potential of an antivenom. In 1979, the World Health Organisation (WHO) proposed the adoption of standard methods for assaying the biological activity of venoms and antivenoms (WHO, 1981). These standard assays, some of which used mice, included the LD50 assay as well as tests for the assessment of necrotizing, haemorrhagic, and defibrinogenating activities of venom (Theakston and Reid, 1983), and neutralisation by antivenoms of the toxins responsible for these pathologies. WHO have recently reviewed the procedures recommended for the production and standardisation of antivenoms (Theakston et al., 2003) but animals continue to be used today in antivenom research because they are considered indispensable. For example, the vast majority of antivenom producers currently use horses (African Health Laboratory Service (previously known as South African

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Institute for Medical Research [SAIMR]), Haffkine Biopharmaceutical Co. Ltd, India, Instituto Butantan, Brazil, Commonwealth Serum Laboratories Australia and the Thai Red Cross Society (Theakston and Warrell, 1991). Sheep (Protherics, UK and Micropharm, UK), rabbits, goats and chickens (Antiven Pty Ltd, Australia) are also used to a lesser extent. The routine procedures required, such as injection and blood sampling, do not compromise these domestic and farm animals, providing that adjuvants are used with care and that the welfare of the animals is a priority. The lethality of venom batches and of laboratoryseparated venom fractions is measured by the LD50 test which also provides a baseline against which the ED50 test is used. Both these tests, which necessarily involve inflicting pain and death, are closely monitored by the Home Office in the UK and the LD50 test has been identified as a priority for replacement by ECVAM (European Centre for the Validation of Alternative Methods) in the European Union (Balls and Straugham, 1996). Tests to measure particular pathologies caused by envenoming, and their neutralisation with antivenoms, such as necrosis, myonecrosis, haemorrhage, neurological damage and disorders of haemostasis, although not as common as the LD50 and ED50 tests, are invariably carried out in animals. Alternatives to all these uses of animals are urgently sought and current progress on this is the main subject of this review. Relatively little funding is directed towards the development of new antivenoms which are the only medically effective treatment for systemic envenoming by snakes and other venomous animals, such as scorpions and spiders. Each year, hundreds of thousands of landworkers, upon whom rural communities depend, suffer significant mortality and morbidity as a result of snakebite (Chippaux, 1998; Trape et al., 2001; WHO, 1981; Theakston et al., 2003). Snakebite is basically a problem of the rural tropics with farmers, hunter gatherers, herdsmen and children being the major groups at risk. Antivenom, if available at all, is unaffordable by those who need it most (Theakston and Warrell, 2000; Lalloo et al., 2002) and there are few lucrative markets for producers. It is certain that this lack of competitive commerce reduces the incentives for introducing progressive methods in the production of new antivenoms. Despite the difficult economics of supply and demand, it is the responsibility of all those who work towards developing effective and cheap antivenoms to also design humane or non-sentient quality control assays and to recruit the resources needed to validate them. The scientific will demonstrated by those working in medical research to find in vitro alternatives was first expressed by Russell and Burch (1959) using the principles of Reduce, Refine and Replace. However, since then, progress has generally been slow, perhaps partly due to conservative attitudes in the laboratory which tend to prevail until other factors, such as cost and a new awareness of society’s values, cause change.

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Attitudes towards the acceptability of using experimental animals have altered radically and there are now many safeguards for animal welfare including legislation for laboratory procedures, ethical committees for research proposals and editorial policies for the publication of animal-dependent research (Toxicon, 1991), all of these are concerned with the reduction or abolition of in vivo tests. In addition, research proposals in the US must include proof of literature searches showing that any animal work proposed has not been superceded by alternatives. In 1997, the UK Home Office initiated the establishment of mandatory Ethical Review Processes (ERP) in all designated premises (i.e. areas approved for the carrying out of animal experimentation). The ERP committees scrutinise the ethics of projects, advise on animal welfare and assist participants in the practical implementation of projects. A recent review of the ERP (Home Office: Animals (Scientific Procedures) Inspectorate, 2001) reports encouraging progress in all these aspects. Public pressure and economic strategy also encourage a departure from conventional animal experimentation. For example, shareholders may favour pharmaceutical companies which demonstrate responsible practice and such ‘ethical investments’ may have a market advantage. Investment policies of this type are growing rapidly world wide and consumers wield considerable power. The UK Society for Investment Forum (UKSIF) (and its European counterpart (EUROSIF)) comprise the secretariat for an allparty parliamentary group on socially responsible investment strategies and endorse the need for government and research institutes to be increasingly ‘transparent’ in their activities. However, before antivenoms can be used in human victims of snake bite, it is obviously vital that they first be accurately assessed preclinically. The challenge is to design relevant and humane in vitro assays; the goal is to progressively reduce the use of animals to the point where they are no longer necessary. In this article, techniques have been grouped under some of the major pathologies of envenoming for ease of reference. Where possible, non-sentient alternatives have been compared and contrasted with the standard animal assays.

2. Estimation of venom lethality (LD50 testing) The measurement of the lethality of venoms and their fractions underpins much venom research and, as stated previously, is essential for the estimation of the preclinical efficacy of an antivenom. Since venoms are a mixture of different proteins, peptides, enzymes and other toxins, many of which have not been characterised in detail, it is necessary to measure the net pathological effect in an in vivo model to obtain an accurate assessment of the overall toxicity (LD50).

2.1. In vivo rodent test for LD50 The standard test is carried out in 18 – 20 g mice over a 24 h period. The test determines the least amount of venom needed to kill 50% of a statistically significant sample. Various concentrations of venom, in a volume of 200 ml physiological saline are injected intravenously (i.v.) (and occasionally subcutaneously (s.c.), see Section 4, paragraph 5) and the LD50 is calculated from the number of mouse deaths by probit analysis (Theakston and Reid, 1983). To establish the dose-response curve for the probit analysis (Finney, 1971) upon which the LD50 of a venom is based, a number of groups of mice (each comprising five mice) is injected with different amounts of venom. A range of effects is sought so that the least amount of venom injected results in 100% mouse survival and the most amount of venom results in 100% mouse death. The three remaining groups have a varying number of mouse deaths from which the standard curve is determined. 2.2. In vivo egg test for LD50 Recently, an alternative in vivo test has been developed which has the advantage of being pain-free (Sells et al., 1998). Fertile hens’ eggs incubated for less than 10 days do not have a complete nervous reflex system and therefore cannot experience pain (Rosenbruch, 1989). The eggs have a vascularised yolk sac membrane with a normal blood circulation and display a primitive embryonic beating heart, the arrest of which provides a clear end point for lethality testing. Briefly, on day 4 of incubation at 37 8C, eggs are broken out of their shells into containers and incubated for a further three days. On day 6, different amounts of venom in physiological saline (total volume 2 ml) are applied to a 2 mm diameter filter paper disc which is placed over the vitelline vein on the exposed yolk sac membrane of each egg (five eggs per group). After 6 h the number of embryo deaths is recorded from a range of groups, each group having been injected with a different amount of venom. The results are statistically analysed and the LD50 calculated as in Section 2.1. Results of the mouse and egg assays were compared (using the Spearman rank correlation coefficient test) and showed a strong correlation; rs ¼ 1:00; p , 0:01: (Sells et al., 1998).

3. Estimation of antivenom preclinical efficacy (ED50 testing) The assessment of antivenom efficacy is central to the treatment of snakebite. The WHO specifies that each batch of antivenom must be tested for its capacity to neutralise the lethal effect of corresponding venom(s) using an assay method approved by the national control authority of

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the country in which the antivenom is manufactured (WHO, 1971). In the UK, the approved pharmacopoeial method (European Pharmacopoeia, 2002) is the mouse ED50 test. The corresponding monograph in the British Pharmacopoeia (2002) is identical. 3.1. In vivo rodent test for ED50 The ED50 test determines the least amount of antivenom needed to protect half the animals in a statistically significant group of animals from five times (usually) the amount of venom needed to kill 50% of the group (LD50). Venom and varying amounts of antivenom are incubated in a total of 200 ml at 37 8C for 30 min and injected i.v. into the tail vein. The test is usually carried out on groups of five mice over a period of 24 h. The number of groups establishes a range of antivenom protection from 100% survival to 100% death and includes three intermediate groups with varying numbers of deaths. The ED50 is calculated on the number of mouse deaths using probit analysis. Thus the lower the ED50 value, the higher the neutralising ability of the antivenom (Laing et al., 1992). 3.2. In vivo egg test for ED50 The fertile hen’s egg assay, which is an in vivo but insensate model, shows a good correlation ðr ¼ 0:95; 9 df, p , 0:01Þ with mouse ED50 tests (Sells et al., 2000). Nine medically important venoms and appropriate antivenoms were each compared in both rodent and egg models. Venom samples of 5 £ LD50 were incubated with different amounts of antivenom (total volume 2 ml/egg for 5 eggs/group) for 30 min at 37 8C, applied to filter paper discs and placed over the vitelline vein on the yolk sac membrane. After 6 h, the number of embryo deaths was counted in each group, the groups comprising a range of antivenom protection as for the rodent assay, and the ED50 value calculated as in 3.1. Egg and rodent assay ED50 values were compared using the Spearman rank correlation coefficient test. 3.3. In vitro ELISA test for ED50 The ELISA (Enzyme Linked Immunosorbent Assay) depends on specific antigen –antibody binding in which one or other is first adsorbed to a 96-well, polystyrene microtitre plate. For the detection of specific antibody, for example in a test sample of human serum, plates are first coated with the appropriate venom and incubated with the test sample. After washing, an enzyme-linked, antihuman antibody (i.e. directed towards the species in which the putative antibody was generated) is added and, following further incubation and washing, a chromogenic substrate for the linked enzyme is added. A resulting coloured solution indicates the presence of bound antigen – antibody complexes which are quantified spectrophotometrically. Normal and positive serum samples as controls should be included on each plate.

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For the detection of venom in a test sample, the plate is first coated with specific antivenom IgG, incubated with the test sample and followed with enzyme-linked specific antivenom IgG, to which substrate is added after incubation and washing as above. The ELISA is a sensitive assay (detecting 1 – 400 ng protein/ml) and its specificity is determined by the quality of the reagents used. In 1979, Theakston and Reid published an adaptation of the ELISA (Theakston et al., 1977) as a possible in vitro alternative to the mouse ED50 test. The ED50s of reference antivenoms to four venoms medically important in Africa were established in mice. The same venoms were used to coat ELISA plates and reference curves were obtained relating the ED50 results of the antisera to their optical densities. Test antivenoms may be compared with 1 £ ED50 of reference antivenom against a particular venom so that the point of colour match enables the ED50 of the unknown sample to be calculated. A good correlation ðp , 0:001Þ was observed between in vitro and in vivo methods. Reference antivenoms (those which have had an ED50 established in mice) should be tested on each ELISA plate as a positive control, and keep for many years as sterile aliquots at 4 8C. Usually, opacities develop before potency is lost. Freeze-dried or frozen antivenoms also demonstrate long-term stability. Despite this, the standardisation and distribution of reference antivenoms for every venom which may be encountered is not practical and this is a significant disadvantage. 3.4. In vitro haemolytic test for ED50 A strong correlation ðp , 0:001Þ was observed between the ability of 15 samples of polyvalent equine antivenom to neutralise lethal (ED50) and indirect haemolytic activities of Bothrops asper (Gutie´rrez et al., 1988). Indirect haemolytic activity was tested by using as a standard the haemolytic halo induced by a known amount of venom added to wells containing an agarose gel consisting of egg yolk and sheep erythrocytes. Different amounts of antivenoms were incubated with the venom standard, the mixture added to the wells and the size of the halo compared with the venom-only control. The amount of antivenom that neutralised 50% of the halo was recorded and the value compared with the ED50 of the same antivenom used in a standard rodent ED50 test. This sensitive test allows the testing of serum from individual horses immunised with phospholipase A2containing venom and provides a screening test which reduces the number of mice otherwise needed to monitor the activity of serum batches.

4. General comments on LD50 and ED50 tests ED50 tests (for the efficacy of antivenoms) based on testing the antivenom against a set multiple of the venom

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LD50 value, are calculated for each venom/antivenom mixture from a dose response curve using probit analysis. The number of mice required by the European Pharmacopoeia using this method to test the activity of one European viper venom antiserum (LD50 and ED50 tests combined) against five venoms is 374 mice per batch of antivenom (Weisser and Hechler, 1997). This large number is in contrast to the testing of other antitoxins (to tetanus, diptheria, botulinum) where an endpoint titration is conducted in comparison to a reference preparation. The reference preparations enable the concept of ‘relative potency’ to be applied to toxicity testing in which toxins and antitoxins are compared to the reference preparations of known activity. This has two major advantages; first, the lethal end point can be replaced with other measurable in vivo effects which are significantly less distressing; for example, the flaccid muscle paralysis test and second, the number of mice required for an ED50 test may be reduced by approximately 30% (Sesardic et al., 1996). Although venoms each contain several toxins (while diptheria and tetanus contain one major toxin each and botulinum usually three) some reference venoms and antivenoms are beginning to be established (see below). The WHO Workshop Report (Theakston et al., 2003) recommended that national or regional venom and antivenom reference preparations should be established and activities should be expressed in units related to those standards. International reference venoms were unsuitable as these would not reflect their geographical variations. Reference venoms for Daboia russelii, Naja naja, Echis carinatus and Bungarus caeruleus are available to all antivenom producers in India and are distributed by the Indian National Control Laboratory. China has already established a bank of Chinese National Antivenom standard preparations. Apart from in China and India, no standard reference venoms or antivenoms are available at this time. The current measurement of absolute potency in venom studies, rather than a potency relative to a reference preparation, means that LD 50 and ED50 values are susceptible to variations between laboratories and there is no effective standardisation. Inconsistency of these assays between different laboratories is well documented (Schantz and Kautter, 1978; Theakston et al., 2003). However, it is possible that this may be due to all the interlaboratory variables (e.g. size/weight of mice, etc.) not being fully standardised. Unpublished results performed by the WHO Collaborative Centre for the Control of Antivenoms at the Liverpool School of Tropical Medicine between 1994 and 1996 indicated that if all known variables were minimised between laboratories, the results were fairly consistent. In this study, the possible presence of inter-lab differences in estimates of LD50 results was investigated using General Linear Modelling on SPSS v11. The eight snake species were fitted as a fixed effect and the nine testing laboratories as a random effect. There was no significant effect due to

inter-laboratory differences ðp ¼ 0:525Þ: Only mean LD50 results for each species/lab were analysed. Incorporation of individual measurements (rather than their crude mean) would have made the statistics more powerful but were unfortunately unavailable. The design of the conventional rodent ED50 tests for antivenoms requires premixing of the venom with the test antivenom before i.v. injection into the animal. This does not simulate the clinical situation in humans which occurs as two distinct events; envenoming by the s.c., intradermal (i.d.) or intramuscular (i.m.) route followed eventually by antivenom therapy. The efficacies of the same antivenom compared experimentally in the two situations showed that premixing is 2.5 times more effective in neutralising venom (Laing et al., 1992). The s.c. LD50 value is much greater than that of the i.v. value, as expected, and LD50 and ED50 testing is usually done by i.v. injection for a practical reason; s.c., i.d. and i.m. routes of administration of venom in mice lead to a wider range of 95% confidence limit values which would thus require greater numbers of mice for statistical analysis. The UK Home Office limits animal numbers through the requirement to use preclinical range-finding studies with small numbers of animals. This permits the establishment of the approximate range of the lethality assay, thus decreasing the total number of animals required. It is also possible to reduce considerably the numbers of animals used from over 30 per assay to 8 – 10 per assay (Meier and Theakston, 1986), but this gives only an approximate result. The numbers of mice or eggs used within each group per dilution of test venom or antivenom – venom solution vary slightly; numbers have been reduced from 6 (Theakston and Reid, 1983) to 5 per group (Laing et al., 1992; Sells et al., 2000) and still maintain statistical validity for probit analysis. WHO and European Pharmacopoeia each have slight variations in recommendations for LD50 and ED50 tests (Weisser and Hechler, 1997). Most importantly, the confidence limits and statistical analysis should be validated on the least number of animals necessary. Antivenoms, are required by law to undergo preclinical testing in animals, usually in mice. Generally, the results of ED50 (and other pathological venom tests in mice on the comparative efficacies within a group of antivenoms) have approximated to clinical efficacy (Warrell et al., 1986; Laing et al., 1992; Cardoso et al., 1993; Theakston et al., 1995), although this is not universally so (Keegan et al., 1964, 1965; Warrell et al., 1980). The overall conclusion is that the preclinical data obtained following murine lethality testing is of real use in selecting appropriate antivenoms for clinical studies (Theakston et al., 1995). The heterogeneity of venom, both between and within snake species, makes it difficult to establish any refinements of the LD50 test (i.e. to find a more reliable humane endpoint than lethality). The toxic principals (e.g. haemorrhagins, procoagulants or post-synaptic neurotoxins) in the broad categories of venoms, usually represent the major

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rate-limiting step in the in vivo pathological process. However, a correlation between the LD50 of the purified toxic principal and the LD50 of its whole venom would need to be established before in vitro ‘mode of action’ assays using purified fractions could offer an alternative. Attempts to find a correlation between protection against the lethality of Naja naja kaouthia whole venom and its principal postsynaptic neurotoxin (CM3) which comprises 20% of the whole venom, were unsuccessful (Sells et al., 1994b). AntiCM3 antibodies were protective in mice challenged with CM3 but not with whole venom. It is possible that CM3 interacts synergistically with other venom components in vivo or that the purified toxin may lose stability upon separation. The LD50 and ED50 tests require large numbers of mice. As mentioned earlier, unless the variables are strictly controlled from laboratory to laboratory, the results are likely to vary widely from centre to centre and may not reliably predict the clinical efficacy of new antivenoms. However, the lack of any validated alternative continues to perpetuate these tests in the legal statutes.

5. Estimation of haemorrhagic activity Envenoming may result in local or systemic haemorrhage. It is usually caused by zinc metalloproteinases (MPs) which attack the vascular matrix; these are the principal lethal components in most viperine and crotaline venoms. Assays are based on measuring the amount of haemorrhage in the skin after venom is injected in vivo such as in the mouse, or applied to blood vessels via a filter paper disc as in the egg tests, or on the amount of degradation in vitro of extracellular matrix components. The main targets for MPs in venom are thought to be the collagens (particularly Type IV), laminin, fibronectin and other components of the extracellular matrix (Rucavado et al., 1998). MPs (or haemorrhagins) are almost all proteolytic enzymes and are destroyed by chelating agents such as EDTA.

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humanely killed and the extent of haemorrhage in the skin is measured (Laing et al., 1992). The above intradermal method of Kondo et al. (1960) has been compared with a method quantitating the amount of haemorrhage resulting from an i.m. injection of venom into the thigh muscles of mice (Ownby et al., 1984). Biopsies were homogenised and centrifuged and the amount (g%) of haemoglobin released into the tissues following injection either with Crotalus viridis viridis or Crotalus atrox was estimated by spectrophotometry. Both methods showed a high correlation between dose and the amount of haemorrhage measured. However, the photometric method has the advantage of being less subjective and may be used with a variety of routes of administration of the venom. 5.2. In vivo egg test (a) for haemorrhagic activity An alternative to the mouse assay is the insensate, in vivo test in fertile hens’ eggs which correlates well with the rodent test ðp , 0:01Þ and is approximately thirty times more sensitive than the mouse assay (Sells et al., 1997). Haemorrhage and its neutralisation may be clearly demonstrated in eggs. The venom (total volume 2 ml/egg) is applied to a 2 mm diameter filter paper disc placed over the vitelline vein, midway between the outer margin of the egg and the embryo and incubated for 4 h. A previously calculated amount of venom, known to cause a ring (halo) of haemorrhage 2 mm wide is accepted as the standard haemorrhagic dose (SHD). The neutralising activity of antivenom or antidotes is estimated by incubating 1 £ SHD with various amounts of antidote at 37 8C for 30 min before being applied to the egg as above. A discrete corona of haemorrhage spreads out beyond the disc in positive controls and is entirely absent when neutralisation is achieved. The minimum amount of antidote required to prevent haemorrhage is recorded as the Minimum Effective Neutralising Dose. The end point of local haemorrhage in this test does not normally result in the death of the embryo during the experiment. 5.3. In vivo egg test (b) for haemorrhagic activity

5.1. In vivo rodent test for haemorrhagic activity The conventional preclinical test used to measure the efficacy of antivenoms for neutralising haemorrhage is carried out on rodents, usually rats or mice, but sometimes rabbits may be used (Kondo et al., 1960). The Minimum Haemorrhagic Dose (MHD) is defined as the least amount of venom (mg dry weight) which, when injected intradermally into mice or rats, results in a haemorrhagic lesion of 10 mm diameter, 24 h later (Theakston and Reid, 1983). To determine the amount of antivenom (ml or mg) which completely neutralises 1 £ MHD, various amounts of antivenom are mixed with the MHD of the venom, incubated at 37 8C for 30 min and injected intradermally in a total volume of 0.1 ml. After 24 h, the animals are

A simplified version of the above method has been described (Glunder et al., 2001) which is more economical in terms of time and equipment and is suitable for qualitative haemorrhagic tests. Instead of a total of six days incubation of the fertile eggs, the test is performed on the eggs, still in their shells, on day 4. The shell covering the air sac is removed to expose the vascularised yolk sac membrane and venom/antidote solutions are applied to a paper disc placed over a vein as before. The yolk sac membrane surface area in this method is limited by the confines of the shell and is approximately one third of the area of the membrane surface which is available in the shell-less preparation (Sells et al., 1997). Thus measurement of the haemorrhage is difficult, but the method does provide a useful qualitative screening

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assay. Observation of the accompanying pathology can be performed microscopically.

(Howes J.-M., personal communication). It is therefore necessary to double check degradation activity with haemorrhagic activity in mice or eggs.

5.4. In vitro ELISA Li and Ownby (1996) compared the ELISA reactivity of a rabbit antiserum with its in vivo neutralisation of the haemorrhagic fraction of Crotalus viridis viridis venom in mouse skin. No correlation between the in vitro and in vivo tests was found.

6. Advantages and disadvantages of the egg embryo assay system

5.5. Gelatin-degradation ELISA for haemorrhagic activity

† The egg yolk sac membrane model uniquely provides an insensate, in vivo assay for measuring the net outcome of venom activity or its neutralisation. † The test is cheap. One egg costs about 35 pence compared with a mouse costing about £4.00, a factor of about 10. † The vascular yolk sac membrane is a convenient model for the study of vasoactive venoms; for example that of the mole viper, Atractaspis enggadensis. Sections of the vitelline vein vasoconstricted or dilated by venom may be fixed and removed for further examination (West et al., 2001). † Haemorrhagic activity can be quantified by measurement with a ruler although the area of haemorrhage is small; a total volume limit of 2 ml/egg restricts the dose response range of concentrations compared with 200 ml/mouse. For example, Echis venom concentrations of approximately 0.5 – 3.0 ug/egg produce haemorrhagic coronas from 1.0 to approximately 4.0 mm, respectively. The model is particularly convenient for the qualitative screening of the neutralisation of haemorrhagic activity which is easy to observe. † Sensitivity of the egg assay is greater than the rodent assay; approximately one thirtieth of the amount of venom and antivenom was used in the egg ED50 assays. † Variation in growth rate or experimental results between the eggs of different breeds of hens is not significant. Commercial flocks are routinely immunised against Salmonella enteriditis and IgY in the egg yolk will be directed against this and any other pathogens or vaccines used for immunisation. Crossreactions between shared epitopes used for immunisation of the hen and test samples (e.g. venom) used in laboratory tests on the eggs, are theoretically possible but this problem has not been encountered so far. † The technique is semi-sterile (70% alcohol is used to wipe eggshells before breaking contents into hammocks for incubation) and no autoclaved equipment is required. Contamination during incubation is not a significant problem. † It is possible to obtain blood samples from the heart and to inject the vitelline vein. For heart puncture samples, a heparinised capillary, drawn to a fine but patent point over heat and a rubber bulb to withdraw

The degradation ELISA is an in vitro test with potential as a screening assay for haemorrhagic activity (Bee et al., 2001). This adaptation is designed to measure venom MP activity (Clegg et al., 1997) using gelatin (denatured collagen) or other extracellular matrix components as the target substrate. Briefly, the ELISA plate is coated with gelatin, incubated with venom (0.01 mg/ml) then washed and incubated with rabbit antigelatin antibody. The amount of undegraded gelatin remaining is measured by colour development with antirabbit, enzyme-linked antibody after incubation with substrate, using a spectrophotometer. The degradation ELISA is a promising alternative to the rodent test. The specific activity of gelatinase constituents of venom fractions can be evaluated, using collagen or laminin, and large sample numbers can be screened easily. Correlations with existing tests are yet to be demonstrated. 5.6. In vitro gelatin zymography Zymography follows the same principles as the gelatin degradation ELISA but is applied to gelatinPAGE (polyacrylamide gel electrophoresis), where a substrate (gelatin, Type IV collagen, laminin, etc.) is incorporated into the gel. MP activity shows as a clear band in the final Coomassie Blue-stained gel where the substrate has been degraded. Destained gels show both clear bands (with activity) and dark stained protein bands (lacking activity). Both the degradation ELISA and gelatin zymography have potential as screening assays, particularly for venom fractions, which would otherwise be tested in mice. Both tests depend on the intact tertiary structure of the venom/fractions. The ELISA has the advantage, in a comparison of the two tests, of being quantitative and of requiring very small amounts of substrate to coat the plate. The degradation PAGE method has the advantage of computerised comparison with parallel samples run in SDS-PAGE so that active protein bands may be identified and their molecular weights recorded. However, substrates can be difficult to incorporate and may interfere with the electrophoretic process. Also, not all degradation bands in a gelatin PAGE represent haemorrhagic activity, though those seen in Type IV collagen PAGE gels are more likely to be

6.1. Advantages

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blood was the most effective. A Hamilton syringe was used for venous injection (Sells, unpublished). † Avian and mammalian tissues appear to share enough common characteristics for chick embryo assays to be relevant models for venom activity. Integrin receptors and their ligands (which determine the structural architecture of blood vessels), as well as the collagens, show a high degree of homology across species (Nakashima et al., 1992). In addition, another avian assay, the chick biventer cervicis nerve-muscle preparation, is well- established for the measurement of neurotoxic venom activity (Harvey et al., 1994). 6.2. Disadvantages Egg assays are not suitable for neurotoxic or cardiotoxic venoms because the receptor targets are absent at day 6 of incubation; 1 £ LD50 of N. kaouthia purified neurotoxin (CH3) had no effect on the embryo. Myotoxic, cardiotoxic and PLA2 venom fractions are untried. 6.3. General points † In view of the small total volume applied to the disc, it is important that the venom is made up as a concentrated solution in saline (diluted as necessary) and which must be completely soluble. † Variability of venom activities on the eggs occurs, as in all biological, uncloned models but is encompassed by the use of statistically valid numbers of eggs, for example, in ED50 tests usually five eggs per dilution are used and each test is repeated twice. No false positives for haemorrhagic activity have been observed. † We assume that venom constituents diffuse out from the disc and bind to the vascular wall of the vessel beneath the disc as their first target. Some venom constituents will also enter the lumen depending on their size, charge and the local permeability of the vascular membrane. Venous flow will carry the venom towards the vital organs (chick embryo) which will become the second target, presumably behaving in parallel to natural envenoming.

7. Estimation of coagulant activity Proteases present in venom, especially in the Viperidae and Crotalidae, interfere with the clotting cascade through multifactorial pathways.

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blood. The Minimum Defibrinogenating Dose (MDD) is defined as the minimum amount of venom which, when injected i.v. into mice, causes incoagulable blood one hour later. Neutralisation of this activity was estimated as the amount of antivenom (ml or mg) which prevented defibrinogenation (incoagulable blood) by the MDD of the venom. Various amounts of antivenom are mixed with the MDD of venom and incubated for 30 min at 37 8C before injection (Laing et al., 1992). 7.2. In vitro clotting test The in vitro tests for the Minimum Coagulant Dose (MCD) measure either (i) the thrombin-like activity of the venom on bovine fibrinogen as a substrate or (ii) the overall activity of the venom (thrombin-like enzymes, Factor II, V and X activators, etc.) on the coagulation cascade using human plasma as the substrate. MCD, defined as the least amount of venom that clots human plasma (MCD-P) or bovine fibrinogen (MCD-F) in 60 s at 37 8C is estimated as described by Theakston and Reid (1983). Briefly, different amounts of venom are added to 0.2 ml human plasma or a standard solution of human fibrinogen (2.8 mg/ml) and the clotting time recorded. Neutralisation of the procoagulant activity is estimated by mixing the MCD-P or MCD-F with various amounts of antivenom until clotting is completely prevented (Laing et al., 1992).

8. Estimation of neurotoxic activity Neurotoxins are found chiefly in the venoms of elapids (kraits, mambas and Australasian land snakes) and sea snakes, although they are occasionally present in the venoms of Viperidae and Crotalidae species. They may have presynaptic targets (b-bungarotoxin, dendrotoxin) interfering with the release of acetylcholine and acting on potassium channels or post synaptic targets (a-bungarotoxin, acobratoxin) binding to the neuromuscular nicotinic acetylcholine receptor (nAChR) which results in flaccid paralysis (Chang, 1979; Harris, 1984). k-neurotoxins bind to neuronal nicotinic receptors. In cases of fatal envenoming, death is due to asphyxia following the paralysis of respiratory muscles. Through their specific binding activities, experiments with these toxins have helped to reveal the structure and function of nicotinic and muscarinic receptors. a-bungarotoxin is widely used in the biochemical and pharmacological characterisation of nAChR ligands (Bixel et al., 2000).

7.1. In vivo rodent test 8.1. In vivo rodent estimation of neurotoxicity The in vivo test for defibrinogenating activity does not distinguish between many contributing activities; for example, thrombin-like enzymes and plasminogen activators. It estimates the total fibrinogen consumption of the venom in vivo which results in the end point of incoagulable

In vivo venom neurotoxicity and its neutralisation are determined by LD50 and ED50 tests where venom and/or antivenom is administered to mice (Theakston and Reid, 1983; Laing et al., 1992) as described in Sections 2 and 3.

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8.2. In vitro chick biventer cervicis muscle-nerve preparation In vitro nerve-muscle preparations can characterise the potency and target of purified individual neurotoxic and myotoxic constituents as well as whole venom. The in vitro assay of the chick biventer cervicis preparation is the method of choice used to assess both pre- and post-junctional neurotoxic and myotoxic venom activity (Ginsberg and Warriner, 1960; Harvey et al., 1994,). The innervated biventer cervicis muscle is dissected from the back of the neck of anaesthetised 4 – 14 day old chicks (which are sacrificed without regaining consciousness). The preparation is placed in a Petri dish containing Ringer solution and secured to a ring electrode and transducer around the upper tendon where the nerve supplying the muscle is located. Stimulation is either indirect (via the nerve) or direct (to the muscle) and venom/test solutions are added to the bathing medium which is maintained at room temperature or higher (34-37 8C) and aerated with oxygen containing 5% CO2. Antivenom neutralisation is assessed by adding the test sample to the tissue bath 10 min before addition of the venom (Barfaraz and Harvey, 1994). The relative effectiveness of different batches of antivenom may be compared against a standard venom. This is a practical approach to determining the comparable activity of these antivenoms since a particular antivenom in vivo may neutralise neuromuscular blocking effects of venom but not the myotoxic effects and vice versa. Myotoxic damage in both the rodent and chick preparations, is assumed to be a consequence of direct contracture (or unresponsiveness to direct stimulation) of the observed skeletal muscle, though microscopic examination (preferably electron microscopy) provides unequivocal evidence. Many venoms (e.g. Naja kaouthia) contain a mixture of toxins (cardiotoxin [cytotoxin]), myotoxin, postsynaptic a-cobratoxin, and three minor neurotoxins have been identified) but by testing at lower venom concentrations for neurotoxic activity and at higher concentrations for myotoxic activity, discrimination broadly consistent with clinical findings is possible (Campbell, 1979). 8.3. In vitro mouse hemidiaphragm phrenic nerve preparation The rat or mouse hemidiaphragm phrenic nerve assay (Bulbring, 1946; Kitchen, 1984) is still in widespread use for testing the efficacy of neutralising antibodies to postsynaptic neurotoxins (Jones et al., 1999). It has been shown to be more sensitive than the chick biventer cervicis muscle preparation in studies on Papuan taipan (Oxyuranus scutellatus canni) venom (Crachi et al., 1999). The rat or mouse is killed by stunning and exsanguination and a length of 3 – 4 cm of the left phrenic nerve is dissected from within the thoracic cavity together with the rib attached to the diaphragm. The preparation is suspended in an organ bath

with the nerve attached to an electrode and the muscle to the transducer. The Ringer solution in the bath is kept at 22 or 37 8C and is well aerated during subsequent testing. With experience, successful dissection should take about 40 min and biopsies are viable for use in the assay for a few hours. 8.4. In vitro frog rectus abdominis muscle preparation A simple preparation uses the rectus abdominis muscle (RAM) dissected from the length of the abdomen of a pithed frog. No nerves are included but direct recordings of nAChR antagonist activity may be made by attaching the RAM to a transducer in an organ bath filled with Ringer solution (Perry, 1970). 8.5. In vitro guinea pig isolated ileum preparation This smooth muscle preparation uses segments of guinea pig ileum mounted on tissue holders in an organ bath containing carbogenated Krebs solution at 37 8C. It has been shown to identify PLA2 activity by concentration-dependent contractile responses to Papuan taipan venom (Crachi et al., 1999) as well as indicating the presence of PLA2 and cyclooxygenase metabolites in the venom of the inland taipan (Bell et al., 1998) and Vipera ammodytes (Sket and Gubensek, 1976). 8.6. In vitro estimation of neurotoxicity using cultured cells As an alternative to animal biopsies of nervous tissue, functional tests may be carried out with precision on cultured cells, though these are usually for the elucidation of receptor activity rather than for venom/antivenom assays. Cultured cells offer great versatility, either through recombinant technology or constitutive expression of antigens/receptors, and provide a consistent and easily manipulated source for functional assays, the disadvantage being the capital cost of electrophysiological and software equipment needed to interpret the data. A large number of tumour-derived cell lines are available which express specific receptors in vitro. For example, PC12, IMR32 and BC3H-1 naturally express mammalian nAChRs of various subtypes. Genetically engineered, mammalian clonal cell lines, with stable receptor expression, which may be altered by site-directed mutagenesis, are used for pure research where venom provides a specific binding probe. Such cell lines have extended the pioneering work of Changeux et al. (1970) who used nAchR isolated from Torpedo marmorata. Voltage clamp recordings may be obtained from endogenous Xenopus oocyte receptors and expression of venom receptor target cDNA in oocytes provides a correctly folded source of the human subunit epitope (Boorman et al., 2000). Yields are adequate for functional studies (which may be automated using ion imaging) but may have limitations for biochemical applications.

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Tissue slices from rat brain have been used to investigate the in vitro binding of venoms (e.g. Dendroaspis venom), to neuronal nAchR and muscarinic AchR (Jerusalinsky et al., 1992). Human nAchRs, naturally expressed by the foetalmuscle-like tumour cell line, TE671, have the potential to be used as an assay to measure the efficacy of antibody against a post-synaptic toxin using the patch clamp method (Schoepfer et al., 1988; Shao et al., 1998; Yamamoto et al., 1991). The efficacy of an ovine antibody raised against the purified a-neurotoxin from N. kaouthia venom was tested against the purified fraction on a single cell preparation of the TE671 cell line. The decline in response to acetylcholine, caused by the addition of purified a-neurotoxin from N. kaouthia venom, was significantly restored (by 66%) by prior incubation of the toxin with the antineurotoxin antibody (Mellor and Sells, unpublished).

fibres which is caused by the specific activity of a toxin. Damage is characterised by the disruption of plasma membranes, local infiltration of inflammatory cells and oedema as well as the appearance of myoglobin in the urine. Presynapically acting phospholipase A2 (PLA2) enzymes are found in the crotalids, viperids, sea snakes and many elapids. These PLA2 s may be primarily myotoxic (e.g. Enhydrina schistosa PLA2) or neurotoxic (e.g. b-bungarotoxin) or both (Crotalus durissus terrificus PLA2 [crotoxin]. Assessment of activity by in vitro methods should take into account the differing specific targets of individual PLA2 enzymes which ideally should be tested against a range of substrates. For example, notexin is more myotoxic to rat and chicken muscle than to mouse muscle (Harris, 1991) and immature muscle fibres may be relatively resistant to myotoxins (Harris and Johnson, 1978).

8.7. In vitro estimation of neurotoxicity and its neutralisation by ELISA

9.1. In vivo rodent estimation of myotoxicity

Non-radioactive binding studies of post-synaptic neurotoxins with purified AchRs (T. californica), in both indirect and competitive ELISAs, have provided the basis for tests to study the specificity and cross-reactivity of mouse monoclonal antibodies raised against neurotoxins (Stiles, 1991, Stiles et al., 1994). Microtitre plates were coated with toxin (coating with AchRs was unsuccessful). Sufficient neurotoxin in the correct orientation appears to bind to microtitre plates for the initial adsorption phase to produce positive results with added AchR although this may only be 90 ng of 5 mg cobrotoxin initially added to the well (Stiles, 1991). Monoclonal antibodies inhibited up to 71% of toxin binding to AchR in vitro but afforded only a slight protective effect in vivo; an a-bungarotoxin/antibody molar ratio of 1:1.5 resulted in a ,2.5-fold increase for the time to death relative to control animals. Conventional ED50 tests were not carried out. The ELISA may be used to evaluate antibody binding directly to neurotoxins adsorbed on the microtitre plate (Sells et al., 1994a) although in a further study, positive in vitro binding results again did not correlate with protective in vivo activity (Sells et al., 1994b). There may be several explanations for this but the lack of correlation illustrates a general principle which is that immunoassays cannot always distinguish between neutralising and non-neutralising antibodies. Good correlations are usually achieved when the immunodominant epitope of the antigen coincides with the neutralising epitope to which the protective antibody binds. Correct orientation of the antigen during coating of the microtitre plate to expose reactive epitopes is also an important consideration in the sensitivity of an assay.

9. Estimation of myotoxic activity Myotoxicity leads to the degeneration of skeletal muscle and the breakdown of the normal architecture of muscle

Mice or rats may be injected with toxin intramuscularly in the right gastrocnemius muscle. At intervals after injection, blood samples may be taken and tested for plasma creatinine kinase, the presence of which indicates muscle damage. (Gutie´rrez et al., 1992; Alam et al., 2002). A biopsy of the injected muscle can be fixed and stained with haematoxylin and eosin and examined microscopically for disorganised myofilaments, hypercontraction and necrotic cells. 9.2. In vitro measurement of myotoxicity on cultured cells Cytolysis and the release of lactic dehydrogenase (LDH) into the supernatant by cultured cells is thought to reflect the activity of class II myotoxic viperid PLA2 toxins in vivo (Lomonte et al., 1999). Murine myoblast cells (C2C12), differentiated into myotubes, were exposed to PLA2 toxins (purified from a variety of snake venoms) and released LDH into the supernatant where it was colourimetrically quantified. Microscopic evaluation confirmed the complete disappearance of myotubes. The authors also demonstrate that C2C12 differentiated cells are more susceptible in the cytolytic assay than cultured tEnd murine endothelial cells. Creatine kinase (CK) activity was not found to be a practical quantitative correlate of myotoxic activity as it was unstable and the Triton X-100 used in the assay interfered with its measurement. Furthermore, no relationship between CK levels and the degree of muscle damage has been established (Rowlands, 1980). Neither notexin (class I) or crotamine (‘small myotoxin’) demonstrated cytolytic activity on the C2C12 cells, presumably reflecting their different lipid targets. In vitro studies of ammodytin L on the viability of rat skeletal muscle cells (Incerpi et al., 1995) and of cultured rat fibroblasts (Rufini et al., 1996) both support the concept that in vivo activity of class II myotoxic PLA2s can be measured by in vitro cytotoxicity on appropriate cell lines (i.e. where

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the lipid constitution of the membrane is susceptible to hydrolysis by the enzyme under test). The mode of action in vitro studies on the effects of B. asper PLA2 on rabbit sarcoplasmic reticulum (s.r.) may have the potential to become a more routine assay for myotoxins. The hydrolysis of s.r. phospholipids due to B. asper myotoxin was measured as follows. Horseradish peroxidase added to s.r. was sonicated, incubated and washed before the addition of different doses of myotoxin. After incubation and the addition of peroxidase substrate, the release of peroxidase entrapped in the vesicles was measured by absorbance at 490 nm and found to be dose dependent. Gutie´rrez et al. (1987) further showed that purified B. asper myotoxin inhibited Ca-ATPase activity in the s.r. following incubation in vitro (at a toxin concentration of 4.7 mg/ml, ATPase activity decreased 50%). Although practical and quantitative these tests are affected by the presence of albumin, Triton X-100 and the specificity of the myotoxin for the target substrate.

10. Estimation of necrotising activity Necrosis is the end point of a process started by inflammation which may involve cytolysis and apoptosis, culminating in the death of cells in an area of tissue. It may be caused by a number of different venom toxins, particularly the haemorrhagins and phospholipases. Haemorrhagic zinc MPs occur mainly in viper venoms and, in the case of the Bothrops, Echis and other viperid and crotalid species, are associated with necrosis at the bite site or point of injection of venom. 10.1. In vivo rodent estimation of necrosis The WHO standard test defines the minimum necrotizing dose (MND) as the least amount of venom which causes a necrotic skin lesion of 5 mm diameter 3 days after being injected intradermally into mice or rats (Theakston and Reid, 1983). 10.2. In vitro estimation of necrosis on cultured cells Investigations into the mode of action of MPs in certain venoms have been carried out using in vitro tests. Although these tests do not measure actual cytolytic activity, the potential of a venom to cause necrosis via another pharmacological pathway may be estimated. For example, the in vitro induction and cleavage of host pro-TNF-a by jararhagin (a purified MP from Bothrops jararaca venom) resulted in the release of mature TNF-a which is implicated in the development of in situ necrosis (Moura-da Silva et al., 1996). In vitro tests were employed as follows; the induction of TNF-a (and other pro- inflammatory cytokines; e.g. IL1b, IL-6) into the supernatant was assayed by ELISA after

incubation of jararhagin with murine peritoneal adherent cells. Induction of cytokines following incubation was confirmed by analysis of mRNA expression (Clissa et al., 2001) and the cleavage of proTNF-a to TNF-a by jararhagin was demonstrated by SDS-PAGE. Activity of cleaved TNFa was assayed by the viability of an actinomycin-treated, TNF-sensitive cell line (WEHI) following incubation (Moura-da-Silva et al., 1996). Thus venom-induced necrosis may be partly caused by MP activation of endogenous TNFa, though it should be noted that the cytolytic activity of PLA2 present in some venoms, for example, Bothrops and Crotalus, may also cause release of TNF-a from murine cells in vitro (Chisari et al., 1998). However, such indirect in vitro assays, when rigorously designed, have the potential to identify and evaluate necrotising venoms. 10.3. In vitro assays for phospholipase activity Direct in vitro assays for phospholipase include the original pH stat method using egg lipoprotein as substrate (Nieuwenhuizen et al., 1974), the inhibition of ouabainsensitive Naþ Kþ ATPase activity (Khelif et al., 1985) and the hydrolysis of fluorescent phospholipids (Thuren et al., 1986; Bougis et al., 1986). A colourimetric assay (using a dye as the indicator of pH variation) is particularly convenient as a qualitative assay for the presence of PLA2 in column fractions of whole venom (De Araujo and Radvanyi, 1987). For example, using phenol red dye and lecithin-cholate as a substrate, 60 samples were tested for the presence of PLA2 (30 – 50 ng easily detected) in 30 min (compared with 8 h for the pH stat method). This assay is simpler to use than another rapid method based on the haemolysis of red blood cells in the presence of cardiotoxin (Vogel et al., 1981), but quantitative data must be interpreted within the limitations of this technique. Venom PLA2 activity may also be assessed by measuring the release of free fatty acids from a phosphatidylcholine (egg yolk) substrate using cresol red as a pH indicator (adapted from De Araujo and Radvanyi, 1987). Venom and antivenom are premixed for 30 min and the optical density of the solution is then measured 10 min after the addition of substrate. This test has been used for antibody neutralisation studies of PLA2 in bee venom (Jones et al., 1999) but attempts to relate in vivo toxicity of b-bungarotoxin PLA2 to in vitro hydrolytic potency failed to correlate LD50 values and the hydrolysis of phosphatidylcholine (Kondo et al., 1982) and it is likely that the binding sites for these two activities occupy different parts of the molecule. Venom fractions used for mode of action studies should be rigorously tested for purity (usually by SDS-PAGE or twodimensional gel electrophoresis). For example, PLA2 may coelute with cytotoxins (cardiotoxins) during ion-exchange chromatography (Dufton and Hider, 1991) and k-neurotoxin (from Bothrops multicinctus venom) may co-purify with abungarotoxin (Ravdin and Berg, 1979). Usually, reversephase high-pressure liquid chromatography RP-HPLC) using

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a dual separation system, such as hydrophobic interaction on phenyl-Superose followed by anion exchange on Mono Q (Pharmacia), is sufficient for a clean separation. However, extra controls could be usefully included in in vitro assays (e.g. samples prepared in calcium-free conditions to inhibit PLA2 activity or treated with 1 mM EDTA to eliminate MP activity). It is also important in the context of in vitro tests not to confuse the passive release of a substance through cytolysis (cell death) with the specific induction and subsequent exocytosis from the cell of the same substance, both of which would give an equally positive ELISA reading from the culture supernatant. The distinction can usually be made by the detection of specific mRNA in the cells (in situ hybridisation) by the reverse-transcriptase polymerase chain reaction (which has replaced metabolic labelling and autoradiography techniques) assuming there are no significant post-translational modifications.

11. Validation of new procedures and trends in animal use Regulated animal studies began in the UK with the British Cruelty to Animals Act 1876 and research today is governed by The Animals (Scientific Procedures) Act 1986. In the UK, the Home Office regulates the issue of personal and project licences for animal studies and is responsible for ensuring compliance with the 1986 Act. The validation of a new assay is a graduated process requiring endorsement of repeatability following standardisation in several different participating laboratories. It may take up to 10 years from the time of development of the new test to its implementation in regulations. However, new editions of the European Pharmacopeia (2004) and interim addendums will include specific recommendations from the European Directorate for the Quality of Medicines to draw attention to alternative in vitro assays which have not been validated but which show potential. Several supporting organisations such as FRAME (Fund for the Replacement of Animals in Medical Experiments, Nottingham, UK) founded in 1969, and ECVAM founded by the European Commission in 1992, aim to promote the scientific and regulatory acceptance of alternative methods. For example, ECVAM publishes procedures for the validation of nonanimal assays, organises conferences and training and offers limited funding (Halder et al., 2002). The number of rodents used in venom research and antivenom production is not specified in UK Home Office statistics (Home Office, 2000) or the US. Overall, numbers of procedures in normal animals have dropped but there is a rise in the use of genetically engineered mice. For example, in 1995 in the UK, 1,932,000 procedures (in fundamental biological research and applied studies) were carried out on normal animals compared with 1,511,000 in 2000. In contrast, in 1995 in the UK, 87,000 procedures

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were carried out on genetically modified animals compared with 186,000 in 2000 (Home Office, 2000).

12. Future prospects Future venom research is likely to include the increasing use of molecular and analytical techniques. While molecular studies may increase the use of animals (e.g. genetically engineered mice) in the short term, they should help to reveal the detailed pathophysiology of envenoming upon which simple in vitro assays may be designed for future use in the field or laboratory. For example, DNA immunisation and epitope mapping may focus the generation of protective antibodies to pathologically-dominant venom epitopes so that in vitro assays could replace the conventional ED50 test. Recent advances in protein engineering, proteomics (the study of proteins coded for by the genome) and analytical techniques are likely to improve our understanding of venom-target topographical binding and function and to offer new scope for the design of in vitro assays. Antibody binding has been studied with a view to producing ‘universal’ antivenoms which neutralise the epitopes common to a group of different venoms. Apart from obvious clinical advantages, such an achievement would significantly reduce the need for the development and testing of individual antivenoms. However, the use of monoclonal antibodies (Kohler and Milstein, 1975) and a variety of non-animal analytical techniques directed to this end (reviewed by Menez, 1991), met with limited success. Increasing automation may permit this objective to be revisited. For example, direct parallel coupling of HPLC (high pressure liquid chromatography) systems to mass spectrometers and NMR (nuclear magnetic resonance), known as LC –NMR and LC – NMR –MS, are able to analyse complex mixtures in solution as well as providing structural information (Hammadi et al., 1998). The success of the genome projects has spurred on many new techniques used in proteomics; for example, 2D gel electrophoresis for high resolution/purification of proteins which are subsequently ‘fingerprinted’ by MALDI or SELDI-TOF laser analysis and matched to a database for identification (Petrocoin et al., 2002). In future, identification of the snake species responsible for envenoming patients may be made from a blood sample using this technique, as well as the monitoring of in vivo titres of neutralising antivenoms and venom metabolites which would replace antigen/antibody detection by ELISA. Analytical assays for toxins are used routinely in the shellfish industry and are more rapid and accurate than the mouse bioassays for toxins they have superceded (Goto et al., 2001; Quilliam, 2001). This progress has occurred because of incentives provided by the food safety industry

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and demonstrates the effectiveness of commercial competition in combination with funding specifically for new toxin assays (Asian Pacific Economic Cooperation). Analytical techniques, such as liquid chromatography and mass spectrometry (LC – MS), used for the identification and quantitation of proteins in tissue, could be adapted to analyse biopsies/cells from envenomed tissue, provide correlations with myotoxic/necrotic damage and to establish reference standards for sublethal end points in vivo for relative potency studies. New ideas under development also include molecular imprint polymers (MIPS) (Andersson et al., 1995) which have the potential to replace antibodies (Vlatakis et al., 1993). Briefly, synthetic polymers are ‘imprinted’ by an antigen and, following polymerisation, retain a tertiary complementary structure to the desired epitope from the ‘print’ antigen. MIPS are currently used for techniques based on immobilised antibodies e.g. ELISA (Piletsky et al., 2001) but in combination with nanotechnology may eventually be suitable for clinical treatment. Advantages of MIPS would include the replacement of animal immunisation to generate antibodies, a long shelf-life without the need for refrigeration, simple scale-up for bulk manufacture and the provision of more standardised reagents for use in in vitro diagnostic tests (e.g. ELISA).

13. Conclusion In reviewing the data for this article, a distinction was made between (i) experiments in venom research which investigate the ‘mode of action’ in physiological studies related to envenoming and (ii) relatively simple techniques based on known specific reactions which are suitable for routine assays, (e.g. ELISA). Sometimes the distinction was difficult to make and some of the more exploratory techniques, which suggested potential for a good assay, have been included for future consideration. Ultimately, it is the results of mode of action research which drive the design of precise and relevant in vitro assays and reduce the need for animals (Sesardic et al., 2000a,b). Assays may be qualitative or quantitative. In the context of clinical envenoming, a quantitative assay is only useful if the degree of pathological damage in vivo can be correlated with an amount of the causative substance detected in vitro. There should be a linear correlation between dose and effect (a clear physiological end point) which may be measured easily. This may not be possible where synergy, inhibition and host responses interfere with activity of venom constituents in vivo. Qualitative or functional in vitro assays are therefore probably the most useful but also have limitations. They may depend on the assumption that the animal or pharmacological substrates used in the test have human

counterparts and extrapolation from in vitro results to the in vivo clinical situation may be perilous. The most acceptable in vitro assays, both scientifically and ethically are those in which human tissue is used. For example, an in vitro kit using human cells develops a vascular network in 12 – 14 days (similar to the chick chorionic allantoic membrane (CAM)) on which the mechanisms of haemorrhage and permeability may be studied (TCS Biologicals, Buckingham, UK). Funding for studies on alternative assays is always difficult in spite of its ‘political correctness’. In the absence of strong commercial competition and dedicated funding, which has so enhanced progress in non-animal testing for toxins in the shellfish industry, there is an economic shortfall in venom research which must be addressed. Organisations such as the Dr Hadwen Trust and the Lord Dowding Fund will not, and the Humane Research Trust generally do not, support projects using any animal tissues, (including cell lines and fertilised eggs) and instead use human cell lines and biopsies. Nevertheless, such organisations also appreciate that new non-animal assays may have to be compared with tests in animals for comparative purposes at a later stage before the new test can be established, using funding from alternative sources. This continuum is the only practical way to introduce non-animal tests in the longer term. The establishment of Ethical Review Processes in 1997 by the government in the UK, which provide evaluation and advice at a local level, effectively endorse the principles of Reduce, Refine and Replace (Russell and Burch, 1959). However, until alternative tests are officially validated, the mouse ED50 will remain as the statutory preclinical test for antivenoms. European Agency for the Evaluation of Medicinal Products (EMEA) (2002) recommended that ‘it is highly desirable to avoid use of animals by substituting in vitro methods’, but most venoms contain several active components, some of which may act synergistically in vivo during envenoming thus the development of in vitro methods for antivenom assessment may be difficult. The mouse ED50 test therefore remains the only one that can be accepted by the authorities as satisfying the requirements of the regulations. Progress is dependent not just upon incentive, legislation and funding but also upon the development of appropriate technology and advances in mode of action studies. Interdisciplinary collaboration in analytical chemistry, physics and molecular genetics is accelerating the dissection of the mechanisms of action in envenoming. It is to be hoped that the new information this will provide will encourage the design of precise in vitro assays to replace the animal assays currently necessary to measure ‘net effect’ pathology. A tripartite approach, incorporating clinical (human) studies, animal experimentation and in vitro studies has been very productive in medical research, for example, the development of polio vaccine which relied on all three at different stages of research (Zurlo et al., 1994). By

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rigorously implementing the three R’s as follows, there is hope for a significant reduction in the role of sensate animal models in future; Reduce. Use the minimum number of animals compatible with a statistically valid result. Reserve definitive animal testing for promising samples already identified by in vitro or insensate screening assays. Refine. Ensure thorough justification of use and clarity of experimental design to achieve an unequivocal result. Adopt venom and antivenom reference preparations and humane alternatives to lethal end-points where possible. Replace. Develop alternatives where possible and applicable, such as in vitro studies using human tissue and cell lines.

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The factors determining progress towards the significant reduction of animal experimentation, with particular regard to snake venom research, are an integral part of a wider context. Policy makers and organisations which espouse humanitarian principles should foster progress not only in ethical research but also on facilitating supplies of affordable antivenom to where they are urgently needed.

Acknowledgements I am most grateful to Professor R.D.G. Theakston for his comments and advice on the manuscript.

Appendix A. Summary of some of the major in vivo and in vitro tests used in reply to: snake venom research

Rodent Route Endpoint

LD50 (venom lethality)

ED50 (antivenom potency)

Haemorrhagic Coagulant venom activity venom activity

Neurotoxic venom activity

Myotoxic venom activity

Necrotic venom activity

i.v. Death

i.v. Survival

i.d. or i.m. Haemorrhage

i.v. Death

i.d. Necrosis

24 hours Laing et al. (1992)

24 hours Laing et al. (1992) and Ownby et al. (1984)

i.m. Creatinine kinase Variable Gutie´rrez et al. (1992), Alam et al. (2002)

Topical Death 6 hours Sells et al. (2000)

Topical Haemorrhage 4 hours Sells et al. (1997)

Time 24 hours Reference Theakston and Reid (1983) Egg Route Endpoint Time Reference

Topical Death 6 hours Sells et al. (1998)

i.v. Blood clotting 1 hours Laing et al. (1992)

24 hours Laing et al. (1992) and Theakston and Reid (1983)

3 days Theakston and Reid (1983)

In vitro Gelatin ELISA Clotting time ELISA (Laing et al., (Theakston (Bee et al., 1992) 2001) and Reid, 1979), haemolysis (Gutie´rrez et al., 1988)

Biopsies from: chick (Harvey et al., 1994), mouse (Kitchen, 1984), frog (Perry, 1970), guinea pig (Crachi et al., 1999), oocytes (Boorman et al., 2000), ELISA (Stiles et al., 1994; Sells et al., 1994a)

LDH release from cells (Lomonte et al., 1999), cytotoxiciy (Incerpi et al., 1995; Rufini et al., 1996)

PLA2 activity (De Araujo and Radvanyi, 1987)

i.v.: intravenous, i.d.: intradermal, i.m.: intramuscular, ELISA: enzyme-linked immunosorbent assay, LDH: lactic dehydrogenase, PLA2: phospholipase A 2. LD50: median lethal dose (mg) of venom necessary to kill 50% mouse sample. ED50: median effective dose (ml) of antivenom required to protect 50% mouse sample from a defined venom challenge.

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