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Toxicity testing in the development of anticancer drugs
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Animal toxicity testing
Toxicity testing in the development of anticancer drugs
John Double Director, Cancer Research Unit, The Tom Connors Cancer Research Centre, University of Bradford, UK Is toxicity testing of new anticancer compounds necessary before proceeding to clinical trials? It is not possible to give a simple yes or no to this question. To some extent, any answer needs to be qualified, because of the particular problems posed by cancer. Some of these problems are very different from those encountered in other serious medical conditions. It is also important to define what toxicology testing entails. By the time many common cancers are diagnosed they are already life threatening ie, there has been metastatic spread and although in many instances removal of the primary tumour produces significant benefit, it may have little effect on lifespan. Since the introduction of cytotoxic agents for the systemic treatment of disseminated disease there has been a major improvement in survival for many patients with cancer. However, the figures still show that for many solid cancers this may only represent, at best, a 2–3 year improvement and with many of the agents that are currently available, this benefit must also be balanced against a number of unpleasant, and sometimes dangerous, side-effects. There is, therefore, a compelling need to develop more effective and less toxic therapies for cancer. Historically, the development of anticancer drugs was largely an empirical process, with new chemical entities screened against various cell lines and experimental tumours in animals thought to represent human cancers. The majority of agents identified in this process were ‘cytotoxic’, but only tended to be active at the ‘maximum tolerated dose’. Regulations have always required toxicity testing before clinical trials. Arguably, because of the known cytotoxic properties of potential anticancer agents, excessive demands for extensive preclinical toxicology have been made on anticancer drug development without due regard for their potential clinical use. Although there is some justification for determining acute toxicity and maximum
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tolerated dose—as a guide to a safe starting dose in clinical trials—chronic toxicity studies, particularly in non-rodent species, seem harder to justify when many cytotoxic agents will not be used in this setting. In an ideal world, acute dosing would produce a cure; sadly, in a majority of cases, treatments only produce modest effects and patients die from their disease. With this qualification, and on the basis of current experience, it would seem that there is no justification for the current demands on preclinical toxicology testing of anticancer agents using non-rodent models and extensive chronic studies. A recent article by Newell and colleagues,1 and an accompanying editorial by Leyland-Jones and Grieshaber,2 reported on the experience of the former Cancer Research Campaign in the UK (now Cancer Research UK). Data on 25 new drugs clearly showed that, in terms of a safe starting dose and the prediction of common dose-limiting toxicities, rodent-only toxicology was a safe and rapid way to progress new agents into phase I trials. If this is the case with ‘cytotoxic’ agents, which, by their very nature, will only be poorly selective towards cancer, then with future agents or therapies that will be more selective towards cancer cells, a serious reappraisal of preclinical toxicology testing will be needed. It is hoped that our increased knowledge of the molecular basis of carcinogenesis will lead to the development of non-toxic cancer-specific agents. If such drugs are to be rapidly progressed into clinical use, then we will have to move away from the concept of the maximum tolerated dose in animals being a safe starting dose in clinical trials. We will have to think in terms of an effective dose as a new yardstick. Furthermore, it is likely that the dose–response relation seen with many cytotoxic agents, ie, the higher the dose, the higher the cell kill, may not hold for therapies tailored to specific molecular targets. For example, if the target were a receptor, once this had been blocked, then additional dosing would not further improve the therapeutic effect. Although such dosing may produce added toxicity it may not be directly related to the mechanism of action and would be irrelevant in terms of potential clinical use. Again, we will have to think in terms of determining an effective, rather than maximum, tolerated dose as one objective of early clinical trials.
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Animal toxicity testing
There is a major public demand for better cancer therapies; at the same time, our knowledge of the molecular steps in the cancer process is increasing rapidly. The research community and the pharmaceutical industry are optimistic that they will be able to meet this demand and will produce highly selective anticancer agents. However, unless there is a change in the understanding and attitude of the regulatory bodies, and to a certain extent the toxicology community, current demands on preclinical toxicology will delay the use of these new therapies clinically. This has to be immoral on the grounds that it involves unnecessary animal suffering and denies cancer patients access to more effective therapies. Ultimately, the efficacy of any new therapy can only be determined in the clinic—in the laboratory we can only attempt to model clinical disease. In all areas of medicine, our increasing knowledge of molecular biology will lead to more selective treatments, so the necessity for extensive toxicology testing before clinical trials has to be questioned and as far as cancer is concerned, toxicology could and should become an integral part of therapeutic evaluation. There can be no real justification for toxicity testing of cancer-specific therapies in non-tumour-bearing animals. References
1 Newell DR, Burtles SS, Fox BW, et al. Evaluation of rodent-only toxicology for early clinical trials with novel cancer therapeutics. Br J Cancer 1999; 81: 760–68. 2 Leyland-Jones B and Grieshaber CK. Of (only) mice and men. Br J Cancer 1999; 81: 753–55.
Nigel Barrass AstraZeneca, Macclesfield, Cheshire, UK The primary objectives of any toxicology studies done to support anticancer drug development are to help guide candidate drug selection and subsequently to promote worldwide clinical development as quickly and as safely as possible, ensuring rapid introduction of new medicines. Currently, the information provided when a new drug is first given to humans usually includes details of target-organ toxicity in animals, the potential for reversibility of any lesions, and data on the dose that causes toxicity. This information is important not only for setting a reasonably safe dose at the start of clinical trials but also to help justify the dosing schedule and to identify any factors which may require special monitoring in the clinic. It is not yet possible, however, to provide all these data without using animal models, so the issue focuses on what experimentation is necessary to ensure patient (or volunteer) safety, and when it is most appropriate to conduct these experiments within the context of the overall development of a new anticancer agent. The majority of (non-hormonal) anticancer drugs developed in the past few decades have had a broad spectrum of cytotoxic effects and have needed acute intravenous administrations at toxic doses interspersed with long periods of recovery. Although these have had a variety
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of pharmacological activities against a range of molecular targets, the overriding biological effect has been generally similar ie, killing of proliferating cells. It is perhaps not surprising therefore, that fairly simple animal models have been used to provide preclinical information about target organ toxicities and their reversibility with a certain degree of confidence based on comparisons with clinical experiences with drugs of similar classes. Thus, with cytotoxic drugs, the main issue is to get the first clinical dose correct for the target population rather than identifying new toxicities. Hence I have no quarrel with the conclusion, presented by Newell and his colleagues in 1999 and referenced in the introduction,1 that, for some examples of cytotoxic drugs, appropriately designed rodent-only toxicology models are adequate to define safely the first dose given to patients. However, I would add a note of caution that physiological, pharmacodynamic, and metabolic considerations should all be included in the design of the toxicology programme.2 More recently, the emphasis of drug developers has shifted towards newer classes of agents targeted towards biological processes that may affect tumours selectively in different ways. For example, tyrosine-kinase inhibitors, anti-angiogenic agents, or anti-invasive drugs all target the growth and regulation of tumours rather than killing proliferating cells. These drugs have a different mode of action from cytotoxic agents, and their toxicity profile is less predictable from their basic pharmacology. Many of them are intended for daily oral therapy with no “drug holiday” and therefore no inbuilt recovery period from any adverse effects. In an ideal world, these agents will have a clear therapeutic margin for efficacy versus toxicity in clinical use, but, as yet, this wish is not backed up by substantial hard evidence. In addition, due to their intrinsically lower toxicity towards normal tissues, some of these drugs are initially given to healthy volunteers rather than cancer patients, which tips the balance between the risks and the benefits firmly towards risk, since volunteers do not derive any clinical benefit. Volunteers are used in order to obtain important information on the pharmacokinetics and tolerability of any new medicine in a tightly controlled environment as quickly as possible, thus accelerating the drug development process. Clearly, we are duty bound to ensure that every effort has been made to confirm the safety of the drug before administration in clinical trials, and that means using the best available models. Currently, the international consensus (and the regulatory position) is that this requires the use of pharmacologically relevant nonrodent and rodent models, dosed by the clinical route and schedule, for at least as long as the clinical trial, and at multiples of the intended initial clinical exposure. Even in cases where volunteer studies are ruled out of the clinical development plan it seems odd that we should be advocating a “minimal” programme of safety evaluation when we really have little or no knowledge of what models are the best predictors for man and therefore the most appropriate method of judging clinical safety. Just because the rodent-only concept seems to work well for certain classes of cytotoxic drugs doesn’t mean it will necessarily
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Forum work as well for other agents. Indeed, taken across the broad range of new drug approvals in recent years, including the anticancer medicines, the International Life Sciences Institute database suggests that animal studies in rodents and dogs predict many of the toxicities seen in man and that of the two species, the dog is the most effective model; especially for prediction of toxicity to specific organs such as the cardiovascular system.3 If in future, the majority of anticancer drugs are directed at chronic oral administration in patients with generally improved prognosis, they will require supporting data much more in keeping with other pharmaceutical agents. The hope is that the tenets established for the support of phase I trials with cytotoxic drugs may be extended to encompass global phase I development of novel anticancer agents, but it is clear that much more data is needed before we can make this step in a suitably informed manner, and ultimately the data may not support it. Currently, there is little preclinical or clinical information available on the safe use of the various classes of novel anticancer agents. It has been suggested that non-rodent toxicology studies cause delay to clinical trials. Generally, this is not the case; toxicology studies supporting the different phases of clinical development (ie, safety pharmacology, rodent and nonrodent studies) generally run in parallel, not in series, and rodent studies often take just as long as non-rodent. Undertaking both rodent and non-rodent toxicology in advance of phase I increases the chance of identifying severe toxic effects before risking the health of people enrolled in clinical trials. These studies also broaden the available safety database before the first human dose thus providing reassurance about the suitability of the clinical-dose selection, and accelerate the clinical trials programme by enabling trials to take place anywhere in the world. Further, this approach avoids a regulatory hiatus later on, should the drug be successful in phase I/II and therefore move into phase III trials and an eventual launch into the marketplace. Another suggestion is that the use of animals, and particularly non-rodents, early in anticancer drug development programmes specifically constitutes wastage. Wasting the lives of any animals, either rodent or nonrodent, is unacceptable in any circumstances. The onus is therefore on the toxicologist to ensure that the use of animals to develop any drug candidate is supported by sound scientific justification. If the use of non-rodents adds further justification and solidifies good scientific practice in the evaluation of a new drug then so be it; if it does not, we should re-examine how we give an appropriate level of assurance to patient safety. Finally, a brief comment about the regulatory environment: without going into details, there are subtle differences in the regulatory requirements of the USA, Japan, and western Europe with regard to toxicological support for anticancer drug development. This diversity causes the default position to veer towards satisfying all the authorities all the time, which is not satisfactory. There are ongoing harmonisation processes and the pharmaceutical industry hopes these will ultimately lead to better clarity of guidance worldwide. However, irrespective of regulatory
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demands, one must not lose sight of the main reason for the use of animals: patient safety. So, to answer the question: is toxicity testing of new anticancer compounds necessary before proceeding to clinical trials? Yes—for the foreseeable future; but we must continue to refine our approaches to reflect the nature of many new drugs in development. References
1 Newell DR, Burtles SS, Fox BW, et al. Evaluation of rodent-only toxicology for early clinical trials with novel cancer therapeutics. Br J Cancer 1999; 81: 760–68. 2 Colombo P, Gunnarsson K, Iatropoulos M, Brughera M. Toxicological testing of cytotoxic drugs. Int J Oncol 2001; 19: 1021–28. 3 Olsen H, Betton G, Robinson D, et al. Concordance of the toxicity of pharmaceuticals in humans and animals. Reg Tox Pharm 2000; 32: 56–67.
Neal D Barnard Physicians Committee for Responsible Medicine, Washington DC, USA How shall we investigate the safety of drugs that are, by their very definition, toxic? The question has so far confounded toxicologists and research oncologists. Animal tests in hazard assessments of new drugs raise three thorny issues: ethical considerations, the inability of animal tests to accurately predict toxicity to humans, and the sluggishness with which newer test methods are accepted. The use of animals has been made ever more troubling by the findings of physiological and behavioral research. It is now clear that both the sensory nerves and the brain centres that perceive pain stimuli are as efficient and sensitive in rats and mice as they are in non-rodent species, or, for that matter, humans. It is also certain that rodents, dogs, primates, and all other animals, are acutely stressed by routine laboratory procedures, from confinement and disruption of normal social and exercise patterns to gavage and phlebotomy, let alone more invasive procedures. Although some investigators have taken comfort in the idea that rats or mice are “lower species”, the more we understand an animal’s ability to sense the rigors of the laboratory, the more the rationale for using animals in toxicological testing wears thin. The need for better methods is further highlighted by the increasingly apparent limitations of current toxicological methods. In 1990, the US General Accounting Office reported important data on the fallibility of premarket testing of drugs in general, and of oncology drugs in particular. Pre-market tests included animal studies and limited human trials that led to drug approval. Of the 198 new drugs marketed in the USA from 1976 to 1985, 52% (102 drugs) had serious postapproval risks that were not predicted by premarket tests. In this context, risks were defined as adverse reactions that could lead to hospitalisation, increases in the length of hospitalisation, severe or permanent disability, or death, leading to the relabeling or the withdrawal of the drug from the market.
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Animal toxicity testing
Of nine oncology drugs marketed during this period, five had unanticipated postapproval risks. These included carmustine, cisplatin, cyclosporine, lomustine, and tamoxifen citrate, all of which required serious label changes. The unexpected incidence of health problems does not mean that these drugs have no place on the market. But it does mean that testing, via the conventionally accepted methods, does not provide anything resembling the full drug toxicity profile. It was not until these drugs were in clinical use that their dangers became clear. Very often, animals do not respond to drugs in the same way as their human counterparts. Sometimes the problem is identifiable, such as a fundamental difference in hepatic detoxification pathways or excretion mechanisms that allow the accumulation of toxic byproducts in one species and not in another. Many times, however, we are left scratching our heads, wondering why animal test results do not mirror the human experience. Rodent bioassays for carcinogenicity have been especially notorious, and it has been shown that results between these two species can diverge by as much as 30%.1,2 The error in estimating human risk is certainly higher. It is likely that the high rate of false positives in animal carcinogenicity studies is due to increased cell division resulting from administration of high test doses.3 For example, Ennever and co-workers found that 19 out of 20 human non-carcinogens tested positive in rodent bioassays.4 False negatives are of even greater public health concern, yet are common in typical rodent assays.5 But even simple acute toxicity tests have a dismal record. Such tests in rats and mice are only about 65% accurate in predicting lethal blood concentrations of chemicals in humans.6 What shall we do instead? It is not entirely clear how to predict the toxic effects of new drugs. But we do have a good idea of where the science of toxicology will be moving. Animal tests are currently used to provide preclinical information on potential health hazards and to determine a safe starting dose for human studies. The Multicentre Evaluation of In Vitro Cytotoxicity programme found that, while rat and mouse acute toxicity tests were only 65% accurate in predicting lethal blood concentrations in humans, a combination of human cell-culture tests predicted acute toxicity with considerably greater precision. 29 independent laboratories studied a set of 50 chemicals by use of 61 different in vitro assays; four tests emerged which, if used in combination, could predict lethal blood concentrations with an accuracy of about 80%.6 Because some cancer-fighting drugs increase the risk of later malignancies, subsequent tumour development is an important endpoint to examine when evaluating potential adverse effects of new drugs. Genetic toxicity can be evaluated with structure–activity information, computer modeling, and in vitro toxicity testing. Common structural features of genotoxic compounds have been identified, particularly from the results of the National Toxicology Program (National Institutes of Health, MD, USA) collaborative study on short-term tests for carcinogenicity, and have been useful in predicting response.7
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Several non-animal assays for genotoxicity have achieved worldwide regulatory acceptance because of their high degree of sensitivity and specificity to some endpoints which include reverse mutation, chromosomal aberration, cell gene mutation, and sister chromatid exchange. Bacterial reverse mutation tests, such as the Ames assay, have been used for more than 20 years to detect point mutations.8 Unfortunately, animal tests continue for each of these toxicological endpoints, despite serious limitations in their predictive value, and the availability of suitable alternatives. Until we improve testing methods for cancer drugs, their true measure of toxicity will continue to be apparent only in clinical use, whereas animal tests serve as little more than a distasteful regulatory formality. A transition to human cellular testing methods and other non-animal assays is a medical necessity. References
1 Lave LB, Ennever FK, Rosenkranz HS, Omenn GS. Information value of the rodent bioassay. Nature 1988; 336: 631–33. 2 Gold LS, Bernstein L, Magaw R, Slone TH. Interspecies extrapolation in carcinogenesis: prediction between rats and mice. Environ Health Perspect 1989; 81: 211–19. 3 Gold LS, Slone TH, Ames BN. What do animal cancer tests tell us about human cancer risk? Overview of analyses of the carcinogenic potency database. Drug Metab Rev 1998; 30: 359–404. 4 Ennever FK, Noonan TJ, Rosenkranz HS. The predictivity of animal bioassays and short-term genotoxicity tests for carcinogenicity and non-carcinogenicity to humans. Mutagenesis 1987; 2: 73–8. 5 Salsburg D. The lifetime feeding study in mice and rats—an examination of its validity as a bioassay for human carcinogens. Fundam Appl Toxicol 1983; 3: 63–7. 6 Clemedson C, McFarlane-Abdulla E, Andersson M, et al. MEIC evaluation of acute systemic toxicity. ATLA 1996; 24: 273–311. 7 Ashby J, Paton D. The influence of chemical structure on the extent and sites of carcinogenesis for 522 rodent carcinogens and 55 different human carcinogen exposures. Mutat Res 1993; 286: 3–74. 8 Ames BN, Durston WE, Yamasaki E, Lee FD. Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc Natl Acad Sci USA 1973; 70: 2281–85.
Vyra Navaratnam Animals (Scientific Procedures) Inspectorate, London, UK. Across the world, animal research is regulated in a number of ways. These may be nationwide with penalties for non-compliance (UK), locally administered (USA and Australia), or locally without powers of enforcement (Japan). In 1986, the European Economic Community adopted a Council Directive for protecting animals used for scientific purposes.1 In the UK, the Animals (Scientific Procedures) Act of 19862 implemented these requirements and regulates all procedures that may cause animals pain, suffering, distress, or lasting harm. The act prohibits animal experiments where other practicable and satisfactory methods, not requiring animals, are available. Under the act, a conviction for an offence carries the maximum penalty of 2 years imprisonment, or a fine, or both. The UK’s Home Office has issued guidance on the operation of the act3 explaining how it is implemented.
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Forum Briefly, a project licence authorises a programme of scientific work using animals. Experiments defined within the project licence are then carried out only by persons holding a personal licence detailing all the relevant in vivo procedures that they are personally authorised to do. The work must be carried out in an approved place and there are codes of practice on the care and housing of all animals used.4,5 A project licence may last for 5 years, but only for purposes specified by the act ie, the prevention, diagnosis, or treatment of disease, ill health, or abnormality in man, animals, or plants. Applicants for project licences for safety evaluation studies must exercise judgement and flexibility in designing test protocols and show an awareness of alternatives to animal tests and be prepared to revise their practices as alternative methods gain regulatory acceptance. They must review existing knowledge to address any gaps in that knowledge and to avoid unnecessary duplication, and they must be willing to participate in data sharing opportunities to reduce animal tests. Project licence applicants should try to predict the likely toxic effects and thus minimise the need for new animal data by, for example, use of computer modelling, structure– activity associations, physicochemical properties, in vitro studies, and non-regulatory screening tests. Where more than one approved method exists, the best in vivo approach is that which uses animals of the lowest neurophysiological sensitivity, causes the least pain, suffering, distress, or lasting harm, and uses the least number of animals. More severe tests that seem to exceed the legal minimum need special justification. Scientific considerations, rather than custom and practice, should dictate the choice of species. Special justifications are needed for cats, dogs, equidae, and non-human primates. Experimental protocols must cause the minimum amount of animal suffering; consistent with the study objectives. Suffering must stop at once if: it is not justified by the study objective; the objective has been achieved; it becomes clear that the objective cannot be achieved; or the data has been compromised. Further details of the UK’s Home Office requirements for project licence applications for regulatory testing are detailed in an additional document entitled “Guidance on the conduct of regulatory toxicology and safety evaluation studies”.6 National and international guidelines vary with regard to test requirements, detail, and degree of discretion they provide. Although it is better to have testing strategies that satisfy several regulatory bodies to avoid duplication and extra tests, it would be preferable to harmonise national requirements. This would enable mutual acceptance of data worldwide and, ultimately, a reduction in animal testing. The UK Home Office supports the international conference on harmonisation of technical requirements for
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registration of pharmaceuticals for human use (ICH), which seeks to eliminate redundant or duplicate data requirements and thereby expedite the availability of new medicines and reduce or refine animal testing. It has been agreed to phase out the LD50 single-dose toxicity test for pharmaceutical drugs and to reduce the duration of repeat-dose chronic rodent studies. Harmonised guidelines have been adapted on reproductive toxicology and carcinogenicity testing. The Organisation of Economic Co-operation and Development (OECD) has produced guidelines on humane endpoints7 and a draft document on alternative methods of validation.8 It would be helpful if the ICH produced similar guidelines to improve animal welfare, and to reduce, refine, and replace animal tests. The European Centre for the Validation of Alternative Methods (ECVAM), supported by the EU, recently validated three in vitro assays for testing potential corrosivity, irritancy, and phototoxicity of chemicals to the skin. These have been approved in Annex V of the European Council Directive 67/548.EEC, and subsequently, animal tests for these purposes have not been authorised in the UK. National regulations controlling the use of animals in drug testing vary and appear to be influenced by culture, institution, and individual preference. Furthermore, some international companies impose worldwide standards on drug development. Ideally, harmonised universal regulations to improve animal welfare is desirable, but in the short-term it might be easier and quicker to first define agreed humane endpoints, and other animal welfare measures, within the ICH guidelines.
References
1 Council Directive 86/609/EECD on the approximation of laws, regulations, and administrative provisions of the member states regarding the protection of animals used for experimental or other scientific purposes. Official J Eur Communities 1986; L358: 1–29. 2 Animals (Scientific Procedures) Act 1986. London: HMSO, 1986. 3 Guidance on the operation of the Animals (Scientific Procedures) Act 1986. London: HMSO, 2000. 4 Code of practice for the housing and care of animals used in scientific procedures. London: HMSO, 1986. 5 The code of practice for the housing and care of animals used in designated breeding and supplying establishments. London: HMSO, 1995. 6 Guidance on the conduct of regulatory toxicology and safety evaluation studies. London: HMSO, 2001. 7 Guidance document on the recognition, assessment, and use of clinical signs as humane endpoints for experimental animals used in safety evaluation. Series on testing and assessment No. 19. Paris: The Organisation of Economic Co-operation and Development (OECD), 2000. 8 Draft guidance document on the development, validation, and regulatory acceptance of new and updated internationally acceptable test methods in hazard assessment. Series on testing and assessment No. 34. The Organisation of Economic Co-operation and Development (OECD), 2001.
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Animal toxicity testing
Toxicity testing in the development of anticancer drugs
John Double Director, Cancer Research Unit, The Tom Connors Cancer Research Centre, University of Bradford, UK Is toxicity testing of new anticancer compounds necessary before proceeding to clinical trials? It is not possible to give a simple yes or no to this question. To some extent, any answer needs to be qualified, because of the particular problems posed by cancer. Some of these problems are very different from those encountered in other serious medical conditions. It is also important to define what toxicology testing entails. By the time many common cancers are diagnosed they are already life threatening ie, there has been metastatic spread and although in many instances removal of the primary tumour produces significant benefit, it may have little effect on lifespan. Since the introduction of cytotoxic agents for the systemic treatment of disseminated disease there has been a major improvement in survival for many patients with cancer. However, the figures still show that for many solid cancers this may only represent, at best, a 2–3 year improvement and with many of the agents that are currently available, this benefit must also be balanced against a number of unpleasant, and sometimes dangerous, side-effects. There is, therefore, a compelling need to develop more effective and less toxic therapies for cancer. Historically, the development of anticancer drugs was largely an empirical process, with new chemical entities screened against various cell lines and experimental tumours in animals thought to represent human cancers. The majority of agents identified in this process were ‘cytotoxic’, but only tended to be active at the ‘maximum tolerated dose’. Regulations have always required toxicity testing before clinical trials. Arguably, because of the known cytotoxic properties of potential anticancer agents, excessive demands for extensive preclinical toxicology have been made on anticancer drug development without due regard for their potential clinical use. Although there is some justification for determining acute toxicity and maximum
438
tolerated dose—as a guide to a safe starting dose in clinical trials—chronic toxicity studies, particularly in non-rodent species, seem harder to justify when many cytotoxic agents will not be used in this setting. In an ideal world, acute dosing would produce a cure; sadly, in a majority of cases, treatments only produce modest effects and patients die from their disease. With this qualification, and on the basis of current experience, it would seem that there is no justification for the current demands on preclinical toxicology testing of anticancer agents using non-rodent models and extensive chronic studies. A recent article by Newell and colleagues,1 and an accompanying editorial by Leyland-Jones and Grieshaber,2 reported on the experience of the former Cancer Research Campaign in the UK (now Cancer Research UK). Data on 25 new drugs clearly showed that, in terms of a safe starting dose and the prediction of common dose-limiting toxicities, rodent-only toxicology was a safe and rapid way to progress new agents into phase I trials. If this is the case with ‘cytotoxic’ agents, which, by their very nature, will only be poorly selective towards cancer, then with future agents or therapies that will be more selective towards cancer cells, a serious reappraisal of preclinical toxicology testing will be needed. It is hoped that our increased knowledge of the molecular basis of carcinogenesis will lead to the development of non-toxic cancer-specific agents. If such drugs are to be rapidly progressed into clinical use, then we will have to move away from the concept of the maximum tolerated dose in animals being a safe starting dose in clinical trials. We will have to think in terms of an effective dose as a new yardstick. Furthermore, it is likely that the dose–response relation seen with many cytotoxic agents, ie, the higher the dose, the higher the cell kill, may not hold for therapies tailored to specific molecular targets. For example, if the target were a receptor, once this had been blocked, then additional dosing would not further improve the therapeutic effect. Although such dosing may produce added toxicity it may not be directly related to the mechanism of action and would be irrelevant in terms of potential clinical use. Again, we will have to think in terms of determining an effective, rather than maximum, tolerated dose as one objective of early clinical trials.
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Animal toxicity testing
There is a major public demand for better cancer therapies; at the same time, our knowledge of the molecular steps in the cancer process is increasing rapidly. The research community and the pharmaceutical industry are optimistic that they will be able to meet this demand and will produce highly selective anticancer agents. However, unless there is a change in the understanding and attitude of the regulatory bodies, and to a certain extent the toxicology community, current demands on preclinical toxicology will delay the use of these new therapies clinically. This has to be immoral on the grounds that it involves unnecessary animal suffering and denies cancer patients access to more effective therapies. Ultimately, the efficacy of any new therapy can only be determined in the clinic—in the laboratory we can only attempt to model clinical disease. In all areas of medicine, our increasing knowledge of molecular biology will lead to more selective treatments, so the necessity for extensive toxicology testing before clinical trials has to be questioned and as far as cancer is concerned, toxicology could and should become an integral part of therapeutic evaluation. There can be no real justification for toxicity testing of cancer-specific therapies in non-tumour-bearing animals. References
1 Newell DR, Burtles SS, Fox BW, et al. Evaluation of rodent-only toxicology for early clinical trials with novel cancer therapeutics. Br J Cancer 1999; 81: 760–68. 2 Leyland-Jones B and Grieshaber CK. Of (only) mice and men. Br J Cancer 1999; 81: 753–55.
Nigel Barrass AstraZeneca, Macclesfield, Cheshire, UK The primary objectives of any toxicology studies done to support anticancer drug development are to help guide candidate drug selection and subsequently to promote worldwide clinical development as quickly and as safely as possible, ensuring rapid introduction of new medicines. Currently, the information provided when a new drug is first given to humans usually includes details of target-organ toxicity in animals, the potential for reversibility of any lesions, and data on the dose that causes toxicity. This information is important not only for setting a reasonably safe dose at the start of clinical trials but also to help justify the dosing schedule and to identify any factors which may require special monitoring in the clinic. It is not yet possible, however, to provide all these data without using animal models, so the issue focuses on what experimentation is necessary to ensure patient (or volunteer) safety, and when it is most appropriate to conduct these experiments within the context of the overall development of a new anticancer agent. The majority of (non-hormonal) anticancer drugs developed in the past few decades have had a broad spectrum of cytotoxic effects and have needed acute intravenous administrations at toxic doses interspersed with long periods of recovery. Although these have had a variety
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of pharmacological activities against a range of molecular targets, the overriding biological effect has been generally similar ie, killing of proliferating cells. It is perhaps not surprising therefore, that fairly simple animal models have been used to provide preclinical information about target organ toxicities and their reversibility with a certain degree of confidence based on comparisons with clinical experiences with drugs of similar classes. Thus, with cytotoxic drugs, the main issue is to get the first clinical dose correct for the target population rather than identifying new toxicities. Hence I have no quarrel with the conclusion, presented by Newell and his colleagues in 1999 and referenced in the introduction,1 that, for some examples of cytotoxic drugs, appropriately designed rodent-only toxicology models are adequate to define safely the first dose given to patients. However, I would add a note of caution that physiological, pharmacodynamic, and metabolic considerations should all be included in the design of the toxicology programme.2 More recently, the emphasis of drug developers has shifted towards newer classes of agents targeted towards biological processes that may affect tumours selectively in different ways. For example, tyrosine-kinase inhibitors, anti-angiogenic agents, or anti-invasive drugs all target the growth and regulation of tumours rather than killing proliferating cells. These drugs have a different mode of action from cytotoxic agents, and their toxicity profile is less predictable from their basic pharmacology. Many of them are intended for daily oral therapy with no “drug holiday” and therefore no inbuilt recovery period from any adverse effects. In an ideal world, these agents will have a clear therapeutic margin for efficacy versus toxicity in clinical use, but, as yet, this wish is not backed up by substantial hard evidence. In addition, due to their intrinsically lower toxicity towards normal tissues, some of these drugs are initially given to healthy volunteers rather than cancer patients, which tips the balance between the risks and the benefits firmly towards risk, since volunteers do not derive any clinical benefit. Volunteers are used in order to obtain important information on the pharmacokinetics and tolerability of any new medicine in a tightly controlled environment as quickly as possible, thus accelerating the drug development process. Clearly, we are duty bound to ensure that every effort has been made to confirm the safety of the drug before administration in clinical trials, and that means using the best available models. Currently, the international consensus (and the regulatory position) is that this requires the use of pharmacologically relevant nonrodent and rodent models, dosed by the clinical route and schedule, for at least as long as the clinical trial, and at multiples of the intended initial clinical exposure. Even in cases where volunteer studies are ruled out of the clinical development plan it seems odd that we should be advocating a “minimal” programme of safety evaluation when we really have little or no knowledge of what models are the best predictors for man and therefore the most appropriate method of judging clinical safety. Just because the rodent-only concept seems to work well for certain classes of cytotoxic drugs doesn’t mean it will necessarily
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Forum work as well for other agents. Indeed, taken across the broad range of new drug approvals in recent years, including the anticancer medicines, the International Life Sciences Institute database suggests that animal studies in rodents and dogs predict many of the toxicities seen in man and that of the two species, the dog is the most effective model; especially for prediction of toxicity to specific organs such as the cardiovascular system.3 If in future, the majority of anticancer drugs are directed at chronic oral administration in patients with generally improved prognosis, they will require supporting data much more in keeping with other pharmaceutical agents. The hope is that the tenets established for the support of phase I trials with cytotoxic drugs may be extended to encompass global phase I development of novel anticancer agents, but it is clear that much more data is needed before we can make this step in a suitably informed manner, and ultimately the data may not support it. Currently, there is little preclinical or clinical information available on the safe use of the various classes of novel anticancer agents. It has been suggested that non-rodent toxicology studies cause delay to clinical trials. Generally, this is not the case; toxicology studies supporting the different phases of clinical development (ie, safety pharmacology, rodent and nonrodent studies) generally run in parallel, not in series, and rodent studies often take just as long as non-rodent. Undertaking both rodent and non-rodent toxicology in advance of phase I increases the chance of identifying severe toxic effects before risking the health of people enrolled in clinical trials. These studies also broaden the available safety database before the first human dose thus providing reassurance about the suitability of the clinical-dose selection, and accelerate the clinical trials programme by enabling trials to take place anywhere in the world. Further, this approach avoids a regulatory hiatus later on, should the drug be successful in phase I/II and therefore move into phase III trials and an eventual launch into the marketplace. Another suggestion is that the use of animals, and particularly non-rodents, early in anticancer drug development programmes specifically constitutes wastage. Wasting the lives of any animals, either rodent or nonrodent, is unacceptable in any circumstances. The onus is therefore on the toxicologist to ensure that the use of animals to develop any drug candidate is supported by sound scientific justification. If the use of non-rodents adds further justification and solidifies good scientific practice in the evaluation of a new drug then so be it; if it does not, we should re-examine how we give an appropriate level of assurance to patient safety. Finally, a brief comment about the regulatory environment: without going into details, there are subtle differences in the regulatory requirements of the USA, Japan, and western Europe with regard to toxicological support for anticancer drug development. This diversity causes the default position to veer towards satisfying all the authorities all the time, which is not satisfactory. There are ongoing harmonisation processes and the pharmaceutical industry hopes these will ultimately lead to better clarity of guidance worldwide. However, irrespective of regulatory
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demands, one must not lose sight of the main reason for the use of animals: patient safety. So, to answer the question: is toxicity testing of new anticancer compounds necessary before proceeding to clinical trials? Yes—for the foreseeable future; but we must continue to refine our approaches to reflect the nature of many new drugs in development. References
1 Newell DR, Burtles SS, Fox BW, et al. Evaluation of rodent-only toxicology for early clinical trials with novel cancer therapeutics. Br J Cancer 1999; 81: 760–68. 2 Colombo P, Gunnarsson K, Iatropoulos M, Brughera M. Toxicological testing of cytotoxic drugs. Int J Oncol 2001; 19: 1021–28. 3 Olsen H, Betton G, Robinson D, et al. Concordance of the toxicity of pharmaceuticals in humans and animals. Reg Tox Pharm 2000; 32: 56–67.
Neal D Barnard Physicians Committee for Responsible Medicine, Washington DC, USA How shall we investigate the safety of drugs that are, by their very definition, toxic? The question has so far confounded toxicologists and research oncologists. Animal tests in hazard assessments of new drugs raise three thorny issues: ethical considerations, the inability of animal tests to accurately predict toxicity to humans, and the sluggishness with which newer test methods are accepted. The use of animals has been made ever more troubling by the findings of physiological and behavioral research. It is now clear that both the sensory nerves and the brain centres that perceive pain stimuli are as efficient and sensitive in rats and mice as they are in non-rodent species, or, for that matter, humans. It is also certain that rodents, dogs, primates, and all other animals, are acutely stressed by routine laboratory procedures, from confinement and disruption of normal social and exercise patterns to gavage and phlebotomy, let alone more invasive procedures. Although some investigators have taken comfort in the idea that rats or mice are “lower species”, the more we understand an animal’s ability to sense the rigors of the laboratory, the more the rationale for using animals in toxicological testing wears thin. The need for better methods is further highlighted by the increasingly apparent limitations of current toxicological methods. In 1990, the US General Accounting Office reported important data on the fallibility of premarket testing of drugs in general, and of oncology drugs in particular. Pre-market tests included animal studies and limited human trials that led to drug approval. Of the 198 new drugs marketed in the USA from 1976 to 1985, 52% (102 drugs) had serious postapproval risks that were not predicted by premarket tests. In this context, risks were defined as adverse reactions that could lead to hospitalisation, increases in the length of hospitalisation, severe or permanent disability, or death, leading to the relabeling or the withdrawal of the drug from the market.
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Animal toxicity testing
Of nine oncology drugs marketed during this period, five had unanticipated postapproval risks. These included carmustine, cisplatin, cyclosporine, lomustine, and tamoxifen citrate, all of which required serious label changes. The unexpected incidence of health problems does not mean that these drugs have no place on the market. But it does mean that testing, via the conventionally accepted methods, does not provide anything resembling the full drug toxicity profile. It was not until these drugs were in clinical use that their dangers became clear. Very often, animals do not respond to drugs in the same way as their human counterparts. Sometimes the problem is identifiable, such as a fundamental difference in hepatic detoxification pathways or excretion mechanisms that allow the accumulation of toxic byproducts in one species and not in another. Many times, however, we are left scratching our heads, wondering why animal test results do not mirror the human experience. Rodent bioassays for carcinogenicity have been especially notorious, and it has been shown that results between these two species can diverge by as much as 30%.1,2 The error in estimating human risk is certainly higher. It is likely that the high rate of false positives in animal carcinogenicity studies is due to increased cell division resulting from administration of high test doses.3 For example, Ennever and co-workers found that 19 out of 20 human non-carcinogens tested positive in rodent bioassays.4 False negatives are of even greater public health concern, yet are common in typical rodent assays.5 But even simple acute toxicity tests have a dismal record. Such tests in rats and mice are only about 65% accurate in predicting lethal blood concentrations of chemicals in humans.6 What shall we do instead? It is not entirely clear how to predict the toxic effects of new drugs. But we do have a good idea of where the science of toxicology will be moving. Animal tests are currently used to provide preclinical information on potential health hazards and to determine a safe starting dose for human studies. The Multicentre Evaluation of In Vitro Cytotoxicity programme found that, while rat and mouse acute toxicity tests were only 65% accurate in predicting lethal blood concentrations in humans, a combination of human cell-culture tests predicted acute toxicity with considerably greater precision. 29 independent laboratories studied a set of 50 chemicals by use of 61 different in vitro assays; four tests emerged which, if used in combination, could predict lethal blood concentrations with an accuracy of about 80%.6 Because some cancer-fighting drugs increase the risk of later malignancies, subsequent tumour development is an important endpoint to examine when evaluating potential adverse effects of new drugs. Genetic toxicity can be evaluated with structure–activity information, computer modeling, and in vitro toxicity testing. Common structural features of genotoxic compounds have been identified, particularly from the results of the National Toxicology Program (National Institutes of Health, MD, USA) collaborative study on short-term tests for carcinogenicity, and have been useful in predicting response.7
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Several non-animal assays for genotoxicity have achieved worldwide regulatory acceptance because of their high degree of sensitivity and specificity to some endpoints which include reverse mutation, chromosomal aberration, cell gene mutation, and sister chromatid exchange. Bacterial reverse mutation tests, such as the Ames assay, have been used for more than 20 years to detect point mutations.8 Unfortunately, animal tests continue for each of these toxicological endpoints, despite serious limitations in their predictive value, and the availability of suitable alternatives. Until we improve testing methods for cancer drugs, their true measure of toxicity will continue to be apparent only in clinical use, whereas animal tests serve as little more than a distasteful regulatory formality. A transition to human cellular testing methods and other non-animal assays is a medical necessity. References
1 Lave LB, Ennever FK, Rosenkranz HS, Omenn GS. Information value of the rodent bioassay. Nature 1988; 336: 631–33. 2 Gold LS, Bernstein L, Magaw R, Slone TH. Interspecies extrapolation in carcinogenesis: prediction between rats and mice. Environ Health Perspect 1989; 81: 211–19. 3 Gold LS, Slone TH, Ames BN. What do animal cancer tests tell us about human cancer risk? Overview of analyses of the carcinogenic potency database. Drug Metab Rev 1998; 30: 359–404. 4 Ennever FK, Noonan TJ, Rosenkranz HS. The predictivity of animal bioassays and short-term genotoxicity tests for carcinogenicity and non-carcinogenicity to humans. Mutagenesis 1987; 2: 73–8. 5 Salsburg D. The lifetime feeding study in mice and rats—an examination of its validity as a bioassay for human carcinogens. Fundam Appl Toxicol 1983; 3: 63–7. 6 Clemedson C, McFarlane-Abdulla E, Andersson M, et al. MEIC evaluation of acute systemic toxicity. ATLA 1996; 24: 273–311. 7 Ashby J, Paton D. The influence of chemical structure on the extent and sites of carcinogenesis for 522 rodent carcinogens and 55 different human carcinogen exposures. Mutat Res 1993; 286: 3–74. 8 Ames BN, Durston WE, Yamasaki E, Lee FD. Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc Natl Acad Sci USA 1973; 70: 2281–85.
Vyra Navaratnam Animals (Scientific Procedures) Inspectorate, London, UK. Across the world, animal research is regulated in a number of ways. These may be nationwide with penalties for non-compliance (UK), locally administered (USA and Australia), or locally without powers of enforcement (Japan). In 1986, the European Economic Community adopted a Council Directive for protecting animals used for scientific purposes.1 In the UK, the Animals (Scientific Procedures) Act of 19862 implemented these requirements and regulates all procedures that may cause animals pain, suffering, distress, or lasting harm. The act prohibits animal experiments where other practicable and satisfactory methods, not requiring animals, are available. Under the act, a conviction for an offence carries the maximum penalty of 2 years imprisonment, or a fine, or both. The UK’s Home Office has issued guidance on the operation of the act3 explaining how it is implemented.
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Forum Briefly, a project licence authorises a programme of scientific work using animals. Experiments defined within the project licence are then carried out only by persons holding a personal licence detailing all the relevant in vivo procedures that they are personally authorised to do. The work must be carried out in an approved place and there are codes of practice on the care and housing of all animals used.4,5 A project licence may last for 5 years, but only for purposes specified by the act ie, the prevention, diagnosis, or treatment of disease, ill health, or abnormality in man, animals, or plants. Applicants for project licences for safety evaluation studies must exercise judgement and flexibility in designing test protocols and show an awareness of alternatives to animal tests and be prepared to revise their practices as alternative methods gain regulatory acceptance. They must review existing knowledge to address any gaps in that knowledge and to avoid unnecessary duplication, and they must be willing to participate in data sharing opportunities to reduce animal tests. Project licence applicants should try to predict the likely toxic effects and thus minimise the need for new animal data by, for example, use of computer modelling, structure– activity associations, physicochemical properties, in vitro studies, and non-regulatory screening tests. Where more than one approved method exists, the best in vivo approach is that which uses animals of the lowest neurophysiological sensitivity, causes the least pain, suffering, distress, or lasting harm, and uses the least number of animals. More severe tests that seem to exceed the legal minimum need special justification. Scientific considerations, rather than custom and practice, should dictate the choice of species. Special justifications are needed for cats, dogs, equidae, and non-human primates. Experimental protocols must cause the minimum amount of animal suffering; consistent with the study objectives. Suffering must stop at once if: it is not justified by the study objective; the objective has been achieved; it becomes clear that the objective cannot be achieved; or the data has been compromised. Further details of the UK’s Home Office requirements for project licence applications for regulatory testing are detailed in an additional document entitled “Guidance on the conduct of regulatory toxicology and safety evaluation studies”.6 National and international guidelines vary with regard to test requirements, detail, and degree of discretion they provide. Although it is better to have testing strategies that satisfy several regulatory bodies to avoid duplication and extra tests, it would be preferable to harmonise national requirements. This would enable mutual acceptance of data worldwide and, ultimately, a reduction in animal testing. The UK Home Office supports the international conference on harmonisation of technical requirements for
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registration of pharmaceuticals for human use (ICH), which seeks to eliminate redundant or duplicate data requirements and thereby expedite the availability of new medicines and reduce or refine animal testing. It has been agreed to phase out the LD50 single-dose toxicity test for pharmaceutical drugs and to reduce the duration of repeat-dose chronic rodent studies. Harmonised guidelines have been adapted on reproductive toxicology and carcinogenicity testing. The Organisation of Economic Co-operation and Development (OECD) has produced guidelines on humane endpoints7 and a draft document on alternative methods of validation.8 It would be helpful if the ICH produced similar guidelines to improve animal welfare, and to reduce, refine, and replace animal tests. The European Centre for the Validation of Alternative Methods (ECVAM), supported by the EU, recently validated three in vitro assays for testing potential corrosivity, irritancy, and phototoxicity of chemicals to the skin. These have been approved in Annex V of the European Council Directive 67/548.EEC, and subsequently, animal tests for these purposes have not been authorised in the UK. National regulations controlling the use of animals in drug testing vary and appear to be influenced by culture, institution, and individual preference. Furthermore, some international companies impose worldwide standards on drug development. Ideally, harmonised universal regulations to improve animal welfare is desirable, but in the short-term it might be easier and quicker to first define agreed humane endpoints, and other animal welfare measures, within the ICH guidelines.
References
1 Council Directive 86/609/EECD on the approximation of laws, regulations, and administrative provisions of the member states regarding the protection of animals used for experimental or other scientific purposes. Official J Eur Communities 1986; L358: 1–29. 2 Animals (Scientific Procedures) Act 1986. London: HMSO, 1986. 3 Guidance on the operation of the Animals (Scientific Procedures) Act 1986. London: HMSO, 2000. 4 Code of practice for the housing and care of animals used in scientific procedures. London: HMSO, 1986. 5 The code of practice for the housing and care of animals used in designated breeding and supplying establishments. London: HMSO, 1995. 6 Guidance on the conduct of regulatory toxicology and safety evaluation studies. London: HMSO, 2001. 7 Guidance document on the recognition, assessment, and use of clinical signs as humane endpoints for experimental animals used in safety evaluation. Series on testing and assessment No. 19. Paris: The Organisation of Economic Co-operation and Development (OECD), 2000. 8 Draft guidance document on the development, validation, and regulatory acceptance of new and updated internationally acceptable test methods in hazard assessment. Series on testing and assessment No. 34. The Organisation of Economic Co-operation and Development (OECD), 2001.
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