ing, and will only replace animal testing when it is ... Summary â In the pharmaceutical industry, toxicology testing is normally done by preclinical scientists.
ATLA 30, Supplement 2, 221–223, 2002
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ECVAM and Pharmaceuticals Philippe Vanparys Department of Genetic and In Vitro Toxicology, Johnson & Johnson Pharmaceutical Research & Development (a Division of Janssen Pharmaceutica NV), Turnhoutseweg 30, 2340 Beerse, Belgium Summary — In the pharmaceutical industry, toxicology testing is normally done by preclinical scientists during the Development phase. In the last decade, the implementation of high-throughput screens during the Discovery phase has resulted in an ever-increasing number of lead candidates to be selected for drug development. The low throughput of the conventional safety tests is a bottleneck in the drug-development process. The pharmaceutical industry needs new techniques, down-scaled tests and in vitro alternative test models to determine the absorption, distribution, metabolism, and excretion (ADME) and toxicology profiles of compounds in the late-Discovery phase and/or early in the Development phase. Medium-throughput ADME and toxicity tests will enhance the selection of safer new chemical entities for animals and/or humans. Consequently, this testing strategy will not only reduce the use of resources and the overall development time, but will also result in a substantial decrease in animal use. Key words: alternatives, animal testing, drug discovery, preclinical toxicology.
Introduction The pharmaceutical industry is strongly committed to the highest safety standards for drugs intended for use in humans. It strongly relies on animal testing, and will only replace animal testing when it is scientifically and legally justified, without compromising human safety. Although there is a lot of controversy on the predictivity of animal studies for human toxicity, a review on 131 pharmaceutical agents from several pharmaceutical companies revealed a 69% prediction rate of human toxicity by animal models (1). It is naive to believe that all in vivo animal tests needed to prove safety of new chemical entities (NCEs) can be replaced by alternative models. However, the pharmaceutical industry is committed to the reduction, refinement and replacement of animal models. At the Johnson & Johnson Pharmaceutical Research & Development test facility in Beerse, a substantial decrease in the number of animals per full-time researcher and per compound tested has been achieved over the last two decades. The company strongly encourages scientific research into the development of in vitro alternative methods. The ultimate goal of the pharmaceutical industry is to bring better and safer drugs onto the market in as short a time as possible. An increase in the efficiency of development will be reached by better and earlier selection of NCEs during discovery screening and, in particular, during early lead optimisation. Pharmacokinetics and toxicology should already play a major role at these stages. Stopping the development of an unsuitable drug as early as possible is a major factor in saving money and time, but also
animal use. Therefore, most of the large pharmaceutical companies put a lot of effort into integrating toxicity testing into the discovery phase. At Johnson & Johnson Pharmaceutical Research & Development, two groups for toxicity testing have been created: the ADME/TOX group in Discovery, and the preclinical toxicology group in Development.
ADME/TOX in Discovery Traditionally, safety evaluation has been based mainly on animal experimentation. The huge number of chemicals that have to be screened in discovery for their ADME/TOX (absorption, distribution, metabolism and excretion/toxicity) profile makes it impossible to screen them in animals. In silico/in vitro models such as (quantitative) structure–activity relationship ([Q]SAR), physicochemical characterisation and alternative tests are employed, in order to enhance the selection of safer NCEs for animals and/or humans. The first generation of these tests are mainly low-throughput but, once internally validated, these tests are miniaturised and automated to a second generation of in vitro tests, taking into account the low availability of test compounds and the need for a high throughput. These in vitro studies bridge the gap between the high-throughput discovery screens and the conventional safety-assessment studies. Consequently, this testing strategy leads to a reduction in the number of animals used for safety evaluation testing. The ultimate goal for in silico prediction models is to accurately predict the in vivo toxicological and pharmacokinetic properties of NCEs in humans. Until now, in silico testing has mainly been used to
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prioritise NCEs under consideration for testing. Companies should better integrate knowledge on the ADME/TOX properties of series of compounds in in silico models, to gain a more accurate prediction of toxicological endpoints, before these NCEs enter animal studies. Better knowledge management and expansion of the databases on long-term animal studies will surely lead to improvements in in silico prediction models. SAR methods tailored to the problem of interest should result in a decrease in the testing of compounds that are toxic to animals. For reproductive toxicology, there is a need for screening models that select out embryotoxic or teratogenic NCEs. An in vitro embryotoxicity test, which relies on the capacity of embryonic stem cells (ESCs) to differentiate into embryonic bodies, has been developed by Spielmann et al. (2), and this test was one of three tests successfully validated in an ECVAM-sponsored formal validation stusy (3). Johnson & Johnson Pharmaceutical Research & Development have made the commitment to fund research in the further development and implementation of the ESC test. With regard to carcinogenicity testing, the pharmaceutical industry is interested in an in vitro test model that detects genotoxic as well as nongenotoxic carcinogens. Until now, the Syrian hamster embryo (SHE) cell transformation assay is the only available method (3). However, this test is not widely applied and, when used, has varying success. A more reliable and simpler test should be developed. As the end users of the NCEs are mainly humans, human cells should ideally be used for this carcinogenicity screening model. For some areas of toxicological assessment, more research is needed. No widely accepted in vitro systems are available for repeated-dose toxicity testing. The development of a reliable and relevant in vitro method, preferably using human cell lines, would be very useful for the pharmaceutical industry, to screen late-selected candidates for their toxicological properties.
Preclinical Toxicology Testing The role of the preclinical scientist in the drugdevelopment area is to perform toxicological studies to support regulatory submissions. Reduction in animal use for regulatory studies has been achieved through harmonisation of the study protocols and the test battery by the International Conference on Harmonisation (ICH) process (5). However, additional work is needed. By introducing new techniques, such as flow cytometric enumeration of micronucleated erythrocytes, an additional saving in animal use can be obtained. The first step of a validation study, in which 14 lab-
P. Vanparys
oratories participated, showed that the transferability of this flow cytometric method is high when the instrumentation is guided by a biological standard (6). Following the recommendations of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM; 7) and the International Workshop on Genotoxicity Test Procedures (IWGTP; 8), a second step in the validation process is in progress. By using this technique, a negativecontrol group can be omitted, as each animal can serve as its own control and the same animal can be followed over time for micronucleus induction. For more than 25 years, positive-control animals were needed in in vivo micronucleus tests to satisfy the regulatory guidelines. A survey taken over 10 years revealed that the positive-control animals always showed a uniform response. At the IWGTP, held in Washington in 1999, I proposed an adapted test design which would make possible the omission of a positive-control group. With the inclusion of proof of target exposure in in vivo genotoxicity testing (9), the need for positive-control animals is less necessary. It is sufficient to include a few positivecontrol slides obtained in a previous study, in order to have a control on the reliability of the readings (10). Although there was willingness at the IWGTP to reconsider the use of a positive control-group in micronucleus testing, we still need to include a positive-control group. Against this background, ECVAM or the European Consensus Platform on Alternatives (ecopa) could coordinate an independent peer review on the need for positive controls in in vivo studies. The omission of a positive-control group would substantially reduce the number of animals required. At Johnson & Johnson Pharmaceutical Research & Development, we do not perform Draize eye irritation tests unless requested to do so by regulatory authorities. Compounds are tested for eye irritation in the bovine corneal opacity–permeability (BCOP) test and/or the hen’s egg test–chorioallantoic membrane (HET-CAM). The BCOP test has been improved by developing a new opacitometer (Van Goethem, F., Hansen, E., Sysmans, M. & Vanparys, P. Development of a new opacitometer for the bovine corneal opacity and permeability [BCOP] assay. Abstract from the 4th World Congress on Alternatives and Animal Use, New Orleans, 2002), which works with laser light. Because of the problem of bovine spongiform encephalopathy (BSE), we were faced with the fact that bovine eyes were temporarily unavailable. As a consequence, we looked for another alternative method. An in vitro human corneal epithelium model was established. A prevalidation (partially funded by the Belgian Platform for Alternative Test Methods) was set up by Johnson & Johnson Pharmaceutical Research & Development (Lead Laboratory), with Pfizer, Novartis and SkinEthic (Van Goethem, F., Hansen, E., De Wever, B., Rosdy, M., Straube, F., Catoire, S.,
ECVAM and pharmaceuticals
Romeike, A., Alépée, N. & Vanparys, P. Evaluation of a new in vitro human cornea model as an alternative to the in vivo rabbit eye Draize test. Abstract 4th World Congress on Alternatives and Animal Use, New Orleans, 2002). During the prevalidation study, protocol refinements were made. The report on this prevalidation study will be submitted to ECVAM, for consideration as to whether a formal validation study is appropriate. The pharmaceutical industry is also active in the domain of refinement. Endoscopy, laparoscopy and ultrasonography are techniques used in veterinary surgeries, which are now also applied in toxicology testing. As these tools are non-invasive, and do not require surgery, they are of great value in the refinement of animal testing. Furthermore, the three techniques allow subsequent follow-up in the same animal. Consequently these tools also result in a reduction in non-rodent use.
Conclusion The pharmaceutical industry is still willing to fund research on in vitro alternatives, if the industry can be convinced that the in vitro tests have an added value in the selection of drug candidates, and if they give high-quality data that allow the companies to make good and reliable risk assessments. Although only a few validated and regulatory-accepted alternative methods are available at present, the pharmaceutical industry has been able to make significant progress in reducing and refining animal testing. However, it is quite clear that, to gain more-pronounced progress in the replacement of animal tests, we must continue working effectively with other industries, academia and governmental organisations. Progress in biological research, drug screening and drug development will surely increase the number of in vitro alternative test models entering the validation process.
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