Alternative testing in drug research and development—The validation issue

Toxic.

Pergamon

0887-2333(%)OOO!%I

in

Vim

Vol.

9.

No.

6, pp.

789-793.

1995

Copyright 0 1995 Elsevicr Scmce Ltd Printed in Great Britain. All rights reserved 0887-2333/95 59.50 + 0.00

Alternative Testing in Drug Research and Development-The Validation Issue* B. GARTHOFF Bayer AG Leverkusen, PF-Centre, D-51368 Lcverkusen, Germany ECVAM (the European Centre for the Validation of Alternative Methods) intensifies its work, it is worth discussing the aspects of alternative testing in drug research and development as well as the implication of validating tests-and the possible role of ECVAM in this. Substituting animal in ho tests with alternative testing has always been a major target in the pharmaceutical industry, for ethical and practical reasons. In vim tests have an important role. especially in the first phase of drug discovery (the substance-finding phaSe) but lo a lesser extent in safety testing. In the further development of a new drug, validation becomes more important for safety tests than for the initial screening and substance-linding tests. That also implies that diverse safety and toxicity testing of pharmaceuticals should be based on worldwide accepted and validated protocols. ECVAM has a role here, in informing the scientific and regulatory community about promising (validated) tests in drug discovery or development and pressing for worldwide harmonization, especially of safety testing. At&act-As

Introduction

As ECVAM intensifies its work, it is worth discussing alternative

testing in drug research

and development,

as well as the implication of validating alternative tests and the possible role of ECVAM in this. Substituting in oivo tests with alternative tests has always been a major target for the pharmaceutical industry, for ethical and practical reasons. Even though there has already been a considerable reduction in animal use in the past decade (Bundestagsdrucksache, 1993), of late even fewer animals have been used, especially in the development and safety testing of drugs. Taking the example of the Federal Republic of Germany, the gradual decrease in the use of animals over the years might serve as an indicator of the general reduction; in Germany, the decrease has been maintained in 1993, according to the recent official information of the BMELF (Federal Ministry of Alimentation and Agriculture; BMELFInformationen, 1994), in which an additional lowering of 7.6% was noted (see Fig. 1). This was again particularly marked in the category of drug development and safety testing: a total of 1016 x lo3 animals used in 1993 versus 1171 x IO’ in 1992-a further reduction of 13.2%. Although during the substancediscovery phase the issue of validation of alternatives is less important, it has always been an issue of major concern in the development phase including toxicology and safety testing of new drugs. As has previously been pointed out, the root of the word ‘validation’ is the Latin validus meaning ‘power*Presented on the occasion of the opening of the European Centre for the Validation of Alternatives Methods (ECVAM), 17 October 1994 in Ispra, Italy, at the Joint Research Centre of the European Union.

ful’ (Flint, 1991). Alternative tests in the discovery phase or development phase have been developed and were (or are being) adapted by industry, whenever recognized by the scientific community or regulatory agencies as ‘powerful’ enough to support evidence. A definition of validation according to Webster’s Dictionary (Gove, I98 I ) is ‘. . . the process of determining the degree of validity of a measuring device or method’, whereas the CAAT/ERGATT Workshop defined it as ‘the process by which the validity and relevance of a test are established’ (Balls et al., 1990). For discussion of the current and future potential in alternative testing during the phases of drug research and development, the dictionary’s broader definition of ‘valid’ alternatives is used here because, at least for the discovery phase, it does not necessarily require an official worldwide multicentre study for an assay to be accepted and introduced. Phases in drug research and development To follow the issue of in tlico testing in drug research and development, it is essential to identify the phases of creating a new therapeutic. When the pharmaceutical development time of more than 8-l 2 years for a therapeutic is reviewed, the major time (and cost) factor lies in the clinical evaluation, that is, in clinical phases I-III and the period needed for registration (see Fig. 2). Figure 2 also indicates in which phase of discovery and development in vice testing has to be used in order to comply with the legal requirements of the Declaration of Helsinki, requesting basic information for the clinician, before testing in healthy volunteers can take place. The size of the in oiuo bars in the Figure also indicates approximately how many 789

B. Garthoff

790

Total number in Mio

forum for the diverse groups and help to accelerate the process. Discovery phase

1993 1992 1990 1991 1989 Fig. 1. Reduction of laboratory animals used in the Federal Republic of Germany [Bundestagsdrucksache, 1993, and data of BMELF (Federal Ministry of Alimentation and Agriculture)].

animals are used at the different stages; formerly, the first bar would have been much larger. Owing to the introduction of new methodology and techniques, in vivo testing has been markedly reduced in the first part of the preclinical phase, which involves identification of a novel mechanism and a new substance class, followed by optimization to identify drug candidate(s). In the second part of the preclinical phase, in which safety testing or toxicological trials and studies on kinetics or metabolic fate predominate, in uiuo testing is in most cases required by law. Alternatives-if existing and validated-sometimes take too long to be established; this seems strange, as all parties involved should be interested in establishing valid alternatives as soon as possible-industry, because of ethical and practical considerations (financial as well, as in vivo experiments are the most expensive choice), regulatory bodies, because of public opinion and pressure, and animal welfare groups, because of their obvious concern. In this context, ECVAM can represent the

costs Mio DM A

350 -

Preclinical

Phase

During the discovery phase, in vitro tests are the rule, and in vivo tests the exception, in the meantime, despite the fact that some of the early in vitro screens are not ‘officially validated’. On the other hand, validation cannot be expected, as innovative tests leading to the detection of a novel mechanism and new substance classes become validated in the followup of this new compound by virtue of its activity as a new therapeutic. In addition, they comprise part of the proprietary position of a company, leading to a patent and then being published as a reproducible manual and system, so that it can be easily investigated by everybody. Meaningful and powerful techniques, assay systems or screens are rapidly adopted-and therefore become ‘validated’. This is also one of the reasons for the incredibly high percentage reduction in laboratory animal numbers that is still achieved. The introduction of radioimmunoassays, robot screens, transcription factor testing and biotechnology/gene-technology techniques have contributed to this reduction. The increased number of in vitro tests for substance screening during the past few years is depicted in Fig. 3, giving as examples the figures for single in vitro tests in the cardiovascular research institute of a pharmaceutical company (Garthoff, 1993). The basis for this development is simply the new technology that has become available or that has been developed in the pharmaceutical industry. A hypothetical example is the target organ, the liver, and some of its functions (Fig. 4). The sequence from the whole animal to isolated liver enzymes in a test-tube seems obvious-and even trying to substitute rat hepatocytes in culture with human hepatocytes is purely logical. However, prerequisites such as

m

in viva

m

in vitro

Clinics

15 %

300 250 8,402

458 1987

Fig. 2. Phases in creation of a new therapeutic: in viuo testing (I), time and approximate distribution of costs.

Fig. 3. Reduction of number animals (m) used in drug research and increased number of in vitro tests (I) for substance screening in a pharmaceutical industry cardiovascular research institute.

Alternative testing in drug R&D In vitro tests in drug finding and development (example)

m

Isolated

perfused

Isolated

hepatocytes

Hepatocyte microsomeslmembranes

Cultured hepatocytes

4

kd

Isolated enzyme

::.’

Fig. 4. Reduction of number of animal tests by progress in basic research and methods development: possibilities of in vitro tests in drug finding and drug development, using the

liver as an example.

culture conditions, nutrients and organ bath solutions had to be developed first and the limits of the information gained have to be taken into account. In distributing information on the basic methodology of such tests (standard operation procedures), ECVAM will be of use. Although the number of laboratory animals used is smaller and that of substances tested is higher in a test system in vitro, the usefulness of a new therapeutic cannot be deducted from a single isolated enzyme test-tube assay. That still requires in viuo conflrmation, and thereby extension of information base, in order to supply the clinician with more valid details than merely the information that ‘it works on the enzyme or the isolated cell’ before giving it to humans for the first time. A few examples from cardiovascular research, may help to demonstrate the complexity of in vitro versus in vivo testing. A standard still used in pharmacology is the isolated perfused Langendorff heart preparation, in which several substances can be tested, for example for inotropy, with the use of only one rabbit. ‘Validation’ is inbuilt, because reference drugs such as ouabain and digitalis have been in use for a century. Even better, one might switch to human atria1 tissue, obtained when hearts are connected to a heart-lung machine (Hebisch et al., 1994). However, availability is limited, and ethical concerns make ‘validation’ difficult. Nevertheless, human tissue is being used whenever possible and feasible. The further development of ion channel recording techniques in cellsthe patch clamp method, developed by the Nobel prize-winning team led by Neher and Sakmann (Hamill et al., 1981)-have simplified mechanism studies, but validation of single channel recordings and screening of hundreds of substances in this system is not feasible. In addition, as heart muscle

791

cells are differentiating in culture it cannot be determined which is the optimum phase of cell growth for test assaying and for reflecting the in vivo wholeorganism situation. Furthermore, it must be remembered that only one parameter is being analysed-ion flux leading to muscle cell contraction. New technology, such as the use of dyes as indicators for ion movements in cell systems cultured in a 96-well plate, facilitates automatic large-scale screening systems that can be validated and optimized. In contrast to the past, where isolated organs dominated testing in the first discovery phase, almost all the channels, enzymes, receptors, etc. produced by the help of gene technology methods, can now be identified and used in new test systems in pharmaceutical high-throughput screening. Specifically, cellular assay systems have been developed that make use of coupling to a luciferase reporter gene and thereby ensure fully automated robot screens for an enormous amount of test compounds. The future for additional early in vitro testing lies with such technology as testing for influencing transcription factors-‘a new frontier in pharmaceutical development’ (Peterson and Tupy, 1994). This simplifies the initial elimination of inactive substance classes (with the limitation of ‘losing’ substances that have to be metabolized to become active). Similarly, as soon as proteins, factors and receptors are structurally identified the possibility exists of bringing molecular modelling into play and the introduction of targeted synthesis of chemical compounds. However, molecular modelling has been of greatest use in the optimization phase, that is, once an active molecule has been identified. Once again, ECVAM may be beneficial in making new technology known to the community. The significance (and validity) of the abovementioned tests has to be verified in in vivo assaysand even there, new technology has also helped to limit the number of animals, for example by use of transgenic animals (Garthoff, 1994). Although it seems to be a contradiction in itself to create transgenie laboratory animal strains to reduce the total number of experimental animals used, reductions consistent with the three ‘R’s of Russell and Birch can be achieved by this route. Transgenic animal models reduce the number of animals necessary for in vivo assays in the preclinical phase, because (a) they offer better transferability (for example, if they incorporated the respective human ‘sick gene’), (b) they allow for a more targeted approach, and (c) they replace experiments that can otherwise only be done in higher species, specifically primates (an example of this being testing for the neurovirulence of poliovirus type 3 vaccines; Dragunsky et al., 1993). Less obvious, perhaps, is the potential use of transgenic animals as uniform and renewable sources of immortalized cell lines for primary screening programmes. Mice that carry a transforming oncogene (such as the large T-antigen gene of SV 40) can be

B. Garthoff

192 Table I. Key differences in regional

recommendations for toxicity studies 10 support develooment’

various

stages of clinical

Region Japan

USA

Initial Phase I studies

14-day repeat-dose toxicity, genetic toxicology

I

month repeat-dose, genetlc toxicology. male fertility studies

14-day repeat-dose toxicity

Greater than 30 days clinical study

6 month toxicity

h month toncity

Tox = clinical duration for Phase II or To? = 3 x clinical duration for Phase III

Inclusion of women in clinical investigation

Segment II (embryoifoetal toxicity)

Total reproductwe toxicology package

Segment II and female fertility

Clinical

*Griffith

stage

(1994); Parkinson

Europe

er al. (1994).

used to generate cell lines that are, for example, expressing the synthesis of a particular cell surface receptor target for a new therapeutic. It is here that ECVAM has a potential role, although there are concerns that ‘. this rapidly developing field affords great scientific opportunities. but also threatens to greatly increase laboratory animal suffering’ (Balls, 1994), the potential offered by transgenic technology should not be underestimated. Besides the obviously improved-and therefore ‘refined’--in t!it~ models (see Field, 1993; Garthoff, 1994; Lathe and Mullins, 1993; Liggitt et al., 1992) this technique also provides embryonic stem cells and immortalized transgenic cell lines for in vitro screening, as discussed above. ECVAM can help to introduce the methodology and transfer it to laboratories performing validation studies, when needed.

Development phase In development, where most of the safety and toxicity testing is done, the highest level of standardization is required, and classically this has required extensive use of animal models. It should be remembered that toxicity testing starts early in the preclinical phase of drug development. Nevertheless, the industry has pursued new approaches to acute toxicity testing, substituting the classical LDSo test with the approximate median lethal dose (e.g. Lorke, 1983). The result is that many laboratory animals have been spared (about one-third). However, the introduction process took far too long. For the future, there is room for improvement, also under the aspects of worldwide harmonization. Before the first clinical study in humans (which is the fundamental reason for the necessity for toxicology studies), complete toxicity and safety testing has to be presented. Table 1 lists the diverse recommendations in different regions (Parkinson et al., 1994). Depending on the studies envisaged, different nations require widely differing toxicity studies. Even worse, judging by a survey recently performed by the UK Centre for Medicines Research (CMR)

(Parkinson et al.. 1994), the pharmaceutical companies questioned in different continents gave quite different views of what, according to the Helsinki Declaration. is deemed appropriate preclinical testing before first administration to humans. If women with child-bearing potential were to be included, there was even the conviction in Japan that a complete carcinogenicity study had to be available. This would mean that numerous laboratory animals would be used in safety testing even before the start of clinical testingwith the risk that the compound would subsequently fail for other reasons. ECVAM, with the support of industry, could help to harmonize opinion worldwide on the prerequisites for clinical trials. In the carcinogenicity testing area, where two species are required at present (mice and rats), there seem to be opportunities to limit the total usage, for example by using one sex/species only. As exemplified in Fig. 5, it might be sufficient to test a potential new therapeutic on male rats and female mice and be almost as safe as with both sexes in both species. In a recent study by Griffith (1994), the screening of 49 compounds

cf

mouse and 9

rat-

33

I

I

I

I

I

t

0

10

20

30

40

50

Number of Compounds

1 60

(n=49)

Fig. 5. Compounds flagged as rodent carcinogens used in preliminary analysis for carcinogenicity testing. When studied in both the rat and the mouse, 46 of the 49 compounds studied (i.e. 94%) would have been assumed to be rodent carcinogens, if only the male rat and female mouse had been used for the study (from Griffith, 1994).

Alternative testing in drug R&D (assumed to be rodent carcinogens) demonstrated that at least 46 of them could be identified in a ‘two cell system’ (i.e. male rat/female mouse). The necessity for a second rodent species, also questioned by the US FDA, should be further addressed, especially, as the relevance of mouse to man with regard to carcinogenicity is questionable in view of the fact that liver and lung tumours are more prevalent in mice. This is where ECVAM might have an informative role.

193

BMELF-Informationen (1994) Zahl der Versuchstiere ging 1993 weiter zuriick. BMELF-Informationen, No. 44, (31 ocq. Bundestagsdrucksache (1993) Tierschutxbericht 1993, Bericht iiber den Stand der Entwicklung des Tierschutxes. Bundestagsdrucksache 12, 4242 (1 Feb). Dragunsky-E., Gardner D., Taffs R. and Levenbook I. (1993) Transgenic PVR Tg- 1 mice for testing of poliovirus Type 3 neurovirulence: comparison with monkey test. Biologicals

21, 233-237.

Field L. J. (1993) Transgenic mice in cardiovascular research. Annual Review of Physiology 55, 97-114. Flint 0. P. (1991) In vitro test validation. ATLA 19, 140-142.

Conclusions In summary, alternative testing by ‘validated’ assays has always been, and will be in future, an issue of major concern to the pharmaceutical industry. To ensure a rapid and optimal introduction process, the scientific and regulatory community has to be informed of promising ‘validated’ tests. Worldwide harmonization is an absolute prerequisite for registration requirements-and the most important issue for pharmaceutical companies that are operating on a global basis. The role of ECVAM, therefore, consists of (a) providing information on new validated tests and checked screens, and (b) pressing for worldwide harmonization. in vitro

author wishes to thank Professor Peter Rae, Miles Inc., West Haven, Connecticut, USA, Acknowledgements-The

for information opportunities.

regarding

transgenic

methodology

and

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