Preclinical studies in animals10

In general, the following tests are undertaken:

Pharmacodynamics: to explore actions relevant to the proposed therapeutic use, and other effects at a range of doses

Pharmacokinetics: to discover how the drug is distributed in and disposed of by, the body.

Toxicology: to see whether and how the drug causes injury (in vitro tests and intact animals) in:

— single-dose studies (acute toxicity)

— repeated-dose studies (subacute, intermediate, and chronic or long-term toxicology) General toxicology studies are performed in two species, usually a rodent and dog. Regulatory requirements differ around the world but significant alignment has been made. Single and repeat dose study requirements are given in Tables 3.1 and 3.2. The dosing regimens are selected to produce a range of plasma concentrations, the highest of which will be several times greater than that achieved in man.

Special toxicology involves areas in which a particularly horrible drug accident might occur on a substantial scale; all involve interaction with genetic material or its expression in cell division.

Mutagenicity (genotoxicity) tests are designed to identify compounds that may induce genetic damage. A standard battery of tests is conducted and include:

• A test for gene mutation in bacteria, e.g. Ames test

10 Mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey (not all used for any one drug).

TABLE 3J Single and repeated dose toxicity requirements co support studies in healthy normal volunteers (Phase 1) and in patients (Phase 2} in the European Union (EUj.and Phases 1. 2 & 3 the U5A and Japan1

Duration of clinical trial

Minimum duration of repeated dose toxicity studies

Rodents Non-rodents

Single dose Up to 2 weeks Up to 1 month Up to 3 months Up to 6 months >6 months

2 weeks2 2 weeks

2 weeks 2 weeks 1 month 1 month

3 months 3 months 6 months 6 months 6 months chronic'

1 In Japan, if there are no Phase 2 clinical trials of equivalent duration to the planned Phase 3 trials, conduct of longer duration toxicity studies is recommended as given in Table 3.2. 2ln the USA, specially designed single dose studies with extended examinations can support single dose clinical studies. 3Regulatory authorities may request a 12-month study or accept a 6-month study, determined on a case-by-case basis. See p. 56 for a description of a clinical trial.

1 In Japan, if there are no Phase 2 clinical trials of equivalent duration to the planned Phase 3 trials, conduct of longer duration toxicity studies is recommended as given in Table 3.2. 2ln the USA, specially designed single dose studies with extended examinations can support single dose clinical studies. 3Regulatory authorities may request a 12-month study or accept a 6-month study, determined on a case-by-case basis. See p. 56 for a description of a clinical trial.

TABLE 3*2 Repeated dose toxicity requirements to support Phase 3 studies in the EU,and marketing in all regions'

Duration of clinical trial

Minimum duration of repeated dose toxicity studies

Rodents Non-rodents

Up to 2 weeks Up to 1 month Up to 3 months >3 months

1 month I month 3 months 3 months 6 months 3 months 6 months Chronic

'When a chronic non-rodent study is recommended if clinical use > I month.

'Regulatory authorities may request a 12-month study or accept a 6-month study, determined on a case-by-case basis.

'When a chronic non-rodent study is recommended if clinical use > I month.

'Regulatory authorities may request a 12-month study or accept a 6-month study, determined on a case-by-case basis.

• An in-vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in-vitro mouse lymphoma thymidine kinase (tK) assay

• An in-vivo test for chromosomal damage using rodent haematopoietic cells.

Usually the first two tests are performed before human exposure, but all must be complete prior to Phase II studies. Additional tests may be required.

Definitive carcinogenicity (oncogenicity) tests are often not required prior to the early studies in man unless there is serious reason to be suspicious of the drug, e.g. if the mutagenicity test is unsatisfactory; the molecular structure, including likely metabolites in man, gives rise to suspicion; or the histopathology in repeated-dose animal studies raises suspicions.

Full scale (most of the animal's life) carcinogenicity tests will generally be required only if the drug is to be given to man for above one year, or it resembles a known human carcinogen, or it is mutagenic (in circumstances relevant to human use) or it has major organ-specific hormonal agonist action.

It may be asked why any novel compound should be given to man before full-scale formal carcinogenicity studies are completed. The answers are that animal tests are uncertain predictors,11 that such a requirement would make socially desirable drug development expensive to a seriously detrimental degree, or might even cause potentially valuable novel ventures to cease. For example, tests would have to be done on numerous compounds that are eventually abandoned for other reasons. This may seem right or wrong, but it is how things are at present.

Toxicology testing of biotechnology-derived pharmaceuticals. The standard regimen of toxicology studies is not appropriate for biotechnology-derived pharmaceuticals. The choice of species used will depend on the expression of the relevant receptor. If no suitable species exists, homologous proteins or transgenic animals expressing the human receptor may be studied. Additional immunological studies are also required, and the genotoxicity and carcinogenicity studies are modified.

Reproduction studies have to be extensive because of the diversity of physiological processes that may be affected, and because the consequences of error in this field are potentially horrific. Tests include

11 A sardonic comment on the relevance for man or carcinogenicity tests in animals was made by investigators who induced cancer in animals using American 'dimes' (10 cent coin) and the plastic of credit cards. They advised the US Government to consider banning money as unsafe for humans (Moore GE et al 1977 Journal of the American Medical Association 238: 397)

effects on fertility, reproductive performance, fetal organogenesis, and peri- and postnatal development. Studies are in mammals, usually the rat. Embryo-fetal development studies are conducted in a non-rodent, usually the rabbit. Later development studies include growth, behaviour and intellectual function of progeny, and their fertility (second generation effects).

Local tolerability studies. In most acute and repeat dose studies, the test drug is administered by the oral route. Additional studies are required when the clinical route of administration is parenteral. There are two objectives. First, to determine if the drug is absorbed in sufficient quantities, e.g. by inhalation, and second to test for local tolerability, e.g. by the percutaneous or intravenous routes.

It is plain that all the above tests constitute a major laboratory exercise requiring great and diverse scientific skills and significant financial resource.

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