Mutagenicity carcinogenicity and other tests

Mutagenicity tests aim to determine whether the proposed drug is capable of inducing DNA damage, either by inducing alterations in chromosomal structure or by promoting changes in nucleotide base sequence. Although mutagenicity tests are prudent and necessary in the case of chemical-based drugs, they are less so for most biopharmaceutical substances. In many cases, biopharmaceutical mutagenicity testing is likely to focus more so on any novel excipients added to the final product, rather than the biopharmaceutical itself. (Excipient refers to any substance other than the active ingredient that is present in the final drug formulation).

Mutagenicity tests are usually carried out in vitro and in vivo, often using both prokaryotic and eukaryotic organisms. A well-known example is the Ames test, which assesses the ability of a drug to induce mutation reversions in E. coli and Salmonella typhimurium.

Longer-term carcinogenicity tests are undertaken, particularly if (a) the product's likely therapeutic indication will necessitate its administration over prolonged periods (a few weeks or more) or (b) if there is any reason to suspect that the active ingredient or other constituents could be carcinogenic. These tests normally entail ongoing administration of the product to rodents at various dosage levels for periods of up to (or above) 2 years.

Some additional animal investigations are also undertaken during preclinical trials. These include immunotoxicity and local toxicity tests. Again, for many biopharmaceuticals, immunoto-xicity tests (i.e. the product's ability to induce an allergic or hypersensitive response, or even a clinically relevant antibody response) are often impractical. The regulatory guidelines suggest that further studies should be carried out if a biotechnology drug is found capable of inducing an immune response. However, many of the most prominent biopharmaceuticals (e.g. cytokines) actually function to modulate immunological activities in the first place.

Prediction and preclinical assessment of the immunogenic potential of any biopharmaceutical in humans is by no means straightforward. The use of animal models is inappropriate, as the human protein will be automatically seen as foreign by their immune system, almost certainly stimulating an immune response. Some factors, such as the extent and nature of post-translational modifications, the mode and frequency of administration and whether the protein sequence is of human origin or not (Box 4.1), provide pointers but are by no means accurate predictors. One potential predictive approach entails the development and use of transgenic animals that are made immuno-tolerant for the human protein under development. Such animals can then provide some basis for the study of breaking immunological tolerance.

Many drugs, including many biopharmaceuticals, are administered to localized areas within the body by, for example, s.c. or i.m. injection. Local toxicity tests appraise whether there is any associated toxicity at/surrounding the site of injection. Predictably, these are generally carried out by s.c. or i.m. injection of product to test animals, followed by observation of the site of injection. The exact cause of any adverse response noted (i.e. active ingredient or excipient) is usually determined by their separate subsequent administration.

Preclinical pharmacological and toxicological assessment entails the use of thousands of animals. This is both costly and, in many cases, politically contentious. Attempts have been made to develop alternatives to using animals for toxicity tests, and these have mainly centred around animal cell culture systems. A whole range of animal and human cell types may be cultured, at least transiently, in vitro. Large-scale and fairly rapid screening can be undertaken by, for example, microculture of the target animal cells in microtitre plates, followed by addition of the drug and an indicator molecule.

The indicator molecule serves to assess the state of health of the cultured cells. The dye neutral red is often used (healthy cells assimilate the dye, dead cells do not). The major drawback to such systems is that they do not reflect the complexities of living animals and, hence, may not accurately reflect likely results of whole-body toxicity studies. Regulatory authorities are (rightly) slow to allow replacement of animal-based test protocols until the replacement system is proven to be reliable and is fully validated.

The exact range of preclinical tests that regulatory authorities suggest be undertaken for bi-opharmaceutical substances remains flexible. (Generally, only a subgroup of the standard tests for chemical-based drugs is appropriate. Biopharmaceuticals pose several particular difficulties, especially in relation to preclinical toxicological assessment. These difficulties stem from several factors (some of which have already been mentioned). These include:

• the species specificity exhibited by some biopharmaceuticals, e.g. GH and several cytokines, means that the biological activity they induce in man is not mirrored in test animals;

• for biopharmaceuticals, greater batch-to-batch variability exists compared with equivalent chemical-based products;

• induction of an immunological response is likely during long-term toxicological studies;

• lack of appropriate analytical methodologies in some cases.

In addition, tests for mutagenicity and carcinogenicity are not likely required for most biopharma-ceutical substances. The regulatory guidelines and industrial practices relating to biopharmaceuti-cal preclinical trials thus remain in an evolutionary mode, and each product is taken on a case-by-case basis. An overview of the main preclinical tests undertaken for a sample biopharmaceutical (Myozyme) is provided in Box 4.2.

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