Screening for genotoxicity

Every new chemical entity (NCE) has to pass through genotoxicology tests in order to get regulatory approval. A 'negative' on this crucial test takes the sample drug one step closer the market. Sachin Jagdale analyses

Drug discovery has remained one of the most challenging aspects for pharmaceutical companies. It is the process of taking new chemical leads, the output of which is the emergence of new chemical entities (NCEs). Though NCEs are the precursors for any drug discovery, their actions on biological targets and subsequent genetic changes still baffle researchers and the medical fraternity.

Few decades ago, 'chemical mutagenicity' was central to drug discovery and the term 'genotoxico-logy' was struggling to find a place in medical textbooks. However, intervening period saw chemical mutagenicity getting replaced by genotoxicology. Today, there are many organisations and journals that are completely dedicated to genotoxi-cology. Indeed, genotoxicology has left a strong impression on many organisations concerned with detrimental health effects of toxic chemicals. It is a general belief that the field of genotoxicology was discovered by H J Muller and C Auerbach, who had narrated the first chemical and physical mutagens by using sub-mammalian species.

"From a risk management point of view, it is not advisable to proceed to development phase without any prior information on genetic toxicity" - Dr Vimal Sanghavi Head Clinical Services, Intas Biopharmaceuticals Limited (IBPL)

"Registration of pharmaceuticals requires a comprehensive assessment of their genotoxic potential. It is clear that no single test is capable of detecting all relevant genotoxic agents" - Dr Prabhakar Naik Director and CEO Intox Labs

Why genotoxicology?

"We have learnt from history that if a drug is not tested properly, it can have disastrous results as was seen in pregnant females receiving thalidomide for pregnancy induced nausea and vomiting. Hundreds of kids were born with phocomelia, and subsequently this drug was banned" - Dr Kumar Prabhash Oncologist Tata Memorial Hospital

The regulatory submission of any new drug demands a back up of series of tests, and genotoxicology tests are one of this set. Traditionally, genotoxicity tests have been used to detect possible ill effects of NCEs on genetic factors. Genotoxicology tests do not reveal any information on the safety, metabolism, toxicity and pharmacokinetics of the tested compounds. The importance of these tests has been cited by the medical fraternity as well. Dr Kumar Prabhash, Oncologist, Tata Memorial Hospital, opines, "Genotoxicology is an important part of drug development. We have learnt from history that if a drug is not tested properly, it can have disastrous results, as was seen in pregnant females receiving thalidomide for pregnancy induced nausea and vomiting. Hundreds of kids were born with phocomelia, and subsequently, this drug was banned." He adds, "There has been lots of data regarding side effects of drugs leading to various chromosomal changes in animals and human beings. It is important for all drugs to undergo genotoxicology testing before further development. This ensures that patients are not exposed to drugs which cause these side effects."

Besides regulatory testing, genotoxicity assays are used to assess safety of excipients, metabolites, degradants etc. Pharma companies are well aware of the necessity of the genotoxicology. According to Dr Vimal Sanghavi, Head Clinical Services, Intas Biopharmaceuticals Limited (IBPL), in recent years many companies involved in drug discovery have started screening for genotoxicity in early stage of discovery to reduce the chance of failure at the development stage.

Testing period

It is difficult to predict the boundaries of this fertile field, especially in light of new technologies that guarantee more precise and accurate details of effects of NCEs on genetic material and the manner in which that particular chemical functions.

A basket of supplemental assays have been developed to determine the genotoxicity of the drug. The most common tests normally include a bacterial mutagenesis assay (Ames test) that is used to detect frameshift and point mutations and an in vitro cytogenetics assay that is commonly used in cultured human lymphocytes. In vivo cytogenetics assay are also most often used to evaluate rodent bone marrow.

Miniaturised version of Ames and in vitro micronucleus assays (IVMN) have been given the vote of confidence by researchers for early screening of samples. The smaller version of the Ames assay, the 'mini-Ames', is done in the same way as the standard Ames, and hence, results obtained by these two assays are considered reliable. IVMN detects chromosome loss as well as breakage, though it does not generally evoke the same trust as the mini-Ames assay. It has been widely accepted as a test for early clastogenesis detection.

Since last few years, 'omics' technologies have given competition to the older genotoxicology tests. These technologies have shown great potential to detect quantitative and qualitative cellular changes in gene expression induced by chemicals. Researchers have been enthused with these new techniques, because the parameters followed by these technologies allow a better understanding of the processes at play. This helps to subsequently avoid unacceptable toxicity levels in NCEs. In this respect, omics technologies have a slight advantage over older genotoxicology tests.

"Registration of pharmaceuticals requires a comprehensive assessment of their genotoxic potential. It is clear that no single test is capable of detecting all relevant genotoxic agents. Therefore, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. Such tests are complemen-tary rather than representing different levels of hierarchy," says Dr Prabhakar Naik, Director and CEO, Intox Labs.

All is not well

As genotoxicology tests are required to follow Good Laboratory Practice (GLP) guidelines, the test generally calls for multigram quantities of compounds. For this reason, genotoxicology testing was historically pushed to later stages of drug development as such large sample quantities were not available at the early stages of development. This meant that pharma companies had to abandon an NCE at later rather than preliminary stages. This resulted in loss of time as well as financial resources, not to mention the loss of face in the industry.

Therefore, as Sanghavi says, "It is essential that screening of the drug candidate for genotoxicity is done early in the discovery phase. The newer in vitro techniques usually require only few milligrams of the sample drug. From a risk management point of view, it is not advisable to proceed to development phase without any prior information on genotoxicity."

The debate over early testing has another angle as well. Most often, researchers resist early testing for genotoxicity, as it could mean losing fruitful research areas in case of positive genotoxicology findings. Besides this there is a risk of false positive tests, which could arise from impurities present in the sample. Most samples get purified later in the development process. To avoid such dangers, proper use of screening tools is needed to add value to the drug discovery process.

Is the test trustworthy?

It is important to check the credibility of the test before deciding which tests have to be used. It is said that nothing is perfect and there is always scope for improvement. Is the same principle applicable to genotoxicology tests as well? If genotoxicology tests are not hundred percent reliable then it is an issue of patient safety and global concern. Doctors and pharma professionals have their own take on this crucial debate. Prabhash says, "Genetic long term clinical effects of genotoxicity need to be studied. It is also a possibility that clinical implications of genotoxicity observed in labs may not always correlate completely with that observed in clinics. Genotoxicology is very important in identifying drugs with genetic toxicity but a better methodology may be needed to identify the clinical implications of these effects observed in the lab."

Sanghavi puts his views. He says, "Data accumulated over past few years has shown that sensitivity and specificity of widely used genotoxicology tests is not high. The positive in vitro results require in vivo testing to eliminate false positives. Only one third of the positive results are real." IBPL is trying to make genotoxicology tests more reliable. In addition to employing screening for genetic toxicity early in research, the company is planning to develop practical biomarkers of genetic damage like measures of adduct formation, DNA strand break etc for incorporation in animal studies, which may be used in human studies as well.

Advances in genetics and genotoxicology have flourished almost at the same time. It is foreseeable that by accepting changing methodologies and technology, genotoxicology will revolutionise the process of discovery. However, the field of genotoxicology has not changed much, maybe due to strict regulatory requirements or GLP guidelines. It is a well accepted fact that genotoxicology has the potential to bring a noticeable change in the drug discovery process. Today, the role of genotoxicology maybe be confined not by its own limitations but the limited vision of genetic toxicologists.

sachin.jagdale@expressindia.com