LI Hui, HUANG Zhe(School of Business Administration, Shenyang Pharmaceutical University, Shenyang 110016, China)

Risks and Risk Control Measures of in vivo Genotoxicity Test in New Drug R&D

LI Hui, HUANG Zhe
(School of Business Administration, Shenyang Pharmaceutical University, Shenyang 110016, China)

Objective To provide risk identifying factors for false positive or false negative risk identification in vivo genotoxicity test of new drug research and development and to propose the relevant risk control measures. Methods Risk factors were identified through literature review and in combination with the “Drug Registration Management Measures” and toxicology studies guiding principles in ICH. Results and Conclusion Risks of in vivo genotoxicity test are analyzed in view of the risks of false positive result or false negative result. And the risk control measures are proposed according to the features of in vivo genotoxicity test. The false negative risk of in vivo genotoxicity test is drug pharmacokinetic risk, the false positive risk is the risk of experimental animal physiological conditions change. The failure risk of in vivo genotoxicity test can be reduced by conducting more in vivo tests and monitoring the biological index of the experimental animals frequently to remove the interferences.

genotoxicity; risk identify; risk control

1 Introduction

In new drug research and development the fate of a new drug depends on three key factors, i.e. validity, safety and the controllability of the quality. New drug research and development is a complex systematic project, and the genotoxicity test runs through the entire process as the key factor for safety evaluation of the drugs. And in nonclinical safety evaluation of the drugs, genotoxicity test is of significant importance, and it is closely related to the reproductive toxicity and carcinogenicity evaluation. New drug research and development is a high risky and costly program, and the failure means a big waste, so risk analysis and risk control of genotoxicity test are very important. The risk factors of in vivo genotoxicity test in new drug research and development are analyzed and risk control measures are proposed in this paper.

2 Risk identifying in vivo genotoxicity test

Usually in the in vivo genotoxicity test we can defect the genotoxicity of the drugs effectively and verify the result of the in vitro test. Some carcinogens with genotoxicity that can’t be defected in vitro test can be identified in vivo genotoxicity. But if there is something wrong with the in vivo genotoxicity test result, then it will be a major blow to the new drug research and development. So risk factors of in vivo genotoxicity test are analyzed in view of the possible false negative and false positive results.

2.1 Analysis of false negative risk for in vivo genotoxicity test

Rodent bone marrow or peripheral blood micronucleus test is the most widely used in vivo genotoxicity test, but not all the rodent carcinogens can be tested with positive results in these tests. Target cells must be exposed to test samples for enough time, so if genotoxicity is not found in test drugs with genotoxicity, the reason may be the pharmacokinetics behavior of the drugs[1]. The risk factors for in vivo genotoxicity test will be analyzed in details below.

2.1.1 The influence of pharmacokinetics of drugs on the test result

Pharmacokinetics is a subject that studies the absorption, diffusion, metabolism and excretion of drugs inbody. And the in vivo test result will be influenced by drug’s pharmacokinetic quality. The bone marrow hematopoietic cell is the target of in vivo test in standard test, but its biotransformation ability is low. The active reaction component generated in liver or other organs with short period couldn’t reach bone marrow hematopoietic cell. So the exposure time and level of genotoxic may be different because of pharmacokinetics of the drugs and the positve results may depend on the stability of the metabolites.

2.1.2 Species-sex-tissue and organ specificity of the drugs

Some chemicals cause significant genotoxic response only in one organ or cell type due to the imbalanced distribution of the test drugs in body or the different activation and detoxicification ability of the organs. Some tissue specific genotoxicity reaction is caused by the higher concentration and longer aggregation time of the genotoxic in specific tissue or better proportion of activate and detoxic. So the choice of the right tissue for detection is very important for the result and species specificity can also affect the test which is confirmed by Purchase[1]. Therefore, species-sex-tissue and organ specificity of the drugs should be taken into account when the human use condition is inferred from the animal test result.

2.1.3 The route of administration influence the biotransformation of test drugs

The administration route also has some effect on the test of genotoxicity. When the drugs need to be activated by gut flora, the test with intraperitoneal injection may be useless. However, those insoluble drugs with intraperitoneal injection will be effective for genotoxicity detection. The pH of the rodent stomach is different from that of human beings, then for the chemicals with amino or acylamino, rodents need to be injected with histamine to lower the pH to 1.5-2.0 so that the nitrotlon reaction can happen and generate high genotoxic chemicals N-nitroso derivatives. If histamine isn’t given to the test animals, the test results will be false negative. So the route of administration is of great importance. Choosing the right way may effectively avoid the failure of in vivo genotoxicity test.

2.2 Analysis of false positive risk for in vivo genotoxicity test

In the in vivo genotoxicity test rodent micronucleus test is used to detect the genotoxicity of candidate drugs. Micronucleus test evaluates the induction of chromosome damages on nucleated erythrocytes. The chromosome fragments that being produced in the process of cell division of erythrocytes or the chromosomes that don’t enter any daughter cell nucleus after erythrocytoschisis are still visible in the immature erythroplastid as micronucleus. Micronucleus formation mechanism is associated with induction of chromosome aberration. Therefore, rodent micronucleus test is applied to in vivo genotoxicity test to detect the genotoxicity of candidate drugs.

The positive result of mironucleus test is considered as the sign of long-term unhealthy effect of the drugs, including carcinogenic effect and genetic effect. But more and more data indicate that sometimes the positive result of in vivo genotoxicity may be unrelated to the genotoxicity of the test drugs. When the physiological environment of the rodents is disturbed, the amount of micronucleus may rise, but this rising micronucleus is not caused by genotoxicity. The possible risk factors for false positive result of in vivo genotoxicity test will be discussed below.

2.2.1 The amount of micronucleus rise caused by temperature change from the drugs

Drug acting on organisms can produce physiological effect through direct action on target cells, and at the same time drugs can also indirectly affect the organisms by influencing various physiological functions. Temperature works as an important indicator for organism physiological function, the change of temperature will inevitably cause the functional effect on biological systems[2]. But sometimes the temperature change caused by drugs, which in turn leads to the increase in the number of micronucleus unrelated to intrinsic genotoxicity of test samples, will have certain effect on the health evaluation of people.

Henning Hintzsche and Thorsten Riese studied the hyperthermia’s effect on animal cells, and the results indicated that for human cells or rodent cells the micronucleus of experimental group was significantly higher than that of control group and the hyperthermia can cause genotoxic damage[3,4]. Similarly, lowering the environment temperature can also cause micronucleus[5]. S.Asanami and some people studied the effect of hypothermia on micronucleus emerging from mice bone marrow cell. They found that temperature lower than 33°C for 40h would induce micronucleus, and the possible mechanism was the disturbing on mitotic apparatus. When some drugs such as E-5842[6,7], haloperidol and chlorpromazine[8]are given to experiment animals, the temperature of rectum declines by 10°C and the amount of micronucleus goes upsignificantly. These all show that hypothermia can increase the micronucleus and may disturb the micronucleus test result.

So when the in vivo genotoxicity test is positive, if there is the possibility that the test drug may cause the temperature change, the validity of the result should be judged rationally to avoid the waste of the subsequent experiment.

2.2.2 The amount of micronucleus rise caused by increasing erythrocyte from the drugs

Except temperature there are also many other factors may interfere the micronucleus producing false positive result. The study from Steinheider[9]shows that bleeding can lead to micronucleus rise of PCEs and NECs. Giving EPO to stimulate the erythrocyte form has been proved to increase the amount of micronucleus in rodent erythrocyte[10-12]. The increasing EPO and the subsequent erythrocyte rise are considered as the basic mechanism to induce micronucleus to bleed, anemia caused by chemicals and exposure to hypoxia environment. The increase of cell division and shortening DNA damage repair time, the spindle formation damage in rapid cell division and the errors in the process of denucleating or erythrocyte differentiation are all possible sources of increasing micronucleus formation. Therefore, if the test drug triggers micronucleus formation increase to generate false positive result, whether the drug causes the increased blood cells should be taken into account when the test result is positive.

3 The risk factors of in vivo genotoxicity test

The possible risk factors in in vivo genotoxicity can be gotten from the risk identify process above. The risk factors are summarized from the risk of false negative and false positive results of in vivo genotoxicity test.

3.1 The risk factors of false negative of in vivo genotoxicity test

The pharmacokinetic behaviors of the drugs may lead to false negative results in in vivo genotoxicity, and the risk factors can be summarized as three risk factors below:

(1) The pharmacokinetic quality of drugs can influence the test result;

(2) Species-sex-tissue and organ specificity of the drugs;

(3) The route of administration influence the biotransformation of test drugs.

Because of these factors genotoxic substance can’t reach the detected tissue or test drugs can’t convert into genotoxic materials effectively. So the test result is false negative. But these drugs are genotoxic substances actually, so these factors may lead to the failure of the test.

3.2 The risk factors of false positive of in vivo genotoxicity test

The physiological condition of experiment animals can be changed by drugs to lead to false positive result. The risk factors of false positive result in in vivo micronucleus test are analyzed as two aspects in details in this paper:

(1) The amount of micronucleus rise caused by temperature change from the drugs;

(2) The amount of micronucleus rise caused by erythrocyte increase from the drugs.

The test drugs lead to animal physiological conditions change, and the test index rises in the experiment. But this result has nothing to do with the genotoxicity, and may lead to the failure of the test.

4 The risk control measures to in vivo genotoxicity test

Risk control measures must be done to reduce the risk of the test failure to the risk factors identified above. The risk control measures are proposed respectively to the false negative risk and false positive risk.

4.1 Risk control measures to false negative result risk of in vivo test

The pharmacokinetic quality of drugs or the speciessex-tissue and organ specificity of the drugs can lead to false negative result. But these factors are difficult to detect in advance. So more in vivo test can be done with different tissues, organs, species or administration route. By comparing the consistency of different in vivo genotoxicity test, the genotoxicity of the candidate drugs can be judged comprehensively. UDS, TG and Comet assay are compared by David Kirkland[13], and the results showed that TG and Comet assay are better than UDS. So the recommended additional in vivo tests are TG and Comet assay.

4.2 Risk control measures to false positive result risk of in vivo test

Drugs can disturb the physiological condition of experiment animals and may lead to false positive result.But the hyperthermia and hypothermia or erythrocyte increase all can be detected by measuring. So in the process of test the physiological index and physiological changing should be paid more attention. At the same time other factors may lead to micronucleus increase such as bleeding should be excluded. When the drugs do have some influence on the physiological conditions, some other tests can be done to comprehensively analyze the size of the disturbance.

5 Conclusions

The factors that may lead to the failure of in vivo genotoxicity test are analyzed in this paper. The risk factors of false negative of in vivo genotoxicity test include: (1) The pharmacokinetic quality of drugs can influence the test result; (2) Species-sex-tissue and organ specificity of the drugs; (3) The route of administration influences the biotransformation of test drugs. The risk factors of false positive of in vivo genotoxicity test include: (1) The amount of micronucleus rise caused by temperature change from the drugs; (2) The amount of micronucleus rise caused by erythrocyte increase from the drugs. And the risk control measures to these risks are proposed, including adding more in vivo test, monitoring physiological index frequently to exclude the disturbances. The recommended additional in vivo tests are TG and Comet Assay. The risks of in vivo genotoxicity in new drug research and development are identified and risk control measures are proposed and appropriate adjustments and corrections are welcome.

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Project supported by: Public relation plan of social development of science and technology agency in Liaoning Province “platform of technical transfer service in biomedical technology industry” (2012225095) 2012.04-2014.12

Author’s information: HUANG Zhe, Associate professor. Major research areas: Knowledge management, e-commerce. Tel: 13998874590, E-mail: huangzhe2000@sina.com