There may be differences in the metabolic characteristics of small molecule drugs in experimental animal species and humans, and there may be unique or high-level metabolites in humans. If these metabolites are not identified or fully evaluated in non-clinical safety trials, the safety of human medications may not be fully guaranteed. Depending on the specific circumstances, non-clinical safety studies of these metabolites may be required. This article elaborates the requirements of the International Coordination Committee for the Registration of Technical Requirements for Drugs for Human Use and the relevant guiding principles of regulatory agencies, combined with case studies, to discuss the key points of non-clinical research on metabolites in the development of small molecule drugs.
During the development of new drugs, non-clinical safety trials need to be conducted to indicate the potential risks of clinical drug use and provide information for risk management and control of clinical trials. In non-clinical safety trials, it is important that the original drug and its metabolites achieve sufficient exposure in animals. Due to the differences between species, there may be qualitative and quantitative differences in the metabolic characteristics of small molecule drugs in different animal species and humans. After the development of small molecule drugs enters the clinical trial stage, human-specific or high-level metabolites may be found in human pharmacokinetics (PK) studies. If these metabolites are not found in non-clinical safety animal trials. Being identified or fully evaluated may lead to insufficient protection of the safety of human medication.
Therefore, in the non-clinical safety evaluation of small molecule drugs, it is not only necessary to pay attention to whether the exposure of the original drug is sufficient, but also whether the exposure of the metabolites is sufficient. According to the specific situation, additional non-clinical safety trials should be carried out when necessary. To evaluate the safety of metabolites. This article discusses the relevant requirements of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) and the relevant guidelines of regulatory agencies, combined with case analysis, to discuss the metabolism of small molecule drug development The focus of product non-clinical research.
Requirements of drug regulatory agencies for non-clinical studies of metabolites
The M3(R2) guidelines issued by ICH in June 2009, “Guidelines for Non-clinical Safety Research to Support Drugs for Clinical Trials and Marketing” and the M3(2) Guidelines for Questions and Answers (R2) on Metabolism, were issued in March 2012. The necessity of non-clinical safety evaluation of the product and the test items are described. The US Food and Drug Administration (FDA) first issued the “Guidelines for the Safety Test of Drug Metabolites” in February 2008. Based on this, my country issued the “Safety of Drug Metabolites” in 2012. Test Technology Guiding Principles, which are translations based on the FDA’s 2008 Guiding Principles. Based on the continuous recognition of the safety research of metabolites, the FDA issued the second edition of the “Guidelines for the Safety Testing of Drug Metabolites” in March 2020. The guidelines are based on compliance with the ICH M3(R2) guidelines. Recommendations are provided on when and how to identify and characterize drug metabolites that need to be assessed for non-clinical toxicity.
1.1 ICH M3(R2) guidelines and Q&A requirements for non-clinical studies of metabolites
ICH M3(R2)  Recommendations: Before starting human clinical trials, in general, animal and human in vitro metabolism, plasma protein binding rate, and systemic exposure data of animal species used in repeated dosing toxicity tests should be evaluated. Further animal PK information (such as absorption, distribution, metabolism, and excretion), as well as in vitro biochemical information related to potential drug interactions, should be obtained before large-scale or long-term clinical trials (usually before phase III). These data can be used to compare the differences between human and animal metabolites and determine whether additional tests are needed.
ICH M3(R2) proposes: Only when the exposure of human metabolites exceeds 10% of the total drug-related exposure, and the exposure of metabolites in humans is significantly higher than the maximum exposure in the toxicology test, it is necessary to Non-clinical evaluation of metabolites is carried out to support phase III clinical trials. For drugs with a daily dose of less than 10 mg, when the proportion of drug metabolites is greater, it may be more appropriate to test the metabolites. Some metabolites (such as most glutathione conjugates) have no toxicity concerns and do not need to be tested. For metabolites that do have reasons for safety concerns (such as human-specific metabolites), non-clinical characterization should be considered based on the principle of specific analysis.
ICH M3(R2) Questions and Answers  refined the requirements for metabolites in the form of question and answer. In the text of ICH M3(R2), “significantly higher than” in the “human body exposure level is significantly higher than the maximum exposure level in the toxicology test, the metabolites need to be non-clinically evaluated” is interpreted as: In the kinetic evaluation, the difference of ≥2 times (average) area under the curve (AUC) is usually regarded as having toxicological significance. Therefore, when the exposure in animals is more than 50% of human exposure, it is generally considered that the toxicity characterization of metabolites is sufficient. However, when a metabolite accounts for most of the total human exposure, the exposure of this metabolite in animals should exceed that of humans.
Regarding “when the toxicity of metabolites needs to be evaluated, it is important in which type of in vivo non-clinical tests metabolites achieve sufficient systemic exposure”, which is explained as: in general toxicology tests, carcinogenicity tests (or no need to perform In carcinogenicity evaluation, in vivo micronucleus test) and embryo-fetal developmental toxicity test, the metabolite in each animal species needs to be fully exposed.
Regarding “when the human metabolite exposure is compared with the maximum exposure of the metabolite in the toxicity test, should the maximum exposure reached in the animal test be used, or in some cases the unseen adverse reaction dose shall be used? (No observed adverse effect level, NOAEL), no observed effect level (NOEL) or maximum tolerated dose (MTD) exposure is more appropriate”, explained: because of the original drug and metabolism The product contributes to the target organ toxicity observed in animals under MTD. Assuming that toxicity concerns can be fully monitored in humans and will not cause unacceptable risks, the animal’s exposure at the MTD level should be compared with the human’s therapeutic dose The maximum exposure level is compared between species. If the toxicity at the time of MTD cannot be monitored in the human body, or an unacceptable risk is caused, then the exposure comparison should be carried out on the NOAEL where the toxicity is concerned.
Regarding “when should the non-clinical data of metabolites be obtained during the development period”, the requirement in the main body of ICH M3(R2) is again clarified: animal and human in vitro metabolism data should be evaluated before the start of human clinical trials. Before a large sample of subjects or long-term exposure (usually before Phase III), the in vivo metabolism data of the test species and humans should be obtained.
Requirements for genotoxicity and safety pharmacology tests: For the genotoxicity of metabolites, the ICH M3(R2) question and answer clearly “this question is beyond the scope of ICH M3(R2)”, so ICH M3(R2) and ICH M3(R2) ) Q&A did not clarify the genetic toxicity test requirements for metabolites. Regarding safety pharmacology, the ICH M3(R2) Q&A believes that: Since clinical trials to evaluate safety pharmacology endpoints are usually carried out in Phase I, and safety pharmacology endpoints have been evaluated in humans, it is generally not necessary to evaluate metabolites. Non-clinical safety pharmacology test. However, if safety pharmacology signals that were not predicted in non-clinical trials of the original drug are observed in humans, in order to better understand the mechanism, additional safety pharmacology tests can be considered for these human metabolites.
Regarding “how to design non-clinical trials for metabolites (species selection, duration, test type, etc.)”, the interpretation is: beyond the scope of ICH M3 (R2); the trial design should be considered based on the principles of specific analysis of specific issues, and Regulatory agencies negotiate and make scientific judgments.
Regarding “whether the guiding principles of metabolites are applicable to prodrugs”, it is explained that if the prodrug can be converted into active metabolites in animal species similar to the human body, the standard test recommended by ICH M3(R2) can be used method. If the active metabolites produced in the animal species are insufficient, the target molecule for toxicological evaluation should be the active metabolite. Therefore, additional tests other than the recommended metabolite test are appropriate. In this case, the schedule of non-clinical trials of active metabolites should follow the general schedule in ICH M3(R2).
1.2 Requirements for non-clinical studies of metabolites in relevant guiding principles in my country
The “Technical Guidelines for Non-clinical Pharmacokinetic Research of Drugs”  issued by my country in 2014 requires that: The differences in metabolism between experimental animals and humans used in drug efficacy and toxicity tests should be investigated as soon as possible. There are two situations for this difference: one is the difference in quantity. The metabolites of animals and humans are the same, but the amount or proportion of each metabolite is different; the other is the difference in quality, that is, the difference between animals and humans. The metabolites are inconsistent. At this time, we should consider whether this metabolic species difference will affect its efficacy and toxicity, and use this as the basis for the selection of efficacy and toxicity test animals. It is recommended to conduct in vitro drug metabolism tests (such as animal and human liver tissue homogenates, primary hepatocytes, liver S9 and liver microsomes, etc.) in early non-clinical PK studies to predict whether there is a difference in metabolism between animals and humans. The guiding principle also points out that various in vivo and in vitro pharmacokinetic tests must be carried out at different stages of new drug development to clarify the absorption, distribution, metabolism, and excretion (absorpotion, distribution, metabolism, excretion, ADME) and other properties of the candidate drug. Especially for metabolites that exist only in the human body, or metabolites whose body exposure level at steady state is higher than 10% of the total exposure of all drug-related substances and much higher than the level in any toxicological test animal species, There are hidden dangers in drug safety, and metabolite safety studies are required.
In addition, my country issued the “Technical Guidelines for the Safety Testing of Drug Metabolites” in 2012. The guidelines are translations based on the FDA’s “Technical Guidelines for the Safety Testing of Drug Metabolites”, so the main content is similar. . Because the FDA has updated the guidelines, it is more meaningful to refer to the current version of the FDA “Technical Guidelines for the Safety Testing of Drug Metabolites”. For details, see the next section.
1.3 FDA’s requirements for non-clinical studies of metabolites
In the “Technical Guidelines for the Safety Testing of Drug Metabolites” , the FDA encourages early confirmation of the differences in drug metabolism between animals and humans used in non-clinical safety assessments in the drug development process. The discovery of differences in drug metabolites in the late stages of drug development may lead to delays in drug development. Since the research on human metabolism is usually relatively late in the drug development process, the FDA strongly recommends that applicants conduct human metabolism assessments as soon as feasible.
When drug metabolites are disproportionate drug metabolites (that is, metabolites that only occur in humans, or metabolites whose concentrations in humans are higher than those in any animal species used in non-clinical toxicology trials) Product), a safety assessment should be considered. Human metabolites higher than 10% of the total drug-related exposure at steady state can cause safety concerns. The exposure of metabolites in humans and animals is usually AUC, but sometimes the peak concentration (maximum plasma concentration, Cmax) may be more appropriate.
For assessing the safety of metabolites, the FDA proposes to consider two methods: The first method is to determine an animal species commonly used in toxicology tests that can form an appropriate exposure level of the metabolite (and human exposure Equivalent or higher), and then study drug toxicity in the animal species; the second method is to synthesize the metabolite and give it directly to the animal if it is not possible to determine a related animal species that forms the metabolite. Further safety assessment. In this method, an analytical method that can identify and measure the level of the metabolite in non-clinical toxicity tests should be developed.
General considerations for non-clinical trial design: When designing non-clinical trials for disproportionate drug metabolites, the following factors should be considered: the similarity between the metabolite and the parent molecule; pharmacological or chemical category; solubility; gastric pH Stability; Phase I or Phase II metabolites; The relative amount of exposure levels in humans and animals. Other factors that need to be considered include the intended indications of the drug and the patient population (for example, non-clinical trials for serious disease indications can be simplified, such as amyotrophic lateral sclerosis (ALS)), and the planned course of medication (short-term, intermittent) Or long-term medication) and the exposure level at the therapeutic dose.
Non-safety testing of metabolites should follow good laboratory practice (GLP). Non-clinical safety trials of metabolites include the following aspects.
General toxicity test: The administration period of the general toxicity test for the direct administration of metabolites should follow the recommendations of ICH M3 (R2); the drug metabolism should be studied at a dose that is several times the human exposure or at least equivalent to the human exposure Toxicity of the product; the clinical route of administration of the original drug is recommended. Other routes of administration can be used to achieve sufficient exposure if there are normal reasons. If the route of administration is oral, it is important to verify the stability of the metabolites in the gastric environment. It is essential for this type of test to pass toxicokinetics to ensure proper exposure.
Genotoxicity test: The potential genotoxicity should be evaluated in in vitro tests for detecting point mutations and in vitro chromosomal aberration tests. If the results of one or both of the tests are uncertain and/or positive, a complete standard combination test of genotoxicity may be required.
Reproductive toxicity test: If the drug is intended to be used in a population of women with childbearing potential, embryo-fetal toxicity test should be performed on the metabolites. Based on the results of general toxicity and embryo-fetal developmental toxicity tests, other reproductive toxicity tests may be required. Sometimes, it is reasonable to conduct embryo-fetal developmental toxicity tests on only one species that forms drug metabolites.
Carcinogenicity test: For the treatment of chronic or recurrent diseases, the drug should be administered continuously for at least 6 months or intermittently. When the potential carcinogenicity of metabolites cannot be fully assessed in the carcinogenicity test of the parent drug, metabolism should be carried out Carcinogenicity test of the product. A carcinogenicity test or an alternative test should be conducted with reference to the ICH S1 guidelines.
Regarding when to conduct the safety assessment of drug metabolites: If toxicity testing of drug metabolites is necessary, the sponsor should complete the test and provide the FDA with a test report before the start of a large-scale clinical trial. In order to optimize and accelerate drug development for serious or life-threatening diseases other than cancer (such as ALS, human immunodeficiency virus), the number and types of non-clinical trials for drug metabolites will be modified according to specific conditions.