Q1: What is the significance of metabolic enzyme/metabolic reaction phenotype research?

A1: Metabolic enzyme/metabolic reaction phenotype research, also known as metabolic zymogram/metabolic reaction spectrum research, is an indispensable part of the process of new drug development. Through the study of enzyme phenotype, it is not only possible to identify the metabolic enzymes/reactions and the contribution of each metabolic enzyme/reaction involved in the biotransformation of a drug, but also of great significance in evaluating the possibility and extent of drug interactions and the participation of gene polymorphic enzymes.

Q2: How to select drug concentration in metabolic enzyme phenotype test? How to select the concentration of human liver microsome/recombinase? How to set incubation time?

A2: In the metabolic enzyme phenotype test, only one drug concentration needs to be set, which is usually consistent with the drug concentration in the metabolic stability test; the concentration of human liver microsome protein shall be controlled within 1mg, i.e. ≤ 1mg/mL; the concentration of recombinant enzyme is generally 10~30pmol/mL; generally, 1 or 2 incubation times are selected, which can be set according to the steps of metabolic stability experiment.

Q3: Which enzymes are mainly concerned in enzyme phenotype research?

A3: In the study of enzyme phenotype, the CYP enzyme subtypes mainly concerned include CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4/5, which generally need to be studied; the major UGT enzyme subtypes are UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B6 and UGT2B15. There is evidence that UGT enzyme is the main metabolic pathway that needs to be studied.

Q4: Is it necessary to use chemical inhibition method and recombinant enzyme method for CYP450 enzyme phenotype study?

A4: Phenotype study of metabolic enzymes is qualitative study, The current regulations and industry views both believe that a single method cannot draw reliable conclusions. Chemical inhibition method and recombinant enzyme method are both needed. However, it is generally believed that the results of chemical inhibition method are dominant, and the results of recombinant enzyme method are auxiliary.

Q5: In CYP450 enzyme phenotype study, how to explain when the results of chemical inhibition method and recombinant enzyme method are inconsistent?

A5: First of all, this situation should not be called inconsistency, because it is the result of metabolism of two different systems. The difference between the results of these two metabolic systems should not be called inconsistency, but a normal phenomenon caused by the difference of the metabolic system itself; Secondly, liver microsome is a component of directly extracted liver, which is a homologous metabolic system, while recombinant enzyme is expressed through the protein expression system, which is a non homologous test system. Therefore, the results of chemical inhibition method are the main and the results of recombinant enzyme method are the auxiliary in the conclusion.

Q6: The results of metabolic stability showed that the remaining amount of drug was close to 100%. Is it necessary to study the metabolic phenotype of CYP enzyme? How?

A6: The low metabolic rate in vitro means that the metabolic rate in vivo is also likely to be low, so no metabolic phenotype study is required. It should be noted that the low metabolic rate does not mean that the metabolic ratio is necessarily low (for example, when the excretion rate is lower). Therefore, it is still necessary to know the specific subtype pathway of drug metabolism through CYP enzyme, unless there is evidence that CYP enzyme is not the main metabolic pathway.

   Technically, if the residual amount of the drug is close to 100%, it is really impossible to judge by the metabolic rate of the drug, but it can be judged by the amount of metabolites generated. In this case, it is better to have the main metabolite synthesized. If there is no reference substance of the target metabolite, the ion pair of the main metabolite found in the metabolite identification can also be used for rough qualitative detection.

Q7: When to consider UGT phenotype research, and how to do UGT phenotype research?

A7: When there is evidence (in vitro or in vivo data) that UGT is the main metabolic enzyme, UGT phenotype study should be considered. The study of UGT phenotype only uses recombinant enzyme method, because there is no specific inhibitor of UGT subtype for workers in academia at present. However, it should be noted that CYP and UGT, even other metabolic enzymes, may be the main metabolic pathways at the same time.