Q: Why do we need to study metabolic stability?

A: First, the metabolic stability reflects the sensitivity of compounds to biotransformation. If the metabolic stability is low, it means that compounds are easy to be metabolized in the body, which often indicates bad pharmacokinetic properties, such as low oral availability and short action time; secondly, the in vitro and internal clearance rate “Clint” obtained from the metabolic stability test can be used to predict the human clearance rate; thirdly, the consistency information of metabolic rates among various genera can provide certain data support for the selection of species of safety assessment animals.

Q: How to select drug concentration in metabolic stability test? Why?

A: In the metabolic stability test, one drug concentration can meet the requirements, generally 1μM, and it is not recommended to be too high. The higher the incubation concentration, the lower the Clint measured, and the more distorted; theoretically, when the incubation concentration is as low as<<K_m, the measured Clint is basically unchanged, while the K_m of most compounds is>>1μM.

Q: In the study of metabolic stability, which one to choose? Liver microsomes or hepatocyte?

A: In the study of metabolic stability, the choice of liver microsomes or liver hepatocyte mainly depends on the metabolic characteristics of the compound and who has the highest metabolic rate. In general, liver microsomes are preferred, especially when the water solubility of compound molecules is strong and phase Ⅰ metabolism is the main metabolic pathway (especially via CYP). When there is evidence that phase Ⅱ metabolism is the main pathway, hydrolysis is the main metabolic pathway, non-specific protein binding in liver microsome is very high, and metabolism in liver microsome is not obvious, liver cells can be used for the test.

Q: How to select the microsomal protein concentration in the metabolic stability test? Why?

A: In the metabolic stability test, the microsomal protein concentration is usually 0.1 mg/mL~1 mg/mL, The specific protein concentration should be selected according to the metabolic characteristics of the compound itself. If the concentration of microsomal protein is too high, it will lead to non-specific binding of drugs and microsomal protein.

Q: What is the effect of non-specific protein binding on the test results?

A: Drug molecules can bind to microsomal proteins in incubation systems. The higher the protein concentration, the smaller the fu value of the free fraction, which will result in a greater difference between the measured Clint value and the theoretical "true value" (the measured value is smaller than the theoretical value). If necessary, it can be corrected by measuring the fu value in the incubation system.

Q: How to select incubation time in metabolic stability test?

A: Generally, if liver microsome is selected as the test system, the incubation time is within 60min, and 3-5 non-zero time points are set; if hepatocytes are selected as the test system, the incubation time shall be controlled within 120min, and 3-5 non-zero time points shall be set.

Q: What effect does the addition of organic solvent in the incubation system have on the test results? Why?

A: The content of organic solvent in the incubation system should be controlled within 1%. The component that plays a metabolic role in the incubation system is enzyme, and the essence of enzyme is protein. Too much organic solvent will lead to denaturation of enzyme, decrease or loss of activity. Therefore, the amount of organic solvent added in the incubation system should be controlled.

Q: How to calculate the half-life period and clearance rate?

A: Take the concentration of the test substance in 0min as 100%, and compare the concentration of the test substance at each time point with the concentration in 0 0min to obtain the residual percentage. The natural logarithm of the remaining percentage of the test substance at each time point and the incubation time were linearly regressed to obtain the slope (k). The elimination half-life t_1/2 in various microsomes and the intrinsic clearance rate Clint in vitro in vitro liver microsomes were obtained from the formula.

Q: What are solution A and solution B in the phase Ⅱ metabolic stability kit? What is the purpose?

A: Solution A and solution B in the kit are components of the NADPH regeneration system, and NADPH can be obtained

through reaction. Because some compounds are first transformed through phase Ⅰ reaction, if the polarity is still weak, the phase Ⅱ reaction will be started, Therefore, the kit includes both the phase I and phase II cofactors. However, if you only want to investigate the glucuronylation reaction, you can not use solution A and solution B.

Q：Only phaseⅠmetabolism can metabolize 10% of the drug, and only phase Ⅱ metabolism can metabolize 10%, but using the two-phase metabolism kit can metabolize 50%. How to explain it?

A: Biotransformation of drugs can be divided into phase Ⅰ metabolic reaction and phase Ⅱ metabolic reaction. The drug mainly undergoes oxidation, reduction and hydrolysis in the first phase metabolic reaction, after phase Ⅰ metabolic reaction, the drug may have some polar groups, such as hydroxyl, carboxyl, etc. Phase Ⅱ metabolism, also known as binding reaction, refers to the reaction in which the primary metabolite or prototype drug combines with endogenous small molecules under the influence of enzymes to reduce the toxicity and activity of drugs or increase their polarity, which is easy to be discharged. Generally speaking, drugs are first converted by phase Ⅰ reaction. If the polarity is still weak, phase Ⅱ reaction will be initiated. However, some drugs can directly conduct phase Ⅱ reaction. Therefore, the above phenomena exist.

Q：Metabolic reaction always occurs at the metabolism point of 0 min, how to deal with it？

A：It is recommended to add the termination solution first, and then start the metabolic reaction.

Q：The solubility of the compound in the water phase is not good, how to study the metabolic stability?

A：It is suggested to use liver hepatocytes as the metabolic system for stability study. If liver microsomes is selected as the test system, the method of single tube sampling and shock incubation can be used for the test.

Q：The compound can be detected in organic solvent, but can not be detected after adding the kit?

A: The preliminary judgment may be caused by the poor solubility of the compound in the water phase or the easy hydrolysis of the compound in the aqueous environment. If it is caused by poor solubility in the water phase, the sample pretreatment process can be optimized; if the response of compounds of the same concentration in aqueous solution and organic solvent is more than 50% difference, it is judged that the compounds are easy to hydrolyze (0.1 M PBS may have matrix effect on some compounds, and the matrix effect should be excluded before judgment), and it is not easy to conduct in vitro metabolism test. It is recommended to confirm whether the efficacy of the compounds is the compounds themselves or their hydrolysates.

Q: When the substrate elimination method is used for the test, no metabolic reaction occurs to the positive substrate in the kit? Why?

A： The concentration of positive substrate assembled in the kit is 10mM, even add 1μL, the final concentration can reach 50μM, this concentration is suitable for detecting the generation of metabolites. The content of microsomal enzyme in the incubation system is limited, it needs to be diluted before the test. If it needs to detect the reduction of positive substrate. The recommended final concentration is 1μM.

Q：What is the role of the positive substrate in the metabolic stability study kit? How should the incubation time be set?

A：The positive substrate in the kit is mainly used to verify the effectiveness of the kit. The setting of incubation time point mainly depends on the test purpose, if it is only to verify whether the incubation system works normally, just set 0 point and a non-zero time point; if the metabolic stability characteristics of positive substrate need to be investigated, 0 point and 3-5 non-zero time points need to be set.

Q: What should I do if I can't use up the kit at one time? How should I save it? Why?

A：Microsome, solution A, solution B, UDPGA and other components in the kit all contain enzyme components. Repeated freezing and thawing will result in reduction or loss of enzyme activity. If it cannot be used up at one time, it is recommended to repackage according to the amount used and make marks, and store according to the storage temperature marked on the kit.

Q: Why is the control group necessary?

A：The purpose of the control group is to investigate the stability of the compound itself in different components, and the stability of the control group is the precondition for the establishment of the whole metabolic experiment.