Inventory of the three major in vitro pharmacokinetic research methods

 

The metabolic properties of a compound are an essential factor in whether or not it can be used as a drug in the clinical setting, so pharmacokinetic studies of newly synthesized compounds are required in drug development. In vitro incubation with liver microsomes, recombinant CYP450 enzyme lines, and in vitro incubation with hepatocytes are some of the more common in vitro drug metabolism methods.

1. In vitro incubation method with liver microsomes

The metabolic stability and metabolic phenotypes of candidate compounds in different species of liver microsomes are good predictors of the metabolic properties of compounds in vivo. They are practical tools for evaluating candidate compounds in the pre-development phase of drug development. Liver microsomes include rat liver microsomes, human liver microsomes, canine liver microsomes, monkey liver microsomes, and mouse liver microsomes.

In in vitro incubation of the liver, microsomes are the "gold standard" for in vitro drug metabolism in drug discovery, drug metabolic characterization, and drug interaction studies of new chemical entities, and is currently the most widely used method for clinical and preclinical pharmacokinetic studies.

Medicilon has rich experience in vitro research on pharmacokinetics, including metabolic stability, P450 induction and inhibition, metabolic pathway, and metabolite identification, with animals involved, including rats, mice, rabbits, dogs, and monkeys.

Microsomes are vesicles formed by endoplasmic reticulum fragments obtained by homogenization through ultracentrifugation, containing all components of CYPs. They are widely used for their simple preparation technique, fast metabolic process, good reproducibility of results, easy operation in large quantities, and long-term stability at -80°C.

According to online reports, researchers at other companies investigated the in vitro metabolism of Chrysanthemum bicolor in human liver microsomes, and the in vitro co-incubation of liver microsomes blank control, ethanolic solution of Chrysanthemum bicolor alcoholic extract, inactivated liver microsomes plus Chrysanthemum bicolor alcoholic extract and liver microsomes plus Chrysanthemum bicolor alcoholic extract was performed by high-performance liquid chromatography[1]. The results showed that the content of each component of the alcoholic extract of Chrysanthemum bicolor was reduced under the action of human liver microsomal drug-metabolizing enzymes, and four substances were metabolized but not detected. The alcoholic extract of Chrysanthemum bicolor could be metabolized and eliminated more rapidly under the action of human liver microsomal drug-metabolizing enzymes.

Two 、Gene recombination CYP450 enzyme system technology

The cytochrome P450 (CYP450) enzyme family has enzyme proteins encoded by gene superfamilies involved in the biotransformation of many endogenous and exogenous substances. The effect of drugs on cytochrome P450 enzyme activity is one of the leading causes of drug interactions. Recombinant enzymes are a reliable method for studying the metabolic characterization of CYPs due to their single composition and explicit drug metabolic characterization, and genetically recombinant P450 enzyme lines are increasingly used in in vitro hepatic metabolism studies of drugs.

Genetically recombinant P450 enzyme lines, i.e., using genetic engineering and cell engineering, the genes regulating the expression of P450 enzyme lines are integrated into E. coli or insect cells are cultured to express high levels of P450 enzyme lines and purified to obtain a purer single P450 isoenzyme. Genetically recombinant P450 enzyme lines for in vitro hepatic metabolism studies of drugs have unique advantages in identifying the enzyme isoforms that induce drug metabolism and studying drug-drug interactions and thus have further penetrated various fields of drug metabolism studies.

3. In vitro incubation method with liver microsomes

In vitro incubation of hepatocytes is similar to the liver microsome method, i.e., a system in which prepared hepatocytes are supplemented with redox coenzymes to carry out metabolic reactions under simulated physiological and environmental conditions. The hepatocytes in the incubation system can reasonably simulate the physiological environment of the liver in vivo and have many advantages in studying the biological activity, toxicity, toxicological mechanism, metabolic fate, and carcinogenicity detection of exogenous compounds and are considered a reliable model for preclinical toxicity testing of drugs. For example, some researchers have established an in vitro warm incubation model of rat primary hepatocytes: After warm incubation of serpentine with rat primary hepatocytes, the serpentine content in the warm incubation solution was determined by applying HPLC method to study its metabolic characteristics in vitro.

 The liver is an essential organ of drug metabolism and is the leading site of biotransformation in the body. It is rich in an extensive system of cytochrome P450-dependent mixed-function oxidative enzymes involved in drug metabolism. Most of phase I and phase II reactions of drugs depend on the hepatic enzyme system for their occurrence. The elucidation of critical enzymes of drug metabolism and their metabolic pathway realization provides a significant reference value for the rationale and safety of clinical drug use.

[1] In vitro metabolism study of Chrysanthemum bicolor in human liver microsomes [J].