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What are the three major in vitro pharmacokinetic research methods?

2017-09-28
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The metabolic properties of a compound are an important 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 research 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 and are effective tools for the evaluation of candidate compounds in the pre-development phase of drug development. In vitro metabolism was done in liver microsomes from the mouse, rat, dog, monkey and human. The data show that all preclinical species produced similar major metabolites to humans.Liver microsomes include rat liver microsomes, human liver microsomes, canine liver microsomes, monkey liver microsomes, and mouse liver microsomes. 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.

what are the three major in vitro pharmacokinetic research methods?

Microsomes are vesicles formed by endoplasmic reticulum fragments obtained by homogenization through ultracentrifugation, containing all components of CYPs, and 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.
CYP inhibition studies using human liver enzymes are important screens in drug development. The major human CYPs are available as cloned, expressed purified enzyme preparations and a number of highly specific substrates have been identified and characterized. It is therefore possible to examine the test compound’s ability to inhibit a CYP enzyme in isolation, or in crude preparations such as microsomes or S9 fraction.
According to reports, some researchers 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. 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[1].

2、Gene recombination CYP450 enzyme system technology

The cytochrome P450 (CYP450) enzyme family has enzyme proteins encoded by gene superfamilies that are involved in the biotransformation of many endogenous and exogenous substances. The effect of drugs on cytochrome P450 enzyme activity is one of the main causes of drug interactions. Many drugs require CYP450 metabolism for conversion to the active metabolite, are metabolized to inactive by-products, or conversely to reactive intermediates, and thus expression differences arerelevant for drug efficacy and possible toxicity.
Recombinant enzymes are a reliable method for studying the metabolic characterization of CYPs due to their single composition and clear 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, which 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 environmental conditions. The hepatocytes in the incubation system can well 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 content of serpentine in the warm incubation solution was determined by applying the HPLC method to study its metabolic characteristics in vitro.
The liver is an important organ of drug metabolism and is the main site of biotransformation in the body. It is rich in a large system of cytochrome P450-dependent mixed-function oxidative enzymes involved in drug metabolism, and most of the phase I and phase II reactions of drugs depend on the hepatic enzyme system for their occurrence. The elucidation of key enzymes of drug metabolism and their metabolic pathway realization provides an important reference value for the rationale and safety of clinical drug use.
[1] In vitro metabolism study of Chrysanthemum bicolor in human liver microsomes [J].
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