The metabolic properties of a compound are an important factor related to whether it can be turned into a drug and actually used in the clinic. Therefore, it is necessary to conduct pharmacokinetic studies on newly synthesized compounds in drug development. Among them, the in vitro incubation of liver microsomes, the gene recombination CYP450 enzyme system technology, and the in vitro incubation of hepatocytes are relatively common in vitro drug metabolism research methods.
1. In vitro incubation of liver microsomes
Metabolic stability and metabolic phenotype studies of candidate compounds in different species of liver microsomes have a good predictive effect on the metabolic properties of compounds in vivo, and are an effective means to evaluate candidate compounds in the early stage of drug development. Liver microsomes include rat liver microsomes, human liver microsomes, canine liver microsomes, monkey liver microsomes, mouse liver microsomes, and the like. In the drug discovery of new chemical entities, drug metabolism characteristics and drug interaction studies, the in vitro incubation of liver microsomes is the “gold standard” for in vitro drug metabolism, and is currently the most widely used method in clinical and preclinical pharmacokinetic studies. broad method. Medicilon has extensive experience in the in vitro study of pharmacokinetics. In vitro studies refer to research projects such as metabolic stability, P450 induction and inhibition, metabolic pathway research, and metabolite identification. The animals involved include rats and mice. , rabbits, dogs, monkeys, etc.
Microsomes are small vesicles formed from endoplasmic reticulum fragments obtained by homogenization and ultracentrifugation. They contain all the components of CYPs. They have the advantages of simple preparation technology, fast metabolic process, good reproducibility of results, easy to operate in large quantities, and in- It is widely used due to the advantages of long-term stability at 80 °C.
Some researchers investigated the in vitro metabolism of two-color coreopsis in human liver microsomes, and mixed liver microsome blank control, two-color coreopsis alcohol extract ethanol solution, inactivated liver microsomes plus two-color coreopsis alcohol extract and liver. Microsomes were co-incubated with two-color coreopsis alcohol extracts in vitro, and were determined by high performance liquid chromatography. By comparing the chromatographic peaks, the in vitro metabolism rules of two-color coreopsis in liver microsomes were clarified . The results showed that under the action of drug-metabolizing enzymes in human liver microsomes, the content of each component of the two-color coreopsis alcohol extract was reduced, and 4 substances were metabolized without being detected. It can be rapidly metabolized and eliminated under the action of drug-metabolizing enzymes.
2. Gene recombination CYP450 enzyme system technology
Cytochrome P450 (CYP450) enzymes are composed of enzyme proteins encoded by a superfamily of genes and 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 reasons for drug-drug interactions. Recombinase is a reliable method for studying the metabolism of CYPs because of its single composition and clear characterization of drug metabolism. P450 enzyme system of genetic recombination has been more and more widely used in the study of in vitro liver metabolism of drugs.
Gene recombination P450 enzyme system is the use of genetic engineering and cell engineering to integrate the genes regulating the expression of P450 enzyme system into E. coli or insect cells, and after cell culture, express a high level of P450 enzyme system. After purification, a purer single P450 can be obtained. isoenzyme. Genetically recombined P450 enzyme system is used for in vitro hepatic metabolism study of drugs, and has unique advantages in determining the enzyme isoforms that induce drug metabolism and studying drug-drug interactions, so it has further penetrated into various fields of drug metabolism research.
3. In vitro incubation of hepatocytes
The in vitro incubation method of hepatocytes is similar to the liver microsome method, that is, the prepared hepatocytes are supplemented with redox coenzymes, and the metabolic reactions are carried out under the simulated physiological environment conditions. Hepatocytes in the culture system can well simulate the physiological environment of the liver in vivo, and have many advantages in the study of the biological activity, toxicity, toxicological mechanism, metabolic fate and carcinogenicity detection of exogenous compounds, and are considered to be the most important in clinical medicine. A reliable model for protoxicity detection. If a researcher has established an in vitro incubation model of rat primary hepatocytes: after incubation of osthole and rat primary hepatocytes, the content of osthole in the incubation solution was determined by HPLC, and its metabolic characteristics in vitro were studied. .
In conclusion, liver-based in vitro metabolic models have been widely used with their unique advantages in pharmacokinetic studies. The liver is an important organ of drug metabolism and the main site for biotransformation in the body. It is rich in a huge cytochrome P450-dependent mixed-function oxidase system involved in drug metabolism. Occurs in the liver enzyme system. Elucidating the key enzymes of drug metabolism and the realization of their metabolic pathways provides important reference value for the rationality and safety of clinical drug use.
Bioanalytical Projects for In Vitro ADME
Drug metabolism stability and metabolite determination
-Drug liver microsome metabolic stability test
-Drug liver cell metabolism stability test
-S9 metabolic stability assay
-Plasma and whole blood metabolic stability test
-Drug metabolism pathway and metabolite identification
-CYP450 inhibition assay
-CYP450 induction assay
-CYP450 phenotype experiments
-Plasma protein binding assay
-Blood red blood cell distribution assay
-Caco-2 drug intestinal permeability test
-MDR1-MDCK cell permeability and Pgp-mediated drug efflux assay
Physical and chemical properties
-Solubility and Log D determination
-Chemical stability test
In vitro toxicity
-Non-GLP Mini-Ames experiment
AxisPharm is a San Diego based bioanalytical LC/MS/MS service provider with more than 25 years experience in the field. Our bioanalytical chemistry department specializes in developing and validating robust bioanalytical methods for PK/TK sample analysis of small molecules, proteins, peptides, and metabolites using LCMS/MS (HPLC, UPLC, on-line SPE), HPLC/UV, and HPLC/FL. We have experience analyzing API and metabolites in various biological matrices and can provide bioanalytical support throughout all the stages of drug development.