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].
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