Liver Microsome Model of Drug Metabolism Model in Vitro

For those drugs with low metabolic rate, high toxicity, or lack of sensitive detection methods, in vitro drug metabolism studies can eliminate the interference of internal factors and provide reliable theoretical data for the overall experiment. The liver microsomal model is one of the common in vitro models for studying drug metabolism in laboratories. It has the advantages of fast metabolism and easy mass manipulation. It can be widely used in the research of enzyme activity and in vitro metabolic clearance.

Biological macromolecular drugs have large molecular weights and complex structures. Compared with small molecule drugs, they have unique and complex PK characteristics, such as target receptor-mediated elimination and FcRn transcytosis, and their in vitro metabolism studies are also different. For macromolecular drugs, they are generally not metabolized by CYP 450 enzymes. The main elimination pathways in the body are glomerular filtration, enzymatic hydrolysis, receptor-mediated endocytosis elimination and anti-drug antibody-mediated elimination.

The liver is the main metabolic organ of drugs, rich in mixed-function oxidase systems involved in the one-phase and two-phase metabolism of drugs. 90% of the drugs are mainly biotransformed by cytochrome P450 enzymes. Microsomes extracted from the liver or intestines are added to the reduced coenzyme II (NADPH) regeneration system to carry out metabolic reactions in a simulated physiological environment in vitro. The original drugs and metabolites are measured by high-performance liquid chromatography-mass spectrometry and other methods. Among them, the liver microsome method is the most common.

The liver microsome method for in vitro drug metabolism studies has the advantages of convenient and easy preparation, good reproducibility, easy storage of enzyme mixtures, easy optimization of incubation conditions, recognized subenzyme substrates, inhibitors, and sensitivity and effectiveness. Shanghai Medicilon has rich experience in in vitro pharmacokinetic research. In vitro research refers 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.

Preparation of Rat Liver Microsomes

(1) Purchase experimental animal SD rats, 20, male and female, weight (180-220) g.

(2) Rat liver is perfused. The purchased rat is intraperitoneally injected with 3% sodium pentobarbital (30 mg²kg-1) on the same day to make it anesthetized. After routine disinfection with alcohol, the abdominal cavity is opened to expose the liver, and the hepatic portal vein is inserted. Tube and fix. The superior vena cava was ligated, and the inferior vena cava was cut and perfused with phosphate buffer solution until the liver color was khaki.

(3) After the perfused liver is taken out, put it into a homogenization tube containing phosphate buffer (containing 1mM EDTA, 0.25M sucrose) at a ratio of 1:4, cut and homogenize.

(4) Pour the homogenate into a 50mL high-speed centrifuge tube and centrifuge at 9000g for 20 min at 4°C.

(5) Save the supernatant and transfer it to an ultracentrifuge tube, centrifuge at 100000g for 60 minutes at 4°C.

(6) Keep the pellet and resuspend it in phosphate buffer (containing 0.9% NaCL), centrifuge at 100,000g at 4°C for 60 min, and the pellet obtained is the liver microsome.

(7) Resuspend the liver microparticles in 0.1M phosphate buffer (pH7.4, 20% glycerol, 1mM EDTA, 0.25M sucrose) and store in the refrigerator at -70℃, and save a small tube for protein concentration determination.

(8) The determination of liver microsomal protein concentration is based on the Lowry et. al. method (Lowry et al., 1951). First, prepare a standard solution of bovine serum albumin into protein standard solutions of different concentrations. After mixing with Fehling reagent, the protein concentration is determined. Linear regression is performed based on absorbance A and protein concentration C and a standard curve is drawn. The absorbance of liver microsomes was measured in the same way, and the protein concentration of liver microsomes was calculated according to the linear regression equation of the standard curve.

(9) Determination of p450 enzyme content in liver microsomes, according to the method of omura and Sato. Dilute the liver microsome sample to 0.3-0.5mg/ml with Tris-HCL buffer, take two liver microsomes and place them in the reference pool and sample pool respectively, and scan the baseline at 400~500nm; add both reference pool and sample pool A small amount of Na2S2O4 was stirred slightly, and CO gas was applied to the sample for 30 seconds, and then scanned again, recording the absorption light at 450nm and 490nm, and calculating the content of CYP450 according to Beer’s law.

The Role of Liver Microsomal Drug Metabolism Model in Vitro

(1) The in vitro metabolism model of liver microsomal drugs can be used for drug clearance research

To predict the clearance rate of a drug in the body, the steps are to first obtain Vmax and Km by measuring the enzyme kinetic parameters of drug metabolism, and then use a reasonable kinetic model to infer the clearance rate of drug metabolism. For example, researchers studied the intestinal metabolism of the anti-AIDS drug candidate 3-cyanomethyl-4-methyl-DCK (CMDCK) in the human intestinal microsome incubation system, and measured the elimination half-lives, and applied the WellStirred model to the intestinal microsomes. The kinetic parameters are extrapolated, and the intestinal clearance rate in the body is predicted to be 3.3 mL·min-1·kg-1, which is close to the average blood flow of the human intestine (4.6 mL·min-1·kg-1) . It is speculated that the intestinal metabolism of CMDCK may have a significant impact on its oral first pass effect.

(2) The in vitro metabolism model of liver microsomal drugs can be used for high-throughput drug screening research

In the early stage of new drug development, liver particle models are often used to screen the pharmacokinetic characteristics of candidate compounds in order to determine whether the candidate compound has the value of continued development in the early stage of research and development. For example, using liver microsome incubation model (in vitro model) and isochronous sample mixing (in vivo model) methods, establish an LC-MS/MS analysis method for combined detection of multiple compounds, and quickly screen the pharmacokinetic properties of the compounds , Through in vitro metabolic stability studies, the binding compound can be further developed.

(3) The in vitro metabolism model of liver microsomal drugs can be used to study drug interactions

Drugs that inhibit or induce P450 enzymes (such as CYP3A4) can affect the pharmacokinetics of other drugs in combination, thereby affecting the exposure of the drug in plasma (expressed as changes in AUC) and leading to DDI in pharmacokinetics. The enzymatic reaction kinetics of ligustilide metabolism was studied by rat liver microsomes, and the main isoenzymes of ligustilide metabolism were explored through specific inhibition experiments. The results showed that ketoconazole, trimethoprim and α-naphthoflavone significantly inhibited the metabolism of ligustilide in vitro, and it was concluded that CYP3A4, CYP2C9, and CYP1A2 are the main metabolic enzymes involved in the metabolism of ligustilide, CYP2C19, CYP2E1, and CYP2E1. CYP2D6 has no obvious involvement.

Disadvantages of in Vitro Metabolism Model of Liver Microsomal Drugs:

(1) Due to the destruction of the complete structure during the preparation process, non-specific reactions are more likely to occur in the in vitro incubation system;

(2) The removal of the outer cell membrane leads to the loss of transport proteins (such as Pgp), which has a great impact on the study of intestinal metabolism;

(3) The lack of a complete enzymatic reaction system required for metabolism requires the addition of an appropriate amount of cofactor NADPH;

(4) Some drug-metabolizing enzymes are removed during the preparation process, such as the metabolic enzymes located in the cytoplasm.