CN112691110B

CN112691110B - Application of pharmaceutical composition in preparation of medicine for treating viral hepatitis B

Info

Publication number: CN112691110B
Application number: CN202110036546.2A
Authority: CN
Inventors: 王广基; 张经纬; 陈倩莹; 周芳
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2016-01-13
Filing date: 2016-01-13
Publication date: 2022-09-13
Anticipated expiration: 2036-01-13
Also published as: CN105687220A; CN112691110A

Abstract

The invention relates to a novel medicine composition, in particular to application of diammonium glycyrrhizinate in improving the curative effect of entecavir on hepatitis B. The pharmacokinetic results show that glycyrrhizic acid/glycyrrhetinic acid increases the drug concentration in entecavir plasma/liver tissue and the amount of drug taken in the liver cells/sub-cells at the whole animal and cell/sub-cell levels, respectively. Pharmacodynamic results also find that glycyrrhizic acid/glycyrrhetinic acid increases entecavir antiviral efficacy in HBV transfected in vitro cell models and HBV mouse models.

Description

Application of pharmaceutical composition in preparation of medicine for treating viral hepatitis B

The application is a divisional application with the application date of 2016, month 01 and day 13, the application number of 201610027329.6, and the name of the invention is 'a novel drug combination for treating viral hepatitis B'.

Technical Field

The invention relates to a novel medicinal composition, in particular to application of glycyrrhizic acid which is a main component of a traditional Chinese medicine liquorice in improving the curative effect of a nucleoside medicament entecavir for treating hepatitis B.

Background

Hepatitis B is an infectious disease caused by Hepatitis B Virus (HBV), mainly by hepatitis B virus, and can cause multiple organ damage, and chronic hepatitis B can even lead to cirrhosis or liver cancer. At present, hepatitis B is widely spread in all countries of the world, the incidence rate is increased year by year, and the number of hepatitis virus carriers is continuously increased, so that the hepatitis virus becomes one of the most popular and most serious diseases in China. Because the hepatitis B virus has rapid mutation, the drug resistance of the anti-hepatitis B virus drug is easily caused; meanwhile, the hepatitis B patients have long course of disease and long administration time, so the liver injury is easily caused. The current treatment method related to hepatitis B is mainly antiviral treatment, and the aim of delaying the disease progression is achieved by inhibiting the continuous replication of HBV. Entecavir (Entecavir, ETV) is a first-line drug against hepatitis b virus, inhibits the initiation, reverse transcription and DNA plus strand synthesis of HBV polymerase, has a strong antiviral effect, has few side effects and low virus variability, and has been widely used for the treatment of chronic hepatitis b. Glycyrrhizic acid is an extract of the effective components of the traditional Chinese medicine liquorice, and has certain effects of resisting inflammation, protecting liver cell membranes and improving liver functions. Clinical data show that when entecavir and Diammonium Glycyrrhizinate (GLN) are used in combination, the entecavir has stronger viral hepatitis treatment effect, and simultaneously improves liver function and delays liver fibrosis. Entecavir is used as antiviral medicine and diammonium glycyrrhizinate is used as liver protecting medicine, and the research on the combined application mechanism of these two medicines becomes one of the keys in further developing medicine composition for treating viral hepatitis.

The licorice root is a plant of leguminosae, has mild nature and sweet taste, and has the effects of tonifying qi and spleen, regulating the middle warmer and relieving urgency, harmonizing the other drugs and the like. Glycyrrhizic acid is the main component of liquorice, is a pentacyclic triterpene series saponin, can be hydrolyzed into Glycyrrhetinic Acid (GA) and two molecules of glucuronic acid, and has a plurality of pharmacological activities including anti-inflammatory, immunoregulation, anti-liver injury and the like. The structural formula is as follows:

patent CN200610018367.1 discloses the use of glycyrrhizic acid or glycyrrhetinic acid in the preparation of drugs for treating inflammatory bowel diseases. Glycyrrhizic acid or glycyrrhetinic acid can inhibit inflammatory reaction and resist oxidation to achieve anti-inflammatory bowel disease effect. Patent CN200510045377.X discloses a pharmaceutical composition mainly used for liver diseases, which is prepared from medlar, angelica and glycyrrhizic acid, and has the efficacy of treating viral hepatitis, drug-induced liver injury, fatty liver and the like together. Patent CN98805037.4 discloses an antiviral pharmaceutical composition containing glycyrrhizic acid and at least one protein having antiviral activity.

Disclosure of Invention

The invention aims to provide a novel medicinal composition of diammonium glycyrrhizinate-entecavir for treating hepatitis B diseases, namely, the medicinal combination has a synergistic effect compared with entecavir. The pharmacokinetic results show that glycyrrhizic acid/glycyrrhetinic acid increases the drug concentration in entecavir plasma/liver tissue and the amount of drug taken in the liver cells/sub-cells at the whole animal and cell/sub-cell levels, respectively. Pharmacodynamic results also find that glycyrrhizic acid/glycyrrhetinic acid increases entecavir antiviral efficacy in HBV transfected in vitro cell models and HBV mouse models.

The research scheme of the invention is that (1) after the combined administration of diammonium glycyrrhizinate and entecavir is inspected at the whole animal level, the plasma pharmacokinetics and the change of the concentration in tissues of entecavir are inspected, and the possible mechanism of the combined synergistic effect of diammonium glycyrrhizinate and entecavir is judged from the whole animal perspective. (2) A hepatocyte model is used for investigating the cell pharmacokinetic behaviors of entecavir in hepatocytes, such as uptake, subcellular distribution and the like, and the influence of glycyrrhetinic acid on the processes. (3) By using the HepG2215 cell model transfected by HBV, whether the cell supernatant virus copy number is reduced compared with that of the single-use group after the combination of the medicaments is examined. (4) By applying the HBV transgenic mouse model, whether the HBV virus copy number in the plasma of the mouse is reduced compared with that in a single-use group after long-term drug combination is examined.

The research result of the invention mainly comprises that the combined use of diammonium glycyrrhizinate has no significant influence on the plasma pharmacokinetics process of the entecavir, but the distribution quantity of the entecavir in liver tissues is increased; the distribution quantity of the entecavir in cell nucleus and cytoplasm is improved after the glycyrrhetinic acid is combined; the virus copy indexes in the HepG2215 cell model and the HBV transgenic mouse model in vitro are reduced in the combined group compared with the single group.

Drawings

FIG. 1: combined use of entecavir after diammonium glycyrrhizinate for rat pharmacokinetic change

FIG. 2: rat tissue distribution quantity change of combined use of diammonium glycyrrhizinate

FIG. 3: effect of Glycyrrhetinic acid on Entecavir hepatocyte uptake

FIG. 4: entecavir subcellular distribution change after combined use of glycyrrhetinic acid

FIG. 5 is a schematic view of: combined use of glycyrrhetinic acid to Entecavir for virus inhibition IC in HepG2215 model ₅₀ Influence of (2)

FIG. 6: influence of long-term combined diammonium glycyrrhizinate on inhibition of replication process of HBV mouse viruses by entecavir

Detailed Description

Example 1: determination of single and multiple combined use of entecavir diammonium glycyrrhizinate in plasma concentration of rat

The method mainly comprises the following steps:

male SD rats, 20, were randomly assigned to weight into four groups of 5 rats each. 1. Single combined administration: rats were gavaged (i.g.) with 0.09mg/kg ETV (control group), 100mg/kg GLN +0.09mg/kg ETV (test group)2. chronic combination: rats were gavaged (i.g.) daily for 10 consecutive days with 0.09mg/kg ETV (control group), 100mg/kg GLN +0.09mg/kg ETV (test group), and on day 11 with 0.09mg/kg ETV in both groups following normal GLN administration. Heparin was previously added to the blood collection tubes used in the experiment as a plasma anticoagulant at a ratio of 1. mu.l/20. mu.l (v/v, anticoagulant/whole blood). Collecting 200 mu l of venous blood of 0.167, 0.5, 1, 1.5, 2, 4, 8 and 24h before and after administration from orbital venous plexus into a blood collection tube, centrifuging at 8000rpm for 5min, transferring upper plasma, measuring the temporal concentration of the entecavir in each group of plasma by LC-MS/MS after treatment, and calculating main pharmacokinetic parameters, wherein the results are as follows:

TABLE 1 major pharmacokinetic parameters of entecavir after diammonium glycyrrhizinate for long and short term use

Example 2: determining the drug concentration of single and multiple combined use of entecavir after diammonium glycyrrhizinate in rat tissues

The method mainly comprises the following steps:

60 male SD rats were randomly assigned to twelve groups of 5 rats by body weight. 1. Single combined administration: rats were either gavaged (i.g.) followed by 100mg/kg GLN6h, 0.09mg/kg ETV (test group) or directly gavaged (i.g.) followed by 0.09mg/kg ETV (control group), and the rat femoral artery was exsanguinated and sacrificed after 1, 2, 4h and the tissue was harvested. 2. Long-term combined administration: rats were gavaged (i.g.) daily for 10 consecutive days with 100mg/kg GLN6h followed by 0.09mg/kg ETV (test group) or 0.09mg/kg ETV (control group) directly by gavage (i.g.) and 0.09mg/kg ETV in both groups after 11 days of normal GLN administration. Heparin is added into a blood collection tube used in the experimental process according to the proportion of 1 mu l/20 mu l (v/v, anticoagulant/whole blood) in advance to serve as a plasma anticoagulant, the whole blood is collected and is centrifuged at 8000rpm for 5min, then upper plasma is transferred, and the concentration of the entecavir in each group of plasma and tissues is measured.

Example 3: determining the effect of Glycyrrhetinic acid on Entecavir hepatocyte uptake

The method mainly comprises the following steps:

HepG2 cells were selected for culture in high glucose DMEM medium containing 10% Fetal Bovine Serum (FBS), penicillin 100U/ml and streptomycin 100. mu.g/ml at 37 ℃ in an environment containing 5% CO2, and were digested and seeded into 24-well plates when the cells reached 90% confluency. After 90% confluency of the cells cultured on the 24-well plate, the drug-containing ETV (20. mu.M), GA (10. mu.M) + ETV (20. mu.M) was added. Timing, respectively incubating in a shaking table at 37 ℃ for 0.167, 0.33, 0.5, 1, 2 and 4 hours. After the cells are subjected to ultrasonic disruption, the drug concentration is determined by an LC-MS/MS method, and the BCA legal protein is taken out of the cell suspension. Drug absorption is generally expressed as the amount of drug absorbed per mg of protein in the cell.

Example 4: combined use of glycyrrhetinic acid, the distribution quantity of entecavir subcellular changes

The method mainly comprises the following steps:

HepG2 cells were inoculated in T-75 flasks and administered when the cells reached 90% confluence. The combined administration group is pre-administered with Hank's solution containing glycyrrhetinic acid (10 mu M), after 30min, the solution is sucked dry, Hank's solution containing glycyrrhetinic acid (10 mu M) + entecavir (20 mu M) is added again, meanwhile, entecavir single administration group with the same concentration is taken as a parallel control (n is 3), and the combined administration group is respectively incubated in a shaking table at 37 ℃ for 0.167, 0.33, 0.5, 1, 2 and 4 h. After collection, the cells were transferred to a centrifuge tube and collected by centrifugation at 1000rpm for 5 min. Adding lysis solution into cell precipitate, separating out cell nucleus, mitochondria and cytoplasm according to the operation of cell nucleus/mitochondria separation kit (Kaikyi organism) instruction, and storing in refrigerator at-80 deg.C. The LC-MS/MS method measures drug concentration and takes cell nucleus, mitochondria and cytoplasm suspensions as BCA legal protein.

Example 5: combined use of glycyrrhetinic acid to Entecavir for virus inhibition IC in HepG2215 model ₅₀ Influence of (2)

The method mainly comprises the following steps:

HepG2215 cells were selected for culture in high glucose DMEM medium containing 10% Fetal Bovine Serum (FBS), penicillin 100U/ml, streptomycin 100. mu.g/ml, at 37 ℃ in an environment containing 5% CO2, and when the cells reached 90% confluency, they were digested and seeded in 48-well plates. After 90% fusion of cells on a 48-well plate, respectively adding 0.5ml of culture medium containing entecavir (0.01, 0.1, 0.5, 1, 5, 20 μ M) with different concentrations, glycyrrhetinic acid (10 μ M) + entecavir (0.01, 0.1, 0.5, 1, 5, 20 μ M), incubating for 72h in a cell incubator, rapidly collecting 0.2ml of cell supernatant, extracting total DNA according to the requirement of a DNA extraction kit (Takara), purifying, performing fluorescence quantitative PCR analysis, comparing the copy number of viruses in each group, and calculating IC ₅₀ The value is obtained.

Example 6: influence of long-term combined diammonium glycyrrhizinate on inhibition of replication process of HBV mouse viruses by entecavir

The method mainly comprises the following steps:

30 HBV transgenic mice are selected and randomly distributed into six groups according to different virus copy numbers, and each group comprises 5 HBV transgenic mice. The groups comprise a blank control group, an entecavir single-use group (0.15mg/kg), a diammonium glycyrrhizinate single-use group (200mg/kg), an entecavir combined low-dose diammonium glycyrrhizinate group (100mg/kg), and an entecavir combined high-dose diammonium glycyrrhizinate group (200mg/kg), wherein the administration groups are continuously used for 22 days, the entecavir in the administration groups is administrated by intragastric administration (i.g.) every day, and the diammonium glycyrrhizinate is administrated every other day. Small amounts of orbital blood were collected at

days

0, 3, 7, 15, and 22 after dosing, total DNA was extracted according to DNA extraction kit (Takara) instructions, and virus copy number in plasma was determined by fluorescent quantitative PCR. Heparin is added into a blood collection tube used in the experimental process according to the proportion of 1 mu l/20 mu l (v/v, anticoagulant/whole blood) in advance to serve as a plasma anticoagulant, the whole blood is collected and is centrifuged at 8000rpm for 5min, and then the upper plasma is transferred.

Claims

1. The application of the glycyrrhetinic acid and the entecavir in preparing the medicine for treating the viral hepatitis B is characterized in that the molar ratio of active ingredients of the glycyrrhetinic acid to the entecavir in the medicine is 1: 2.

2. Application of glycyrrhetinic acid in preparation of a pharmaceutical composition for improving antiviral drug effect of entecavir.