WO2014071712A1

WO2014071712A1 - Medicinal composition for treating liver injury, and method thereof

Info

Publication number: WO2014071712A1
Application number: PCT/CN2013/072269
Authority: WO
Inventors: 李小羿; 张国辉; 戴向荣; 凌娟; 胡代娣; 周冠群
Original assignee: 兆科药业（合肥）有限公司
Priority date: 2012-11-08
Filing date: 2013-03-07
Publication date: 2014-05-15
Also published as: CN102895649A

Abstract

Disclosed are a medicinal composition for treating a liver injury, and a method thereof. The medicinal composition in the present invention is formed by oxidized glutathione disodium and inosine.

Description

Pharmaceutical composition for treating liver injury and preparation method thereof The present application claims to be submitted to the Chinese Patent Office on November 8, 2012, the application number is 201210443808.8, the invention name is "a pharmaceutical composition for treating liver damage and a preparation method thereof" The priority of the Chinese Patent Application, the entire contents of which is incorporated herein by reference. Technical field

The invention relates to the field of medicine, in particular to a pharmaceutical composition for treating liver damage and a preparation method thereof. Background technique

The drug-induced liver disease tube is called the drug liver. It refers to the damage of the function and structure of the liver caused by the action of drugs and their metabolites in the therapeutic amount of the drug. It can occur in healthy people who have no history of liver disease or have serious problems. Patients with the disease. Different degrees of liver damage occur after the use of a certain drug, which is called drug-induced liver disease. At least 600 drugs are currently available to cause drug-induced liver disease. Drug-induced liver damage accounts for 10% to 15% of all drug reactions. Drug-induced liver disease can even lead to disability and death.

Clinically, drug-induced liver injury involves almost all types of liver damage, including acute or chronic hepatic parenchymal cell damage, cholestasis, fatty liver, liver fibrosis, cirrhosis, vascular disease, benign and malignant tumors, and the like. Different drugs cause different types of liver damage, such as Parker's heat can destroy liver cells to cause toxic hepatitis, tetracycline, corticosteroids, asparaginase can cause fatty liver, long-term use of danazol can induce primary liver cells Cancer, oral contraceptive, diethylstilbestrol, can induce benign tumors. In the acute phase, the performance is similar to viral hepatitis, that is, 80% to 90% of patients are sick within 8 weeks of medication, with fatigue, anorexia, nausea, dark urine, hepatomegaly, and tenderness. Drugs that can cause jaundice, such as sputum testosterone, rifampicin, and novobiocin. Long-term use of drugs such as remi-salt, sulfa drugs, furantan, diacetin, etc., often cause drug-induced chronic active hepatitis.

The mechanism of drug-induced liver injury generally includes two aspects: one is the damage of the liver to the liver, that is, the essential liver poisoning, and the other is the liver damage related to the patient's specific constitution. Essential hepatotoxic drugs have a hepatotoxic effect on most people and are predictive of poisoning. This type of liver injury can be divided into direct liver injury and indirect liver injury. Direct liver damage drugs not only cause liver damage, but also cause damage to multiple organs such as the gastrointestinal tract, kidney, lung, pancreas, brain, and heart.

Indirect liver injury drugs interfere with certain aspects of normal metabolism of liver cells, such as inhibition of enzyme activity or a certain secretory process. Hepatic lesions vary widely and may resemble hepatitis-like lesions, cholestasis or mixed lesions. Liver cell necrosis and degeneration occur several hours to several days after administration, and the incidence is high, and the damage is related to the dose. Indirect hepatocyte injury types can be further divided into cytotoxic and cholestatic forms. The cytotoxic type is mainly to selectively interfere with a certain part of the metabolism of liver parenchyma cells, which affects protein synthesis and causes hepatic steatosis. Cholestatic mainly interferes with the excretion of bilirubin into the bile duct or interferes with the uptake of bilirubin by the liver. The jaundice may be obstructive or hepatocellular.

Liver damage associated with idiosyncratic conditions occurs only in patients who are particularly sensitive to drugs. The incidence is low, the incubation period is long, and the injury is not related to the dose. Therefore, it is also called unpredictable liver injury. For example, anti-tumor drug-induced liver damage is most common in essential liver poisoning, and liver damage due to idiosyncratic is rare.

Drug-induced liver disease can be asked to receive a history of treatment, clinically in addition to liver damage or jaundice, often accompanied by other systemic symptoms, such as kidney damage, myelosuppression, nervous system dysfunction, may occur before or during liver damage Systemic drug allergy signs, such as fever, rash, joint pain, increased peripheral white blood cell count, and eosinophilia. Drug-induced intrahepatic cholestatic syndrome, mainly jaundice, itchy skin, mild systemic weakness, moderately elevated blood bilirubin, elevated blood cholesterol and alkaline phosphatase, mild alanine aminotransferase activity or Moderately increased, the peak of transaminase rise appears after the peak of bilirubin rise. When the jaundice is deepened, the liver does not shrink, but the condition is not heavy.

When there are signs of liver damage such as elevated serum transaminase levels or decreased albumin levels, different liver protection drugs may be administered. Reduced glutathione (GSH) is a tripeptide compound composed of glutamic acid, cysteine and glycine residues. It is a biologically active peptide widely present in mammals and is an important cell. Metabolic regulators. The liver is the main synthesis and consumption site of GSH in the body. When the liver is damaged by toxic substances, it can induce a large amount of free radicals such as superoxide ions and peroxides. GSH provides active sulfhydryl groups or is directly catalyzed by enzymes. In combination with free radicals, oxygen free radicals in tissues can be removed, thereby reducing damage to the liver. In addition to enhancing the clearance of free radicals and protecting hepatocytes, GSH also loads unbound bilirubin into microsomes under the action of GSH thiol transferase, and binds it to glucuronidase to form conjugated bilirubin. Excreted in vitro, the methionine content of the liver is maintained by the γ-glutamine cycle, which ensures the conversion of thiol and transamination, improves liver synthesis, detoxification, fat metabolism, bilirubin metabolism and inactivated hormones, and promotes gallbladder. Acid metabolism, thereby accelerating the recovery of liver function. During chemotherapy, GSH can also directly combine with the metabolites of chemotherapeutic drugs to form low-toxic products, and enhance the tolerance of hepatocytes to oxygen free radicals, thereby protecting and restoring liver cells, but at the same time due to large consumption. Directly leads to a decrease in GSH concentration.

Oxidized glutathione (GSSG) is formed by the oxidation of GSH and the incorporation of a sulfhydryl group on two GSH monomers to form a disulfide bond. In biological media, GSSG is metabolized by NADP*H ⁺ dependent glutathione reductase, which cleaves the two-gram bond of GSSG to form two molecules of GSH. GSSG is a natural derivative that acts at lower concentrations and is a component of the GSH pathway. It is an important cellular system component that regulates the redox state of cells. The main role of this redox coupling is to regulate protein function by forming a disulfide complex (reversible) between the protein cysteine and GSH, a process known as glutathionization. Thus, changes in the ratio of intracellular GSH/GSSG can affect and participate in a variety of physiological responses to signaling pathways (including gene expression, cell proliferation, growth arrest, and apoptosis). The role of gene expression, including regulation of gene transcription factors such as NFKB and AP-1, has been shown to play an important role in the regulation of many genes involved in immune and inflammatory responses, including cytokines and growth factors. The activity of other immune/inflammatory regulatory proteins is also sensitive to GSH/GSSG (eg, mitogen-activated protein kinases, MAPK's). In addition, changes in GSH/GSSG regulate protein phosphorylation in signaling pathways, further expanding the effects of reduction-oxidation changes on cell function. GSSG induces glutathionization of proteins and changes in signaling pathway protein functions in cell-free and intracellular systems. Although not permeable to the cell membrane, the addition of GSSG allows a rapid change in the intracellular GSH concentration by altering the equilibrium of the disulfide mixture with the protein thio group.

There is still a lack of good drug control methods for liver damage caused by most drugs. Summary of the invention It is an object of the present invention to provide a pharmaceutical composition for treating liver damage and a process for the preparation thereof.

The pharmaceutical composition provided by the present invention consists of oxidized glutathione disodium and inosine.

Oxidized glutathione disodium, its chemical name: di-(γ-L glutamyl)-L-cysteine-d-glycine disodium, molecular formula: C ₂ . H ₃ . N ₆ 0 ₁₂ Na ₂ S ₂ , molecular weight: 656.59, the chemical structural formula is as follows:

Preferably, the ratio of the amount of the substance of oxidized glutathione disodium and inosine is 1: (5 - 5). More preferably, the ratio of the amount of the substance of oxidized glutathione disodium to inosine is 1:1. The invention also provides a pharmaceutical formulation comprising the pharmaceutical composition and a pharmaceutically acceptable excipient or carrier.

Preferably, it is an injection or a lyophilized powder injection.

More preferably, the content of the main component per ml of the preparation is 21.3 mg of oxidized glutathione disodium and 8.7 mg of inosine.

Another object of the present invention is to provide the use of the pharmaceutical composition for the preparation of a medicament for the treatment of liver damage.

The composition of the present invention is preferably administered at a dose of 10 mg to 40 mg per day (based on oxidized glutathione disodium) in the treatment of drug-induced liver damage, and is administered by the injection route.

The invention also provides a preparation method of the pharmaceutical composition, comprising the following steps: Step 1: Mixing reduced glutathione with water for injection, adding sodium hydroxide to adjust the pH of the solution at 10 ° C ~ 25 ° C To 4 ~ 6;

Step 2: Add oxidant to the solution at 10 ° C ~ 25 ° C, and continue to stir the reaction; add glycosides to the solution, mix and filter with a filter, and freeze-dry.

Preferably, the oxidizing agent in step 2 is a 2% to 5% hydrogen peroxide solution, and the ratio of the amount of reduced glutathione to hydrogen peroxide is 2:1. Preferably, the membrane pore size of step 2 is 0.22 μηι.

The obtained oxidized glutathione disodium salt solution is mixed with inosine, filtered, lyophilized, reconstituted, and sterile-filtered to constitute the pharmaceutical composition of the present invention.

The pharmacodynamic test showed that the pharmaceutical composition of the present invention is useful for treating liver damage caused by drugs and immune liver fibrosis, and has liver protection effects such as liver damage caused by drugs, and the curative effect is remarkable. The preparation process of the invention is reasonable and reliable, the quality is stable, the safety is high, and the invention has broad application prospects. detailed description

The invention discloses a pharmaceutical composition for treating liver injury and a preparation method thereof, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The products, methods, and applications of the present invention have been described in terms of the preferred embodiments thereof, and it is obvious that the methods and applications described herein may be modified or appropriately modified and combined without departing from the spirit, scope and scope of the invention. The techniques of the present invention are implemented and applied.

In order to make those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments. Example 1: Preparation Process of Oxidized Glutathione Disodium

Using reduced glutathione as a starting material, use a sodium hydroxide solution (5% by mass to 5% to 20%) to adjust the pH value of the solution (to ρΗ is 4 to 6), and then in the oxidant (preferably 2% ~) Under the action of 5% hydrogen peroxide solution, the reaction produces oxidized glutathione disodium (the reaction temperature is about 10 ° C ~ 25 ° C when reacting with hydrogen peroxide). The yield was determined to be 98% or more by high performance liquid chromatography. The preparation process is reasonable and reliable, the tube is easy to operate, the impurity content is low, and the product quality is stable and controllable. Example 2: Preparation process of the pharmaceutical composition of the present invention

Weigh 666.5g of reduced glutathione raw material, add 1000 ~ 2000ml of water for injection, mix Evenly suspended. Add sodium hydroxide solution to the solution at 10 ° C ~ 25 ° C to clarify the solution and adjust the pH of the solution to 4 ~ 6. Add 10% ~ 5% hydrogen peroxide solution 1000 ~ 2000 ml to the solution at 10 ° C ~ 25 ° C, stirring continuously to make the reaction. After the oxidation was completed, 291.7 g of the glycoside and an appropriate amount of water for injection were added to the solution, mixed, filtered through a 0.22 μηη filter, and lyophilized in a lyophilizer. The lyophilized powder is reconstituted and diluted with water for injection, filtered and dispensed. Example 3: Experimental study on the drug composition of the present invention inhibiting sputum, Ν-dimercaptonitrosamine-induced liver injury in rats

Sixty male Sprague-Dawley rats, 160g ~ 180g, were randomly divided into 6 groups (10 in each group), which were the normal group, the model group, and the oxidized glutathione disodium drug composition (10.8 mg/kg, among them). Oxidized glutathione disodium and inosine accounted for 71% and 29% of the total mass, respectively, the same as), medium (5.4mg/kg), low (2.7mg/kg) dose group, positive control group (for injection) Reduced glutathione, 108mg/kg, produced by Chongqing Pharmaceutical Pharmaceutical Co., Ltd.). Except the normal group, the other groups were prepared for liver injury model: 0.5% dimercaptonitrosamine solution (199 times saline diluted) was injected intraperitoneally at a dose of 10 mg/kg once every two days for 4 weeks. 14 times. At the same time as the model was prepared, each drug-administered group was injected with the corresponding drug once a day for 4 weeks, and the normal group and the model group were given the same amount of normal saline.

At the end of the fourth week, the animals were sacrificed and blood was taken for detection of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), and total bilirubin levels. The left hepatic lobe tissue was obtained. The homogenate was used to detect the content of hydroxyproline (Hyp), and a part was fixed with 10% furfural for pathological examination.

The results of the effects of the pharmaceutical composition on liver function in rats with liver injury are shown in Table 1.

Table 1

Dose AST (U / ALT (U / TP (g / L ALB (g / Hyp (g / g group n

Mg/kg L) L) ) L) )

Normal 10 - 179.40 soil 100.00 soil 68.89 soil 27.74 soil 0.27 soil group 26.55 27.19 2.40 0.92 0.09 model 9 NS 684.6 soil 558.0 soil 49.03 soil 21.96 soil 0.58 soil group 315.86 229.76 2.91 1.02 0.18 Positive 9 108 377.1士329.56士52·84士21.97士0.41士对照231.03* 89.31* 6.12 4.00 0.11 * Group

High dose 9 10.8 258.22 士 224.6士 53.37士 21.43士 0.41士量组 48.27** 112.18* 5.43 2.90 0.10*

*

Medium agent 9 5.4 466.4士 286.67士 48.38士 19.16士 0.41士量组 232.33 68.71** 6.39 2.25 0.13* low agent 6 2.7 634.00 士 503.67± 48.73 士 21.52士 0.56士量组 392.12 204.80 9.49 7.43 0.14

Note: * indicates that compared with the model group, P < 0.05; ** indicates that compared with the model group, P < 0.01. The high, medium and low dose groups refer to the high, medium and low dose groups of the oxidized glutathione disodium pharmaceutical composition.

Table 1 shows that compared with the normal group, the levels of ALT, AST, and Hyp in the model group were significantly increased, and the contents of TP and ALB were significantly decreased, indicating that the liver function was significantly decreased after modeling. Compared with the model group, the ALT and AST levels were significantly lower in the high-dose group, and the ALT level was significantly lower in the middle-dose group. The Hyp content in the high- and medium-dose groups was significantly lower.

Liver tissue fibrosis lesions can be divided into five stages according to their severity: SO, no fibrotic lesions; S1, fibrosis around the portal area, local sinus or intralobular fibrosis, does not affect the integrity of the lobular structure; S2, portal area Peripheral fibrosis, fibrous interstitial formation, but still retain most of the lobular structure; S3, a large number of interfiber spaces, segmentation and destruction of hepatic lobule, resulting in lobular structural disorder, but no cirrhosis; S4, early cirrhosis, extensive destruction of liver parenchyma Diffuse fibrosis, segmented hepatocyte masses with varying degrees of regeneration, and pseudolobule formation.

The pathological stage of liver fibrosis in each group in the experiment is shown in Table 2.

Table 2

Group n dose liver fibrosis pathological staging

Mg/kg SO SI S2 S3 S4 normal group 10 - 10 0 0 0 0 model group 9 NS 0 1 2 3 3 Positive control 9* 108 1 5 3 0 0 group

High dose group 9* 10.8 2 5 2 0 0 medium dose group 9* 5.4 1 5 3 0 0 low dose group 6 2.7 1 2 3 0 0

Note: * indicates that compared with the model group, P < 0.05. The high, medium and low dose groups refer to the high, medium and low dose groups of the oxidized glutathione disodium drug composition.

In the normal group, the hepatocyte bundles were arranged neatly, the structure of the hepatic lobule was clear, the morphology of the hepatocytes was intact, no hepatocyte degeneration, necrosis and fibrosis were observed, and the hepatic sinus was not dilated and hemorrhagic. Only a small amount of collagen fibers were seen in the portal area and the central vein wall. In the model group, extensive hemorrhage occurred in the liver tissue of rats, and there were a large number of hepatocytes necrosis. There were a large number of inflammatory cells in the necrotic foci, a large number of connective tissue hyperplasia, and collagen fibers were enlarged. Most of the rat liver lobular structures were destroyed, and pseudolobule formation was observed. The above-mentioned pathological changes were significantly alleviated in the high- and medium-dose groups of the pharmaceutical composition as well as in the positive control group. Example 4: In vivo efficacy test of the pharmaceutical composition against rat immunological liver fibrosis

132 Wistar male rats aged 4-5 weeks were randomly divided into normal group and model group. After the successful induction of liver fibrosis by pig serum, the rats were divided into inosine group according to the equilibrium random method (0.1566mg-100g). " ¹ , Guangdong Zhaoqing Xinghu Bio-Technology Co., Ltd.", GSSG group (0.3834mg.l00g ^-1 , homemade), high-dose group of drug composition (1.08mg.l00g ^-1 ), medium dose group (O^Amg .lOOg- ¹ ) Low dose group (OJ mg.lOOg- ¹ ), model group (normal saline, O.lml.lOOg- ¹ , produced by Jinan Limin Pharmaceutical Co., Ltd.), GSH group (10.80mg.100g- ¹ L.lml.100g, the group of the GSH (0.383mg'100g ^-1 ) + inosine ( 0.157mg '100g ^-1 ) and the natural recovery group, a total of 10 groups, the normal group was given 0. lml.100g In addition to the natural recovery group, the other groups were administered once a day for several weeks, for 6 weeks. After the end of the administration, the rats were sacrificed, liver function was measured, and the liver index (the ratio of liver weight to body weight) was calculated. The content of Hyp was determined, and histopathological changes were observed by pathological section. Statistical software SPSS11 was applied. 5 Statistical analysis was performed. The measurement data were expressed as the mean standard deviation. The grade data was expressed by the rank sum test. P < 0.05 indicates that the difference was statistically significant. The serum liver function indexes of each group were ALT, AST, A/G (the ratio of albumin to globulin), and the liver index and Hyp value are shown in Table 3.

table 3

* indicates that compared with the model group, Ρ <0.05; ** indicates that compared with the model group, Ρ < 0.01. The high, medium and low dose groups refer to the high, medium and low dose groups of the oxidized glutathione disodium drug composition. The liver histopathological comparison of each group is shown in Table 4.

Table 4

* indicates P<0.05 compared with the model group; ** indicates P<0.01 compared with the model group. , medium and low dose groups refer to high, medium and low doses of oxidized glutathione disodium drug composition Volume group.

The results showed that the ALT, AST, A/G values of the groups were significantly lower than those of the model group, and the high and medium dose groups of the normal group were significantly lower (PO.01). Except the natural recovery group, the other groups also had some Decrease (PO.05); compared with the model group, the liver index of each group was significantly lower in the high and middle dose groups (PO.01), except for the natural recovery group, the other groups were also reduced (PO .05); Compared with the model group, the normal group, the high and middle dose groups of the drug group were significantly lower (PO.01), and the other groups were also reduced (PO. 05); Compared with the model group, the pathological histology of the normal group and the high- and medium-dose group of the drug group was significantly improved (PO.01), and the improvement effect was better than other groups. In conclusion, under these conditions, high, medium and low doses of oxidized glutathione disodium-containing pharmaceutical compositions have an antagonistic effect on immune liver fibrosis in rats, among which the high- and medium-dose groups are superior. Other groups such as reduced glutathione.

As can be seen, the oxidized glutathione disodium-containing pharmaceutical composition of the present invention can significantly improve drug-induced liver damage. The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. It should be considered as the scope of protection of the present invention.

Claims

Rights request

A pharmaceutical composition comprising oxidized glutathione disodium and gemini.

The pharmaceutical composition according to claim 1, wherein the ratio of the amount of the oxidized glutathione disodium to the glutamic acid is 1: (5 - 5).

The pharmaceutical composition according to claim 1, wherein the ratio of the amount of the substance of oxidized glutathione disodium to inosine is 1:1.

A pharmaceutical preparation comprising the pharmaceutical composition according to any one of claims 1 to 3 and a pharmaceutically acceptable excipient or carrier.

The pharmaceutical preparation according to claim 4, which is an injection or a lyophilized powder injection.

The pharmaceutical preparation according to claim 4, wherein the content of the main component per ml of the preparation is 21.3 mg of oxidized glutathione disodium and 8.7 mg of inosine.

7. Use of a pharmaceutical composition according to claim 1 for the manufacture of a medicament for the treatment of liver damage.

8. The method for preparing a pharmaceutical composition according to claim 1, comprising the steps of: Step 1: mixing the reduced glutathione with water for injection, and adding a sodium hydroxide adjusting solution at 10 ° C to 25 ° C; pH to 4 ~ 6;

Step 2: Add oxidant to the solution at 10 ° C ~ 25 ° C, and continue to stir the reaction for 1 ~ 2 h; add inosine to the solution, mix and filter with a filter, and freeze-dry.

The preparation method according to claim 8, wherein the oxidizing agent is a 2% to 5% hydrogen peroxide solution, and the ratio of the amount of the reduced glutathione to the hydrogen peroxide is 2 :1.

The preparation method according to claim 8, wherein the membrane pore size of the step 2 is 0.22 μm.