CN104042603B - Application of the γ-aminobutyric acid on the drug for the intravascular hemolysis that preparation prevention and treatment puerarin injection induces - Google Patents

Abstract

The present invention relates to the compatibility application methods of γ-aminobutyric acid and Puerarin, the application method is that the injection containing Puerarin is made in γ-aminobutyric acid and Puerarin compatibility, or first use γ-aminobutyric acid, Puerarin is used again, the sporadic haemolysis adverse reaction for effectively overcoming puerarin injection improves the safety of puerarin injection administration.

Description

The role of γ-aminobutyric acid in preventing and treating intravascular hemolysis induced by puerarin injection drug application

technical field

The invention relates to a new medical application of gamma-aminobutyric acid, in particular to application in puerarin injections.

Background technique

γ-Aminobutyric acid (GABA for short) is a four-carbon non-protein amino acid widely present in animals and plants. According to literature records, GABA was discovered from potatoes as early as 1949, and GABA was found in animal brains in 1950. In the nerve center, 1/3 of the nerve synapses use GABA as the transmitter, and there is a close relationship with the cerebral cortex, pituitary, hypothalamus, reproductive organs, liver and kidneys. It has many benefits to the human body, such as direct or indirect effects on cardiopulmonary blood vessels, regulating blood pressure and heart rate; nourishing the development of the nervous system, promoting the synthesis of membrane proteins; widely used in the treatment of epilepsy, Parkinson's, Huntington's disease and other diseases; Promote cell metabolism, improve brain vitality; inhibit glutamic acid decarboxylation, reduce blood ammonia, protect liver; reduce neuron activity, calm nerves, inhibit anxiety and other diseases; also protect kidneys, anti-aging, promote growth hormone secretion, and prevent obesity , Promote alcohol metabolism and other effects. It has multiple physiological activities such as protecting the liver, benefiting the kidney, strengthening the brain, and delaying aging, and is used in the fields of medicine, health food, new resource food, and feed additives (see "γ-aminobutyric acid" written by Yang Jingjing, Qu Yuan, and Cui Xiuming) Research progress on the preparation method and content determination of "Food Industry Science and Technology, 2014, 35(3): 351-356).

Puerarin is a monomer-isoflavone compound, 4',7-dihydroxy-8-β-D glucosyl isoflavone, extracted from the dried root of Pueraria mirifica. Clinically widely used in the treatment of cardiovascular diseases. The pharmaceutical preparations containing puerarin currently used clinically are mainly injections. With the widespread use of puerarin, there have been more and more reports on adverse reactions of puerarin in recent years, which has aroused widespread concern in the medical field. Through the analysis of adverse reactions of traditional Chinese medicine in the past 20 years, puerarin injection ranked 18th. The National Adverse Drug Reaction Monitoring Center reported the adverse reactions of puerarin injection in the third issue of "Adverse Drug Reaction Information Bulletin" in January 2003. Most researchers believe that puerarin injection is formulated with 50% propylene glycol as a solvent, which inevitably leads to insufficient purity due to differences in extraction processes, techniques, etc., and the introduction of impurities leads to various reactions (see "To Analysis of Adverse Reactions in Clinical Use of Puerarin Injection", Shanghai Journal of Traditional Chinese Medicine, 2006, 40(8): 71-72; "Clinical Application and Adverse Reactions of Puerarin" written by Xu Shiguo, Shizhen Guoyi Guoyao, 2005 , 16(12):1307-1308). After analyzing 63 cases of puerarin adverse reactions reported in major domestic medical journals from 2000 to 2004, it was found that there were 40 males and 23 females; the age ranged from 34 to 81 (57.5±23.5) years. All cases were administered by intravenous infusion. The administration dose is 0.4-0.6 g, and the drug diluents are 5% glucose solution, normal saline, 5% glucose saline and the like. The fastest reaction time was 3 minutes, and the slowest was 13 days, all of which occurred during the administration process. There were 47 cases of response to the first administration, and 16 cases of response to repeated administration. Statistics show that common adverse reactions include allergic reactions (heat, trembling, etc., 24 cases), anaphylactic shock (4 cases), hemolytic anemia (13 cases), liver damage, kidney damage (7 cases), drug fever (10 cases ), death (5 cases). All cases had no history of allergies, and all recovered after drug withdrawal after the reaction occurred (except for the death cases). The dosage and diluent used are within the range indicated in the drug instructions, and all reactions must be caused by puerarin. The occurrence of adverse reactions has nothing to do with the disease, age and gender, and little relationship with the drug diluent. It is related to the difference in the constitution of patients, especially the elderly and infirm. The duration of the reaction is related to the slowness of the onset of the traditional Chinese medicine preparation (see "Literature Analysis of 63 Puerarin Adverse Reactions" written by Quan Xinrong, Modern Journal of Integrated Traditional Chinese and Western Medicine, 2005, 14(1): 140). A patient developed acute renal insufficiency and hemolytic anemia after intravenous infusion of puerarin for about 10 minutes (see "Hemolytic Anemia Induced by Puerarin" by Guan Minghua, Journal of Adverse Drug Reactions, 2003, 5: 291). Therefore, the hemolytic anemia caused by puerarin is caused by puerarin itself.

Contents of the invention

The purpose of the present invention is to provide a new application of gamma-aminobutyric acid medicine, that is, the application of gamma-aminobutyric acid in puerarin injection, and gamma-aminobutyric acid can effectively prevent and treat adverse reactions of intravascular hemolysis induced by puerarin.

In fact, the present invention relates to the application of gamma-aminobutyric acid in the preparation of puerarin injection compound preparations, and it can also be temporarily used in combination with puerarin injections in the form of injection and infusion, or in the application of gamma-aminobutyric acid in the form of injection and infusion. GABA was followed by puerarin injection.

In order to achieve the above object, the technical solution adopted in the present invention is: the new application of gamma-aminobutyric acid in the preparation of puerarin injection. γ-aminobutyric acid and puerarin injections are temporarily applied in the form of injection and infusion. After applying gamma-aminobutyric acid in the form of injection and infusion, the application of puerarin injection is followed.

The relevant content in the above-mentioned technical scheme is explained as follows:

1. In the above scheme, the injection contains the following medicines in parts by weight: 1-50 parts of γ-aminobutyric acid, and 1-500 parts of puerarin.

2. In the above scheme, the injection also contains 1-50 parts by weight of excipients and 1-10000 parts by weight of water for injection. The auxiliary material is sodium bicarbonate solution, glucose solution, propylene glycol solution, glucose saline, sodium chloride injection or physiological saline.

3. In the above scheme, the injection can be a clinically acceptable injection, powder injection or infusion.

4. In the above scheme, the preparation method of the injection is as follows:

The powder injection is prepared by mixing and sterilizing γ-aminobutyric acid and puerarin; or mixing γ-aminobutyric acid, puerarin and sodium chloride, dissolving in water for injection, and then adding hydrochloric acid or sodium bicarbonate The solution is adjusted to a pH value of 5.0-8.5, filtered, and the filtrate is sterilized and filled in a powder ampoule to be sterilized again;

The injection is mixed with γ-aminobutyric acid, puerarin and sodium chloride, dissolved in water for injection, then adjusted to pH 5.0-8.5 with hydrochloric acid or sodium bicarbonate solution, filtered, and the filtrate is sealed in an ampoule for sterilization be made of. Or gamma-aminobutyric acid is composed of 5% glucose solution, 5% glucose saline, propylene glycol solution (formed by volume ratio (1:1) of propylene glycol and normal saline, containing propylene glycol 0.5mL/mL), sodium chloride injection solution or saline solution, then mixed with puerarin injection, filtered, and the filtrate was sealed in ampoules and sterilized.

The infusion is mixed with γ-aminobutyric acid, puerarin and sodium chloride, dissolved in water for injection, then adjusted to a pH value of 5.0-8.5 with hydrochloric acid or sodium bicarbonate solution, dissolved, filtered, and the filtrate is potted in saline glass Sterilized in bottles. Or gamma-aminobutyric acid is composed of 5% glucose solution, 5% glucose saline, propylene glycol solution (formed by volume ratio (1:1) of propylene glycol and normal saline, containing propylene glycol 0.5mL/mL), sodium chloride injection solution or saline solution, then mixed with puerarin injection, filtered, and the filtrate was sealed in ampoules and sterilized.

5. In the above scheme, the temporary combined application refers to infusion or injection mixed with γ-aminobutyric acid at the same time as the application of puerarin injection in the hospital.

6. In the above scheme, the application of γ-aminobutyric acid in the form of injection and infusion followed by the application of puerarin injection refers to the application of γ-aminobutyric acid in the form of injection and infusion in the hospital, followed by Puerarin injections are then applied in the form of injections and infusions.

Puerarin has the effects of dilating coronary arteries and cerebrovascular, reducing myocardial oxygen consumption, improving microcirculation and anti-platelet aggregation. It is clinically used as an adjuvant treatment for coronary heart disease, angina pectoris, myocardial infarction, retinal arteriovenous occlusion, sudden deafness, ischemic cerebrovascular disease, viral myocarditis in children, diabetes, etc. Puerarin can also be compatible with γ-aminobutyric acid to make compound puerarin injection for the prevention and treatment of various diseases, such as for the prevention and treatment of diabetic peripheral neuropathy, hypertension combined with diabetes, diabetic nephropathy, acute cerebral infarction, stable angina pectoris, lower extremity Deep vein thrombosis and vertigo due to vertebrobasilar insufficiency. However, puerarin in puerarin-containing injections can easily cause intravascular hemolysis. γ-aminobutyric acid not only has multiple physiological activities such as protecting the liver, benefiting the kidney, strengthening the brain, and delaying aging, but the inventors have confirmed through research that γ-aminobutyric acid also has the ability to antagonize the adverse reaction of intravascular hemolysis induced by puerarin, improve The safety of injection administration of puerarin.

Further illustrate the purpose of the present invention and the effect that can be achieved in conjunction with some tests below.

In this experiment, the hemolysis test was pre-tested with reference to the "Technical Guidelines for the Irritation and Hemolysis Research of Traditional Chinese Medicines and Natural Medicines". It was found that 8mM puerarin could cause 10% of sheep red blood cells to hemolyze, and the probability of hemolysis in vitro was 100%. The hemolysis test results were all reproduced, indicating that the method can be repeated. This method can be used to discover the potential accidental hemolysis of injections, and is a feasible test method for the development of new drugs for injections to determine whether there is occasional hemolysis, which is conducive to reducing the occurrence of adverse reactions and improving the safety of injections.

1. The role and effect of γ-aminobutyric acid in antagonizing the hemolytic adverse reactions of puerarin-containing injections

(1) Hemolysis test of puerarin injection

To study the effect of γ-aminobutyric acid combined with puerarin on sheep erythrocytes. Take the red blood cells of 10 small-tailed Han sheep, add a certain dose of γ-aminobutyric acid and puerarin respectively, observe the state of red blood cells and the incidence of hemolysis after 10 minutes, and use the X2 test to statistically analyze the experimental results to explore the effect of γ-aminobutyric acid on Antagonism of sporadic hemolysis by puerarin injection. The results reported below:

1 Experimental materials

1.1 Test drugs

γ-Aminobutyric acid (GABA) injection ①: γ-aminobutyric acid was purchased from BAOXIN Company, CAS Number: 56-12-2. Accurately weigh 1.031g of GABA with an electronic balance, dissolve it in normal saline solution, and dilute the GABA solution to 10mL with a 10mL volumetric flask. This is injection ① with a concentration of 1M. Sterilize by filtration with a microporous membrane (0.22 μm) and store at 4°C.

Injection ②: Dilute injection ① to 100mM with sterilized normal saline, 10mL, and store at 4°C for later use.

Injection ③: Dilute injection ① to 10mM with sterilized physiological saline, 10mL, and store at 4°C for later use.

Puerarin injection ④: Provided by Zhejiang Kangenbei Pharmaceutical Co., Ltd., 2mL, product batch number 090501, each mL injection contains 50mg of puerarin.

Puerarin injection (250mM)⑤: Accurately weigh 1.041g of puerarin powder (purchased from Jiangsu Tiansheng Pharmaceutical Co., Ltd.) with an electronic balance, dissolve with 40% dimethyl sulfoxide (DMSO, DZ0231, AMRESCO), and ultrasonically assist , diluted to 9mL with normal saline solution, and the volumetric flask was set to 10mL, and the color of the solution was colorless (configuration environment: performed in a clean bench). The solvent is 40% DMSO solution. Sterilize by filtration with a microporous membrane (0.22 μm) and store at 4°C.

Compound Puerarin Injection ⑥: Take 0.04mL of Injection ① and 0.27mL of Injection ④, mix well to make a total of 0.31mL to form Injection ⑥. Each mL injection contains 43.55mg of puerarin and 13.30mg of γ-aminobutyric acid.

Compound Puerarin Injection ⑦: Take 0.04mL of injection ② and 0.27mL of injection ④, mix well to make a total of 0.31mL to form injection ⑦. Each mL injection contains 43.55mg of puerarin and 1.33mg of γ-aminobutyric acid.

Compound Puerarin Injection ⑧: Take 0.04mL of Injection ③ and 0.27mL of Injection ④, mix well to make a total of 0.31mL to form Injection ⑧. Each mL injection contains 43.55mg of puerarin and 0.133mg of γ-aminobutyric acid.

Compound Puerarin Injection ⑨: Take 0.04 mL of Injection ① and 0.13 mL of Injection ⑤, mix well, and make a total of 0.17 mL to form Injection ⑨. Each mL injection contains 79.61mg of puerarin and 24.26mg of γ-aminobutyric acid.

Compound Puerarin Injection ⑩: Take 0.04mL of Injection ② and 0.13mL of Injection ⑤, mix well, and make a total of 0.17mL to form Injection ⑩. Each mL injection contains 79.61mg of puerarin and 2.426mg of γ-aminobutyric acid.

Compound Puerarin Injection : Take 0.04mL of injection ③, 0.13mL of injection ⑤, mix well, a total of 0.17mL to form injection . Each mL injection contains 79.61mg of puerarin and 0.2426mg of γ-aminobutyric acid.

1.2 Experimental animals

Ten small-tailed Han sheep (20-30Kg) were provided by the Animal Experiment Center of Northwest A&F University. Raise them at a room temperature of 15-25°C and a relative humidity of 50%.

1.3 Reagents and instruments

Sodium chloride injection, batch number: 31213042402, produced by Shiyao Yinhu Pharmaceutical Co., Ltd.; water for injection, self-made; TGL-16B high-speed centrifuge, Hunan Xingke Scientific Instrument Co., Ltd.; HPY-01B biochemical incubator, Huangshi Hengfeng Medical Instruments Co., Ltd.; BIO-RAD680 microplate reader; enzyme label strips were purchased from Jiangsu Haimen Sanhe Xingya Medical Equipment Factory and purchased from Shaanxi Yangling Baoxin Equipment Co., Ltd.

2 methods

2.1 Preparation of red blood cell suspension

10 small-tailed Han sheep were subjected to hemolysis experiments. Collect 10 mL of jugular vein blood from each sheep, anticoagulate with 160 IU of sodium heparin (Jiangsu Wanbang Biochemical Pharmaceutical Co., Ltd.), place in a graduated centrifuge tube, centrifuge at 2000 rpm for 10 minutes, discard the plasma, and add an appropriate amount of sodium chloride The injection was washed, and the supernatant and buffy layer were discarded by centrifugation. Add an appropriate amount of normal saline, shake well, and centrifuge. Repeat this process for 3 times until the supernatant is colorless and transparent after centrifugation. The obtained packed erythrocytes were diluted with physiological saline to an 11% (volume ratio) erythrocyte suspension. At this point, the red blood cell suspension is ready.

2.2 In vitro hemolysis experiment design of puerarin solution

Assume that the concentration of puerarin solution acting on red blood cells is 8 mM (0.00333 g·mL ^-1 ). A negative control group, a solvent control group (DMSO was used instead of puerarin solution to act on red blood cells), a drug treatment group and a positive control group were set up. The pH value of the puerarin solution was determined to be 7.16 with a METTLERTOLEDO bench-top pH meter.

According to the table 1 below, add the corresponding ingredients into the test tubes respectively, paying attention to the order of adding the samples. Shake gently after each ingredient is added. After all the components in the system are added to the system, gently shake and mix, and place in a biochemical incubator at 37°C for incubation. Observe hemolysis and coagulation reaction after 10min. Judgment method of agglutination reaction: If there is reddish-brown or brown-red flocculent precipitate in the solution, which does not disperse after shaking, it indicates that agglutination of red blood cells occurs. Those who are shaken are truly agglomerated. Then each tube was centrifuged at 5000rpm/min, and the color of the supernatant was observed visually. The supernatant was taken, and the light absorption value (OD value) was detected at a wavelength of 540 nm. According to the 2005 "Technical Guidelines for the Irritation and Hemolysis Research of Chinese Medicines and Natural Medicines", the hemolysis rate (%) of each tube of each group was calculated according to the formula:

Hemolysis rate (%)=(OD value of drug treatment group-OD value of negative control group)/(OD value of positive control group-OD value of negative control group)

Reference evaluation criteria: hemolysis rate > 5% indicates that hemolysis occurs, and statistical processing is carried out.

Table 1 Grouping design of puerarin solution and γ-aminobutyric acid (GABA) in vitro hemolysis experiment

3 results

This experiment investigates the influence of 10mM, 1mM, 0.1mM γ-aminobutyric acid on 10 sheep red blood cells when the concentration of puerarin is 8mM. At the same time, the occasional hemolysis experiment of puerarin injection ④ and ⑤ is carried out. The experimental results are all recorded in detail. Statistical analysis was performed using the X2 test, and the results are shown in Table ² .

Table 2 Statistical table of 10 sheep hemolysis test results

Note: compared with normal saline negative group, a P<0.01; compared with puerarin injection group, b P<0.01

The results showed that no hemolysis occurred in the normal saline group, but all hemolysis occurred in the water for injection group: compared with the normal saline group, the puerarin injection groups ④ and ⑤ all had hemolysis, and the incidence of hemolysis was significantly different from that of the normal saline group (P<0.01); The hemolysis test results were all reproduced, indicating that the hemolysis test method is stable and reliable, and the test results can be repeated. Compared with puerarin injection ④, ⑤ groups, compound puerarin injection ⑥, ⑦, ⑧, ⑨, ⑩, No hemolysis occurred in each group, compound puerarin injection ⑥, ⑦, ⑧, ⑨, ⑩, The incidence of hemolysis in each group was significantly different from that of puerarin injection ④ and ⑤ groups (P<0.01); The incidence of hemolysis in each group was not different from that in the normal saline group (P>0.05); compound puerarin injection ⑥, ⑦, ⑧, ⑨, ⑩, The concentration of puerarin in each group was the same as that of puerarin injection ④ and group ⑤, both were 8mM, but compound puerarin injection ⑥ and ⑨, ⑦ and ⑩, ⑧ and They contained 10mM, 1mM, and 0.1mM γ-aminobutyric acid respectively, while the groups of puerarin injection ④ and ⑤ did not contain γ-aminobutyric acid. The above results suggest that the combination of different concentrations of γ-aminobutyric acid and puerarin can antagonize the adverse reaction of hemolysis induced by puerarin, and reduce the incidence to the level of normal saline.

The above experimental results show that puerarin can cause hemolysis, and injections containing puerarin may cause hemolysis. After adding γ-aminobutyric acid, the adverse reaction of hemolysis containing puerarin can be eliminated.

The above test results indicated that no hemolytic reaction occurred in the puerarin injection and the injection made from the combination of puerarin and γ-aminobutyric acid.

Therefore, adding gamma-aminobutyric acid when preparing puerarin-containing injection or applying puerarin injection can eliminate the hemolytic adverse reaction caused by puerarin and improve the safety of puerarin injection.

The advantages of the present invention are: when preparing puerarin-containing injections or applying puerarin injections, adding γ-aminobutyric acid can better antagonize the adverse reaction of intravascular hemolysis induced by puerarin, and it is cheap and free. Toxic and side effects, safe.

The present invention will be further described below in conjunction with embodiment:

Detailed ways:

Embodiment 1: (preparation of compound puerarin injection)

Take 10.31g of γ-aminobutyric acid and 10g of puerarin, add 9g of sodium chloride, add water to 1000mL, adjust the pH to 5.0-8.5 with 1M hydrochloric acid or sodium bicarbonate solution, filter, and seal the filtrate in 2, 5 or 10mL In an ampoule, sterilize at 100°C for 30 minutes to obtain an injection. This product can be used as an auxiliary treatment for coronary heart disease, angina pectoris, myocardial infarction, retinal artery and vein occlusion, sudden deafness, ischemic cerebrovascular disease, viral myocarditis in children, diabetes, etc. This product can be injected intravenously or intramuscularly, 1-500mL each time, 1-3 times a day, 10-20 days as a course of treatment, and can be used continuously for 2-3 courses of treatment.

Embodiment 2: (preparation of compound puerarin powder injection)

Take 25.78 g of γ-aminobutyric acid and 1 g of puerarin, fill them in a 10 mL powder injection ampoule, and sterilize at 100° C. for 30 minutes to obtain a powder injection. This product can be used as an auxiliary treatment for coronary heart disease, angina pectoris, myocardial infarction, retinal artery and vein occlusion, sudden deafness, ischemic cerebrovascular disease, viral myocarditis in children, diabetes, etc. When applying, mix 1-5000 mg of the prepared powder with sodium chloride injection or saline injection, and then inject intravenously or intramuscularly, 1-500 mL each time, 1-3 times a day, for 10-20 days It is a course of treatment and can be used continuously for 2-3 courses.

Embodiment 3: (preparation of compound puerarin infusion)

Take 4.12g of γ-aminobutyric acid, 2g of puerarin, and 9g of sodium chloride, add water to 1000mL, adjust the pH to 5.0-8.5 with 1M hydrochloric acid or sodium bicarbonate solution, filter, and seal the filtrate in a 250mL saline glass bottle. Sterilize at 100°C for 30 minutes to obtain an infusion solution. This product can be used as an auxiliary treatment for coronary heart disease, angina pectoris, myocardial infarction, retinal artery and vein occlusion, sudden deafness, ischemic cerebrovascular disease, viral myocarditis in children, diabetes, etc. In application, it can be directly injected intravenously or intramuscularly, 1-500 mL each time, 1-3 times a day, 10-20 days as a course of treatment, and can be used continuously for 2-3 courses of treatment.

The above-mentioned embodiments are only to illustrate the technical concept and characteristics of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and not to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention shall fall within the protection scope of the present invention.

Claims (6)

1. Application of gamma-aminobutyric acid in the preparation of medicines for preventing and treating intravascular hemolysis induced by puerarin injection. 2. The application according to claim 1, characterized in that: after the application of γ-aminobutyric acid, the puerarin injection is then used. 3. The application according to claim 1, characterized in that: the puerarin injection and the γ-aminobutyric acid injection are mixed evenly before application. 4. The application according to any one of claims 1-3, characterized in that it contains the following medicines in parts by weight: 1-50 parts of γ-aminobutyric acid, and 1-500 parts of puerarin. 5. The application according to any one of claims 1-3, characterized in that it contains 1-50 parts by weight of auxiliary materials. 6. The application according to claim 5, characterized in that: the auxiliary material is sodium bicarbonate solution, glucose solution, propylene glycol solution or sodium chloride injection.