CN102133215B - Application of butylphthalide in preparation of medicine for treating chronic alcoholism - Google Patents

Abstract

The invention relates to an application of butylphthalide or medicinal salts thereof or a medicinal composition containing any one of the butylphthalide and the medicinal salts thereof in preparing a medicament for preventing or treating chronic alcoholism. The medicine or the medicine composition has the characteristics of high efficiency, low toxicity and no addiction in the process of preventing or treating chronic alcoholism.

Description

Application of butylphthalide in preparation of medicine for treating chronic alcoholism

technical field

The invention relates to the application of butylphthalide or its medicinal salt or a pharmaceutical composition containing any one of them in the prevention and treatment of chronic alcoholism, belonging to the field of medicine.

Background technique

Chronic alcoholism refers to severe central nervous system poisoning caused by long-term excessive drinking. Alcohol is ethanol, with the chemical formula C ₂ H ₅ OH, which is a neurotropic substance. Since ethanol that enters the human body cannot be digested and absorbed, it will enter the brain along with the blood, destroy neuron cell membranes, weaken the central nervous system, and cause brain activity to slow down by activating inhibitory neurons and inhibiting activating neurons. A large number of nerve cells die. Excessive drinking can also lead to alcoholic coma.

Chronic alcoholism, also known as alcohol dependence or addiction, is a compulsive mental state of compulsion to drink, which can occur continuously or periodically, which can manifest as a craving for alcohol and a compulsive experience of often needing to drink, stop drinking After that, I often feel uncomfortable, restless, or have withdrawal symptoms such as limb tremors, nausea, vomiting, and sweating. These symptoms disappear quickly when drinking resumes. Due to long-term drinking, most of them are combined with physical damage, especially the heart, liver, and nervous system. The most common ones are liver cirrhosis, peripheral neuropathy, and epileptic seizures. In recent years, the number of patients with chronic alcoholism has been increasing, which has attracted the attention of the medical and sociological circles.

Current treatment of chronic alcoholism may involve one or more medications. Disulfiram (Disulfiram) can interfere with the metabolism of alcohol, drinking small amounts of alcohol can cause nausea, vomiting, confusion and difficulty breathing. Naltrexone can reduce dependence on alcohol, but it has relatively large side effects and should be used under the guidance of a doctor. Benzodiazepines are anti-anxiety drugs commonly used to treat alcohol withdrawal symptoms such as anxiety and insomnia, and are also used to prevent seizures and delirium. Be careful when using them because they can also be addictive. Tricyclic antidepressants can be used to control anxiety and depression from any cause, but because these symptoms may disappear with alcohol withdrawal, they are not used until after alcohol withdrawal if symptoms persist.

Contents of the invention

Therefore, the purpose of the present invention is to find a kind of highly efficient, low toxicity, non-addictive medicine that can effectively prevent or treat chronic alcoholism.

The compound involved in the present invention is butylphthalide (Butylphthalide, referred to as NBP, which can be left-handed, right-handed and racemic), with a chemical formula of C ₁₂ H ₁₄ O ₂ and a relative molecular weight of 190.24. The chemical structural formula is as follows:

Butylphthalide can protect mitochondrial function, improve brain energy metabolism, inhibit Ca ²⁺ influx, and is clinically used to treat mild and moderate acute ischemic stroke. According to the pharmacology, toxicology and clinical test results of butylphthalide in the treatment of ischemic stroke, it is known that butylphthalide is not addictive.

Unexpectedly, the inventors discovered through research that the compound butylphthalide has an effect on preventing or treating chronic alcoholism. In addition, after oral administration, the compound has good curative effect and is non-addictive, thereby overcoming the defects of current drugs for treating chronic alcoholism. Therefore, the present invention relates to the application of butylphthalide or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them in the preparation of a medicament for preventing or treating chronic alcoholism.

The butylphthalide of the present invention can exist in the form of L-isomer and/or D-isomer or racemate when applied.

Preferably, butylphthalide or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them can be formulated together with a pharmaceutically acceptable diluent or a pharmaceutically acceptable carrier before application.

Preferably, butylphthalide can be formulated into a soft capsule for oral application with, for example, an oily carrier. Butylphthalide soft capsules and a preparation method thereof are described in Chinese patent ZL 200310119336.1. The butylphthalide soft capsule is composed of capsule material and medicinal liquid oil, wherein the medicinal liquid oil is mainly composed of butylphthalide and vegetable oil as a diluent, and the weight ratio of the two is 1:1-10. The capsule material is mainly composed of rubber material, plasticizer and water, and the weight ratio of the three is 1:0.2-0.4:0.8-1.3.

Chinese patent ZL 200510136358.8 describes butylphthalide dropping pills and a preparation method thereof. Butylphthalide drop pills are composed of active ingredient butylphthalide, matrix, dispersant and coating material. The matrix is selected from polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 20000, poloxamer. The dispersant is selected from light micro silica gel and cross-linked povidone. The coating material is selected from hypromellose, hydroxypropyl cellulose, ethyl cellulose, Eudragit E ₃₀ D.

The preparation method of butylphthalide freeze-dried powder injection is described in Chinese patent ZL 200410012533.8. The freeze-dried powder injection of butylphthalide is mainly made of butylphthalide, emulsifier and co-emulsifier, excipient and water for injection, and its raw material weight ratio is butylphthalide 5-8: emulsifier 1-5: co-emulsifier Agent 0-1: excipient 4-10: water for injection 5-11. The preparation method is as follows: weigh the raw materials of each component according to the proportioning ratio, first add butylphthalide, emulsifier and co-emulsifier together with part of water for injection and stir evenly at high speed; another part of water for injection is taken to dissolve the excipient, adding 0.1% of the total weight Activated carbon, decarbonized after heating at 100°C for 15 minutes, then mixed the two solutions, added 0.01% activated carbon by total weight, filtered, decarbonized, added water for injection to the full amount, measured pH value, content, finely filtered drug The liquid is divided into bottles and freeze-dried to obtain the freeze-dried injection.

Butylphthalide tablet and preparation method thereof are described in Chinese patent ZL 200710062273.9. Butylphthalide tablets are mainly composed of butylphthalide solid powder and other pharmaceutical excipients, wherein the butylphthalide solid powder consists of 9-30% butylphthalide, 0.01-1.2% emulsifier, 70-90% Composition of cyclodextrin or cyclodextrin derivatives.

In addition, butylphthalide can also be reacted with an acid to form an ester, wherein the acid can be a pharmaceutically acceptable inorganic acid or organic acid. In addition, some esters can continue to react with acids or bases to form salts, which can increase water solubility and enhance drug efficacy, and can be prepared into injection preparations.

On the premise of ensuring the efficacy of the drug, butylphthalide is used as the main drug, and butylphthalide can also be used in combination with drugs or nutritional ingredients that have the same or similar functions in the treatment of chronic alcoholism to exert their synergistic effect and enhance the drug effect. Examples include vitamin A, B complex vitamins, vitamin C, carnitine, magnesium, selenium, zinc, and essential fatty acids and antioxidants, with the preferred nutrient being thiamine (vitamin B1).

In a preferred embodiment of the present invention, studied NBP to the alcohol dependent rat hippocampus hydrogen sulfide (H ₂ S) content and N-methyl-D-aspartic acid (NMDA) receptor 2B subunit (NR2B ) expression. 84 SD male rats were randomly divided into 6 groups. Except the normal group, each group drank 6% (v/v) alcoholic aqueous solution for 28 days, and after drinking 6% (v/v) alcoholic aqueous solution for 14 days, the NBP was low (10mg /kg), medium (20mg/kg), high (40mg/kg) dose group rats were fed with different doses of NBP, and the withdrawal symptom score, hippocampal tissue H2S content, cystathionine β-synthase (CBS) activity and Expression of NR2B. The results showed that the withdrawal symptom score (12.27±1.19), H ₂ S content (30.25±8.82), CBS activity (72.44±7.46) and NR2B mRNA expression (19.47±0.86) in the NBP middle-dose group were significantly higher than those in the experimental control group. Compared with rat withdrawal symptom score (14.09±2.21), H2S content (44.50±6.65), CBS activity (79.06±4.57) and NR2B mRNA expression (29.13±1.39), they were all lower, with significant difference (P< 0.05). Withdrawal symptom score (12.18±1.08), H ₂ S content (33.00±5.38), CBS activity (67.81±9.37) and NR2B mRNA expression (23.12±1.86) in the NBP high-dose group were comparable to those in the experimental control group. ratio, both decreased, with significant difference (P<0.05). Therefore, it can be concluded that NBP can alleviate the withdrawal symptoms of alcohol-dependent rats, which is related to NBP inhibiting the expression of H ₂ S/CBS system and inhibiting the expression of NR2B mRNA.

In animal experiments, the effective dose for the treatment of chronic alcoholism is about 20-40 mg/kg body weight. According to the conventional calculation method in this field, the amount is converted into 5-10 times when applied to the human body. In terms of butylphthalide, the dosage is 2 - 8 mg/kg body weight; preferably, the dosage is 5-7 mg/kg body weight; more preferably, the dosage is 7 mg/kg body weight.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Example 1 Effect of NBP on _H2S content and NR2B expression in the hippocampus of alcohol-dependent rats

Ca ²⁺ can increase the production of H ₂ S, especially the signal transduction pathway mediated by calcium ions/calmodulin can rapidly regulate the production of H ₂ S. Long-term alcohol exposure experiments in vivo and in vitro showed that the expression of NR2B subunits was increased, especially in cultured hippocampal and cortical neurons. In this experiment, the effects of NBP on alcohol-dependent rats were studied by observing the changes of hippocampal H ₂ S content, CBS activity, NR2B mRNA expression and withdrawal symptom score after NBP acted on alcohol-dependent rats.

1. Materials

1. Main instruments and reagents

The Bio-Tek ELx800 microplate reader was provided by American Bio-Tek Company, and the PCR amplification instrument was produced by American MyCycler Company. L-cysteine was produced by Sigma, USA. Trizol reagent was produced by Invintrogen Company of the United States. The butylphthalide preparation is produced by NBP Pharmaceutical Co., Ltd. of CSPC, and its trade name is NBP (hereinafter referred to as NBP).

2. Animals

Zhengzhou University Animal Center provided 84 clean-grade healthy Sprague-Dawley male rats (permit number: scxk (Yu) 2005-0001), weighing 120-160 g.

2. Method

1. Grouping and handling of experimental animals

84 rats were randomly divided into A: normal group, B: alcohol dependence model group, C: experimental control group, D: NBP low dose group, E: NBP medium dose group, F: NBP high dose group, 14 rats in each group . Except the normal group, each group drank 6% (v/v) alcohol solution for 28 days. After 14 days, NBP was diluted with vegetable oil and intragastrically administered once a day. The dosage was NBP 10mg/kg in the low-dose group, 20mg/kg in the middle-dose group, 40mg/kg in the high-dose group, and 5ml/kg of vegetable oil in the experimental control group. kg, the normal group had a normal diet, and the same amount of water was administered to the stomach.

2. Withdrawal score

Animals in each group were determined according to Erden et al. 1054.) Withdrawal Score Scale Observe withdrawal symptoms (stereotyped behavior, irritability, stiff tail, abnormal posture, audiogenic seizures) for 18 minutes 6 hours after the last drinking.

3. Indirect determination of H ₂ S content in hippocampal tissue by spectrophotometry

Measure the absorbance at a wavelength of 670nm with a fully automatic microplate reader, and calculate the _H2S content in the solution according to the _H2S standard curve. Ren Caili, Li Dongliang, Zhao Honggang, et al. Dynamic changes of endogenous hydrogen sulfide in brain tissue of rats with global cerebral ischemia-reperfusion. Chinese Journal of Cerebrovascular Diseases, 2008, 5: 177-181).

4. Indirect measurement of CBS activity in hippocampus by spectrophotometry

Use a fully automatic microplate reader to measure the absorbance at a wavelength of 670nm, and calculate the CBS content in the solution according to the CBS standard curve. The CBS activity in the tissue is represented by the amount of hydrogen sulfide generated per unit weight of tissue per unit time (nmol/g tissue/h) (Ren Caili, Li Dongliang, Zhao Honggang, et al. Dynamic changes of endogenous hydrogen sulfide in brain tissue of rats with global cerebral ischemia-reperfusion. Chinese Journal of Cerebrovascular Diseases, 2008, 5: 177-181).

5. RT-PCR detection of NR2B mRNA expression

According to the instructions of Trizol (invitrogen) reagent, the total mRNA of hippocampal tissue was extracted. cDNA first-strand synthesis by reverse transcription. Using cDNA as template, carry out PCR reaction. NR2B primer design: NR2B primers were designed by Primer 5.0 program, the primer sequence is: upstream primer: 5′-CTT ACT GAA GGC AAT CCT CG-3′, downstream primer: 5′-TCCTCA GAA CAC CTT CGC TT-3′, β -actin primer sequence is: upstream primer: 5′-ATGGAT GAC GAT ATC GCT GCG-3′, downstream primer: 5′-TCG TCC CAG TTGGTG ACA ATG-3′. Synthesized by Shanghai Sangon Bioengineering Company. After the PCR amplification products were subjected to 2.0% agarose gel electrophoresis, the grayscale analysis was performed with BandScan gel image processing software, and the mRNA level of the target gene was expressed as the ratio of the grayscale value of the target gene to the amplified product band of β-actin.

6. Statistical processing

表示，采用《中国医学百科全书·医学统计学》统计软件包(PEMS 3.1)进行统计学分析，组间比较采用t检验，P＜0.05有统计学意义，为差异具有显著性；P＜0.01为差异具有极显著性。Data are presented as mean ± standard deviation

Said that the "Chinese Medical Encyclopedia·Medical Statistics" statistical software package (PEMS 3.1) was used for statistical analysis, and the t test was used for comparison between groups. P<0.05 was statistically significant, and the difference was significant; P<0.01 was The difference is extremely significant.

3. Results

1. Withdrawal behavior signs and audiogenic seizure scores of alcohol-dependent rats (see Table 1)

Six hours after alcohol withdrawal, rats in the alcohol-dependent model group and the experimental control group showed sneezing, irritability, and audiogenic epileptic seizures with increased frequency and intensity. After the intervention of medium and high doses of NBP, the comprehensive score decreased, which was significantly different from the experimental control group; after low-dose NBP intervention, the comprehensive score did not change significantly, and there was no significant difference compared with the experimental control group.

Table 1 Onset score of withdrawal behavioral signs in alcohol-dependent rats

Note: *Compared with the experimental control group, P<0.05. # Compared with the experimental control group, P<0.01.

2. Changes of H ₂ S content and CBS activity in hippocampal tissue (see Table 1)

Compared with rats in the experimental control group, the H ₂ S content and CBS activity in the hippocampal tissue of the rats in the middle and high doses of NBP groups were significantly lower (P<0.05).

3. Changes in the expression of NR2B mRNA in the hippocampal tissue (see Table 1)

Compared with rats in the experimental control group, the expression of NR2B mRNA in the hippocampal tissue of the rats in the middle and high doses of NBP groups was significantly different (P<0.05).

In this study, rats were given free access to 6% alcohol (v/v) aqueous solution for 28 days to establish a rat model of alcohol dependence. Alcohol dependence is a special psychological state caused by repeated drinking, which is characterized by withdrawal syndrome, relapse and tolerance, and the withdrawal symptoms are the most severe 6 hours after alcohol withdrawal. In this study, the alcohol dependence model group and the experimental control group had significantly higher comprehensive scores than the normal group after 6 hours of withdrawal of alcohol, and there was a significant difference, indicating that the alcohol dependence rat model was successfully established. After the intervention of medium and high doses of NBP, the comprehensive score decreased, which was significantly different from that of the experimental control group, indicating that NBP can alleviate the symptoms of alcohol withdrawal.

The results of this study showed that NBP could reduce the H ₂ S content and CBS activity in the hippocampus that were increased by long-term drinking. After the sulfur-containing amino acid (methionine) is metabolized into cysteine in the body, H ₂ S is generated under the catalysis of CBS and CSE. In the central nervous system, CBS is mainly distributed in the hippocampus, cerebellum, cortex, and brainstem. and higher cortical distribution. CBS is the main enzyme that produces H ₂ S in the brain. H ₂ S can hardly be detected in the mouse brain after CBS gene knockout, and the production of H ₂ S can be regulated by changing the activity of CBS. Therefore, this study detected H 2 S ₂ Changes in the S/CBS system. H ₂ S may have affected the NMDA receptor (NMDAR) complex. In vitro experiments have found that H ₂ S higher than physiological concentrations can increase the intracellular Ca ²⁺ concentration of neurons, and can reach neurotoxic levels, eventually causing cell Death due to excitotoxic glutamate injury. Ca ²⁺ can increase the production of H ₂ S, especially the signal transduction pathway mediated by calcium ions/calmodulin can rapidly regulate the production of H ₂ S. Long-term drinking can increase the influx of Ca ²⁺ in hippocampal cells, and bind to a 19-amino acid fragment of CBS, and CBS will be activated immediately, and the activated CBS can catalyze the generation of H ₂ S. Therefore, it can be considered that the generation of H ₂ S is A response to neuronal excitation. Further studies in this experiment confirmed that after long-term drinking in rats, the activity of CBS increased, the generation of H ₂ S increased, and _it had a toxic and damaging effect on cells. 2 times. And it may inhibit cytochrome oxidase C, thereby inhibiting cellular respiration to exert its toxic damage effect on nerve cells.

Medium and high doses of NBP can down-regulate the expression of NR2B mRNA. NBP is able to alleviate intracellular calcium overload induced by glutamate, a particularly important site of alcohol action, in the glutamate-neurotransmitter system. Alcohol is a potent and selective inhibitor of NMDAR, which can lead to a compensatory "upregulation" of NMDAR, and long-term alcohol exposure can cause a hyperexcited state of NMDAR. This is an important reason for alcohol withdrawal symptoms (especially epileptic seizures). NMDAR maintains the normal physiological function of neurons by regulating Ca ²⁺ influx. NMDAR-mediated glutamatergic neurotransmission plays an important role in the formation of alcohol dependence. NMDAR subunits are mainly NR1, NR2A and NR2B, and NR2B subunits It plays a key role in the structure and function of NMDAR, and directly participates in all pathophysiological processes such as the formation, development and maintenance of alcohol dependence by interacting with various proteins in cells. Upregulation of NR2B expression can lead to excessive drinking and relapse, and NR2B can be used as a possible target for drug intervention on alcohol dependence. After NMDAR is activated, it triggers the influx of Ca ²⁺ and other cations. Ca ²⁺ plays an important role in the function of the central nervous system. As a second messenger, it participates in the transmission of nerve signals and the release of neurotransmitters. In drug dependence role has received widespread attention. Compared with the normal group, the expression of NR2B mRNA was up-regulated in the experimental control group and the normal group, and the difference was extremely significant, which was consistent with previous similar experiments; compared with the experimental control group, the expression of NR2B mRNA in the hippocampal tissue of the NBP medium and high-dose groups was significantly reduced, and the difference was significant. This suggests that NBP may reduce the number of NMDARs by down-regulating the expression of NR2B mRNA, thereby inhibiting Ca ²⁺ influx, reducing intracellular Ca ²⁺ overload, down-regulating the activity of CBS, reducing the production of H ₂ S, and alleviating H ₂ S toxicity effect and regulation of NMDAR post-conduction pathway, so that the withdrawal symptoms of rats were alleviated.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the implementation scope of the present invention. Those skilled in the art can make different changes and modifications without violating the spirit and principles of the present invention, but these changes and modifications should be covered within the scope of patent protection defined by the claims of the present invention.

Claims (7)

1. butyphthalide or its pharmaceutical salts or contain the application of pharmaceutical composition any in them in the medicine of preparation prevention or treatment chronic alcoholism.

2. application according to claim 1 is characterized in that, said butyphthalide or its pharmaceutical salts or contain pharmaceutical composition any in them and prepare the back with medicinal diluent or pharmaceutical carrier and use.

3. application according to claim 1 is characterized in that, with butyphthalide or its pharmaceutical salts or contain pharmaceutical composition any in them and pharmaceutical carrier is processed soft capsule, tablet or injection.

4. application according to claim 3 is characterized in that, said injection is a lyophilized injectable powder.

5. according to each described application of claim 1-4, it is characterized in that during application, in butyphthalide, its consumption is the 2-8mg/kg body weight.

6. application according to claim 5 is characterized in that, during application, in butyphthalide, its consumption is the 5-7mg/kg body weight.

7. application according to claim 6 is characterized in that, during application, in butyphthalide, its consumption is the 7mg/kg body weight.