WO2006015546A1 - Use of bicyclol for manufacturing a medicament for preventing and/or treating acute alcoholism and acute or chronic alcoholic liver injury - Google Patents

Abstract

The present invention discloses the use of bicyclol of formula ( I ) for manufacturing a medicament for preventing or treating acute alcoholism and acute, or chronic alcoholic liver injury.

Description

 Application of bicyclol in the preparation of drugs for preventing and/or treating acute alcoholism and acute and chronic alcoholic liver injury

Technical field

 The present invention relates to the use of bicyclol for the preparation of a medicament for the prevention or treatment of acute alcoholism and acute or chronic alcoholic liver injury. Background technique

 Alcoholic liver disease can be divided into alcoholic fatty liver, alcoholic hepatitis, alcoholic cirrhosis and alcoholic cirrhosis with liver cancer according to its pathological changes. In recent years, with the improvement of people's living standards and the increase in drinking consumption, alcoholic liver disease has gradually increased in China, and it is currently the second largest liver disease after viral hepatitis.

 The exact mechanism of the current occurrence of alcoholic liver disease is not fully understood. It is generally believed that the occurrence and development of alcoholic liver disease is closely related to the toxicity of alcohol and its metabolite acetaldehyde and the resulting a series of physiological and biochemical reaction disorders in the body.

 The main clinical measure for the treatment of alcoholic liver disease is abstinence, and the types of drug treatment are very limited. Therefore, it is still necessary to find effective and safe drugs against alcoholic liver disease.

 Bicyclol, .Chemical name: 4, 4'-dimethoxy-2, 3, 2 ', 3 ', - bis(methylenedioxy)-6-hydroxymethyl-6'-methoxycarbonyl linkage Benzene, the structural formula is

为我所研制的我国一类抗肝炎新药。 其肝保护和抗氧化作用在多种化 学性、 药物性、 免疫性实验性肝损伤动物模型上都已得到证实。 双环 醇具有显著保肝作用并具有一定的抗肝炎病毒效果， 对四氯化碳、 D- 半乳糖胺、 扑热息痛引起的小鼠急性肝损伤以及小鼠免疫性肝炎等 4 种动物模型均有显著的降低升高的血清转氨酶作用， 减轻肝脏组织病 理形态学损害。 此外， 对大鼠慢性四氯化碳肝损伤模型， 双环醇除有 降低升高的血清转氨酶作用外， 兼有改善血清白蛋白 /球蛋白比值及肝 脏脯氨酸含量的效果， 即有减轻肝纤维化的作用。 双环醇具有抑制乙 肝病毒核心抗原 （HBeAg)、 乙肝病毒核糖核酸（HBV-DNA)及乙肝病毒 表面抗原（HbsAg)分泌的效果.在长期慢性毒性试验中未发现有毒性， 亦无致畸和致突变的毒性。

A new class of anti-hepatitis drugs developed in China for me. Its liver protection and anti-oxidation effects have been confirmed in a variety of chemical, drug, and immunological experimental liver injury animal models. Bicyclol has significant hepatoprotective effects and has certain anti-hepatitis effects. It is markedly effective in four animal models including carbon tetrachloride, D-galactosamine, acute liver injury caused by paracetamol, and mouse immunological hepatitis. Reduce the effect of elevated serum transaminase, reduce liver tissue disease Morphological damage. In addition, in the model of chronic carbon tetrachloride-induced liver injury in rats, in addition to the effect of lowering serum transaminase, bicyclol has both an effect of improving serum albumin/globulin ratio and liver proline content, ie, reducing liver The role of fibrosis. Bicyclol has the effect of inhibiting the secretion of hepatitis B virus core antigen (HBeAg), hepatitis B virus ribonucleic acid (HBV-DNA) and hepatitis B virus surface antigen (HbsAg). No toxicity or no teratogenicity was observed in the long-term chronic toxicity test. The toxicity of the mutation.

 The results of clinical studies show that: bicyclol has a good effect on clinical symptoms and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in patients with chronic viral hepatitis B and hepatitis C, and can make HBeAg, HBV-DNA is negative. Significantly no significant side effects, good safety and tolerability. Bicyclol was awarded the new drug certificate by the State Drug Administration in 2001 under the trade name "Bai Sainuo 8" and was officially put on the market in October 2001.

 However, there is no description of the protective effect of bicyclol on alcoholism and alcoholic liver injury in the prior art. Summary of the invention

 In order to overcome the deficiencies of the prior art, it is an object of the present invention to provide a compound for preventing or treating acute alcoholism and acute or chronic alcoholic liver injury.

 In particular, a bicyclic alcohol as shown in the general formula (I) is provided.

It is used as a drug for preventing or treating acute alcoholism and acute or chronic alcoholic liver injury. The invention establishes a model of acute alcoholism and acute and chronic alcoholic liver injury, observes the protective effect of bicyclol on alcoholism and alcoholic liver injury and explores its mechanism of action. The results show that bicyclol has a good protective effect on experimental acute alcoholism and acute and chronic liver injury.

 The present invention records the disappearance time and mortality of the righting reflex of the mouse by orally administering a toxic dose of ethanol (50%, 12-24 ml/kg) or a 5% Liber-Decarli feed to the mouse. Alanine aminotransferase (ALT), triglyceride (TG), cholesterol (CHOL), high-density lipoprotein (HDL) and low-density lipoprotein LDL levels, liver lipid peroxidation product malondialdehyde (MDA) production and antioxidant Glutathione (GSH) content, liver mitochondrial membrane fluidity and swelling, liver glutathione-thiol transferase (GST) activity, serum alcohol dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH) As well as serum ethanol concentration, liver NDMA-demethylase activity and pathological changes.

 The results showed that bicyclol (150-300mg/kg) can significantly reduce serum ALT caused by ethanol, increase of liver MDA and TG, CHOL, LDL, reduce the decrease of liver GSH and GST, and inhibit the mitochondrial membrane flow induced by ethanol. Decreased sex and increased mitochondrial swelling, significantly induced HDL, ADH and ALDH, inhibited elevated NDMA-demethylase activity, and decreased blood ethanol concentration. In addition, bicyclol can shorten the disappearance of dysregulation caused by ethanol poisoning and reduce mortality.

 The present invention can draw the following conclusions: Oral administration of bicyclol can significantly reduce mouse death and liver damage caused by ethanol poisoning, improve hepatic steatosis, protect and induce anti-oxidation GST and GSH in vivo, and improve hepatocyte resistance Oxidative capacity, protection against mitochondrial damage and function, regulation of drug metabolism enzymes involved in ethanol metabolism, and reduction of blood alcohol concentration are closely related.

 In particular, the present invention finds the protective effect of bicyclol on acute ethanol poisoning mice. The effect of bicyclol on the prolongation of sleep time in mice with acute alcoholism was examined. Test animals After the administration of 50% ethanol at 16 ml/kg, the disappearance and recovery time of the righting reflex of the mice were observed, and the interval time was determined as the sleep time. The results showed that after oral administration of ethanol, the sleep time of the mice was as long as 3.6 hours, and bicyclol can significantly shorten the sleep time caused by ethanol, and showed a good dose-effect relationship. The average sleep time of the high-dose group was only the model group. 10%.

Bicyclol can reduce the effects of ethanol poisoning on mouse death. The test animals were intragastrically administered with 50% ethanol 24 ml/kg, and the number of deaths of 8, 12, 24 small animals was recorded after ethanol administration. The results indicated that the mortality rate of mice at 8, 12, and 24 hours after oral administration of ethanol was 60%, 80%, and 100%, respectively. Oral bicyclol can significantly reduce the mortality of mice poisoned by alcohol. Bicyclol has a protective effect on acute liver injury induced by ethanol in mice. Test animal irrigation After gastric acid 50% ethanol 12ml/kg, serum ALT was significantly increased (1.8 times), liver TG increased to 1.3 times of normal control, MDA production reflecting lipid peroxidation damage increased, and liver antioxidant GSH and GST fell to 87% and 77% of normal, respectively. Bicyclol can significantly inhibit the elevation of ALT caused by ethanol and the decrease of GSH in the liver, and also reduce the elevated liver TG and MDA to restore it to normal levels. Furthermore, high doses of bicyclol can induce liver GST and improve the body's antioxidant capacity.

 Bicyclol has a protective effect on the pathological changes of acute alcoholic liver injury in mice. After 10 days of infusion of 56% liquor, animal liver cells, mainly 1-2 layers of hepatocytes around the central vein, showed swelling and rounding. Hepatocyte cytoplasm was loose, and some vacuoles were formed in the liver cytoplasm, but the liver cell nucleus was not obvious. change. Nearly half of the model group animals showed necrosis of small focal hepatocytes (3-5 hepatocytes) and small focal inflammatory cell infiltration. However, there was no obvious hepatocyte injury in bicyclol animals, and there was no significant difference compared with the normal control group.

 After oral administration of ethanol in experimental animals, the blood ethanol increased rapidly, which was 35 times that of the normal control. Bicyclol can reduce the elevated blood alcohol concentration by 31%. For the ethanol-metabolizing enzyme, bicyclol significantly induced liver ADH and ALDH activity, which were 215% and 122% of the normal control group, respectively, and inhibited DMA-demethylase activity with an inhibition rate of 21%.

 Free radicals produced during alcohol metabolism can damage mitochondrial membrane lipids, resulting in decreased mitochondrial membrane fluidity. Bicyclol significantly improves the increase in the degree of polarization and microviscosity of the mitochondrial membrane caused by alcohol. The swelling of liver mitochondria is significantly increased under high calcium conditions, and bicyclol can significantly reduce mitochondrial swelling and maintain it at a normal level, thus ensuring the normal function of mitochondria.

 Protective effect of bicyclol on chronic alcoholic liver injury in mice. After 4 weeks of feeding with 5% alcoholic liquid, serum ALT levels increased significantly. After administration of bicyclol, ALT levels returned to normal levels in both the treatment and prevention groups, suggesting that bicyclol has significant anti-alcoholic liver damage.

After 4 weeks of feeding with alcohol-containing liquid diet, the mice showed not only a significant increase in liver TG content, but also a slight increase in serum CHOL, LDL, and HDL. Compared with the model group, the liver TG, serum CHOL, LDL levels in the drug-administered group decreased significantly, and serum HDL levels increased significantly, suggesting that bicyclol can effectively prevent alcohol-induced fat accumulation, accelerating fat-transporting mice after long-term administration of alcohol-containing diet. Not only the GSH content in the liver decreased, but also the GST and glutathione reductase (GR) activities were significantly reduced. After oral administration of bicyclol, the GSH level and GST activity in the liver of the mice returned to normal levels, and the GR activity increased to 1.2 in the control group. Times. The above results suggest that bicyclol has the function of promoting GSH regeneration and enhancing liver free radical scavenging ability.

 Long-term feeding of ethanol to experimental animals induced an increase in the expression of P450 2E1, which further led to lipid peroxidation and aggravated liver damage. In this experiment, P450 2E1 activity was indirectly determined by measuring NDMA-DH. Studies have found that bicyclol can significantly inhibit the increase in P450 2E1 activity caused by long-term alcohol intake, keeping the enzyme at a normal level. Similar to the results of acute alcoholic liver injury, bicyclol significantly induced ALDH activity, especially in cytosolic ALDH activity (2.9 times that of the model group), accelerated acetaldehyde clearance, thereby alleviating liver damage caused by acetaldehyde. In addition, bicyclol has no effect on ADH.

 Bicyclol has a protective effect on pathological changes of chronic alcoholic liver injury. The mice were fed with 5% alcoholic liquid feed for 4 weeks, which caused hepatic steatosis, and the hepatic steatosis of the central lobular lobule was the most severe. Hepatocytes around some central lobular veins are swollen and the cytoplasm is loose. In addition, hepatic venous congestion and other phenomena were observed. After administration of bicyclol, hepatic cell steatosis was significantly alleviated, and hepatocyte swelling did not occur.

The present invention further relates to pharmaceutical compositions comprising bicyclic alcohol conventional pharmaceutical excipients or adjuvants as an active ingredient of a pharmaceutical composition _P compositions of the invention generally comprises 0.5 95% by weight of bicyclic alcohols.

 Pharmaceutical combinations containing the compounds of the invention can be prepared according to methods well known in the art. When used for this purpose, the compounds of the invention may be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants, if desired, in a suitable form or dosage for use as a human or veterinary drug. form.

 The compound of the present invention or a pharmaceutical composition containing the same may be administered in a unit dosage form, which may be enterally or parenterally, such as orally, muscle, nasal cavity, oral mucosa, skin, transdermal, subcutaneous, intradermal, peritoneal. , rectal, intravenous, intramuscular, intrathecal, epidural, intraocular, intracranial, vaginal administration, etc.

 The administration route of the compound of the present invention or a pharmaceutical composition containing the same can be administered by injection. Injections include intravenous, intramuscular, subcutaneous, intradermal, acupoint, intrathecal, and intraperitoneal injections.

The dosage form can be a liquid dosage form or a solid dosage form. For example, the solution properties of the liquid dosage form may be true solutions, colloids, microparticulate forms, emulsion dosage forms, suspension dosage forms. The liquid dosage form may be a syrup, an elixir, an injection solution, a non-aqueous solution, a suspension or an emulsion; a solid dosage form such as a tablet, a lozenge, a capsule, a dropping pill, a pill, a granule, a powder, a cream, a solution Liquid, suppository, dispersible powder such as lyophilized powder injection, aerosol, and the like.

 The compounds of the present invention can be formulated into common preparations, sustained release preparations, controlled release preparations, targeted preparations, and various microparticle delivery systems.

 In order to prepare a unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier include, excipients such as calcium carbonate, lactose, calcium phosphate, sodium phosphate; diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, Calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, dextran, colloidal silica, gum arabic, gelatin, magnesium trisilicate, keratin, etc.; wetting agent and binder such as water, glycerin, Polyethylene glycol, ethanol, propanol, starch syrup, dextrin, syrup, honey, glucose solution, gum arabic, gelatin syrup, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyethylene Pyrrolidone or the like; a disintegrating agent such as dried starch, alginate, agar powder, brown algae starch, sodium hydrogencarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfonate, A Cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, Sodium dialkyl sulfate, etc.; lubricants such as talc, triethylamine magnesium stearate, triethylamine stearic acid, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol Wait. Tablets may also be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer tablets and multilayer tablets to delay disintegration and absorption in the gastrointestinal tract. And thus provide a lasting effect over a longer period of time.

 For example, in order to prepare a drug delivery unit into a pellet, various carriers known in the art can be widely used. Examples of the carrier are, for example, a diluent and an absorbent such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, talc, etc.; binders such as acacia, tragacanth, Gelatin, ethanol, honey, liquid sugar, rice paste or batter; etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, and the like.

 For example, in order to encapsulate the administration unit, the active ingredient compound of the present invention is mixed with the above various carriers, and the mixture thus obtained is placed in a hard gelatin capsule or soft capsule. The active ingredient of the compound of the present invention can also be formulated into a microcapsule, suspended in an aqueous medium to form a suspension, or can be incorporated into a hard capsule or used as an injection.

For example, in order to prepare the administration unit into an oral liquid preparation, it includes an emulsion, a solution, a suspension, a syrup, and the like. Suitable carriers include solutions, suspensions, syrups and the like, and optionally contain additives such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and perfumes. For example, the compound of the present invention is formulated into an injectable preparation such as a solution, a suspension solution, an emulsion, or a lyophilized powder injection, which may be aqueous or non-aqueous, and may contain one and/or more drugs. A pharmaceutically acceptable carrier, diluent, binder, lubricant, preservative, surfactant or dispersing agent. For example, the diluent may be selected from the group consisting of water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, vegetable oils such as olive oil and corn oil, gelatin, and Injectable organic esters such as ethyl oleate, polyoxyethylene sorbitol, fatty acid esters and the like. Such dosage forms may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Further, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and a conventional cosolvent, a buffer, a pH adjuster or the like may be added. These excipients are commonly used in the art.

 Further, if necessary, a coloring agent, a preservative, a flavoring agent, a flavoring agent such as peppermint, wintergreen oil or the like, a sweetener such as sucrose, lactose, saccharin or the like or other materials may be added to the pharmaceutical preparation.

 The sterile medium used in the present invention can be prepared by standard techniques well known to those skilled in the art. They may be sterilized, for example, by filtration through a bacterial filter, by adding a sterilizing agent to the composition, by radiation treatment of the composition, or by heating the composition. They can also be made into sterile injectable media before use.

 The pharmaceutical or pharmaceutical composition of the present invention can be administered by any known administration method for the purpose of enhancing the therapeutic effect for the purpose of administration. Of course, the route of administration of the compounds used in the practice of the invention will depend on the disease and the site in need of treatment. Because the pharmacokinetic and pharmacodynamic characteristics of the compounds of the invention will vary to some extent, the most preferred method of obtaining therapeutic concentrations in tissues is to gradually increase the dose and monitor the clinical effect. For such a gradual increase in therapeutic dose, the initial dose will depend on the route of administration.

The particular therapeutically effective dose level of a pharmaceutical composition of a compound of the invention will depend on a number of factors, such as the nature of the disease to be prevented or treated, the severity of the route of administration, the number of administrations, the purpose of treatment, the clearance of the compound, for any particular patient. Speed, duration of treatment, specific drugs in combination with or in combination with the specific compound, patient or animal gender, age, weight, personality, diet, individual response, and general health status, etc., are well known factors in the field of medical science, and thus the treatment of the present invention The dosage can vary widely. Depending on the condition of the patient being treated, certain changes in dosage may be necessary, and in any event, the physician will determine the appropriate dosage for the individual patient. The dose administered refers to the weight of the compound excluding the weight of the carrier (when the carrier is used). In general, the dosages of the pharmaceutical ingredients employed in the present invention are well known to those skilled in the art. The prophylactic or therapeutic effect of the present invention can be accomplished by appropriately adjusting the amount of the actual drug contained in the final formulation of the compound composition of the present invention to achieve its therapeutically effective amount. It may be administered in a single dosage form or divided into several, for example two, three or four dosage forms; this is limited by the clinical experience of the administering physician and the dosing regimen including the use of other therapeutic means. The compounds or compositions of this invention may be administered alone or in combination with other therapeutic or symptomatic agents and adjusted in dosage.

Liber-Decarli whole nutrient alcohol liquid feed;

 ALDH: acetaldehyde dehydrogenase;

 ALT: alanine aminotransferase;

 TG: triglyceride;

 CHOL: cholesterol;

 HDL: high density lipoprotein;

 DL: low density lipoprotein;

 MDA: malondialdehyde;

 GSH: glutathione;

 GST: glutathione-thiotransferase;

 ADH: alcohol dechlorination enzyme;

 ALDH acetaldehyde dehydrogenase;

 NDMA-DH: dimethyl nitrosamine-demethylase;

 GR: Glutathione Reductase Figure 1 Figure 1. Effect of bicyclol on ethanol-induced sleep in mice

Figure 2 Effect of bicyclol on ethanol concentration in blood of mice with alcohol poisoning

Figure 3. Effect of bicyclol on ethanol-induced mitochondrial membrane swelling in mice. The results are expressed in terms of the mean SD.

Figure 4. Effect of bicyclol on serum ALT elevation in mice with chronic alcoholic liver injury. The results are expressed as mean SD. M: ethanol group T2/3: bicyclol (200/300 mg/kg) treatment group, Y2/3: Bicyclol (200/300 mg/kg) prevention group. P<0.01, **P<0.01 compared with the control group, compared with the model group.

Figure 5 Effect of bicyclol on liver TG elevation in mice with chronic alcoholic liver injury. The result is the average value of SD. M: Ethanol T2/3: (200/300 mg/kg) bicyclol treatment, Y2/3: bicyclol (200/300 mg/kg) prevention. ## P<0.01 Compared with the control group, *P<0.05, **P<0.01 compared with the model group.

Figure 6. Effect of bicyclol on the decline of liver GSH in mice with chronic alcoholic liver injury. The result is the average value of SD. M: ethanol T2/3: (200/300 mg/kg) bicyclol treatment, Y2/3: bicyclol (200/300 mg/kg) prevention, Herp PO.01 compared with the control group, *P<0.05, ** P < 0.01 compared to the model group.

Figure 7. Effect of bicyclol on the decline of liver GST in mice with chronic alcoholic liver injury.

Figure 8 Effect of bicyclol on liver GR decline in mice with chronic alcoholic liver injury. The result is the mean soil SD. M: ethanol T2/3: (200/300 mg/kg) bicyclol treatment, Y2/3: bicyclol (200/300 mg/kg) prevention, ## P<0.01 compared with the control group, * P<0.05 , ** P<0.01 compared to the model group.

Figure 9. Effect of bicyclol on the activity of hepatic cytosolic NDMA-DH in mice with chronic alcoholic liver injury. The result is the average value of SD. M: Ethanol T2/3: (200/300 mg/kg) bicyclol treatment, Y2/3: bicyclol (200/300 mg/kg) prevention. P<0.01 compared with the control group, *P<0.05, **P<0.01 compared with the model group. detailed description

 The following examples are intended to assist those of ordinary skill in the art to understand the invention, but are not intended to limit the invention in any way. I. Protective effect of bicyclol on mice with acute alcoholism

 Example 1. Effect of bicyclol on prolonged sleep time in mice with acute alcoholism

Male Kunming mice, weighing 22-24 g, were randomly divided into 4 groups, 10 in each group, 3 of which were given oral bicyclol 75, 150, 300 mg/kg o in the afternoon of the first day of the experiment and the next day. Give the same volume of excipients. After fasting overnight, the animals in each group were intragastrically administered with 50% ethanol and 16 ml/kg. The disappearance of the righting reflex and the recovery time of the mice after oral ethanol were observed, and the interval was determined as the sleep time. The results showed that after oral administration of ethanol, the sleep time of mice was as long as 3.6 hours, and bicyclol 150, 300 mg/kg could significantly shorten the sleep time caused by ethanol, and showed a good dose-effect relationship, and the average sleep time of the high-dose group. Only 10% of the model group. The results are shown in Figure 1. Example 2. Effect of bicyclol on ethanol death induced by ethanol intoxication

 Male Kunming mice, weighing 22-24 g, were randomly divided into 4 groups, 10 in each group, 3 of which were given oral bicyclol 100, 200 in the afternoon of the first day of the experiment and the afternoon of the next day.

300 mg/kg, the control group was given the same volume of excipients. After fasting overnight, the animals in each group were intragastrically administered with 50% ethanol 24 ml/kg, and the number of deaths of animals after 8, 12, and 24 hours was recorded. Result list

As shown in Figure 1, after oral administration of alcohol, the mortality rate of mice at 8, 12, 24 hours was 60%, 80%, respectively.

 S

 100%. Oral bicyclol (200, 300 mg/kg) significantly reduced mortality in mice poisoned with alcohol.

Table 1 Effect of bicyclol on ethanol-induced death in mice Ethanol bicyclol bicyclic alcohol bicyclol mortality 50% 24ml · kg ¹ 200 mg · kg" ¹ 300 mg · kg" ¹ after administration 8h 60% 50% 30% 0 % ^b time 12h 80% 60% 30% ^a 0%°

24h 100% 70% 40% ^b 10% ° II. Protective effect of bicyclol on acute liver injury induced by ethanol in Example 3. Bicyclol on ethanol caused serum ALT, liver TG, MDA elevation and GSH, GST Impact of change

Male Kunming mice, weighing 22-24 g, were randomly divided into 5 groups, 10 in each group, 3 of which were given oral bicyclol 75, 150, 300 mg/kg on the afternoon of the first day of the experiment and the next day. Give the same volume of excipients. After fasting overnight in each group of animals, except for the normal control group, the other groups were given 50% ethanol 12 ml/kg, and the control group was given 20% glucose solution of the same energy. Animals were sacrificed 6 hours after ethanol administration, serum was taken for ALT determination, and liver was taken for determination of TG, MDA and GSH and GST content. As shown in Table 2, after oral administration of ethanol in mice, liver TG increased to 1.3 times that of normal controls, serum ALT was also significantly increased (1.8 times), and MDA production reflecting lipid peroxidation damage increased, while liver antioxidants GSH and GST fell to 87% and 77%, respectively. Bicyclol (75, 150, 30 s0 mg/kg) significantly inhibited the increase in sALT caused by ethanol and the decrease in liver GSH.

 3

At the same time as this k, bicyclol (150, 300 mg/kg) can also reduce elevated liver TG and MDA,

TO TO

Bring it back to normal levels. In addition, high doses of bicyclol can induce liver GST and increase the body's antioxidant energy S.

Table 1 Liver TG, GSH; GST, MDA and serum ALT levels in mice with liver injury induced by bicyclol

Impact group liver TG / ALT / U · dL" ¹ MDA / GSH / GST / nmol · g" ¹ nmol · g ^_1 liver nmol · min" ¹ · mg" ¹ prot

*

 Normal control 290.8 ±62.3 31.10+ 45.50+6.77 85.57±6.74

 7.58

 Ethanol 50% 377.0±30.0 55.42± 8.37 57.24±6.12 4.51 ±0.48 65.48± 19.46 bicyclol 75 368.8±42.4 31.10+ 53.90±2.56 5.90+0.63

 5.58

 Bicyclol 150 226.6 soil 30.70 soil 41.30 soil 6.41 ±0.84

 **

Mg · kg" ¹ 53.3 7.58 7.62

 Bicyclol 300 225 ± 22.7 28.71 soil 37.31 soil 7.61 ± 0.71 114.58 ± 1 U4

 5.18 4.41

P < 0.05, P < 0.01, PO.001, compared to the model group.

Example 4. Protective effect of bicyclol on pathological changes of acute alcoholic liver injury in mice

After 10 days of infusion of 56° liquor, animal liver cells, mainly 1-2 layers of hepatocytes around the central vein, showed swelling and rounding, hepatocyte cytoplasm was loose, and some vacuoles were formed in the liver cytoplasm. Nearly half of the model group animals were visible. Small focal hepatocytes (3-5 hepatocytes) are necrotic, and small focal inflammatory cells infiltrate. Animals in the bicyclol (300 mg/kg) group showed no obvious hepatocyte injury, and there was no significant difference compared with the normal control group. - Effect of bicyclol on blood ethanol concentration and ethanol metabolism enzyme (1) Effect of bicyclol on serum ethanol concentration in mice

 Male Kunming mice, weighing 22-24 grams, were randomly divided into 3 groups, 7 in each group. The drug-administered group was orally administered bicyclol 300 mg/kg on the afternoon of the first day of the experiment and the next day, and the control group was given the same volume of excipients. Animals in each group were fasted overnight after the last dose. The next day, except for the normal control group, the other animals received oral 50% ethanol 16ml/kg. The animals were sacrificed 1 hour after the ethanol was given, and blood was taken to determine the ethanol content. The results showed that after oral administration of ethanol, the blood ethanol increased rapidly, which is 35 times that of normal control. Bicyclol can reduce elevated blood ethanol concentrations by 31%.

(2) Effects of bicyclol on ADH, ALDH and NDMA-demethylase activities in mouse liver

 Male Kunming mice, weighing 22-24 grams, were randomly divided into 2 groups, 10 in each group. The drug-administered group was orally administered bicyclol 300 mg/kg on the afternoon of the first day of the experiment and the next day, and the control group was given the same volume of excipients. Animals in each group were fasted overnight after the last dose. The animals were decapitated the next morning and the liver was assayed for ADH, ALDH and NDMA demethylase activity. The results showed that bicyclol significantly induced ADH and ALDH activity in the liver, which were 215% and 122% of the normal control group, respectively, and inhibited NDMA-demethylase activity with an inhibition rate of 21%. Table 3 Effect of bicyclol on ADH, ALDH and NDMA-demethylase activity in mouse liver

Group ADH/nmol ·τηίη ¹ 3⁄4" ^! liver NDMA demethylase ALDH

/nmol · min" ¹ · mg" ^l protein /μ g · min ¹ . mg "'protein

Control 6.16± 1.72 2.03 ±0.06 3.27±0·11

* * Bicyclol 13.25 + 0.56 1.61 ±0·13 4.02±0.52 300mg · kg" ¹

 <0.05, **P<0.01 compared with the control group

Example 6. Effect of bicyclol on functional integrity of mouse liver mitochondria

(1) Effect of bicyclol on the decline of hepatic mitochondrial membrane fluidity induced by acute alcoholic liver injury in mice

Male Kunming mice, weighing 22-24 grams, were randomly divided into three groups of 10 animals each. small After the rats were administered with 56° liquor lOml/kg, the free radicals produced during alcohol metabolism could damage the mitochondrial membrane lipids, resulting in decreased mitochondrial membrane fluidity. Bicyclol (200, 300 mg/kg) can significantly improve the increase in the degree of polarization and microviscosity of the mitochondrial membrane from alcohol.

Table 4 bicyclol on ethanol-induced changes in mitochondrial membrane fluidity of the Mouse P n normal control group 0.164 ± 0.02 1.12 ± 0.23 Ethanol 0.183 ± 0.006 ^# 1.33 ± 0.07 ^# bicyclic alcohol ethanol +

 0.170±0.01* 1·17±0·15*

 (200mg/kg)

 Ethanol + bicyclol

 0.173 ±0.007** 1.21 ±0.08** (300mg/kg)

The result is expressed as the mean SD

# Ρ<0.05, compared with the control group

*Ρ<0.05, ** Ρ<0.01 compared with the ethanol group

(2) Effect of bicyclol on hepatic mitochondrial swelling in mice

Male Kunming mice, weighing 22-24 grams, were randomly divided into four groups of 10 animals each. After mice were intragastrically administered with 56° liquor 10ml/kg for 10 days, the swelling of liver mitochondria was significantly increased under high calcium conditions, while bicyclol (200, 300mg/kg) significantly reduced mitochondrial swelling, which was basically maintained at normal levels. Thereby ensuring the normal function of the mitochondria. The results are shown in Figure 3. III. Protective effect of bicyclol on chronic alcoholic liver injury in mice. Example 7 Effect of bicyclol on serum ALT elevation in mice with chronic alcoholic liver injury Male Kunming mice weighing 22-24 g, They were randomly divided into six groups of 10 each. After 4 weeks of feeding in a 5% alcoholic liquid diet, serum ALT levels increased significantly. After administration of bicyclol, both the treatment group (200 mg and 300 mg/kg) and the prevention group (200 mg and 300 mg/kg) returned to normal levels of ALT, suggesting that bicyclol has significant anti-alcoholic liver damage. Results are shown in Figure 4. Example 8 Effect of bicyclol on fat accumulation in chronic alcoholic liver injury in mice

 After 4 weeks of feeding with alcohol-containing liquid diet, the mice showed not only a significant increase in liver TG content, but also a slight increase in serum CHOL, LDL, and HDL. Compared with the model group, liver TG, serum CHOL, LDL levels were significantly decreased, and serum HDL levels were significantly increased, suggesting that bicyclol can effectively prevent alcohol-induced fat accumulation and accelerate fat transport. See Figure 5

Table 5 Effect of bicyclol on serum HDL, LDL and CHOL levels in mice with chronic alcoholic liver injury

 HDL LDL CHOL

 Group

 (mmoI/L) (mmoI/L) (mmol/L) Normal control 5.26±1.22 0.92±0.18 2.61±0.34 Ethanol 5.60±1.33 1.16±0.36 2.98±0.52 Ethanol +bicycloalcohol

 9.56±1.48** 0.84±0.21* 2.22±0.64**

(200 mg/kg, treatment) ethanol + bicyclol

 9.03±1.38** 0.83±0.30* 2.43±0.39* (300 mg/kg, treatment) Ethanol +bicycloalcohol

 8.97±1.23** 0.68±0.26** 2.15±0.43** (200 mg/kg, preventive) Ethanol +bicycloalcohol

 10.54±1.27** 0.76±0.19** 2.38±0.39** (300 mg/kg, prophylaxis) Results were averaged SD*P<0.05, **P<0.01 compared with the ethanol group. Example 9 Effect of bicyclol on antioxidant activity in liver of mice

After long-term administration of an alcoholic diet in mice, not only the GSH content in the liver decreased, but also the GST and glutathione reductase (GR) activities were significantly reduced. After oral administration of bicyclol, the GSH level and GST activity in the liver of the mice returned to normal levels, and the GR activity increased to 1.2 times of the control group. The above results suggest that bicyclol has the function of promoting GSH regeneration and enhancing liver free radical scavenging ability. The results are shown in Figure 6, 7, 8 Example 10 Effect of bicyclol on ethanol metabolism enzymes in mice The amount of alcohol oxidative metabolism by the microsomal alcohol oxidation system (MEOS) accounts for about 10% of the total ethanol intake. The superoxide anion produced by this metabolic process can form hydroxyl groups under the catalysis of magnesium ions. It has been confirmed that the MEOS metabolic alcohol process relies on cytochrome P450 2 E1. The long-term word of experimental animals with ethanol, induced the expression of P450 2E1 enhanced, which can further lead to lipid peroxidation and aggravate liver damage. .

 In this experiment, P450 2E1 activity was indirectly determined by measuring NDMA-DH. Studies have found that bicyclol can significantly inhibit the increase in P450 2E1 activity caused by long-term alcohol intake, keeping the enzyme at a normal level.

 Similar to the results of acute alcoholic liver injury, bicyclol significantly induced ALDH activity, especially in cytosolic ALDH activity (2.9 times that of the model group), accelerated acetaldehyde clearance, thereby alleviating liver damage caused by acetaldehyde. In addition, bicyclol has no effect on ADH. Effects of bicyclol on serum ADH and ALDH levels in mice with chronic alcoholic liver injury

ADH ALDH

 Group

 (U/m protein) (U/mg protein) normal control 7.42±1.73 8.79±0.88 ethanol 6.17±1.04 9.02±0.96 liver homogenate

 Ethanol + bicyclol

 6.95±1.32 10.73±1.65**

(300mg/kg ₃ prevention)

 Normal control 2.60±0.26 3.89±0.81 ethanol 2.62±0.43 ' 2·71±0·91# cytoplasm

 Ethanol + bicyclol

 2.85±0.39 8.09±1.33**

(300mg/kg, prevention)

 The result is the mean soil SD

 # Ρ<0.05 compared with the control group, ** Ρ<0.01 compared with the ethanol group.

Example 11 Protective effect of bicyclol on pathological changes of chronic alcoholic liver injury

After 4 weeks of feeding 5% alcoholic liquid diet, the mice can cause hepatic steatosis, and the hepatic cells in the central lobular veins are most severely fatty. Although the liver cells around the central vein of some small leaves did not undergo steatosis, the hepatocytes were swollen and the cells were loose. In addition, liver was also observed Dirty venous congestion and other phenomena. After administration of bicyclol, hepatic steatosis was significantly alleviated, and hepatocyte swelling did not occur.

 Table 7 Effect of bicyclol on pathological index changes in mice with chronic alcoholic liver injury Groups Pathological index

 Normal control 0.2±0.27

 Ethanol 2·50±0·53# #ethanol +bicyclol (300 mg/kg, prevention) 1.35 1.25*

 Ethanol + bicyclol (300 mg/kg,) 0.65 ± 0.53**

 The result is the average value of SD

 ## P<0.01 Compared with the control group, *PO.05, ** Ρ<0.01 compared with the model group.

Claims

2006/015546

 The use of a bicyclic alcohol of the formula (I) for the preparation of a medicament for preventing or treating acute alcoholism.

A pharmaceutical composition for preventing or treating acute alcoholism, which comprises a pharmaceutically effective amount of a bicyclic alcohol represented by the formula (I) and a pharmaceutically acceptable carrier.

A pharmaceutical composition for preventing or treating acute or chronic alcoholic liver injury, which comprises a pharmaceutically effective amount of a bicyclic alcohol represented by the formula (I) and a pharmaceutically acceptable carrier.

 The pharmaceutical composition according to Claims 3 and 4, wherein the pharmaceutical composition is a tablet, a capsule, a pill, an injection, a sustained release preparation, a controlled release preparation, and various microparticle delivery systems.