TWI393562B - Application of L-butylphthalide in the preparation of drugs for preventing and treating cerebral infarction - Google Patents

Description

Application of L-butylphthalide in the preparation of drugs for preventing and treating cerebral infarction

The present invention relates to the use of a composition comprising levo-n-butylphenylhydrazine and a composition comprising levo-n-butylphenylhydrazine for the preparation of a medicament for the prevention and treatment of cerebral infarction, in particular for focal cerebral ischemia resulting in cerebral infarction.

Cerebrovascular disease has become one of the main causes of death in China. For the treatment of acute ischemic stroke, there is still a lack of better therapeutic drugs at home and abroad. The US FDA approved the first thrombolytic drug t-PA in 1996, which is controversial because of the high adverse reactions of intracranial hemorrhage. Studies on various types of brain protectants have shown that many drugs are not clinically effective or have serious side effects. Several drugs are still in the clinical validation phase.

For more than ten years, we have found that racemic butylphthalide (or butylphthalide, NBP) is a drug that has a significant effect on acute ischemic stroke. Its pharmacodynamics covers many aspects: 1. It can improve the energy metabolism of ischemic brain; 2. Reduce the area of cerebral infarction in rats with local cerebral ischemia and improve the loss of nerve function; 3. Improve the cause of regional cerebral ischemia Cerebral edema; 4. It can significantly improve the regional cerebral blood flow and pia mater microcirculation in the ischemic area; 5 can significantly improve the memory impairment caused by regional cerebral ischemia. The side effects are small. We have applied for and approved two patents for use, respectively (1) "Application of celery A as a drug for the prevention and treatment of diseases caused by cerebral ischemia" (authorized in 1999, patent number ZL93117148, 2, international patent) Classification No. A61K31/34); (2) "Application of Butylphthalide in Preparation of Antithrombotic Drugs" (authorized in 2002, Patent No. ZL981 25618.X, International Patent Classification No. A61K31/34).

After preclinical studies, a total of 542 patients in the phase II and III clinical trials (including a multicenter, randomized, double-blind, placebo-controlled 192 patients) were found to have a total effective rate of 70.7% in the dosing group and placebo in the placebo group. 41.0%, get better therapeutic effect. In September 2002, the State Drug Administration approved a new drug certificate and production approval for a new class of NBP for acute ischemic stroke (Chinese medicine certificate H20020374; H20020375 and 2002H0551; 2002H0552).

We chemically resolved butyl benzoquinone (hereinafter referred to as d1-NBP) to obtain L-butyl phenyl hydrazine (hereinafter referred to as 1-NBP) and dextrobutyl phthalide (hereinafter referred to as d-NBP). The Chinese patent "Method for preparing optically active 3-n-butylphenyl hydrazine" (Application No. 99109673.8, Publication No. CN1283621A) was applied. In the patent "celery A as a drug for the prevention and treatment of diseases caused by cerebral ischemia", the effect of L-butyl benzoquinone is not involved.

In order to overcome the deficiencies of the prior art, the present invention provides as shown in the general formula (I)

The use of L-N-butylphenyl hydrazine (hereinafter referred to as L-NBP) as a drug for preventing or treating cerebral infarction.

The L-n-butyl phenyl hydrazine used in the present invention is chemically synthesized, first obtains racemic n-butyl phenyl hydrazine, and then chemically resolved into a left-handed optical isomer n-butyl benzoquinone, which is subjected to nuclear magnetic resonance, mass spectrometry and infrared spectroscopy. The analysis, especially with Hp 5890 gas chromatograph and chiral gas chromatography column (Chiraldex G-TA), proved that the optical purity and chemical purity of this product is a single optical stereoisomer - L-n-butyl phenyl hydrazine. (Specific optical rotation >-66.49 ⁰ , optical purity >98%, chemical purity >98%), see the Chinese patent "Method for preparing optically active 3-n-butylphenyl hydrazine", application number 99109673.8, publication number CN1283621 . The chemical structure of this product is the same as that of celery A contained in edible celery and its seeds.

The present inventors have found that the optical isomer 1-NBP has a stronger therapeutic effect on cerebral ischemia than d-NBP and is stronger than d1-NBP containing the same 1-NBP dose.

In this study, a rat model of ischemic stroke was induced by reperfusion of rat middle cerebral artery occlusion (MCAO) for 2 hours, and the anti-cerebral ischemia effect of optically active NBP was observed. The transient local cerebral ischemia model in mice was divided into 8 groups, 10 in each group: 1) solvent control group, vegetable oil 1 ml/kg; 2) and 3) d1-NBP40 and 80 mg/kg groups; 4) and 5) were d-NBP20 and 40 mg/kg groups; 6) and 7) were 1-NBP20 and 40 mg/kg groups; 8) were MK801 0.1 mg/kg group. Groups 1-7 were administered orally; groups 8 were administered intraperitoneally. The mouse model of transient local cerebral ischemia was given a butylphthalide solvent control group and a positive control drug 15 minutes after ischemia. Significant differences between groups were tested by one-way ANOVA, indicating that the 1-NBP group (20 mg/kg and 40 mg/kg) reduced the infarct volume significantly and had a dose-response relationship. Among them, the 40 mg/kg group had the strongest effect, and the infarct volume was reduced by 80.4% (P<0.001 compared with the solvent group), and the treatment effect was close to the MK801 group (infarct volume was reduced by 81.8%, P<0.001). The d-NBP and d1-NBP groups had no significant effect (see Figures 1, 2). The above results indicate that 1-NBP is significantly more effective than d-NBP in reducing infarct volume. The infarct volume of d1-NBP (80 mg/kg) and d-NBP (40 mg/kg) was smaller than that of the solvent group, but there was no statistical significance, while 1-NBP was contained in d1-NBP (80 mg/kg). (40 mg/kg). This suggests that the presence of d-NBP in d1-NBP may antagonize the effects of 1-NBP.

By behavioral scoring, the majority of animals in the solvent control group had a neurological score of 2, which showed a contralateral forelimb internal rotation and a lateral compression of the contralateral extensor muscle. A small number of animals showed contralateral rotation symptoms and were rated as 3 points. Individual animal behavior scores were lighter, only showed contralateral forelimb internal rotation, 1 point or severe symptoms, lack of independent activities, rated 4 points. The median score of the vehicle control group was 2.6 ± 0.3. Oral administration of 1-NBP 20 after 15 minutes of ischemia significantly improved neurological symptoms (1.4 points P < 0.01 and 1.1 points P < 0.001), and MK801 had the most significant effect (0.8 points P < 0.001). The d-NBP and d1-NBP groups did not significantly improve neurological symptoms. In the MK801 treatment group, although the symptoms after cerebral ischemia were significantly alleviated, the animals' limbs were weak and weak, and they were sick when crawling, showing ataxia, and often circled to one side. Behavioral scoring showed that the degree of improvement in drug behavior was paralleled by the effect of drugs on reducing infarct volume.

In summary, the present invention has been studied from the biochemical, molecular biology and holistic animal levels, and consistent results have been obtained, that is, 1-NBP has stronger brain protection than d-NBP and d1-NBP, and d-NBP in d1-NBP. The presence antagonizes the action of 1-NBP, so that the effect of d1-NBP against cerebral ischemia is reduced.

Previous studies have shown that 1-NBP antiplatelet aggregation and antithrombotic effects are close to aspirin and spirulina, in addition to 1-NBP also has brain protection and improved brain microcirculation (d-NBP no effect), and these effects It is not available in aspirin and spirulina. Therefore, 1-NBP has a wide therapeutic range, which can not only treat acute ischemic stroke, but also can be used for secondary prevention of stroke, prevention of myocardial infarction and treatment of peripheral vascular disease.

The invention complies with the requirements of the development of new drugs in the world today, and in order to strengthen the curative effect, reduce the side effects, and separate the racemic drug into the monomers of the optical isomer, and to clarify the respective properties, the optical with strong or low toxicity Isomers are developed into chiral new drugs. Accordingly, 1-NBP in butyl benzoquinone has a strong anti-cerebral ischemia effect, and d-NBP not only has a weak effect, but also antagonizes the anti-cerebral ischemia effect of 1-NBP, so it is reasonable to develop L-butyl phenyl hydrazine. Become a new chiral drug for the treatment of acute ischemic stroke.

The invention therefore also relates to a pharmaceutical composition comprising as an active ingredient a compound of the invention and a conventional pharmaceutical excipient or adjuvant. Typically, the pharmaceutical compositions of the invention contain from 0.1% to 95% by weight of a compound of the invention.

Pharmaceutical compositions of the compounds of the invention can be prepared according to methods well known in the art. 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 form for use as a human or veterinary drug.

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, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum.

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, and acupoint injections.

The dosage form can be a liquid dosage form or a solid dosage form. For example, the liquid dosage form may be a true solution type, a colloid type, a microparticle dosage form, an emulsion dosage form, or a suspension dosage form. Other dosage forms such as tablets, capsules, pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, lyophilized powders, 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 form a unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier are, for example, a diluent and an absorbent such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, tannic acid. Aluminum, etc.; wetting agent and binder, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, gum arabic, gelatin paste, sodium carboxymethyl cellulose , shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrating agents, such as dried starch, alginates, agar powder, brown algae starch, sodium bicarbonate and tannic acid, calcium carbonate, polyoxyethylene sorbes Sugar alcohol fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.; absorption enhancer For example, quaternary ammonium salts, sodium lauryl sulfate, and the like; lubricants such as talc, cerium oxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. Tablets may also be further formed into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer tablets and multilayer tablets.

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, diluents and absorbents 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 various carriers described above, and the mixture thus obtained is placed in a hard gelatin capsule or soft capsule. Active Ingredient 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 enclosed in a hard capsule or used as an injection.

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 pharmacodynamics. An 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, polyoxyethylene sorbitan fatty acid ester, and the like. 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 solubilizer, a buffer, a pH adjuster or the like may be added. These excipients are commonly used in the art. In addition, coloring agents, preservatives, perfumes, flavoring agents, sweeteners or other materials may also be added to the pharmaceutical preparations as needed.

The pharmaceutical or pharmaceutical composition of the present invention can be administered by any known administration method for the purpose of administration and enhancing the therapeutic effect.

The dosage of the pharmaceutical composition of the compound of the present invention depends on a number of factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality and individual response of the patient or animal, the route of administration, the number of administrations, For therapeutic purposes, the therapeutic dose of the present invention can vary widely. 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. A suitable daily dose for the compound of the invention is preferably from 0.1 to 100 mg/kg body weight, more preferably from 0.1 to 100 mg/day/person. The above dosages may be administered in a single dosage form or divided into several, for example two, three or four dosage forms, which are limited by the clinical experience of the administering physician and the dosing regimen including the use of other therapeutic means.

The total dose required for each treatment can be divided into multiple or single dose administrations. The compounds or compositions of the invention may be administered alone or in combination with other therapeutic or symptomatic agents and adjusted in dosage.

Term: d1-NBP: butyl-butyl benzoquinone d-NBP: dextro-butyl phenyl hydrazine 1-NBP: L-n-butyl phenyl hydrazine tMCAO: transient focal cerebral ischemia model

Example 1 Effect of optically active butylphthalide on the volume of cerebral infarction in tMCAO rats

Experimental Materials and Methods Reagents and pharmaceuticals 1-, d-, d1-NBP were provided by our synthesis chamber with optical and chemical purity of >99%, and their optical rotations were -66.49, +66.88 and 0 degrees. Formulated with vegetable oil. MK801 is a Sigma product formulated with physiological saline. Chlorotriphenyltetrazolium (TTC): Prepared by using 4% solution in distilled water and produced by Beijing Chemical Plant.

Male Wistar rats were prepared by transient local cerebral ischemia model (tMCAO), weighing 280-320 g, placed in 1 cage every 5 days, kept at 23 ° C at room temperature, free to eat and drink. Rats were anesthetized with sodium pentobarbital (40 mg/kg). Under the operating microscope, the median neck incision exposes the right common carotid artery, and the internal and external carotid arteries (1CA, ECA) are carefully separated and the anastomosis is ligated. The ECA was ligated and cut, straightened in line with the ICA, and a round-neck nylon thread (coated with polylysine) with a diameter of 0.28 mm was inserted into the ICA by ECA for about 20 mm, and the cranial artery was transferred to the middle cerebral artery. The beginning. After 2 hours of ischemia, the nylon thread was carefully pulled out, the ECA opening was ligated, the incision was sutured, placed back in the cage, and reperfused for 24 hours. The whole process was maintained at room temperature of 24-25 °C. During the entire procedure, the rats were placed on a hot plate at 37 ° C to maintain body temperature. Dosing 15 minutes after ischemia.

Grouping and administration were divided into 8 groups, 10 in each group, respectively: 1) solvent control group, vegetable oil 1 ml/kg; 2) and 3) d1-NBP40 and 80 mg/kg group; 4) and 5) For the d-NBP20 and 40 mg/kg groups; 6) and 7) for the 1-NBP20 and 40 mg/kg groups; 8) for the MK8010.1 mg/kg group. Groups 1-7 were administered orally; groups 8 were administered intraperitoneally.

Neurobehavioral scores were followed by behavioral observations 24 hours after ischemia. The rat tail was left about 1 foot away from the ground to observe the condition of the two forelimbs; the shoulders of the rats were pushed to observe whether there was any difference in the resistance between the two sides; the rats were placed on the ground to observe the walking. Score according to the following criteria: 0 points: Both forelimbs are strong and extend symmetrically to the ground: the shoulders have the same resistance and normal walking.

1 point: the operation is performed on the lateral shoulder and the forelimb is adducted; the shoulders have the same resistance and the walking is normal.

2 points: the operation was performed on the lateral shoulder and the forelimb was adducted; when the shoulders were pushed, the resistance on the opposite side of the operation was decreased; the walking was normal.

3 points: the operation is performed on the lateral shoulder and the forelimb is adducted; when the shoulder is pushed, the resistance on the opposite side of the operation is reduced; walking around the circle.

4 points: the operation was performed on the lateral shoulder and the forelimb was adducted; there was no spontaneous activity.

The higher the score, the more serious the behavioral disorder of the animal.

Determination of cerebral infarct volume Rats were anesthetized with sodium pentobarbital (100 mg/kg) and the brain was quickly decapitated 24 hours after surgery. The crown was cut into 6 pieces (the first to the fifth piece was 2 mm thick, and the sixth piece was 4 mm thick), and then the brain piece was quickly placed in a 5 ml solution containing 1.5 ml of 4% TTC and 0.1 ml of 1 M K ₂ HP0 ₄ . Protected from light, incubate at 37 °C for 30 minutes, during which it is flipped every 7-8 minutes. After TTC staining, the normal brain tissue is rose red, and the infarct tissue is white with clear boundaries. After the incubation, the brain slices of each group are arranged neatly, and the digital camera is saved. Each brain slice was processed by a computer image analysis system (SPOT 3.5 software), and the infarct size was superimposed to calculate the infarct volume. The infarct volume is expressed as a percentage of the cerebral hemisphere to eliminate the effects of cerebral edema. The specific calculation formula is: cerebral infarction volume (%) = (volume of the non-operative side hemisphere - volume of the non-infarct portion of the surgical side) / volume of the unoperated side hemisphere.

Statistical analysis All results were expressed as mean ± standard error. The behavioral score, cerebral infarction volume test was performed by one-way ANOVA, and the difference between groups was analyzed by pοsthoc LSD test. P < .05 is a significant difference.

Results The effect of optically active butylphthalide on the volume of cerebral infarction in tMCAO rats was tested by one-way ANOVA. There was a significant difference between the groups. The 1-NBP group (20 mg/kg and 40 mg/kg) reduced the infarct volume. Very significant and dose-dependent. Among them, the 40 mg/kg group had the strongest effect, and the infarct volume was reduced by 80.4% (P<0.001 compared with the solvent group), and the treatment effect was close to the MK801 group (infarct volume was reduced by 81.8%, P<0.001). The d-NBP and d1-NBP groups had no significant effect (see Figures 1, 2). The above results indicate that 1-NBP is significantly more effective than d-NBP in reducing infarct volume. Although the infarct volume of d1-NBP (80 mg/kg) and d-NBP (40/kg) was smaller than that of the solvent group, it was not statistically significant. It is suggested that the presence of d-NBP in d1-NBP may antagonize the effect of 1-NBP.

The behavioral scoring solvent control group had a neurological score of 2 in most animals, which showed a contralateral forelimb internal rotation and a lateral compression of the contralateral extensor muscle. A small number of animals showed contralateral rotation symptoms and were rated as 3 points. Individual animal behavior scores were lighter, only showed contralateral forelimb internal rotation, 1 point or severe symptoms, lack of independent activities, rated 4 points. The median score of the vehicle control group was 2.6 ± 0.3. Oral administration of 1-NBP 20 after 15 minutes of ischemia significantly improved neurological symptoms (1.4 points P < 0.01 and 1.1 points P < 0.001), and MK801 had the most significant effect (0.8 points P < 0.001). The d-NBP and d1-NBP groups did not significantly improve neurological symptoms. In the MK801 treatment group, although the symptoms after cerebral ischemia were significantly alleviated, the animals' limbs were weak and weak, and they were sick when crawling, showing ataxia, and often circled to one side. Behavioral scoring showed that the degree of improvement in behavior was parallel to the effect of the drug in reducing infarct volume (see Figure 3).

In the past, the model we used was permanent local cerebral ischemia (causing the middle cerebral artery). In this study, a transient local cerebral ischemia model (2 hours ischemia + 24 hours reperfusion) was used. The latter model group had a larger cerebral infarction volume than the former. Big. The dose of d1-NBP used in this study was the same as before, but at the dose (40 mg/kg, 80 mg/kg), d1-NBP did not reduce the volume of cerebral infarction. The difference was due to the model. Different caused.

We have studied from the biochemical, molecular biology and holistic animal levels, and have obtained consistent results, that is, the brain protection of 1-NBP is stronger than that of d-NBP and d1-NBP, and the presence of d-NBP in d1-NBP is antagonistic. The action of 1-NBP exerts, so that the effect of d1-NBP against cerebral ischemia is reduced. In the literature reports and in accordance with the requirements of new drug development, in order to enhance the efficacy and reduce toxic and side effects, it is advocated that the racemic drugs should be separated into optical isomers, and their respective properties will be clarified. Isomers are developed into chiral new drugs. Accordingly, 1-NBP in butyl benzoquinone has a strong anti-cerebral ischemia effect, and d-NBP not only has a weak effect, but also antagonizes the anti-cerebral ischemia effect of 1-NBP, so it is reasonable to develop a left-handedness to become a new one. A chiral drug for the treatment of acute ischemic stroke.

Previous studies have shown that 1-NBP antiplatelet aggregation and antithrombotic effects are close to aspirin and spirulina, and it has brain protection and improved brain microcirculation (d-NBP has no effect), and these effects are aspirin and What is not available for spirulina. Therefore, 1-NBP has a wide therapeutic range, which can not only treat acute ischemic stroke, but also can be used for secondary prevention of stroke, prevention of myocardial infarction and treatment of peripheral vascular disease. Expected to have significant social and economic benefits, so prospects for its development as a drug for stroke treatment are promising. The role of L-butylphthalide in the treatment of focal cerebral ischemia has not been reported in the literature at home and abroad.

Figure 1. Digital photograph of the effect of NBP on the volume of cerebral infarction in rats 15 minutes after temporary middle cerebral artery occlusion.

Figure 2. Effect of NBP administration on the volume of cerebral infarction in rats 15 minutes after temporary middle cerebral artery occlusion. Data are expressed as the percentage of volume of the infarcted part of the contralateral (normal) hemisphere, expressed as mean ± standard error.

*P<0.05, **P<0.01, ***P<0.001 and solvent group comparison

Figure 3. Effect of NBP administration on behavioral scores in rats 15 minutes after transient middle cerebral artery occlusion. Data are expressed as mean ± standard error.

*P<0.05, **P<0.01, ***P<0.001 compared to the vehicle group.

Claims (5)

Use of levo-n-butyl benzoquinone represented by general formula (I) for preparing medicine for treating acute ischemic brain injury According to the use of claim 1, it is characterized in that the brain damage is caused by local cerebral ischemia. According to the use of claim 1, it is characterized in that the effective therapeutic dose of the compound is from 0.1 to 100 mg/kg/day. A pharmaceutical composition for treating acute ischemic brain injury, characterized by comprising a therapeutically effective dose of levo-n-butylphenyl hydrazine represented by the general formula (I) and a pharmaceutically acceptable carrier The pharmaceutical composition according to claim 4, characterized in that the pharmaceutical composition comprises a tablet, a capsule, a pill, an injection, a sustained release preparation, a controlled release preparation, and various microparticle delivery systems.