CN1086942C - Application of butyl phthalide in preparing medicines curing thrombosis and thrombocyte coagulation - Google Patents

Abstract

The invention relates to the application of butylphthalide in the preparation of antithrombotic drugs and anti-platelet aggregation drugs. In a preferred embodiment, the butylphthalide is levorotatory.

Description

Application of butylphthalide in the preparation of antithrombotic and antiplatelet aggregation drugs

The present invention relates to a new application of butylphthalide (dl-3-n-butylphthalide, NBP for short), and more specifically relates to the application of the compound in antithrombotic drugs and anti-platelet aggregation drugs.

Cardiovascular and cerebrovascular diseases such as atherosclerosis and high blood pressure can easily cause damage to the intima of blood vessels, slow blood flow, increased blood viscosity, and blood in a state of high viscosity, high coagulation, and high aggregation (StevensMK and Yaksh TL, Time course of release in vivo of PGE ₂ , PGF _2α , 6-keto-PGF _1α , and TXB ₂ into the brain extracellular space after 15min of complete global ischemia in the presence and absence of cyclooxygenase inhibition, J cereb blood flow metab 1988, 8 : 790-94; and Wu JF, Liu TP, Effects of berberine on platelet aggregation and plasma levels of TXB ₂ and 6-keto-PGF _1α in rats with reversible middle cerebral artery occlusion, Acta Pharm Sin 1995, 30(2): 98- 102). It can be seen that antithrombotic therapy is of great significance in the prevention and treatment of thrombotic cerebrovascular diseases.

Butylphthalide can regulate the function of NOS-NO-cGMP system and the metabolism of arachidonic acid in nerve cells after cerebral ischemia (Chong ZZ and Feng YP, Effects of dl-3-n-butylphthalide on production of TXB ₂ and 6 -keto-PGF _1α in rat brainduring focal cerebral ischemia and reperfusion, Acta PharmacolSin, 1997, 18:505-08; and Yan CH and Feng YP, Effects of d-3-n-butylphthalide and 1-3-n-butylphthalide extracellular NO level intracellular cGMP level in primary cultured rat cortical neurons, Acta Pharm Sin, 1998, 33(6):418-423). The metabolism of arachidonic acid and the production of NO have important effects on the formation of thrombus, platelet function and hemodynamics in the body.

Therefore, the object of the present invention is to provide butylphthalide for inhibiting platelet aggregation and its application in the preparation of antithrombotic drugs.

Butylphthalide is a chiral compound, including racemic, left-handed and right-handed butylphthalide, abbreviated as dl-, l- and d-NBP, respectively.

One aspect of the present invention includes the use of NBP in antithrombotic drugs. Preferably, said butylphthalide is l-NBP.

Another aspect of the invention includes the use of NBP in antithrombotic drugs. Preferably, said butylphthalide is l-NBP.

NBP can be administered in pure form or in combination with pharmaceutically acceptable excipients or carriers. For the excipient or carrier, and the dosage form of the composition, those skilled in the art can refer to standard textbooks, such as Remington's Pharmaceutical Sciences ^17th edition, to determine. The dosage of NBP or a composition thereof depends on the severity of the condition to be treated, the route of administration, and the sex, age, health status of the patient, etc., which are obvious to ordinary residents. Furthermore, the appropriate dosage, mode and frequency of administration can be readily determined by one of ordinary skill in the art. Generally speaking, after taking l-NBP 200mg orally, the platelet aggregation rate caused by ADP and AA was significantly lower than normal (P<0.01 or P<0.05), and there was a time-dependent relationship. The effect of l-NBP continued to weaken after 4 hours, suggesting that the effect of l-NBP is reversible, which is very beneficial for clinical safe drug use.

NBP or its composition can be used for inhibiting platelet aggregation and thrombus formation, preventing and treating stroke, coronary heart disease and peripheral vascular disease. In the clinical phase II trial for ischemic stroke in the acute phase and recovery phase, after a randomized, double-blind, placebo-controlled trial, it was found that the NBP group (n=25) was significantly more effective than the placebo group (n=25). Significant difference (P<0.01). This indicates that NBP has a good therapeutic effect on acute ischemic stroke.

Thrombosis is one of the main pathogenic factors in cardio-cerebral ischemic diseases. Moreover, during cerebral ischemia, the ratio of PGI ₂ /TXA ₂ in the brain decreases significantly. At the same time, the platelet function is abnormally hyperactive, and the blood is in a hypercoagulable state, which is one of the important reasons for secondary brain injury. In the following examples of the present invention, the effect of NBP with different optical activity on thrombus formation in vitro was observed by using the common carotid artery-external jugular vein blood flow bypass method in rats, and compared with Asp. The results showed that ip administration of dl and l-NBP both had obvious antithrombotic effect, and the effect was weaker than that of Asp when the dose was small, but the effect of l-NBP was stronger than that of Asp when the dose was increased. After intragastric (ig) administration, dl-NBP and l-NBP can also reduce the wet weight of thrombus in a dose-dependent manner, but it is weaker than Asp at the same dose. Significantly enhanced, similar to high doses of l-NBP or Asp alone. In conclusion, NBP has a strong antithrombotic effect in vivo, and the effect of intragastric administration is significantly weaker than that of intraperitoneal injection, which may be related to the low bioavailability of the drug after intragastric administration.

Since the hyperactivation of platelet function is an important factor of thrombus formation, the effect of NBP on platelet function was further studied. It was found that dl-, d-, and l-NBP could dose-dependently inhibit platelet aggregation induced by AA, collagen (Coll), and ADP, and had relative selectivity to AA-induced platelet aggregation at higher concentrations, among which l-NBP has the strongest effect. dl-NBP and l-NBP can dose-dependently increase the level of cAMP in platelets, and cAMP in platelets can exert anti-platelet effect by inhibiting the increase of intracytoplasmic calcium ion concentration.

In addition, TXA ₂ is also an information molecule with important physiological functions in platelets. AA on platelet membrane phospholipids is metabolized by cyclooxygenase and thromboxane synthase to produce TXA ₂ , which can directly activate PLC in a feedback manner, reduce the level of cAMP in platelets and induce Platelet Aggregation (Edited by Wang Zhong, Zheng Zhiquan, Modern Thrombosis, Beijing: Beijing Medical University, China Union Medical University Press, 1997, pp. 508-509). Zhong Zhaozhong et al found that (Chong ZZ et al., above), dl-, d- and l-NBP can dose-dependently inhibit the release of AA in ischemic brain tissue, promote the synthesis of PGI ₂ in cerebral cortex cells, and inhibit the synthesis of TXA ₂ Synthesis of AA, thereby increasing the ratio of PGI ₂ /TXA ₂ , and it is believed that affecting AA metabolism is one of the main mechanisms for its pharmacological effects.

However, in the study of the present invention, it was found that dl- and d-NBP had no significant effect on the platelet TXA ₂ increase caused by exogenous AA, and l-NBP had a weaker effect only at high concentrations (10 ^-4 mol/L). inhibition. At the same time, the latest research results found that dl-, l-NBP can dose-dependently increase the release of PGI ₂ in cultured endothelial cells, while d-NBP has no obvious effect on the production of PGI ₂ . It can be seen that affecting AA metabolism, especially increasing the production of PGI ₂ , plays an important role in the antithrombotic effect of NBP, and is also one of the possible mechanisms for the stronger effect of l-NBP than d-NBP.

The results of the present invention also found that, unlike Asp, l-NBP can significantly inhibit the release of 5-HT, which has the effect of inducing platelet aggregation and vasoconstriction in platelets, while dl-, d-NBP, and Asp have no significant effect on 5-HT metabolism. Influence.

In summary, dl-, d- and l-NBP can all inhibit platelet function in a dose-dependent manner in vitro, and l-NBP is the most significant. In vivo antithrombotic experiments, l-NBP is also the most effective. l-NBP can inhibit the release of platelet 5-HT and increase the level of cAMP in platelets, and is an important component of dl-NBP to exert antithrombotic effect. However, d-NBP seems to have no antithrombotic effect, which may be related to the stereoselectivity of biomacromolecules in vivo, which makes the metabolic pathways and active products of the two optical isomers different in vivo.

The results of this study show that NBP has antithrombotic effect, suggesting that the research and application of NBP is of great significance to the prevention and treatment of thrombotic cerebral ischemic diseases and peripheral vascular embolism diseases.

The present invention will be described in detail below with reference to the accompanying drawings and embodiments, so that the objects and advantages of the present invention will be more apparent. In the attached picture:

Fig. 1 shows the effect of NBP and Asp(ip) on experimental thrombus formation, wherein, each group n=6, ^* P<0.05, ^** P<0.001 compared with the vehicle group.

Fig. 2 shows the effect of NBP and Asp(ip) on experimental thrombus formation, wherein, each group n=6, ^* P<0.05, ^** P<0.01, ^*** P<0.001 compared with the solvent group.

Figure 3 shows the effects of NBP and Asp on the release of 5-HT from platelets, where n=6 in each group, ^* P<0.05, ^** P<0.01, ^*** P<0.001 compared with the solvent group.

Figure 4 shows the effect of 1-NBP on ADP-induced aggregation of human platelets.

Figure 5 shows the effect of 1-NBP on AA-induced human platelet aggregation.

The following examples will be used: Materials Animals

Sprague-Dawley rats and rabbits were provided by the animal room of the Institute of Drug Control, Ministry of Health. Drugs and Reagents

dl-, l-, and d-NBP were provided by Professor Yang Jinghua, Institute of Materia Medica, Chinese Academy of Medical Sciences, with a purity of >96% and optical rotations of 0, -69.73, and +64.08 degrees, respectively. Aspirin (Asp) was produced by Xinhua Pharmaceutical Factory; collagen (collagen, Coll) was made from rat skin; thrombin was produced by Zhuhai Special Economic Zone Biochemical Pharmaceutical Factory; adenosine diphosphate (ADP) was purchased from Sigma Company; Alkenoic acid (AA) and o-phthalaldehyde (OPT) were purchased from Fluka. OPT was dissolved in HCl (10mol/L) to a 0.004% solution immediately before use, and stored in a refrigerator at 4°C. CAMP and TXB ₂ radioimmunoassay kits were purchased from Beijing Institute of Atomic Energy and Beijing East Asia Institute of Technology, respectively. instrument

TYXN-91 intelligent platelet aggregation instrument (produced by Shanghai General Electromechanical Technology Research Institute).

Platelet aggregation instrument (manufactured by Shimadzu, Japan). statistical analysis

The experimental results were expressed as mean ± standard deviation (SE), and the significance of the difference was analyzed by t test. Example 1: Effect of dl-, l- and d-NBP on experimental thrombus formation in rats

Rats, body weight between 260-300g, male, were divided into random groups, 6 animals in each group. Intraperitoneal administration group (ip): Asp, dl-, d- and l-NBP (5, 10, 20 mg/kg respectively) and the same volume of normal saline (NS). Experimental thrombosis bypass method was carried out after 20min (UmetzuT, Sanai K, Effect of 1-Methyl-2-Mercapto-5-(3-Pyridyl)-Imidazole (KC-6141), an anti-aggregating compound, oneexperimental thrombosis in rats, Thromb Haemost, 1978; 39:74-83): After rats were anesthetized with pentobarbital sodium 30mg/kg ip, three sections of polyethylene tubes with an inner diameter of 0.9mm and a length of about 12cm were taken (with a built-in 6cm long silk thread) ), connect the right common carotid artery and the left external jugular vein, open the blood flow for 15 minutes, then interrupt the blood flow, take out the silk thread and weigh it. At this time, the wet weight of the silk thread minus the dry weight of the original silk thread is the wet weight of the formed thrombus. Intragastric administration group: ig administration of Asp, dl-, d- and l-NBP (doses are 100, 200 mg/kg respectively) and Asp+l-NBP (doses are 100 mg/kg each), after 30 min as described above Perform thrombus bypass surgery. The results are shown in Figures 1 and 2.

The results showed that a low dose of Asp (ip) could produce obvious anti-thrombosis effect, and the inhibitory rates of 5, 10, and 20 mg/kg Asp on thrombus formation were 32%, 48% and 52%, respectively. l-NBP can also inhibit thrombus formation in a dose-dependent manner, and the inhibition rates are 22%, 36% and 69%, respectively. The effect of dl-NBP was significantly weaker than that of Asp and l-NBP at the same dose, while different doses of d-NBP had no significant effect on thrombus wet weight (see Figure 1). After D1, l-NBP (100, 200mg/kg) ig40min, the inhibitory rates to thrombus were respectively 26.4%, 32.7%, 28.0%, 36.1%, while the inhibitory rates to thrombus of the same dose of Asp were 35.6% and 54.8% respectively. %. The combined administration of 100 mg/kg of l-NBP and Asp has obvious antithrombotic effect, and the thrombus inhibition rate is 52.6% (see Figure 2). Example 2: Effects of dl-, l- and d-NBP on platelet aggregation in vitro

Blood was collected from the carotid artery of rats, and platelet rich plasma (platelet rich plasma, PRP) and platelet poor plasma (platelet poor plasma, PPP) were prepared according to conventional methods, and the number of platelets in PRP was adjusted to 4.0-6.0× ¹⁰⁸ /ml by PPP , referring to Born's method (Herbert JM, Bernat A, Samama M, Maffrand JP, The antiaggregating and antithrombotic activity of ticlopidine is potentiated by aspirinin the rat, Thromb Haemost 1996, 76:94-98), take 200 μl of PRP, add dl, d and l-NBP (final concentration of 3, 10, 30, 100 μmol/l) and Asp (100 μmol/l or 3-100 μmol/l) were placed on an intelligent platelet aggregator, prewarmed at 37°C for 5 minutes, and then added Four aggregation inducers: Coll, ADP, thrombin, and AA, the final concentrations were 50 μl/ml, 5 μmol/l, 1 U/ml and 0.5 mmol/l, respectively. The maximum aggregation rate of platelets was measured 5 minutes after the aggregation inducer was added. The results are shown in Tables 1 and 2 below. Table 1: Effects of NBP and Asp on collagen and ADP-induced platelet aggregation in mice (in vitro) Dose (μmol/L) Collagen induction (%) Asp dl-NBP l-NBP d-NBP control 78.4±7.1 3 66.3±8.0 ^* 60.7±2.8 ^*** 74.4±13.6 10 58.7±10.2 ^** 50.2±2.0 ^*** 72.3±6.0 30 49.2±2.2 ^*** 35.7±7.0 ^*** 57.4±4.7 ^*** 100 41.7±7.4 ^*** 34.0±5.2 ^*** 25.0±10.7 ^*** 43.8±8.2 ^*** ADP induction (%) Asp dl-NBP l-NBP d-NBP control 72.4±4.7 3 64.1±3.9 ^** 62.4±3.9 ^** 73.1±5.1 10 59.1±4.1 ^*** 54.6±2.0 ^*** 65.5±3.5 ^* 30 54.4±7.7 ^*** 46.5±3.7 ^*** 65.1±8.8 ^*** 100 69.1±6.0 48.8±2.7 ^*** 37.3±2.8 ^*** 63.6±7.4 ^*** p<0.05, ^** p<0.01, ^*** p<0.001, compared to control. Table 2: Effects of NBP and Asp on AA and thrombin-induced murine platelet aggregation (in vitro) Dose (μmol/L) AA(%) Asp dl-NBP l-NBP d-NBP control 78.9±8.6 3 70.03±13.22 69.3±3.5 ^* 73.9±5.7 66.9±4.2 ^* 10 45.52±12.67 ^*** 48.2±2.6 ^** 59.2±4.5 ^*** 47.1±5.2 ^*** 30 37.2±13.02 ^*** 33.4±5.2 ^*** 33.4±2.7 ^*** 41.2±7.4 ^*** 100 0±0 ^*** 2.6±4.5 ^*** 0±0 ^*** 4.6±4.1 ^*** Thrombin induction (%) Asp dl-NBP l-NBP d-NBP control 72.4±4.7 3 83.7±5.4 85.7±4.5 86.4±4.3 10 84.9±7.9 82.4±7.5 85.9±7.6 30 76.8±16.4 72.7±11.8 82.2±9.5 100 54.2±7.3 ^** 70.2±7.3 70.9±9.9 78.6±11.2 p<0.05, ^** p<0.01, ^*** p<0.001, compared to control.

As can be seen from the results in Tables 1 and 2, dl-, d- and l-NBP at different concentrations in vitro all have the effect of inhibiting the platelet aggregation induced by Coll, ADP, AA, and in a good dose-dependent manner, l- NBP has the strongest effect and can completely inhibit platelet aggregation induced by AA at a high concentration (100 μmol/l), while d-NBP has a weaker effect. Different concentrations of dl-, d- and l-NBP had no significant effect on thrombin-induced platelet aggregation. Asp (100μml/l) has inhibitory effect on platelet aggregation induced by Coll, AA and thrombin, but has no obvious effect on ADP-induced platelet aggregation. Example 3: Effects of dl-, l- and d-NBP on cAMP content in platelets

After the rabbit was anesthetized, blood was collected from the common carotid artery, and PRP was prepared by centrifugation at 120g×10min. The number of platelets was adjusted to 2.4× ¹⁰⁹ /ml, and 0.4ml of PRP was taken, and different concentrations of Asp, dl-, d -Compared with l-NBP and polyethylene glycol (PEG), after prewarming at 37°C for 5 minutes, according to the literature method (Zhang Xiaying, Ren Shuqing, Xiong Jun, a sensitive platelet cyclic adenosine monophosphate (cAMP) assay method, Zhonghua Heart Vascular Disease Journal 1980, 8: 142-43) to measure cAMP content in platelets. The results are shown in Table 3 below. Example 4: Effects of dl-, l- and d-BP on platelet TXA ₂ content

Take blood from rabbits, prepare PRP, adjust the number of platelets to 1.5× ¹⁰⁹ /ml, take 180 μl of PRP, add different concentrations of dl-, d- and l-NBP and PEG control as shown in Table 3, and pre-warm at 37°C for 5 minutes Finally, add AA (final concentration is 0.3mmol/L), after 5min, stop reaction with the indomethazin of 60 μ g/ml, centrifuge at 200g * 15min, draw the supernatant, press the content of TXB ₂ in the determination sample ( Kato K, Sawada S, Toyoda T, Influence of endothelin on human platelet aggregation and prostacycling generation from human vascular endothelial cells in culture, Jpn. Circ. J., 1992, 56: 422-431). Table 3: Effects of NBP on the concentrations of cAMP and TXA2 in AA-induced murine platelets in vitro Dose (μmol/l) cAMP (mol/10 ⁹ platelets) Dl-NBP l-NBP d-NBP control 8.50±3.14 1.0 9.89±2.20 11.54±3.59 9.20±5.71 10 16.52±5.17 ^** 13.92±5.02 ^* 8.56±6.67 100 16.34±2.93 ^*** 19.89±10.44 ^* 12.70±7.89 TXB ₂ (μg/ ¹⁰⁹ platelets) dl-NBP l-NBP d-NBP control 3.88±0.45 1.0 3.60±0.48 3.29±0.33 3.68±0.35 10 3.67±0.28 3.57±0.45 3.41±0.21 100 2.93±0.66 2.94±0.41 ^* 3.29±0.56 Each group n=6, ^* P<0.05, ^** P<0.01, ^*** p<0.001.

The results in Table 3 above show that dl, l-NBP (1, 10, 100 μmol/l) can dose-dependently increase the cAMP content in platelets, while d-NBP has no obvious effect on it. At the same time, dl-NBP and d-NBP had no obvious effect on platelet TXA ₂ content, and l-NBP only had a weak inhibitory effect on the increase of TXA ₂ content at high concentrations. Example 5: Effects of dl-, l- and d-NBP on platelet 5-hydroxytryptamine (5-HT) release

Rabbit PRP was prepared by adding dl-, d- and l-NBP with final concentrations of 0.1, 1.0, 10, and 100 μmol/l, Asp 100 μml/l and PEG control, prewarmed at 37°C for 5 minutes, and induced platelet aggregation in collagen After 5 minutes, the reaction was terminated, and samples were prepared according to the Curzon method (CurzonG, Green AR., Rapid method for the determination of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in small regions of rat brain, Br J Pharmacol, 1970, 39: 653-56) , and use a spectrofluorometer (excitation light 365nm, emission light 480nm) to measure the release rate of 5-HT. The results are shown in Figure 3.

It can be seen from Figure 3 that l-NBP can significantly inhibit platelet 5-HT release in a dose-dependent manner, while dl and d-NBP have no obvious effect. Asp (100μmol/m1) has no obvious inhibitory effect on the release of 5-HT. Example 6: Effect of l-NBP on platelet aggregation in vitro

Healthy subjects, male, 28-30 years old, took l-NBP 200mg/person orally at 8 am. After taking the medicine, 30min, 1h, 2h, 3h and 4h later, blood was taken from the cubital vein, 3ml/time. Prepare platelet-rich plasma (PRP) and platelet-poor plasma (PPP) according to conventional methods, and use PPP to adjust the number of platelets in PRP to 4.0-6.0× ¹⁰⁸ /ml, refer to Born's method (same as above), take 200 μl of PRP, respectively Add dl, d, and l-NBP (final concentrations of 3, 10, 30, and 100 μmol/l) and Asp, place it on an intelligent platelet aggregation instrument, pre-warm at 37°C for 5 minutes, add aggregation inducers: ADP, AA, and The final concentrations were 5 μmol/l and 0.5 mmol/l, respectively. The maximum aggregation rate of platelets was measured 5 minutes after the aggregation inducer was added. The results are shown in Figures 4 and 5.

It can be seen from Figures 4 and 5 that before administration, the platelet aggregation rates induced by ADP and AA were 55.2±6.3% and 55.8±8.0%, respectively. 0.5 hours after taking the medicine, the platelet aggregation rate caused by ADP did not change significantly compared with normal, but from 1 hour to 3 hours, the platelet aggregation rate continued to decrease, which were 37.3±7.5%, 26±15.6% and 24.7±2.1%, respectively. 4 hours after taking the medicine, the platelet aggregation rate recovered to some extent, which was 39.7±5.9%. The effect of l-NBP on platelet aggregation induced by AA was slightly later than that of ADP, and the platelet aggregation rates were 52.8±9.3%, 47.8±9.3%, 28.7±14.5%, 29.3± 5.7% and 43.3±10.7%. Moreover, the subjects reported no discomfort after taking the medicine.

Claims (4)

1, the application of butylphthalide in the preparation anti-thrombosis drug.

2, application as claimed in claim 1, wherein, described butylphthalide is left-handed butylphthalide.

3, the application of butylphthalide in the preparation medicament for resisting platelet aggregation.

4, application as claimed in claim 3, wherein, described butylphthalide is left-handed butylphthalide.

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