CN113855635A - Argatroban injection liposome and preparation method thereof - Google Patents

Abstract

The invention discloses an argatroban liposome injection which is mainly prepared from the following components in parts by weight: the injection comprises the following components, by weight, 1 part of argatroban, 0.1-0.3 part of sodium hydroxide, 10-25 parts of phospholipid, 5-8 parts of phosphatidylglycerol, 5-10 parts of cholesterol and 805-10 parts of tween, wherein the phospholipid is one of soybean lecithin, pelitized phosphatidylethanolamine, dicetyl phospholipid and soybean phosphatidylserine.

Description

Argatroban injection liposome and preparation method thereof

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to argatroban injection liposome and a preparation method thereof.

Background

Cerebral infarction (also called cerebral infarction) and ischemic stroke (cerebral apoplexy) refers to ischemic necrosis or softening of localized brain tissue caused by cerebral blood supply disorder, ischemia and anoxia. The common clinical types of cerebral infarction include cerebral thrombosis, lacunar infarction, cerebral embolism and the like, the cerebral infarction accounts for 80 percent of all cerebral apoplexy, and the cerebral infarction has high morbidity, high mortality, high disability rate, high recurrence rate and more complications and is one of three diseases threatening human health. Data in the Chinese cerebral apoplexy prevention and treatment report show that the cerebral apoplexy is the first cause of death in China, and the morbidity of the cerebral apoplexy tends to rise. With the aging population and the increase of various risk factors, the proportion of ischemic stroke is getting larger and larger. The rate of disability recurrence due to ischemic stroke is higher than hemorrhagic stroke. Therefore, prevention and treatment of ischemic stroke should be the focus of stroke prevention.

The cerebral infarction belongs to the subdivision discipline of neurology, the conventional treatment process usually adopts anticoagulation treatment, thrombin plays a more critical role in the processes of coagulation and platelet activation, the treatment of cerebral infarction mainly inhibits thrombin, heparin is the earliest plasma coagulation system inhibitor and is widely used in clinic, but heparin has no effect on coagulated blood clots, and can be inactivated by platelet factor IV and heparinase to cause heparin-induced platelet reduction, so that thromboembolism can occur to cause the reduction of the application safety of common heparin, therefore, the development of a direct thrombin inhibitor becomes a hotspot of research, the inhibition effect of the direct thrombin inhibitor is independent of antithrombin in vivo, the pharmacological activity is strong, the coagulation effect is more excellent, and hirudin and bivalirudin can be replaced.

Argatroban is an antithrombotic drug studied and synthesized by mitsubishi chemical research institute of japan, and has a chemical name of (2R, 4R) -4-methyl-1- [ N- ((R, S) -3-methyl-1, 2, 3, 4-tetrahydro-8-quinolinesulfonyl) -L-arginyl ] -2-piperidinecarboxylic acid, and a structural formula of:

the argatroban serving as a novel anticoagulant for treating ischemic stroke is an artificially synthesized monovalent small-molecule direct thrombin inhibitor, can be selectively and reversibly combined with a thrombin catalytic site, can inactivate liquid-phase thrombin and thrombin combined with fibrin thrombus, and can not cause other bleeding complications due to influence on normal blood coagulation function in vivo while treating the ischemic stroke. Has the advantages of fast combination with thrombin, direct entering into thrombus, fast recovery after stopping medicine, no dependence, etc.

Argatroban has poor solubility in water and is therefore not suitable for making injections containing argatroban in high concentrations. US patent US5214052 solves this problem by dissolving argatroban in a dissolution medium comprising water, ethanol and sugars. Chinese patent CN101516370A provides an aqueous argatroban preparation, which is prepared into ready-to-administer injectable dosage forms using lactobionic acid and methionine as solubilizers. However, the argatroban injection prepared by the method is unstable in the long-term storage process, and the clinical application of the argatroban injection is influenced

Patent CN102366410 (published 2012 and 03 month 07) discloses an argatroban liposome injection, wherein the liposome is prepared from sphingomyelin, octadecylamine and tween 80, and the preparation method of the scheme is relatively complex and is not suitable for large-scale production.

Therefore, the argatroban liposome injection which is simple in preparation method, high in encapsulation rate and good in treatment effect is urgently needed, the water solubility of argatroban in the injection is improved, the dissolution rate and the stability of the product are improved, and the damage of organic solvents such as sorbitol and ethanol to a human body is solved.

Disclosure of Invention

Aiming at the defects of the prior art, the technical problem to be solved by the invention is that a proper liposome system is constructed, and the components and the component proportion of the liposome are explored, so that the liposome can effectively coat the argatroban active component under the components and proportion of specific phospholipid and cholesterol, the solubility of the argatroban in the solution is obviously enhanced, the use of an organic solution is reduced by relying on the use of the specific liposome, the toxicity of the injection is improved, and the problem of renal toxicity of the traditional argatroban injection is solved, and the specific technical scheme is as follows:

the invention discloses an argatroban liposome injection which is mainly prepared from the following components in parts by weight:

the phospholipid is one of soybean lecithin, phosphatidylethanolamine, dicetyl phospholipid and soybean phosphatidylserine.

The phospholipid is a basic material for preparing the liposome, natural phospholipid and synthetic phospholipid are commonly used, the types of the phospholipid are various, the factors considered comprehensively in the selection process are more, the key for preparing the liposome is how to select proper phospholipid from the various phospholipids, repeated experiments show that the effect of selecting soybean lecithin, peletized phosphatidylethanolamine, dicetyl phospholipid and soybean phosphatidylserine for preparing the argatroban liposome is the best, and particularly the effect of peletized phosphatidylethanolamine and dicetyl phospholipid is the best.

Further preferably, the components are prepared from the following components in parts by weight:

further preferably, the liposome injection further comprises an osmotic pressure regulator and a buffer solution, wherein the osmotic pressure regulator is selected from one or more of sodium chloride, mannitol and glycerol, and the buffer solution is selected from one or more of phosphate buffer solution, citrate buffer solution and acetate buffer solution with the pH value of 6.0-7.5.

Further preferably, the weight ratio of the sum of the weights of the phospholipid and the phosphatidylglycerol to the soybean lecithin is 3:1-6:1, and the weight ratio of the sum of the weights of the phospholipid and the phosphatidylglycerol to the tween 80 is 3:2-6: 1.

Further preferably, the specification of the argatroban liposome injection is that the volume of 20ml injection is 10mg argatroban weight.

The invention also discloses a method for preparing the argatroban liposome injection, which comprises the following steps:

1) dissolving the phospholipid, the phosphatidylglycerol, the cholesterol and the tween 80 with the prescription amount in a buffer solution to obtain the blank liposome;

2) sterilizing blank lipid with steam, and performing ultrasonic treatment;

3) mixing argatroban and sodium hydroxide, dissolving in water, maintaining the temperature of the sterilized blank liposome, slowly adding argatroban water solution under stirring, keeping the temperature for 10-15min, adding osmotic pressure regulator solution, and mixing to obtain argatroban liposome injection.

More preferably, the incubation temperature of the hollow white liposome in the step 3) is 65-75 ℃.

The invention also discloses the application of the argatroban liposome injection obtained by the component scheme and the preparation method in preparing a medicament for treating cerebral infarction diseases.

The invention has the beneficial effects that:

1) by screening proper liposome, the solubility of argatroban in aqueous solution is obviously improved by coating the liposome, the entrapment rate of active components is improved, and the dissolution rate of the product is obviously improved.

2) By selecting proper preparation components, the content of impurities in the product is reduced, the stability of the argatroban injection is obviously improved, the shelf life of the injection product is prolonged, and the toxicity of the injection product is obviously reduced.

3) Provides the argatroban injection with simple preparation method, and is suitable for large-scale industrial production.

Detailed Description

The invention will now be further illustrated by reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention. Further, it should be understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents may fall within the scope of the invention as defined in the appended claims.

The argatroban adopted by the invention is provided by Shandong New times pharmaceutical Co., Ltd, and sodium hydroxide is in medicinal grade and is sold in markets; phosphatidyl ethanol (DSPE-MPEG2000) for injection, purchased from ai Wei Tuo (Shanghai) pharmaceutical technology, Inc.; phosphatidylglycerol, cholesterol, tween 80 was purchased from zibohaijie chemical ltd; the buffer solution and the osmotic pressure regulator are formulated by a conventional method.

Example 1: the argatroban liposome injection is prepared by the following prescription in parts by weight

The argatroban liposome injection is prepared by the following preparation process:

1) dissolving the prescription dose of the culture phosphatidylethanolamine, the phosphatidylglycerol, the cholesterol and the tween 80 in 120ml of sodium chloride solution, stirring and carrying out ultrasonic treatment for 15min to obtain blank liposome;

2) performing ultrasonic treatment on the blank lipid obtained in the step 1) after steam sterilization for 15 min;

3) mixing argatroban and sodium hydroxide according to the prescription amount, dissolving in 300ml of water for injection, heating the sterilized blank liposome to 56 ℃, preserving heat, slowly adding the water solution of argatroban under stirring, continuously preserving heat at 56 ℃ for 10-15min, adding 120ml of citrate buffer solution with pH of 6.5, and uniformly mixing to obtain the argatroban liposome injection.

Example 2: the argatroban liposome injection is prepared by the following prescription in parts by weight

The argatroban liposome injection is prepared by the following preparation process:

1) dissolving dicetyl phosphatide, phosphatidyl glycerol, cholesterol and tween 80 in a prescribed amount in 150ml of ethanol solution, heating and stirring, removing ethanol by rotary evaporation, and performing ultrasonic treatment on the rest components for 15min to obtain blank liposome;

2) performing ultrasonic treatment on the blank lipid obtained in the step 1) after steam sterilization for 15 min;

3) mixing argatroban and sodium hydroxide according to the prescription amount, dissolving in 300ml of water for injection, heating the sterilized blank liposome to 56 ℃, preserving heat, slowly adding the water solution of argatroban under stirring, continuously preserving heat at 56 ℃ for 10-15min, and uniformly stirring to obtain the argatroban liposome injection.

Example 3: the argatroban liposome injection is prepared by the following prescription in parts by weight

The injection comprises the components according to the above (example 3), mannitol as osmotic pressure regulator, acetate buffer solution with pH of 7.5 as buffer solution, and the rest preparation method is the same as example 1

Example 4: the argatroban liposome injection is prepared by the following prescription in parts by weight

The injection comprises the components according to the above formula (example 4), sodium chloride as osmotic pressure regulator, citrate buffer solution with pH of 6.0 as buffer solution, and the rest preparation method is the same as example 1

Comparative example 1: the argatroban liposome injection is prepared by the following prescription in parts by weight

The osmotic pressure regulator and the buffer solution are the same as in example 1, the specific components and contents are prepared according to the above comparative example 1, and the specific preparation method is the same as in example 1.

Comparative example 2: the argatroban liposome injection is prepared by the following prescription in parts by weight

The osmotic pressure regulator and the buffer solution are the same as in example 1, the specific components and contents are prepared according to the above comparative example 1, and the specific preparation method is the same as in example 1.

Comparative example 3: the argatroban liposome injection is prepared by the following prescription in parts by weight

The osmotic pressure regulator and the buffer solution are the same as in example 1, the specific components and contents are prepared according to the above comparative example 1, and the specific preparation method is the same as in example 1.

Comparative example 4: the argatroban liposome injection is prepared by the following prescription in parts by weight

The specific preparation method adopts the following scheme:

(1) dissolving the phosphatidylethanolamine, the phosphatidylglycerol and the cholesterol which are cultured according to the prescription amount in 150ml of ethanol, uniformly mixing, and removing the ethanol on a rotary film evaporator under reduced pressure to obtain a phospholipid membrane;

(2) adding 200ml citric acid-sodium citrate buffer solution with pH of 5.7, shaking, stirring for 25min to completely hydrate phospholipid membrane, homogenizing and emulsifying at high speed for 20min by tissue triturator at 15000r/min, and filtering with 0.45 μm microporous membrane to obtain blank liposome suspension;

(3) the argatroban and sodium hydroxide are mixed and dissolved in 220ml of water for injection, a 0.45 mu m microporous membrane is used for filtering, the filtrate is added into the blank liposome suspension, and the mixture is stirred for 30 minutes at the temperature of 70 ℃ to obtain the argatroban liposome injection.

Test 1: determination of encapsulation efficiency

The argatroban liposome injection preparation prepared in examples 1-4 and comparative examples 1-4 is centrifuged at high speed and 5000r/min for 30min, 1ml of clear solution is taken, dissolved by ethanol, and the argatroban content is measured by high performance liquid chromatography, the encapsulated content M1 is determined, the total amount of argatroban in the liposome preparation is M0, and the encapsulation rate N is:

N＝M1/M0×100％

specific results are shown in Table 1

TABLE 1 encapsulation efficiency results for examples and comparative examples

Test 2: stability test

The samples prepared in the above examples and comparative examples are tested in an accelerated test at 40 ℃ and 75% +/-5% relative humidity for 6 months, and the results are shown in Table 2; the long-term test investigation is carried out under the conditions of normal temperature of 25 ℃ and relative humidity of 60 +/-10% for 18 months, the change of each quality index is detected, and the result is shown in table 2.

TABLE 2 Total impurity content in examples 1-4 and comparative examples 1-4

As can be seen from Table 2, at 6 months of acceleration, the contents of the marketed preparations and the comparative examples decreased, and the related substances increased; the sample property, content and related substance change are not obvious, which shows that the product of the invention has good stability.

While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods and applications described herein, as well as other variations and combinations of the techniques described herein, may be made and used without departing from the spirit and scope of the invention. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention.

Claims (8)

1. An argatroban liposome injection is mainly prepared from the following components in parts by weight:

the phospholipid is one of soybean lecithin, phosphatidylethanolamine, dicetyl phospholipid and soybean phosphatidylserine.

2. The argatroban liposome injection according to claim 1, wherein each component is prepared from the following components in parts by weight:

3. an argatroban liposome injection according to claims 1-2, further comprising an osmotic pressure regulator and a buffer solution, wherein the osmotic pressure regulator is selected from one or more of sodium chloride, mannitol and glycerol, and the buffer solution is selected from one or more of phosphate buffer, citrate buffer and acetate buffer with pH of 6.0-7.5.

4. The argatroban liposome injection according to claim 1, wherein the weight ratio of the sum of the weights of the phospholipid and the phosphatidylglycerol to the cholesterol is 3:1-6:1, and the weight ratio of the sum of the weights of the phospholipid and the phosphatidylglycerol to the tween 80 is 3:2-6: 1.

5. An argatroban liposome injection according to claims 1-2, wherein the specification of the argatroban liposome injection is 20ml injection volume to 10mg argatroban weight.

6. A method of preparing the argatroban liposome injection of claim 1, comprising the steps of:

1) dissolving the phospholipid, the phosphatidylglycerol, the cholesterol and the tween 80 with the prescription amount in a buffer solution to obtain the blank liposome;

2) sterilizing blank lipid with steam, and performing ultrasonic treatment;

3) mixing argatroban and sodium hydroxide, dissolving in water, maintaining the temperature of the sterilized blank liposome, slowly adding argatroban water solution under stirring, keeping the temperature for 10-15min, adding osmotic pressure regulator solution, and mixing to obtain argatroban liposome injection.

7. The method for preparing argatroban liposome injection according to claim 6, wherein the incubation temperature of the hollow white liposome in the step 3) is 65-75 ℃.

8. Use of argatroban liposome injection according to claim 1 for the preparation of a medicament for the treatment of cerebral infarction diseases.