CN102366408B - Monosialotetrahexosyl ganglioside sodium liposome injection - Google Patents

Abstract

The invention discloses a monosialotetrahexosyl ganglioside sodium liposome injection and its preparation method. The liposome injection is made from monosialotetrahexosyl ganglioside sodium, hydrogenated egg yolk lecithin, soybean derived sterol, sodium deoxycholate, poloxamer 188, sodium bisulfite and other auxiliary materials. The liposome injection provided by the invention has good stability; the liposome will not break due to fusion and ice crystals during the freezing process and will also maintain good entrapment rate after stored for a long time. According to the invention, the quality of the preparation product is improved, toxic and side effects are minimized, retention time of the medicament in systematic circulation is increased, bioavailability and blood-brain barrier permeability of the medicament are raised and the curative effect is obviously enhanced. In addition, the preparation method is simple and is suitable for industrial production.

Description

Monosialotetrahexosyl ganglioside sodium liposome injection

Technical Field

The invention relates to a monosialotetrahexosyl ganglioside sodium liposome, an injection thereof and a preparation method thereof, belonging to the technical field of medicines.

Background

The monosialotetrahexosylganglioside sodium is a substance extracted from pig brain and having an effect on nerve cell function damage, and can promote the function recovery of central nervous system damage caused by various reasons. The mechanism of action is to promote "neural remodeling" (including survival of nerve cells, axonal growth, and synaptic growth). The monosialotetrahexosylganglioside has protective effect on secondary nerve degeneration after injury. The monosialotetrahexosyl ganglioside has a positive effect on the parameters of cerebral hemodynamics and on cerebral edema caused by injury. Monosialotetrahexosyl ganglioside reduces neuronal cell edema by improving the activity of cell membrane enzymes. Animal experiments show that the monosialotetrahexosylganglioside can improve the behavioral disturbance caused by the Parkinson disease.

The monosialotetrahexosyl ganglioside can also be used for treating central nervous system diseases by maintaining Na on the cell membrane of the central nervous system⁺-K⁺TTP enzyme and Ca²⁺-Mg²⁺The ATP enzyme activity has the effects of maintaining ion balance inside and outside cells, relieving nerve cell edema, preventing accumulation of intracellular Ca2+, resisting neurotoxicity of excitatory amino acid, and reducing damage of free radicals to nerve cells. The exogenous monosialotetrahexosyl ganglioside can be combined with a nerve cell membrane in a stable manner to cause the functional change of the membrane. Peak radioactivity was measured in the brain andspinal cord 2 hours after dosing. Halving after 4-8 hours. The clearance of the drug is slow and is mainly excreted through the kidneys. The traditional Chinese medicine composition is mainly used for treating vascular or traumatic central nervous system injury in clinic; parkinson's disease.

The length of the hydrophilic end and the hydrophobic end of the ganglioside molecule is similar, so that the ganglioside molecule is easy to form a stable micelle state in water, and is not beneficial to the preparation of injection preparations. Patent WO9317691 prepares monosialotetrahexosylganglioside into sodium salt, uses phosphate as buffer solution for injection preparation, and uses sodium chloride as isoosmotic adjusting agent, thus effectively solving the problems that monosialotetrahexosylganglioside forms micelle state in water and is difficult to prepare medicine. However, the sodium salt of the prescription influences the monosialotetrahexosylganglioside biomembrane and blood brain barrier permeability, so that the blood concentration of the brain is low, the brain cannot be maintained above the treatment concentration for a long time, and the clinical use curative effect is greatly reduced. Therefore, a new method which can improve the rate of monosialotetrahexosylganglioside sodium crossing a biological membrane and a blood brain barrier and has high clinical use curative effect is urgently needed to be found.

Chinese patent CN101899074A discloses a method for preparing monosialotetrahexosylganglioside and a corresponding preparation, which mainly protects the method for preparing monosialotetrahexosylganglioside, but the corresponding preparation is only briefly described, and no special method is adopted for preparing the corresponding preparation, resulting in poor stability of the preparation, because monosialotetrahexosylganglioside itself is an extract, the stability is poor, and if no special method is adopted, the quality of the preparation is definitely poor.

Chinese patent CN101732331A discloses a composition of monosialotetrahexosylganglioside sodium and glutamic acid, wherein glutamic acid is used for replacing phosphate buffer and sodium chloride in patent WO9317691, which is beneficial to the monosialotetrahexosylganglioside sodium passing through biological membranes and blood brain barrier rate, and improves clinical curative effect. However, the glutamic acid can not play a role in protecting monosialotetrahexosylganglioside of a biological extract, and the quality stability of the preparation can not be guaranteed.

Chinese patent CN1799552A discloses a preparation method of a monosialotetrahexosylganglioside sodium preparation, which starts from the original extraction step and mainly protects the whole extraction method, the corresponding preparation is only simply described, no substance is added into the preparation, and the stability is not guaranteed.

In a drug carrier delivery system, research on submicron particles such as microemulsion, microspheres, nanoparticles, liposomes, pharmacosomes and the like has become a very active field in research on new drug formulations. The medicine is encapsulated in the submicron particles, so that the distribution of the medicine in the body can be changed, and the distribution amount of the medicine in a target organ can be increased, thereby improving the curative effect and relieving the toxic and side effects.

In a targeted drug delivery system, the research of the liposome is relatively extensive, and the liposome has good targeting property and biocompatibility in vivo.

As a novel pharmaceutical preparation, the liposome preparation has the following advantages:

(1) has the slow release effect: the active ingredients are slowly released, and renal excretion and metabolism are delayed, so that the action time is prolonged, and the quality effect is improved;

(2) the toxicity of the medicine is reduced;

(3) the solubility of the medicine is increased, and the quality of the preparation is improved;

(4) has targeting property: the drug carried by the liposome maintains high concentration at the local part of the liver and the reticuloendothelial system viscera of the spleen, thereby playing the role of drug organ targeting;

(5) has protective effect on active medicinal components.

Liposomes (liposomes) were originally discovered by the british scholars Bangham and Standlish when phospholipids were dispersed in water for electron microscopy. The phospholipid is dispersed in water to naturally form multilamellar vesicles, and each layer does not contain lipid bilayers; the vesicles are separated by water in the center and between layers, and the bilayer thickness is about 4 nm. Later, such bimolecular vesicles having a structure similar to that of a biological membrane were called liposomes. Liposomes can be divided into multilamellar liposomes and biliary liposomes. Unilamellar liposomes are further divided into small unilamellar liposomes and large unilamellar liposomes. The small unilamellar liposomes are spherical and generally have a size of 20-50 nm; the size of large unilamellar liposomes is in the order of microns.

In 1971 British Leimen et al began to use liposomes as drug carriers, and the main action mechanism was to encapsulate drug powders or solutions in the aqueous phase enclosed by the liposome bilayer lipid membrane or embed them in the liposome bilayer lipid membrane, and the particles had a cell-like structure, and entered into the human body, the main drug was phagocytized by the reticuloendothelial system to activate the autoimmune function of the body, and change the distribution of the encapsulated drug in vivo, so that the drug was accumulated mainly in the tissues and organs such as liver, spleen, lung and bone marrow, thereby improving the therapeutic index of the drug, reducing the therapeutic dose of the drug and reducing the toxicity of the drug.

In recent years, with the continuous progress of biotechnology, the preparation process of liposome is gradually improved, the action mechanism of liposome is further clarified, and in addition, the liposome is suitable for in vivo degradation, has no toxicity and immunogenicity, and particularly, a large amount of test data proves that the liposome can be used as a drug carrier to have the advantages of improving the therapeutic index of drugs, reducing the toxicity and side effects of the drugs, reducing the dosage of the drugs, improving the stability of the drugs and the like.

In view of the physicochemical properties of monosialotetrahexosylganglioside sodium and the problems in terms of bioavailability and blood-brain barrier permeability in the prior art, the present inventors have conducted long-term studies to obtain an injection of monosialotetrahexosylganglioside sodium liposome superior to the prior art after having made creative efforts.

Disclosure of Invention

The invention aims to solve the problems in the prior art, and provides a monosialotetrahexosylganglioside sodium liposome injection and a preparation method thereof, aiming to solve the problem of stability of the existing monosialotetrahexosylganglioside sodium preparation, protect active ingredients by the liposome, improve the bioavailability of the active ingredients, and further effectively promote blood brain barrier permeability of the monosialotetrahexosylganglioside sodium.

In order to form a superior quality monosialotetrahexosylganglioside sodium liposome injection, it is important to find a film-forming material which is well compatible with monosialotetrahexosylganglioside sodium, thereby encapsulating it well and being impermeable, and to find the corresponding components which enable the liposomes to form a stable injection.

In order to achieve the above object, a great deal of research and experiments conducted by the inventors of the present invention have found that monosialotetrahexosylganglioside sodium, hydrogenated egg yolk lecithin, soyasterol, sodium deoxycholate, and poloxamer 188 in a specific weight ratio can be prepared into monosialotetrahexosylganglioside sodium liposome injection, wherein the monosialotetrahexosylganglioside sodium as a pharmaceutically active ingredient has a high encapsulation rate, and the liposome has a small particle size and is uniformly distributed.

The invention aims to provide a monosialotetrahexosylganglioside sodium liposome which is mainly prepared from the following components in parts by weight: 1 part of monosialotetrahexosylganglioside sodium, 3-14 parts of hydrogenated egg yolk lecithin, 0.8-7 parts of soyasterol, 0.3-5 parts of sodium deoxycholate, 0.5-10 parts of poloxamer 188 and 0.01-0.05 part of sodium bisulfite.

In a preferred embodiment, based on parts by weight: 1 part of monosialotetrahexosylganglioside sodium, 4-10 parts of hydrogenated egg yolk lecithin, 1.2-5 parts of soyasterol, 0.5-3 parts of sodium deoxycholate, 0.8-5 parts of poloxamer 188 and 0.02-0.04 part of sodium bisulfite.

As the phospholipid for forming the liposome, natural phospholipid and synthetic phospholipid can be used. In the present invention, monosialotetrahexosylganglioside sodium as a pharmaceutically active ingredient is excellent in water solubility. Aiming at the characteristics of monosialotetrahexosyl ganglioside sodium, the inventors found through research that hydrogenated egg yolk lecithin is particularly suitable as a basal phospholipid film-forming material. When other phospholipids are used, it is difficult to form a liposome having excellent quality, and the properties such as encapsulation efficiency, stability, and leakage rate of the liposome are poor.

In the monosialotetrahexosylganglioside sodium liposome injection, the amount of the hydrogenated egg yolk lecithin is 3 to 14 parts by weight relative to 1 part by weight of monosialotetrahexosylganglioside sodium. If the amount of the hydrogenated egg yolk lecithin is less than 3 parts by weight, stable liposomes cannot be formed; on the contrary, if the amount of the hydrogenated egg yolk lecithin is more than 14 parts by weight, the encapsulation efficiency of monosialotetrahexosylganglioside sodium as a pharmaceutically active ingredient is decreased, and the quality and the therapeutic effect of the injection are decreased.

In the monosialotetrahexosylganglioside sodium liposome injection, soyasterol, sodium deoxycholate and poloxamer 188 are used for regulating the membrane stability of the liposome.

Soyasterol (SS) is a hydrolysate of the de-glucosylation of soyasterol glucosides, which is a mixture of sterol glucosides separated from soybean residue from the extraction of soybean oil. The soyasterol can regulate the stability of the hydrogenated egg yolk lecithin membrane, and has better regulation effect on the stability than cholesterol.

The inventor of the invention has found through research that, in the monosialotetrahexosylganglioside sodium liposome injection, the dosage of the soyasterol is 0.8 to 7 weight parts relative to 1 weight part of monosialotetrahexosylganglioside sodium. If the amount of the soyasterol is less than 0.8 part by weight, the formed liposome has low stability and is easy to leak; on the contrary, if the amount of the soyasterol is more than 7 parts by weight, the membrane fluidity of the monosialotetrahexosylganglioside sodium is too high, the monosialotetrahexosylganglioside sodium encapsulated in the liposome is easily released, and the encapsulation efficiency is decreased.

On the other hand, the inventor researches and discovers that in the monosialotetrahexosylganglioside sodium liposome injection, relative to 1 weight part of monosialotetrahexosylganglioside sodium, the dosage of hydrogenated egg yolk lecithin is 4-10 weight parts, the dosage of soyasterol is 1.2-5 weight parts, the dosage of deoxycholate sodium is 0.5-3 weight parts, and the dosage of poloxamer 188 is 0.8-5 weight parts, so that the formed monosialotetrahexosylganglioside sodium liposome injection has high encapsulation efficiency.

In the monosialotetrahexosylganglioside sodium liposome injection of the present invention, poloxamer 188 is used to further improve the stability of the liposome membrane. Poloxamer 188 is a novel nonionic surfactant, and when used in a double-layer membrane of hydrogenated egg yolk lecithin, the chemical energy between the double-layer membrane can be improved, so that the chemical stability of the liposome in aqueous liquid is improved, and the stability of the monosialotetrahexosylganglioside sodium liposome injection is further improved.

In the monosialotetrahexosylganglioside sodium liposome injection, the amount of poloxamer 188 is 0.5-10 parts by weight relative to 1 part by weight of monosialotetrahexosylganglioside sodium. If the amount of poloxamer 188 is less than 0.5 parts by weight, the stability of the monosialotetrahexosylganglioside sodium liposome injection is not improved sufficiently due to the excessively low amount, whereas if the amount of poloxamer 188 is more than 10 parts by weight, the liposome membrane is liable to leak due to the excessively high amount.

It was found that when the above-mentioned specific amounts of monosialotetrahexosylganglioside sodium, hydrogenated egg yolk lecithin, sodium deoxycholate, soyasterol and poloxamer 188 were used, monosialotetrahexosylganglioside sodium liposomes having excellent quality could be obtained, which had high encapsulation efficiency and stability, low toxicity and high bioavailability.

The invention aims to provide a monosialotetrahexosyl ganglioside sodium liposome injection, which comprises an injection and a freeze-dried powder injection, wherein the specification of the injection is 2ml to 20mg, 2ml to 40mg and 5ml to 100mg, and the specification of the freeze-dried powder injection is 40mg to 100 mg.

The invention aims to provide a monosialotetrahexosylganglioside sodium liposome injection which is mainly prepared from the following components in parts by weight: 1 part of monosialotetrahexosylganglioside sodium, 3-14 parts of hydrogenated egg yolk lecithin, 0.8-7 parts of soyasterol, 0.3-5 parts of sodium deoxycholate, 0.5-10 parts of poloxamer 188, 0.01-0.05 part of sodium bisulfite, 0.4-0.8 part of sodium chloride and a proper amount of water for injection.

In a preferred embodiment, based on parts by weight: 1 part of monosialotetrahexosylganglioside sodium, 4-10 parts of hydrogenated egg yolk lecithin, 1.2-5 parts of soyasterol, 0.5-3 parts of sodium deoxycholate, 0.8-5 parts of poloxamer 188, 0.02-0.04 part of sodium bisulfite, 0.45-0.9 part of sodium chloride and a proper amount of water for injection.

The invention aims to provide a monosialotetrahexosylganglioside sodium liposome freeze-dried powder injection which is mainly prepared from the following components in parts by weight: 1 part of monosialotetrahexosylganglioside sodium, 3-14 parts of hydrogenated egg yolk lecithin, 0.8-7 parts of soyasterol, 0.3-5 parts of sodium deoxycholate, 0.5-10 parts of poloxamer 188, 0.01-0.05 part of sodium bisulfite and 3-12 parts of mannitol.

In a preferred embodiment, based on parts by weight: 1 part of monosialotetrahexosylganglioside sodium, 4-10 parts of hydrogenated egg yolk lecithin, 1.2-5 parts of soyasterol, 0.5-3 parts of sodium deoxycholate, 0.8-5 parts of poloxamer 188, 0.02-0.04 part of sodium bisulfite and 5-9 parts of mannitol.

In the monosialotetrahexosylganglioside sodium liposome injection of the invention, sodium bisulfite is used as an antioxidant for forming a stable injection. Sodium chloride is one of the most commonly used osmotic pressure regulators, and mannitol is one of the most commonly used freeze-drying skeleton agents, so that the stability of the liposome is not affected.

Preferably, the monosialotetrahexosylganglioside sodium liposome injection prepared by the method comprises the following components in parts by weight:

preferably, the monosialotetrahexosylganglioside sodium liposome injection prepared by the method comprises the following components in parts by weight:

preferably, the monosialotetrahexosylganglioside sodium liposome injection prepared by the method comprises the following components in parts by weight:

preferably, the monosialotetrahexosylganglioside sodium liposome freeze-dried powder injection prepared by the method comprises the following components in parts by weight:

preferably, the monosialotetrahexosylganglioside sodium liposome freeze-dried powder injection prepared by the method comprises the following components in parts by weight:

one of the objects of the present invention is to provide a method for preparing a monosialotetrahexosylganglioside sodium liposome injection, which comprises the following steps:

(1) dissolving hydrogenated egg yolk lecithin, soyasterol, poloxamer 188 and sodium deoxycholate in a proper amount of isopropanol-ethanol mixed solvent with the volume ratio of 1:3 to obtain a lipoid solution;

(2) placing the lipoid solution in a pear-shaped bottle, and performing rotary evaporation to remove the mixed solvent in a constant-temperature water bath at 55-65 ℃ to form a uniform lipoid film;

(3) dissolving monosialotetrahexosylganglioside sodium, sodium bisulfite and sodium chloride in water for injection, adding into a pear-shaped bottle, and gently shaking to elute lipid membrane and disperse into hydration medium to dissolve to obtain liposome suspension;

(4) putting the suspension into an ultrasonic instrument, and carrying out ultrasonic treatment to obtain a semitransparent colloidal solution;

(5) filtering the suspension with 0.45 μm microporous membrane, bottling, sealing, and sterilizing to obtain monosialotetrahexosylganglioside sodium liposome injection.

One of the purposes of the invention is also to provide a method for preparing the monosialotetrahexosyl ganglioside sodium liposome freeze-dried powder injection, which comprises the following steps:

(1) dissolving hydrogenated egg yolk lecithin, soyasterol, poloxamer 188 and sodium deoxycholate in a proper amount of isopropanol-ethanol mixed solvent with the volume ratio of 1:3 to obtain a lipoid solution;

(2) placing the lipoid solution in a pear-shaped bottle, and performing rotary evaporation to remove the mixed solvent in a constant-temperature water bath at 55-65 ℃ to form a uniform lipoid film;

(3) dissolving sodium monosialotetrahexosylganglioside, sodium bisulfite and mannitol in water for injection, adding into pear-shaped bottle, and shaking gently to elute lipid membrane and disperse into hydration medium to dissolve to obtain liposome suspension;

(4) putting the suspension into an ultrasonic instrument, and carrying out ultrasonic treatment to obtain a semitransparent colloidal solution;

(5) filtering the suspension with 0.45 μm microporous filter membrane, freezing the filtrate at-45 deg.C for 4 hr, heating to-13 deg.C at a rate of 1.3 deg.C/hr, maintaining for 3 hr, heating to 30 deg.C, and maintaining for 3 hr to dry to obtain the lyophilized preparation of monosialotetrahexosylganglioside sodium liposome.

Advantageous effects

The liposome injection improves the quality of preparation products, reduces toxic and side effects, has good preparation stability, improves the bioavailability of the medicine, and obviously improves the curative effect; and the preparation method is simple and is suitable for industrial mass production. The liposome can not be cracked due to dehydration, fusion, ice crystal and the like in the freezing process, and the liposome can also keep good entrapment rate after long-term storage.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1 shows the plasma concentration-time relationship.

Wherein,

curve 1 represents the plasma concentration time curve of example 1.

Curve 2 represents the plasma concentration time curve of example 2.

Curve 3 represents the plasma concentration time curve of example 3.

Curve 4 represents the plasma concentration time curve of example 4.

Curve 5 represents the plasma concentration time curve of example 5.

Curve 6 represents the plasma concentration time curve of comparative example 1.

Curve 7 represents the plasma concentration time curve of comparative example 2.

Curve 8 represents the plasma concentration time curve of comparative example 3.

Curve 9 shows the plasma concentration time curve of the marketed example.

Detailed Description

The invention is further illustrated by the following specific preferred examples. These examples are illustrative only and should not be construed as limiting the invention.

Example 1Preparation of monosialotetrahexosyl ganglioside sodium liposome injection

Prescription (1000 pieces)

The preparation process comprises the following steps:

(1) dissolving 140g of hydrogenated egg yolk lecithin, 60g of soybean sterol, 50g of poloxamer 188 and 36g of sodium deoxycholate in 2000ml of isopropanol-ethanol mixed solvent with the volume ratio of 1:3 to obtain a lipoid solution;

(2) placing the lipoid solution in a pear-shaped bottle, and performing rotary evaporation to remove the mixed solvent in a constant-temperature water bath at 65 ℃ to form a uniform lipoid film;

(3) dissolving 20g of monosialotetrahexosylganglioside sodium, 0.6g of sodium bisulfite and 18g of sodium chloride in 600ml of water, adding the solution into a pear-shaped bottle, and gently shaking to elute a lipid membrane and disperse the lipid membrane into a hydration medium for dissolving to obtain a liposome suspension;

(4) putting the suspension into an ultrasonic instrument, and carrying out ultrasonic treatment to obtain a semitransparent colloidal solution;

(5) filtering the suspension with 0.45 μm microporous membrane, bottling, sealing, and sterilizing to obtain monosialotetrahexosylganglioside sodium liposome injection.

Example 2Preparation of monosialotetrahexosyl ganglioside sodium liposome injection

Prescription (1000 pieces)

The preparation process comprises the following steps:

(1) dissolving 400g of hydrogenated egg yolk lecithin, 200g of soybean sterol, 200g of poloxamer 188 and 120g of sodium deoxycholate in 6000ml of mixed solvent of isopropanol and ethanol with the volume ratio of 1:3 to obtain lipoid solution;

(2) placing the lipoid solution in a pear-shaped bottle, and removing the mixed solvent by rotary evaporation in a constant-temperature water bath at 55 ℃ to form a uniform lipoid film;

(3) dissolving 40g monosialotetrahexosylganglioside sodium, 1.6g sodium bisulfite and 18g sodium chloride in 600ml water, adding into a pear-shaped bottle, and gently shaking to elute lipid membrane and disperse into hydration medium to dissolve to obtain liposome suspension;

(4) putting the suspension into an ultrasonic instrument, and carrying out ultrasonic treatment to obtain a semitransparent colloidal solution;

(5) filtering the suspension with 0.45 μm microporous membrane, bottling, sealing, and sterilizing to obtain monosialotetrahexosylganglioside sodium liposome injection.

Example 3Preparation of monosialotetrahexosyl ganglioside sodium liposome injection

Prescription (1000 pieces)

The preparation process comprises the following steps:

(1) dissolving 800g of hydrogenated egg yolk lecithin, 360g of soybean sterol, 450g of poloxamer 188 and 190g of sodium deoxycholate in 15000ml of mixed solvent of isopropanol and ethanol with the volume ratio of 1:3 to obtain lipoid solution;

(2) placing the lipoid solution in a pear-shaped bottle, and removing the mixed solvent by rotary evaporation in a constant-temperature water bath at 60 ℃ to form a uniform lipoid film;

(3) dissolving 100g monosialotetrahexosylganglioside sodium, 2g sodium bisulfite and 45g sodium chloride in 800ml water, adding into a pear-shaped bottle, and gently shaking to elute lipid membrane and disperse into hydration medium to dissolve to obtain liposome suspension;

(4) putting the suspension into an ultrasonic instrument, and carrying out ultrasonic treatment to obtain a semitransparent colloidal solution;

(5) filtering the suspension with 0.45 μm microporous membrane, bottling, sealing, and sterilizing to obtain monosialotetrahexosylganglioside sodium liposome injection.

Example 4Preparation of monosialotetrahexosyl ganglioside sodium liposome freeze-dried powder injection

Prescription (1000 bottle)

The preparation process comprises the following steps:

(1) dissolving 160g of hydrogenated egg yolk lecithin, 48g of soybean sterol, 32g of poloxamer 188 and 20g of sodium deoxycholate in 4000ml of mixed solvent of isopropanol and ethanol with the volume ratio of 1:3 to obtain a lipoid solution;

(2) placing the lipoid solution in a pear-shaped bottle, and removing the mixed solvent by rotary evaporation in a constant-temperature water bath at 55 ℃ to form a uniform lipoid film;

(3) dissolving 40g monosialotetrahexosylganglioside sodium, 0.8g sodium bisulfite and 200g mannitol in 1200ml water, adding into a pear-shaped bottle, and gently shaking to elute lipid membrane and disperse into hydration medium to dissolve to obtain liposome suspension;

(4) putting the suspension into an ultrasonic instrument, and carrying out ultrasonic treatment to obtain a semitransparent colloidal solution;

(5) filtering the suspension with 0.45 μm microporous filter membrane, freezing the filtrate at-45 deg.C for 4 hr, heating to-13 deg.C at a rate of 1.3 deg.C/hr, maintaining for 3 hr, heating to 30 deg.C, and maintaining for 3 hr to dry to obtain the lyophilized preparation of monosialotetrahexosylganglioside sodium liposome.

Example 5Preparation of monosialotetrahexosyl ganglioside sodium liposome freeze-dried powder injection

Prescription (1000 bottle)

The preparation process comprises the following steps:

(1) dissolving 500g of hydrogenated egg yolk lecithin, 150g of soybean sterol, 100g of poloxamer 188 and 60g of sodium deoxycholate in 8000ml of isopropanol-ethanol mixed solvent with volume ratio of 1:3 to obtain lipoid solution;

(2) placing the lipoid solution in a pear-shaped bottle, and performing rotary evaporation to remove the mixed solvent in a constant-temperature water bath at 65 ℃ to form a uniform lipoid film;

(3) dissolving 100g monosialotetrahexosylganglioside sodium, 3g sodium bisulfite and 900g mannitol in 2000ml water, adding into pear-shaped bottle, and shaking gently to elute lipid membrane and disperse into hydration medium to dissolve to obtain liposome suspension;

(4) putting the suspension into an ultrasonic instrument, and carrying out ultrasonic treatment to obtain a semitransparent colloidal solution;

(5) filtering the suspension with 0.45 μm microporous filter membrane, freezing the filtrate at-45 deg.C for 4 hr, heating to-13 deg.C at a rate of 1.3 deg.C/hr, maintaining for 3 hr, heating to 30 deg.C, and maintaining for 3 hr to dry to obtain the lyophilized preparation of monosialotetrahexosylganglioside sodium liposome.

Comparative examples 1 to 3Monosialotetrahexosyl gangliosidePreparation of sodium liposome injection

The components and the amounts in comparative examples 1-2 shown in the following table were prepared into monosialotetrahexosylganglioside sodium liposome injection solutions respectively by the same production process as in example 1, and the components and the amounts in comparative example 3 shown in the following table were prepared into monosialotetrahexosylganglioside sodium liposome lyophilized powder for injection by the similar production process as in example 4.

TABLE 1 comparative examples 1-3 compositions

Where "/" indicates not used.

Test example 1Measurement of liposome particle size

Under the condition of room temperature, the monosialotetrahexosylganglioside sodium liposome injection in the examples 1-5 and the comparative examples 1-3 is placed in a sample tube of a Submicron Particle Sizer Model 370 Particle size detector, and the Particle size distribution and the average Particle size are measured; the particle morphology was observed with a projection electron microscope. The results are shown in table 2 below.

TABLE 2 measurement of particle diameter

As can be seen from Table 2, the liposomes obtained in examples 1 to 5 had uniform particle size, spherical shape and uniform size; the liposomes prepared in comparative examples 1 to 3 had non-uniform particle size, indefinite shape, disorder and varied size.

Specifically, even when the same production process was employed, the monosialotetrahexosylganglioside sodium liposomes produced in examples 1 to 5 were significantly superior in particle appearance and average particle diameter to those produced in comparative examples 1 to 3. It is explained that when components other than those used in the present invention are used, or when the amounts of the components are outside the ranges of the amounts of the components defined in the present invention, the obtained monosialotetrahexosylganglioside sodium liposomes are inferior in appearance to the present invention, and the average particle diameter is significantly larger.

Test example 2Determination of encapsulation efficiency

The monosialotetrahexosylganglioside sodium liposome injection prepared in examples 1 to 5 and comparative examples 1 to 3 was centrifuged at a high speed of 5000r/min for 20 minutes, the supernatant was taken, dissolved in methanol, and the content of monosialotetrahexosylganglioside sodium was measured by HPLC method to calculate the encapsulation efficiency, and the results are shown in table 3 below.

TABLE 3 encapsulation efficiency measurement results

As can be seen from Table 3, the encapsulation efficiencies of the liposome formulations prepared in examples 1 to 5 were significantly higher than those of the liposome formulations of comparative examples 1 to 3. It is explained that when ingredients other than the ingredients used in the present invention are used, or when the amount of the ingredients is outside the range of the amount of the ingredients defined in the present invention, the liposome obtained has a lower liposome encapsulation efficiency than that of the present invention.

Test example 3Stability survey

The samples prepared in examples 1 to 5 of the present invention and the samples prepared in comparative examples 1 to 3, and the sodium monosialotetrahexosylganglioside injection (batch No. 20100303-1, Beijing Tetracycline pharmaceutical Co., Ltd.) on the market were each subjected to accelerated test examination for 6 months at a temperature of 40 ℃ and a relative humidity of 75%, and the test results are shown in Table 4.

TABLE 4 accelerated test results

As can be seen from Table 4, at 6 months of acceleration, the contents of the samples prepared in comparative examples 1 to 3 and the preparations on the market are reduced, the related substances are increased, and the pH value is obviously reduced; the sample properties, pH value, content and related substance changes of the samples of the embodiments 1-5 are not obvious, which shows that the product of the invention has good stability.

Test example 4Leak rate test

Samples prepared in test examples 1 to 5 and comparative examples 1 to 3 were taken, and periodically checked at room temperature for 0 day, 30 days, 60 days, 90 days and 180 days, respectively, to determine the encapsulation efficiency, and the leakage rate was calculated in comparison with the amount of the drug encapsulated at 0 day, and the results are shown in table 5 below.

TABLE 5 leak Rate test results

As can be seen from table 5, the leakage rates of the monosialotetrahexosylganglioside sodium liposome injection prepared in examples 1 to 5 of the present invention did not change much during long-term storage, while the leakage rates of the injections prepared in comparative examples 1 to 3 gradually increased and the liposome leakage was severe, which indicates that the monosialotetrahexosylganglioside sodium liposome injection prepared by the method of the present invention had higher stability.

Test example 5Measurement of blood concentration

90 rats were randomly divided into 9 groups, and each group was separately injected with the samples prepared in examples 1 to 5 and comparative examples 1 to 3, and a commercially available monosialotetrahexosylganglioside sodium injection (batch No. 20100303-1, Beijing Tetracycline pharmaceutical Co., Ltd.) in an amount of 20mg of monosialotetrahexosylganglioside sodium. After administration, blood is collected for 0h, 0.5h, 1h, 1.5h, 2h, 3h, 6h and 8h respectively, and blood concentration is determined by HPLC-MS method after blood sample is processed. Blood concentration versus time curves were plotted for the monosialotetrahexosylganglioside sodium liposome injection prepared in examples 1-5, the monosialotetrahexosylganglioside sodium liposome injection prepared in comparative examples 1-3, and the commercially available monosialotetrahexosylganglioside sodium injection, and are shown in fig. 1.

Wherein,

curve 1 represents the plasma concentration time curve of example 1.

Curve 2 represents the plasma concentration time curve of example 2.

Curve 3 represents the plasma concentration time curve of example 3.

Curve 4 represents the plasma concentration time curve of example 4.

Curve 5 represents the plasma concentration time curve of example 5.

Curve 6 represents the plasma concentration time curve of comparative example 1.

Curve 7 represents the plasma concentration time curve of comparative example 2.

Curve 8 represents the plasma concentration time curve of comparative example 3.

Curve 9 shows the plasma concentration time curve of the marketed example.

As can be seen from fig. 1, the monosialotetrahexosylganglioside sodium liposome injection prepared in examples 1 to 5 of the present invention has the following advantages compared to the monosialotetrahexosylganglioside sodium liposome injection prepared in comparative examples 1 to 3 and the commercially available monosialotetrahexosylganglioside sodium liposome injection: the elimination speed in vivo is slowed down, the distribution time in systemic circulation is prolonged, the improved slow release effect is achieved, and the bioavailability is increased.

Test example 6Blood brain Barrier Permeability test

30 New Zealand rabbits were selected, weighed about 2.5kg, and the males and females were divided randomly into 6 groups, 1 to 5 groups were intravenously injected with the monosialotetrahexosylganglioside sodium liposome injection of examples 1 to 5 of the present invention, and 6 groups were intravenously injected with the commercially available monosialotetrahexosylganglioside sodium injection (batch No. 20100303-1, Beijing Tetracycline pharmaceutical Co., Ltd.) in an amount of 20mg of monosialotetrahexosylganglioside sodium. 0.2ml of cerebrospinal fluid is extracted at different times after administration for testing, and the sampling time is 0.5h, 1h, 2h, 4h and 8h after injection of the medicine respectively. Taking 0.1ml of cerebrospinal fluid, adding a proper amount of solvent for suspension for 15min, centrifuging at 10000rpm for 1min, taking 20ul of supernatant, injecting sample, and measuring by high performance liquid chromatography. The results are shown in Table 6.

TABLE 6 measurement of drug concentration in cerebrospinal fluid

Note: indicates significant differences compared to the marketed examples.

The test results in table 6 show that the amount of the monosialotetrahexosylganglioside sodium liposome injection passing through the blood brain barrier is obviously improved compared with the amount of the medicine in the injection in the market, which shows that the amount of the medicine in the monosialotetrahexosylganglioside sodium liposome injection passing through the blood brain barrier is obviously increased, the blood concentration in brain tissues is improved, and the treatment effect is improved. The test result also shows that after the monosialotetrahexosylganglioside sodium liposome injection is administered, the medicine concentration of a target tissue is quickly and stably increased, and the injection has a long-acting effect in the tissue, so that the administration frequency of the monosialotetrahexosylganglioside sodium liposome injection can be reduced. And under the same administration dosage, the effect of the preparation in the embodiment is obviously better than that of the preparation in the market and the comparative example.

Industrial applicability

The results of the above examples and test examples show that the monosialotetrahexosylganglioside sodium liposome of the present invention has the advantages of good appearance, small particle size, uniform particle size, high encapsulation efficiency, high stability, low leakage rate, long retention time in vivo, high bioavailability and good industrial application value.

The present invention has been described in detail with reference to the specific embodiments and the test examples, but it should be understood that the scope of the present invention is not limited thereto, and various modifications, improvements and substitutions can be made to the technical solution and the embodiments thereof without departing from the spirit and the scope of the present invention.

Each reference mentioned or cited in this application is hereby incorporated by reference in its entirety.

Claims (4)

1. The monosialotetrahexosyl ganglioside sodium liposome injection is characterized by being prepared from the following components in parts by weight: 1 part of monosialotetrahexosylganglioside sodium, 4-10 parts of hydrogenated egg yolk lecithin, 1.2-5 parts of soyasterol, 0.5-3 parts of sodium deoxycholate, 0.8-5 parts of poloxamer 188, 0.02-0.04 part of sodium bisulfite, 0.45-0.9 part of sodium chloride and a proper amount of water for injection.

2. A monosialotetrahexosyl ganglioside sodium liposome freeze-dried powder injection is characterized by being prepared from the following components in parts by weight: 1 part of monosialotetrahexosylganglioside sodium, 4-10 parts of hydrogenated egg yolk lecithin, 1.2-5 parts of soyasterol, 0.5-3 parts of sodium deoxycholate, 0.8-5 parts of poloxamer 188, 0.02-0.04 part of sodium bisulfite and 5-9 parts of mannitol.

3. A method for preparing the monosialotetrahexosylganglioside sodium liposome injection of claim 1, characterized by comprising the steps of:

(1) dissolving hydrogenated egg yolk lecithin, soyasterol, poloxamer 188 and sodium deoxycholate in a proper amount of isopropanol-ethanol mixed solvent with the volume ratio of 1:3 to obtain a lipoid solution;

(2) placing the lipoid solution in a pear-shaped bottle, and performing rotary evaporation to remove the mixed solvent in a constant-temperature water bath at 55-65 ℃ to form a uniform lipoid film;

(3) dissolving monosialotetrahexosylganglioside sodium, sodium bisulfite and sodium chloride in water for injection, adding into a pear-shaped bottle, and gently shaking to elute lipid membrane and disperse into hydration medium to dissolve to obtain liposome suspension;

(4) putting the suspension into an ultrasonic instrument, and carrying out ultrasonic treatment to obtain a semitransparent colloidal solution;

(5) filtering the suspension with 0.45 μm microporous membrane, bottling, sealing, and sterilizing to obtain monosialotetrahexosylganglioside sodium liposome injection.

4. A method for preparing the monosialotetrahexosylganglioside sodium liposome freeze-dried powder injection of claim 2, which is characterized by comprising the following steps:

(1) dissolving hydrogenated egg yolk lecithin, soyasterol, poloxamer 188 and sodium deoxycholate in a proper amount of isopropanol-ethanol mixed solvent with the volume ratio of 1:3 to obtain a lipoid solution;

(2) placing the lipoid solution in a pear-shaped bottle, and performing rotary evaporation to remove the mixed solvent in a constant-temperature water bath at 55-65 ℃ to form a uniform lipoid film;

(3) dissolving sodium monosialotetrahexosylganglioside, sodium bisulfite and mannitol in water for injection, adding into pear-shaped bottle, and shaking gently to elute lipid membrane and disperse into hydration medium to dissolve to obtain liposome suspension;

(4) putting the suspension into an ultrasonic instrument, and carrying out ultrasonic treatment to obtain a semitransparent colloidal solution;

(5) filtering the suspension with 0.45 μm microporous filter membrane, freezing the filtrate at-45 deg.C for 4 hr, heating to-13 deg.C at a rate of 1.3 deg.C/hr, maintaining for 3 hr, heating to 30 deg.C, and maintaining for 3 hr to dry to obtain the lyophilized preparation of monosialotetrahexosylganglioside sodium liposome.

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