阅读说明：本技术 一种维生素e琥珀酸酯磷脂化合物及其应用 (Vitamin E succinate phospholipid compound and application thereof ) 是由 熊瑜 万江陵 阮健 盛剑勇 甘祥俊 徐忠瑞 于 2021-08-31 设计创作，主要内容包括：本发明属于药物制剂技术领域,具体公开了一种维生素E琥珀酸酯磷脂化合物及其应用,在该化合物中,磷脂酸分子的两条长脂肪酸链中的至少一条被维生素E琥珀酸酯取代,磷酸基团上的一个氢原子可被聚乙二醇醚取代,另一个氢原子可被胆碱、乙醇胺或肌醇取代,该化合物具有两亲性,能起到生物表面活性剂的作用,在药物制剂中可作为增溶剂、吸收促进剂、乳化剂以及水难溶性和脂溶性药物传递系统载体。本发明维生素E琥珀酸酯磷脂化合物制得的纳米载药系统包封率高、稳定性好,且安全可靠,适合推广应用。(The invention belongs to the technical field of pharmaceutical preparations, and particularly discloses a vitamin E succinate phospholipid compound and application thereof. The nano drug delivery system prepared from the vitamin E succinate phospholipid compound has high entrapment rate, good stability, safety and reliability, and is suitable for popularization and application.)

1. A vitamin E succinate phospholipid compound characterized by: the structural formula of the compound is shown as follows:

wherein R1 is vitamin E succinate;

r2 is vitamin E succinate or hydroxy;

r3 is a polyethylene glycol ether or a hydrogen atom;

x is choline, ethanolamine, inositol or a hydrogen atom.

2. The vitamin E succinate phospholipid compound of claim 1, wherein: the polyethylene glycol ether is mPEG200, mPEG400, mPEG600, mPEG800, mPEG1000, mPEG1500, mPEG2000 or mPEG 4000.

3. A process for the preparation of a vitamin E succinate phospholipid compound as defined in claim 1 or 2, comprising the steps of: dissolving vitamin E succinate and lysophospholipid in an organic solvent, adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine to perform a light-shielding reaction, removing the organic solvent after the reaction is finished, and purifying to obtain the vitamin E succinate-lysophosphatidyl conjugate.

4. The method of claim 3, further comprising the steps of:

s1, dissolving the vitamin E succinate-lysophosphatidyl conjugate in an organic solvent, adding sodium hydride for reaction, removing the organic solvent and excessive sodium hydride after the reaction is finished, and purifying to obtain vitamin E succinate-lysophosphatidyl sodium;

s2, dissolving polyethylene glycol ether in an organic solution to obtain a mixture A, adding paratoluensulfonyl chloride into triethylamine to obtain a mixture B, mixing the mixture A and the mixture B for reaction, extracting after the reaction is finished, removing the organic solution, and purifying to obtain polyethylene glycol ether p-toluenesulfonate;

s3, mixing the vitamin E succinate-sodium lysophosphatide prepared in the step S1 and the polyethylene glycol ether p-toluenesulfonate prepared in the step S2 for reaction, and purifying to obtain the polyethylene glycol ether-vitamin E succinate phospholipid compound.

5. The process for preparing a vitamin E succinate phospholipid compound according to claim 3, wherein: the lysophospholipid is lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol or lysophosphatidic acid.

6. Use of the vitamin E succinate phospholipid compound of claim 1 or 2 in the preparation of an anti-tumor medicament.

7. A nano drug delivery system, which is characterized in that: a vitamin E succinate phospholipid compound according to claim 1 or 2 for use as a pharmaceutical carrier.

8. The nanoplatelet system of claim 7, wherein: the nano drug-carrying system is liposome, nano particles or nano micelle.

9. The nanopharmaceutical system of claim 8, wherein: the nano drug-carrying system is a drug-carrying nano micelle formed by self-assembling the vitamin E succinate phospholipid compound and a water-insoluble drug by a direct dissolution method, a melting method, a solvent evaporation method or a dialysis method.

10. The nanoplatelet system of claim 7, wherein: the particle size of the nano drug-loading system is 10nm-1000 nm.

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a vitamin E succinate phospholipid compound and application thereof.

Background

After the surfactant is put into the solution, the interfacial tension of the solution is rapidly reduced, but the concentration of the surfactant molecules on the surface of the aqueous solution tends to increase along with the increase of the amount, and after the saturation, if the surfactant is continuously added, the interfacial tension of the solution is not changed. The surfactant molecules are then rapidly transferred into the solution, thereby forming surfactant molecule associations, called micelles (micelles), with hydrophilic groups facing outward and hydrophobic groups facing inward. The micelle is a thermodynamic stable system, is widely used for solubilizing insoluble drugs in pharmacy, and the solubilized drugs can reach the concentration required by clinical treatment, so that the bioavailability is improved.

Phospholipids are components of biological membranes, and their associated catabolic enzymes exist in the body and are easily metabolized, so that phospholipids have biodegradability and biocompatibility, and two long fatty acid chains in phospholipid molecules make them hydrophobic. However, phospholipids have small molecular weights, and formed vesicles and micelles have insufficient stability, and thus have great limitations in application as long-circulating and sustained-release materials. The current solution is to add to it components containing long cycles: polyethylene glycol (PEG), polysaccharide, cyclodextrin, etc. are long circulating phospholipids to improve the stability of phospholipid micelles or capsules. The properties of polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) micelles formed by PEGs of different chain lengths have been reported.

The vitamin E polyethylene glycol succinate (TPGS) is a water-soluble derivative of vitamin E, and consists of a hydrophilic polar polyethylene glycol head and an lipophilic non-polar vitamin E succinate tail, the critical micelle concentration is 0.026%, and the HLB (Hydrophile-Lipophile Balance Number) value is 13. TPGS was first developed and marketed by Eastman in 1950 and is collected as a pharmaceutical adjuvant by the United states pharmacopoeia. However, the TPGS has poor drug encapsulation efficiency and micelle stability, and the application of TPGS in insoluble drug delivery system carriers is limited.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a vitamin E succinate phospholipid compound and application thereof, wherein the compound is an amphiphilic biosurfactant, is prepared into nanoparticles with the particle size of 10nm-1000nm, can be used as a solubilizer, an absorption enhancer, an emulsifier and a carrier of a water-insoluble and fat-soluble drug delivery system, and aims to solve the problems of poor drug encapsulation efficiency and poor micelle stability of the existing surfactant.

To achieve the above objects, the present invention provides a vitamin E succinate phospholipid compound, which has a structural formula as follows:

wherein R1 is vitamin E succinate;

r2 is vitamin E succinate or hydroxy;

r3 is a polyethylene glycol ether or a hydrogen atom;

x is choline, ethanolamine, inositol or a hydrogen atom.

Preferably, the polyethylene glycol ether is mPEG200, mPEG400, mPEG600, mPEG800, mPEG1000, mPEG1500, mPEG2000 or mPEG 4000.

According to another aspect of the present invention, there is provided a method for preparing a vitamin E succinate phospholipid compound, comprising the steps of: dissolving vitamin E succinate and lysophospholipid in an organic solvent, adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine to perform a light-shielding reaction, removing the organic solvent after the reaction is finished, and purifying to obtain the vitamin E succinate-lysophosphatidyl conjugate.

Preferably, the preparation method further comprises the following steps:

s1, dissolving the vitamin E succinate-lysophosphatidyl conjugate in an organic solvent, adding sodium hydride for reaction, removing the organic solvent and excessive sodium hydride after the reaction is finished, and purifying to obtain vitamin E succinate-lysophosphatidyl sodium;

s2, dissolving polyethylene glycol ether in an organic solution to obtain a mixture A, adding paratoluensulfonyl chloride into triethylamine to obtain a mixture B, mixing the mixture A and the mixture B for reaction, extracting after the reaction is finished, removing the organic solution, and purifying to obtain polyethylene glycol ether p-toluenesulfonate;

s3, mixing the vitamin E succinate-sodium lysophosphatide prepared in the step S1 and the polyethylene glycol ether p-toluenesulfonate prepared in the step S2 for reaction, and purifying to obtain the polyethylene glycol ether-vitamin E succinate phospholipid compound.

Preferably, the lysophospholipid is lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol or lysophosphatidic acid.

According to another aspect of the invention, the vitamin E succinate phospholipid compound is provided for use in preparing an anti-tumor medicament.

According to another aspect of the present invention, there is provided a drug delivery system comprising the above-described vitamin E succinate phospholipid compound as a drug carrier.

Preferably, the nano drug delivery system is a liposome, a nanoparticle or a nano micelle.

Preferably, the nano drug delivery system is a drug-loaded nano micelle formed by self-assembling the vitamin E succinate phospholipid compound and the water-insoluble drug by a direct dissolution method, a melting method, a solvent evaporation method or a dialysis method.

Preferably, the particle size of the nano drug delivery system is 10nm-1000 nm.

Generally, compared with the prior art, the above technical solution conceived by the present invention has the following beneficial effects:

(1) the compound modifies three places of phosphatidic acid, namely PEG modification, improves the hydrophilicity of the compound and enhances the long circulation function; and thirdly, phosphate groups of phospholipid are modified into lecithin or cephalin and the like to further improve the water solubility of the compound, the compound has low critical micelle concentration, and has high encapsulation rate and good stability when being used as a carrier for encapsulating insoluble drugs.

(2) The amphiphilic surfactant is prepared by utilizing the strong surface activity of lysophospholipid and performing condensation reaction on natural degradable biological materials, namely vitamin E succinate and the lysophospholipid, and the preparation method is simple, adopts biosafety materials, has mild reaction conditions and is suitable for large-scale production and application; meanwhile, the surfactant is used as a drug delivery carrier to improve the safety in vivo and reduce the hemolytic property.

(3) The vitamin E succinate phospholipid compound is adopted as a drug delivery carrier, is suitable for encapsulating various water-insoluble drugs, can be prepared into various dosage forms, has high encapsulation rate and good stability, and can effectively improve the curative effect of the drugs.

Drawings

FIG. 1 is a schematic diagram of a synthetic route for vitamin E succinate diester-lysophosphatidyl conjugate provided in example 1 of the present invention;

FIG. 2 is an electron microscope image of a docetaxel-VES-lyso-mPEG nano-micelle solution prepared in example 4 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The invention provides a vitamin E succinate phospholipid compound, which has the following structural formula:

wherein R1 is vitamin E succinate;

r2 is vitamin E succinate or hydroxy;

r3 is a polyethylene glycol ether or a hydrogen atom;

x is choline, ethanolamine, inositol or a hydrogen atom.

The encapsulation efficiency and stability of the surfactant are closely related to the chemical structure of the surfactant, and the surfactant is used as an amphiphilic substance and contains a hydrophilic group and a hydrophobic group. The compound of the invention modifies three positions of phosphatidic acid: r1 and R2 are modified by vitamin E succinate, so that the lipophilicity of the compound is improved, and hydrophobic substances are favorably entrapped; r3 is modified by polyglycol ether, so that the hydrophilicity of the compound is improved, and the long-circulating effect of the compound is improved; the water solubility of X can be further improved by modifying X with a hydroxyl group-containing substance. The invention modifies the hydrophilic head and the hydrophobic tail of phosphatidic acid respectively, enhances the amphipathy of the surfactant, is beneficial to improving the encapsulation rate and the stability of the surfactant used as a carrier for encapsulating the insoluble medicine, and has low critical micelle concentration.

In some embodiments, R3 in the above formula is preferably polyethylene glycol ether, which may be mPEG200, mPEG400, mPEG600, mPEG800, mPEG1000, mPEG1500, mPEG2000 or mPEG4000, preferably mPEG2000 or mPEG 4000. The invention introduces polyglycol ether as the hydrophilic group of the vitamin E succinate phospholipid compound to improve the hydrophilicity; the longer the polyethylene glycol ether chain, the stronger the binding ability with water, and the better the hydrophilicity.

The invention also provides a preparation method of the vitamin E succinate phospholipid compound, which comprises the following steps: dissolving vitamin E succinate and lysophospholipid in an organic solvent, adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine to perform a light-shielding reaction, removing the organic solvent after the reaction is finished, and purifying to obtain the vitamin E succinate-lysophosphatidyl conjugate.

Lyso-phospholipids are a class of phospholipids having strong surface activity, but they can rupture red blood cells and other cell membranes in blood, causing hemolysis or cell necrosis. The invention prepares the vitamin E succinate-lysophosphatidyl conjugate by carrying out condensation reaction on lysophospholipid and vitamin E succinate, improves the hydrophobicity of the hydrophobic end of the lysophospholipid, reduces the hemolytic property of the lysophospholipid and improves the in vivo safety.

In some embodiments, the condensation of lysophospholipid with vitamin E succinate produces a mixture of vitamin E succinate monoester-lysophosphatidyl conjugate and vitamin E succinate diester-lysophosphatidyl conjugate, which can be isolated and purified by preparative chromatography techniques to yield either vitamin E succinate monoester-lysophosphatidyl conjugate or vitamin E succinate diester-lysophosphatidyl conjugate.

In some embodiments, the molar ratio of vitamin E succinate to lysophospholipid is not less than 2:1, and the vitamin E succinate is in excess due to steric hindrance during the condensation reaction, resulting in an increased yield of vitamin E succinate diester-lysophosphatidyl conjugate in the product.

In some embodiments, the vitamin E succinate phospholipid compound has a polyethylene glycol ether attached to a phospholipid linkage, and is prepared by a method comprising the steps of:

s1, dissolving the vitamin E succinate-lysophosphatidyl conjugate in an organic solvent, adding sodium hydride for reaction, removing the organic solvent and excessive sodium hydride after the reaction is finished, and purifying to obtain vitamin E succinate-lysophosphatidyl sodium;

s2, dissolving polyethylene glycol ether in an organic solution to obtain a mixture A, adding paratoluensulfonyl chloride into triethylamine to obtain a mixture B, mixing the mixture A and the mixture B for reaction, extracting after the reaction is finished, removing the organic solution, and purifying to obtain polyethylene glycol ether p-toluenesulfonate;

s3, mixing the vitamin E succinate-sodium lysophosphatide prepared in the step S1 and the polyethylene glycol ether p-toluenesulfonate prepared in the step S2 for reaction, and purifying to obtain the polyethylene glycol ether-vitamin E succinate phospholipid compound.

In some embodiments, the lysophospholipid is lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, or lysophosphatidic acid.

The invention also provides the application of the vitamin E succinate phospholipid compound in preparing the antitumor drugs, the amphiphilic vitamin E succinate phospholipid compound is used as a carrier of a drug loading system to encapsulate the insoluble drugs, the amphiphilic vitamin E succinate phospholipid compound has an excellent solubilizing effect on the drugs, and has high encapsulation rate and good stability, thereby being beneficial to improving the curative effect of the antitumor drugs.

The invention provides a nano drug delivery system, which takes the vitamin E succinate phospholipid compound as a drug carrier and can be used for loading various water-insoluble drugs, such as docetaxel, adriamycin, voriconazole, paclitaxel, camptothecin and the like, but not limited to the listed drugs.

The nano drug-carrying system can be prepared into various dosage forms, such as liposome, nanoparticles or nano micelles, and the like, and has good self-assembly effect and good stability. Specifically, the vitamin E succinate phospholipid compound and the water-insoluble drug are self-assembled to form the drug-loaded nanoparticle by a solvent evaporation method, a straight emulsification solvent diffusion method, a salting-out emulsification-diffusion method, a nano-deposition method, a coacervation method or an emulsion polymerization method. The vitamin E succinate phospholipid compound and the water-insoluble drug are self-assembled to form the drug-loaded liposome by a thin film dispersion method, a reverse phase evaporation method, a freeze drying method, an ultrasonic dispersion method, a spray drying method, a membrane extrusion method or a high-pressure homogenization method. The vitamin E succinate phospholipid compound and the water-insoluble drug are self-assembled to form the drug-loaded nano micelle by a direct dissolution method, a melting method, a solvent evaporation method or a dialysis method.

In some embodiments, the amphiphilic drug carrier can be prepared into nano drug-carrying systems of different formulations, the particle size of the nano drug-carrying systems can be 10nm-1000nm, specifically, the particle size of the prepared drug-carrying nano micelle or nanoparticle is 10nm-100nm, and the particle size of the drug-carrying liposome is 100nm-1000 nm.

The above technical solution is described in detail below with reference to specific examples.

The raw material medicines and auxiliary materials used in the preparation process of the vitamin E succinate phospholipid compound provided by the invention can be purchased from the market.

Example 1 preparation of vitamin E succinate-lysophosphatidyl conjugate

The lysophospholipid (lyso) used in this example was lysophosphatidylcholine, and a vitamin E succinate-lysophosphatidylcholine conjugate (VES-lyso) was prepared using anhydrous dichloromethane as a solvent, specifically by the following method:

dissolving 0.02mol of Vitamin E Succinate (VES) and 0.01mol of lysophosphatidylcholine in 500mL of dichloromethane, adding 0.02mol of dicyclohexylcarbodiimide and 0.02mol of 4-dimethylaminopyridine, carrying out a light-shielding reaction at room temperature for 3d, filtering after the reaction is finished, washing the filtrate twice with 0.1mol/L hydrochloric acid solution, drying the filtrate with anhydrous sodium sulfate, carrying out rotary evaporation to remove the solvent, purifying the filtrate with a silica gel column (chloroform: methanol: water ═ 65:25:4), removing the solvent, drying, and then obtaining the vitamin E succinate diester-lysophosphatidylcholine conjugate (VES-lyso) by a preparative chromatography technology. The synthesis reaction formula of VES-lyso is shown in FIG. 1.

EXAMPLE 2 preparation of polyethylene glycol monomethyl ether-vitamin E succinate diester phospholipid Compound

In this example, a polyethylene glycol monomethyl ether-vitamin E diester succinate phospholipid compound (mPEG-VES-lyso) is prepared by the following specific preparation method:

dissolving 0.01mol of VES-lyso prepared in example 1 in 500mL of dichloromethane, carrying out ice bath, slowly adding 0.03mol of sodium hydride, heating to room temperature for reaction for 20min, carrying out rotary evaporation to remove the solvent after the reaction is finished, adding absolute ethyl alcohol at room temperature, fully mixing to remove excessive sodium hydride, filtering to remove the ethyl alcohol, washing and precipitating with the absolute ethyl alcohol for multiple times, and then placing in a vacuum drying oven to dry at room temperature for 24h to obtain the vitamin E diester succinate-sodium lysophosphatidate.

6.0g of polyethylene glycol monomethyl ether 2000(mPEG2000) is taken, stirred and dissolved in 20mL of dichloromethane, 3.5mmol of p-toluenesulfonyl chloride is added into 15mL of triethylamine, the triethylamine and the dichloromethane solution are mixed and reacted for 12h, hydrochloric acid solution with the concentration of 1mol/L is dripped into reaction liquid after the reaction is finished, an organic layer is extracted, dichloromethane is used for extracting a water phase, an organic phase is combined, excessive anhydrous sodium carbonate is added into the organic phase, the mixture is fully stirred and filtered, dichloromethane is removed by rotary evaporation, anhydrous ethanol is added to precipitate, the precipitate is obtained after the filtration and is dried in a vacuum drying oven for 24h, and the polyethylene glycol monomethyl ether p-toluenesulfonate 2000 is stored under vacuum drying.

Dissolving 0.01mol of vitamin E diester succinate-sodium lysophosphatidate and 0.01mol of polyethylene glycol monomethyl ether p-toluenesulfonate 2000 in 30mL of toluene, stirring and dissolving at room temperature, heating to 90 ℃, reacting for 4h, cooling the solution to room temperature after the reaction is finished, centrifuging the precipitate with dichloromethane, taking out the precipitate, and drying in vacuum to obtain the target amphiphilic phospholipid compound VES-lyso-mPEG.

Example 3 determination of the critical micelle concentration of VES-lyso-mPEG

In this example, the surface tension of an aqueous solution of VES-lyso-mPEG at each concentration at 25 ℃ was measured by the William hanger method as follows: accurately weighing 10mg of VES-lyso-mPEG, placing into a 100mL volumetric flask, dissolving with deionized water and fixing volume to obtain 1 × 10^-4g/mL of VES-lyso-mPEG standard aqueous solution; after standing and stabilizing, transferring 50mL of the solution to a 100mL volumetric flask by using a pipette, and carrying out constant volume to obtain 5X 10^-5g/mL VES-lyso-mPEG aqueous solution, diluting according to different proportions by analogy, and preparing 1 × 10^-5g/mL、5×10^-6g/mL、1×10^-6g/mL、5×10^-7g/mL、1×10^-7g/mL of an aqueous solution of VES-lyso-mPEG; accurately weighing VES-lyso-mPEG, placing into a 100mL volumetric flask, dissolving with deionized water and fixing volume to obtain 1 × 10^-2g/mL、1×10^-3g/mL VES-lyso-mPEG standard aqueous solution.

The critical micelle concentration of VES-lyso-mPEG is 2.37 x 10 at 25 ℃ as obtained by surface tension test experiment^-5g/mL。

Example 4 preparation of docetaxel-VES-lyso-mPEG nanomicelles

In this embodiment, a film dispersion method is adopted to prepare docetaxel-VES-lyso-mPEG nano micelle, and the specific preparation method is as follows:

weighing 20mg of docetaxel and 80mg of VES-lyso-mPEG, fully dissolving with 2mL of dichloromethane, removing the dichloromethane by rotary evaporation to form a film, and adding 4mL of deionized water to dissolve the film to form the docetaxel-VES-lyso-mPEG nano micelle aqueous solution.

The experiment proves that the average particle size of the docetaxel-VES-lyso-mPEG micelle solution is 28nm, the particle size is uniform, and the entrapment rate of the docetaxel is 98.1%. The micelle electron microscope image is shown in figure 2, and the powder has moderate roundness and uniform size. The micelle solution is still clear and free of turbidity after being placed in a stability test box at 40 ℃ for 1 month, and the encapsulation rate of the docetaxel carrier is 96.9 percent.

Example 5 preparation of Adriamycin-VES-lyso-mPEG Nanoglossicles

In this example, a dialysis method is used to prepare doxorubicin-VES-lyso-mPEG nano-micelle, and the specific preparation method is as follows:

weighing 20mg of adriamycin and VES-lyso-mPEG80mg, fully dissolving with 2mL of absolute ethyl alcohol, pouring the mixed solution into a dialysis bag, dialyzing with 1L of deionized water for 24 hours, and filtering after dialysis to form adriamycin-VES-lyso-mPEG nano micelle aqueous solution.

Experiments show that the average particle size of the drug-loaded nano micelle is 34nm, the particle size is uniform, the distribution is narrow, the micelle is well formed, and the doxorubicin-loaded encapsulation rate is 96.8%. The micellar solution is still clear and free from turbidity after being placed in a stability test box at 40 ℃ for 1 month, and the encapsulation rate of the doxorubicin-loaded solution is 96.2 percent.

Example 6 preparation of Voriconazole-VES-lyso-mPEG Nanoglossicles

In this example, voriconazole-VES-lyso-mPEG nano micelle is prepared by a direct dissolution method, and the specific preparation method is as follows:

weighing 20mg of voriconazole and 60mg of VES-lyso-mPEG, fully dissolving with 4mL of deionized water, stirring for 4h at room temperature, and filtering to form a voriconazole-VES-lyso-mPEG nano micelle aqueous solution.

Experiments prove that the average particle size of the drug-loaded micelle solution in the embodiment is 27nm, the particle size is uniform, and the entrapment rate of the voriconazole-loaded micelle solution is 95.8%. The micelle solution is still clear and free of turbidity after being placed in a stability test box at 40 ℃ for 1 month, and the encapsulation rate of the voriconazole carrier is 94.2 percent.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.