CN111297805A

CN111297805A - Liposome of pimavanserin and preparation process thereof

Info

Publication number: CN111297805A
Application number: CN201911318554.5A
Authority: CN
Inventors: 崔建国; 梁淑芹; 齐洁; 胡梦芬; 赵洁
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-12-19
Filing date: 2019-12-19
Publication date: 2020-06-19

Abstract

The invention relates to the field of medicaments, in particular to a pimavanserin liposome for resisting hallucinations and delusions accompanied with Parkinson's disease treatment and a preparation process thereof. The invention adopts a thin film hydration method to prepare the lipidosome of the pimavanserin, phospholipid and cholesterol are dissolved in an organic solvent, and the lipidosome is decompressed and rotary-evaporated to form a film at a proper temperature; then shaking by phosphate buffer solution and carrying out ultrasonic hydration, and finally extruding through a 0.45 and 0.22 mu m microporous filter membrane to obtain the liposome. The pimavanserin liposome prepared by the method can obviously increase the solubility of pimavanserin, and has the advantages of high entrapment rate, good preparation stability and higher drug-loading rate; and the preparation process is simple, and the auxiliary materials are economical and safer. The liposome of the present invention can be further prepared into liposome powder by techniques such as freeze drying, and the like, and then can be used for preparing capsules and tablets.

Description

Liposome of pimavanserin and preparation process thereof

Technical Field

The invention relates to the field of medicinal preparations, in particular to a pimavanserin liposome for resisting hallucinations and delusions accompanied with Parkinson's disease treatment and a preparation process thereof.

Background

Pimavanserin (Pimavanserin) is the first FDA approved drug for the treatment of hallucinations and delusions accompanying the treatment of parkinson's disease. It was developed by Acadia pharmaceutical company and approved for marketing in 2016 at 4 months. Pimavanserin has poor water solubility, and the tartrate is used as the clinical medication form.

CN107848972A, US20190084935A1 and the like overcome the problem of poor water solubility of pimavanserin by preparing tartrate of pimavanserin and a new crystal form thereof, and although the method solves the problem of poor drug insolubility from the source in one step, on one hand, the pimavanserin is prepared into the tartrate, and the production cost of raw material medicines (API) is greatly increased by preparing the pimavanserin into a specific crystal form in order to ensure the stability of the drug, and the accessibility of the raw material medicines of the specific salt and the crystal form is far lower than that of the pimavanserin; on the other hand, the research on the salt form and the crystal form of pimavanserin which are improved from the source is relatively complete, and an additional solubilization mode is needed to find the breakthrough point of the patent layout. Solubilization of drugs from a formulation level is the most readily contemplated way for drug developers. For example, CN2016102965003 prepared a solid dispersion of pimavanserin hemitartrate, which can improve the solubility and bioavailability of pimavanserin, but potential crystallization and crystal transformation problems of the drug in the solid dispersion may have an influence on the effect of the preparation.

The preparation level has various modes for solubilizing the medicine, such as micronization, cyclodextrin inclusion, liposome and the like, wherein the micronization technology has high energy consumption and is not suitable for some medicines with strong electrostatic action, and the solubilization effect is usually poor and satisfactory only in a physical level; cyclodextrins are generally unstable to acidic conditions, and they rely mainly on physical inclusion, which has a high requirement on the size of drug molecules and poor stability; liposomes are a new type of pharmaceutical preparation that has recently developed, and they utilize the bilayer structure of phospholipids to form vesicles, and encapsulate poorly soluble drugs in the phospholipid bilayer of liposomes, or encapsulate water soluble drugs in the cells enclosed by the bilayer. Because the liposome has good compatibilization effect on insoluble drugs, high entrapment rate, similar structural composition to cell membranes, good biocompatibility, easy endocytosis by cells, and can be subjected to various modifications on the surface to improve the retention time and targeting property of the liposome in vivo, more and more attention is paid.

Common preparation processes of the liposome comprise a thin film hydration method, an ethanol injection method, a reverse evaporation method, an ammonium sulfate gradient method and the like. The film hydration method is the most common method due to mature and simple process and strong repeatability; the ethanol injection method requires that ethanol in which a drug is dissolved is injected into a water phase, and has strict requirements on temperature, stirring and injection speed, so that uniform particle size is not easy to control; compared with the former two methods, the reverse evaporation method has more complex preparation process and is easy to emulsify in the preparation process; the ammonium sulfate gradient method is an active drug loading method, and the drug loading of the prepared liposome can be further improved, but the method has the disadvantage of complex process.

Disclosure of Invention

In order to overcome the technical problem of poor water solubility of the pimavanserin bulk drug, the invention adopts the most common thin film hydration method to prepare the pimavanserin liposome, and optimizes the optimal auxiliary material composition and preparation process conditions by a single-factor test method.

One aspect of the invention provides a pimavanserin liposome, which consists of pimavanserin and liposome materials.

Wherein the liposome material is phospholipid and cholesterol.

Wherein the phospholipid comprises soybean lecithin (SPC), egg yolk lecithin (EPC), hydrogenated soybean lecithin (HSPC), Phosphatidylethanolamine (PE), Phosphatidylglycerol (PG), Dipalmitoylphosphatidylcholine (DPPC); preferably, the phospholipid is selected from the group consisting of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, phosphatidylethanolamine, dipalmitoylphosphatidylcholine, phosphatidylglycerol; further preferably, the phospholipid is selected from one or both of hydrogenated soy lecithin and phosphatidylethanolamine.

Wherein the weight ratio of the phospholipid to the cholesterol is 10:1-1: 5; preferably, the weight ratio of phospholipid to cholesterol is 8:1-1: 1; further preferably, the weight ratio of phospholipids to cholesterol is from 5:1 to 2: 1.

The invention also provides a preparation process of the liposome of pimavanserin, which is a thin film hydration method.

The preparation process comprises the following steps: dissolving pimavanserin, phospholipid and cholesterol in an organic solvent, and performing reduced pressure rotary evaporation at a proper temperature to form a thin film; and adding a phosphate buffer solution prepared in advance into a rotary evaporation bottle, shaking and carrying out ultrasonic hydration to obtain a liposome pre-product, and then sequentially extruding the liposome pre-product through 0.45 and 0.22 mu m microporous filter membranes by using an extruder to obtain a liposome final product.

Wherein the organic solvent is selected from chloroform, dichloromethane, ethanol, acetonitrile, ethyl acetate; preferably, the organic solvent is selected from dichloromethane.

Wherein the dosage weight ratio of the medicine to the liposome material is 1: (1-100); preferably 1: (5-50); more preferably 1: (10-20).

The invention firstly prepares the pimavanserin into the liposome, and screens out the optimal auxiliary material type and preparation process parameters for preparing the liposome through single factor test. The prepared pimavanserin liposome can obviously increase the solubility of pimavanserin, has high entrapment rate which can reach more than 90 percent, and has good preparation stability and higher drug-loading rate. And the preparation process is simple, and the auxiliary materials are economical and safer. In addition, the liposome of the present invention can be further prepared into liposome powder by a technique such as freeze-drying, and then used for the preparation of capsules and tablets.

Drawings

FIG. 1 is a graph showing the distribution of particle sizes of liposomes prepared in example 1 of the present invention.

Detailed Description

I. Test for influence factors on compatibility of auxiliary materials

Soybean lecithin (SPC), egg yolk lecithin (EPC), hydrogenated soybean lecithin (HSPC), Phosphatidylethanolamine (PE), Phosphatidylglycerol (PG), Dipalmitoylphosphatidylcholine (DPPC), and the drugs were thoroughly mixed, and the respective drugs were designated as SPC group EPC, HSPC group, PE group, DPPC group, and PG group, and the pure drug group was used as a control group. The compatibility of the auxiliary materials of the mixture is tested under the conditions of high temperature, high humidity and illumination according to the guiding principle of the stability test of the four portions of 9001 raw material medicaments and the preparation in the Chinese pharmacopoeia 2015 edition. Wherein the weight ratio of the medicine to the auxiliary materials is 1: 5.

TABLE 1 test results of the Effect factors on adjuvant compatibility

As can be seen from the above table, the drug is relatively stable under high temperature and light, and relatively unstable under high humidity conditions. Besides the phosphatidylglycerol, other phospholipids in all auxiliary materials have good compatibility. Although the compatibility of PE and DPPC and pimavanserin was relatively better, there were no significant differences from SPC, EPC, HSPC. Therefore, SPC, EPC, HSPC, PE and DPPC are selected as liposome film forming materials in the follow-up process.

II. Single factor test:

1. screening of solvents

The influence of the types of organic solvents commonly used in the film dispersion method on the particle size and encapsulation efficiency of the preparation was examined. Selecting chloroform, dichloromethane, ethanol, acetonitrile, diethyl ether and ethyl acetate as organic solvent, selecting soybean lecithin as phospholipid, adding cholesterol, and controlling the ratio of phospholipid to cholesterol to be 5: 1.

The preparation processes of the groups are carried out according to the following steps:

dissolving pimavanserin, soybean lecithin and cholesterol in an organic solvent, performing reduced pressure rotary evaporation on a rotary evaporator at a proper temperature, and slowly and spirally drying to form a thin film; adding a prepared phosphate buffer solution (pH 7) into a rotary evaporation bottle, shaking for hydration to obtain a liposome pre-product, and sequentially extruding through 0.45 and 0.22 mu m microporous filter membranes by using an extruder to obtain a liposome final product. The encapsulation efficiency of the liposome is tested by adopting a protamine coacervation method, and the particle size distribution of the liposome is tested by adopting a Malvern nanometer particle size analyzer.

The experimental process and the results show that the solubility difference of different solvents to phospholipid, cholesterol and pimavanserin is large, and diethyl ether can not completely dissolve the phospholipid, the cholesterol and the pimavanserin even after ultrasonic treatment, so that the three can be discarded. The other solvents can better dissolve the pimavanserin and the liposome material. Wherein the film-forming state, liposome state, entrapment rate and particle size data caused by different solvents are shown in Table 2.

TABLE 2 results of the influence of different organic solvents on the particle size, encapsulation efficiency, etc. of the formulations

Note: # indicates that its translucent state is slightly inferior to the other groups.

It is thus clear that different solvents have no significant effect on the particle size distribution and appearance of the liposomes. Although all liposomes were translucent, it was observed after careful observation that ethanol-solubilized liposomes had a slightly less clear appearance than the other groups. Different solvents have great influence on the entrapment rate of the pimavanserin, wherein the entrapment rate of the pimavanserin is the worst when ethanol is used as the solvent, which may cause certain influence on the appearance of the liposome; chloroform was the best solvent for encapsulation, dichloromethane and acetonitrile were the second best, and ethyl acetate was slightly inferior. Since chloroform is easy to decompose under visible light to generate highly toxic phosgene, acetonitrile has a high boiling point, and dichloromethane is preferably used as a solvent in consideration of comprehensive safety, economy and energy consumption.

2. Screening for Phospholipids

Selecting SPC, EPC, HSPC, PE and DPPC which pass through an auxiliary material compatibility test to prepare liposome according to the following process and testing the entrapment rate and the particle size distribution of the liposome:

dissolving pimavanserin and different phospholipids and cholesterol in dichloromethane, performing reduced pressure rotary evaporation on a rotary evaporator at 30 ℃, and slowly and rotatably drying to form a thin film; adding a prepared phosphate buffer solution (pH 7) into a rotary evaporation bottle, shaking and ultrasonically hydrating to obtain a liposome pre-product, and sequentially extruding through 0.45 and 0.22 mu m microporous filter membranes by using an extruder to obtain a liposome final product. The encapsulation efficiency of the liposome is tested by adopting a protamine coacervation method, and the particle size distribution of the liposome is tested by adopting a Malvern nanometer particle size analyzer. Wherein the ratio of phospholipid to cholesterol is controlled to be 5: 1. The film-forming state, liposome appearance, encapsulation efficiency, particle size data for different phospholipids are shown in table 3.

TABLE 3 results of the influence of different phospholipids on the particle size, encapsulation efficiency, etc. of the formulations

The results show that different phospholipids as film-forming materials have great influence on the encapsulation efficiency of the liposome and also influence the particle size distribution of the liposome, wherein the effect of Hydrogenated Soybean Phospholipids (HSPC) in natural phospholipids is optimal, and the effect of PE in synthetic phospholipids is better. In summary, HSPC and PE were selected as preferred film-forming materials.

3. The influence of the dosage ratio of phospholipid and cholesterol on the encapsulation efficiency is researched

The ratio of HPSC to cholesterol was set at 20:1, 10:1, 8:1, 5:1, 2:1, 1:1, respectively, and methylene chloride was used as an organic solvent to prepare liposomes by a thin film dispersion-extrusion process, and the drug encapsulation efficiency and the particle size distribution of the liposomes were tested. The results are shown in Table 4.

TABLE 4 results of the influence of different phospholipid and cholesterol ratios on encapsulation efficiency and particle size, etc

The ratio of phospholipid and cholesterol greatly affects the encapsulation efficiency, and because cholesterol can adjust the fluidity of a liposome membrane, a certain proportion of cholesterol is usually required to be added so that the liposome can more easily encapsulate drugs (especially fat-soluble drugs); table 4 shows that the ratio of (8-1):1 can make the encapsulation rate of the liposome more than 80%, and (3-2): the ratio of 1 can make the encapsulation rate of the liposome adopting HPSC more than 92%.

III preparation example

The following are specific preparation examples, but it should be understood by those skilled in the art that they are merely illustrative and not construed to limit the scope of the present invention.

Example 1:

adding 100mg of pimavanserin, 1g of HPSC and 0.3g of cholesterol into 10mL of dichloromethane, heating to 35 ℃, stirring for dissolving, transferring the liquid onto a rotary evaporator, and removing the organic solvent under reduced pressure to obtain a layer of uniformly distributed film; adding 50ml phosphate buffer solution with pH value of 7, shaking the hydrated film, carrying out ultrasonic treatment for 30min, and then sequentially extruding through 0.45 and 0.22 mu m films to obtain the pimavanserin liposome. The liposome encapsulation efficiency is 92.56%, the drug loading rate is 6.61%, the average particle size is 213.5nm, and the PDI is 0.119.

Example 2:

adding 100mg of pimavanserin, 0.9g of HPSC and 0.3g of cholesterol into 10mL of dichloromethane, heating to 35 ℃, stirring and dissolving, transferring the liquid to a rotary evaporator, and removing the organic solvent under reduced pressure to obtain a layer of uniformly distributed film; adding 50ml phosphate buffer solution with pH value of 7, shaking the hydrated film, carrying out ultrasonic treatment for 30min, and then sequentially extruding through 0.45 and 0.22 mu m films to obtain the pimavanserin liposome. The liposome encapsulation efficiency is 93.89%, the drug loading rate is 7.22%, the average particle size is 211.0nm, and the PDI is 0.112.

Example 3:

adding 100mg of pimavanserin, 1g of PE and 0.3g of cholesterol into 10mL of dichloromethane, heating to 35 ℃, stirring for dissolving, transferring the liquid onto a rotary evaporator, and removing the organic solvent under reduced pressure to obtain a layer of uniformly distributed film; adding 500ml phosphate buffer solution with pH value of 7, shaking the hydrated film, performing ultrasonic treatment for 30min, and then sequentially extruding through 0.45 and 0.22 mu m films to obtain the pimavanserin liposome. The liposome encapsulation efficiency is 95.23 percent, the drug loading rate is 6.80 percent, the average particle size is 209.7nm, and the PDI is 0.102.

Example 4:

adding 100mg of pimavanserin, 0.9g of PE and 0.3g of cholesterol into 10mL of dichloromethane, heating to 35 ℃, stirring for dissolving, transferring the liquid to a rotary evaporator, and removing the organic solvent under reduced pressure to obtain a layer of uniformly distributed film; adding 50ml phosphate buffer solution with pH value of 7, shaking the hydrated film, carrying out ultrasonic treatment for 30min, and then sequentially extruding through 0.45 and 0.22 mu m films to obtain the pimavanserin liposome. The liposome encapsulation efficiency is 95.89%, the drug loading rate is 7.38%, the average particle size is 211.7nm, and the PDI is 0.111.

The pimavanserin is prepared into the liposome, and the preferable conditions in the pimavanserin liposome preparation are obtained through the auxiliary material compatibility test and the single factor test, wherein dichloromethane is preferably selected as the organic solvent; phospholipids are preferably PE or HSPC or a mixture of both; the ratio of phospholipid and cholesterol is preferably (8-1):1, and the preparation process adopts a common thin film hydration-extrusion process. The prepared pimavanserin liposome can obviously increase the solubility of pimavanserin, has high entrapment rate which can reach more than 90 percent, and has good preparation stability and higher drug-loading rate. And the preparation process is simple, and the auxiliary materials are economical and safer. In addition, the liposome of the present invention can be further prepared into liposome powder by a technique such as freeze-drying, and then used for the preparation of capsules and tablets.

Claims

1. The lipidosome of the pimavanserin consists of the pimavanserin and a lipidosome material, wherein the lipidosome material is phospholipid and cholesterol.

2. The liposome of claim 1, wherein the phospholipid comprises soybean lecithin (SPC), egg yolk lecithin (EPC), hydrogenated soybean lecithin (HSPC), Phosphatidylethanolamine (PE), Phosphatidylglycerol (PG), Dipalmitoylphosphatidylcholine (DPPC).

3. The liposome of claim 2, wherein the phospholipid is selected from one or both of hydrogenated soy lecithin and phosphatidylethanolamine.

4. The liposome of claim 1 or 3, wherein the weight ratio of phospholipid to cholesterol is 10:1 to 1: 5; preferably, the weight ratio of phospholipid to cholesterol is 8:1-1: 1; further preferably, the weight ratio of phospholipids to cholesterol is from 5:1 to 2: 1.

5. The process for preparing the liposome of pimavanserin of claim 1, which is a thin film hydration method.

6. The process according to claim 5, wherein pimavanserin, phospholipids and cholesterol are dissolved in an organic solvent and the solution is vacuum-evaporated at a suitable temperature to form a thin film; and adding a phosphate buffer solution prepared in advance into a rotary evaporation bottle, shaking and carrying out ultrasonic hydration to obtain a liposome pre-product, and then sequentially extruding the liposome pre-product through 0.45 and 0.22 mu m microporous filter membranes by using an extruder to obtain a liposome final product.

7. The process according to claim 6, wherein the organic solvent is selected from chloroform, dichloromethane, ethanol, acetonitrile, ethyl acetate; preferably, the organic solvent is selected from dichloromethane.

8. The process according to claim 7 or 8, wherein the ratio by weight of the drug to the liposome material is 1: (1-100); preferably 1: (5-50); more preferably 1: (10-20).