CN108159401B - Apelin liposome and preparation method thereof - Google Patents

Abstract

The invention provides an Apelin liposome and a preparation method thereof, the Apelin liposome comprises Apelin, phosphatidylglycerol, phosphatidylcholine, cholesterol, phosphatidylethanolamine-polyethylene glycol derivatives, and the molar ratio of Apelin to phosphatidylglycerol is 1: 2-1: 10. The Apelin liposome realizes the high-efficiency encapsulation of Apelin, the encapsulation rate reaches more than 90 percent, and the half-life period of Apelin in a living body is obviously prolonged.

Description

Apelin liposome and preparation method thereof

Technical Field

The invention belongs to the field of pharmaceutical preparations, and particularly relates to an Apelin liposome and a preparation method thereof.

Background

Apelin (Apelin peptide) is an important functional peptide in human body. Under the action of in vivo protease, Apelin propeptide containing 77 amino acids is cracked into Apelin fragments with activity, and the fragments can be divided into: apelin-13 (tridecapeptide), pyroglutamic acid type Apelin-13([ Pyrl ] -Apelin-13), Apelin-17 (heptadecapeptide), Apelin-36 (triacontapeptide), Apelin-55 (fifty-pentapeptide), etc. (European Journal of pharmacy 2015, 763: 149-159), and herein, the aforementioned Apelin fragments are collectively referred to as "Apelin".

Apelin is an endogenous ligand for the angiotensin II-like-1 receptor (APJ). The APJ receptor, also known as the Apelin receptor, is a member of the family of orphan G-protein-coupled receptors (GPCRs). The APJ receptor has a structure similar to the angiotensin II type 1 receptor (AT1), has important homology sequences with AT1, but differs in that the APJ receptor does not bind to angiotensin II. The Apelin/APJ system is widely distributed in organisms, is expressed on various organ cells in the organisms, and is particularly higher in the expression level of systems such as cardiovascular systems, central nerves systems, pulmonary vessels and the like. In recent years, many evidences indicate that the Apelin/APJ system plays a role in the pathophysiological processes of cardiovascular diseases, diabetes, tumors, central nervous system diseases and the like, and is a potential important target for treating many diseases (pharmaceutical Reviews 2010, 62: 331-.

However, Apelin has a very short half-life in vivo, and it has been reported in the literature that the Apelin proto drug is undetectable in vivo after 5 minutes after intravenous injection of Apelin solution into animals (Hypertension 2016, 68: 365-377). The defect of short half-life period seriously hinders the development of the medicine for clinical treatment of Apelin.

Aiming at the defect that Apelin has extremely short half-life in organisms, the half-life of Apelin is prolonged by adopting a chemical modification mode in the prior art. For example, WO2012/125408 discloses a polyethylene glycol (PEG) chemically modified Apelin having one or more PEG molecules of molecular weight 5000Da to 50000Da attached by chemical bonds to an amino acid residue at the N-terminus of an Apelin. Compared with Apelin modified by non-PEG, the half-life period of Apelin modified by PEG is prolonged to a certain extent. WO2014/152955 discloses Apelin fusion proteins or Apelin fusion polypeptides comprising an Apelin fusion protein fused to a polyplex component, which upon incubation with plasma or serum in an in vitro environment results in an in vitro plasma or serum half-life of about 1 to 10 hours. WO2015/013169 discloses a bioconjugate of synthetic Apelin polypeptides that chemically links the polypeptides to a half-life extending structure by covalent linkage or fusion, with the purpose of extending the half-life of Apelin. WO2015/013165 discloses a cyclic Apelin derivative, which increases the half-life of Apelin by modifying the sequence or spatial structure of Apelin; WO2015/147641 discloses a synthetic cyclic Apelin analog that has a stability in rat plasma of at least 3 hours and an extended half-life in vitro. WO2016/116842 discloses chemically synthesized Apelin fatty acid conjugates, wherein PEG linker is used to attach groups such as fatty acids, thereby increasing the half-life of Apelin and providing longer circulation time in vivo. However, the chemical modification approach also has the problems of decreased Apelin activity, low yield, difficult industrialization and the like, and the development of the pharmaceutical property and the medicinal product of Apelin is seriously influenced.

In recent years, there have been studies to prolong the action time of Apelin in vivo by physical encapsulation. US2016/0058705A1 discloses an Apelin composition and a preparation method thereof, and Apelin is encapsulated by PEG-modified liposome, so that the slow release of Apelin under physiological conditions is realized, the protein degradation resistance of Apelin is improved, and the action time of a medicament in a body is prolonged. However, the preparation process of the liposome is a thin film dispersion method, belongs to the technology of laboratory stage, and is difficult to realize industrialized mass production. The defect is that the encapsulation efficiency of the liposome disclosed by the patent to Apelin is only about 30%, and the liposome does not realize high-efficiency encapsulation. In addition, the process uses dichloromethane or chloroform, which is easy to generate great toxicity to the central nervous system of an organism and even induce severe side effects such as teratogenesis, mutagenesis and the like, so that the solvent residue must be strictly controlled, and the production cost is indirectly increased.

In conclusion, it is easy to find that, although the existing chemical modification and physical encapsulation technologies can prolong the half-life or the acting time of Apelin in vivo to a certain extent, there are certain problems, such as that the chemical modification of Apelin reduces the original activity of the drug to a certain extent, the industrialization is difficult, and the yield is low; the liposome technology disclosed in US2016/0058705A1 adopts a thin film dispersion method, belongs to a laboratory-stage technology, and also has the problems of difficult industrialization and the like, and the defects that the process has low Apelin entrapment rate and does not meet the practical clinical medication requirement. In addition, organic solvents such as dichloromethane or chloroform and the like used in the process are easy to cause harm to human bodies and environment. Although organic solvents such as methylene chloride or chloroform can be removed from the final formulation by some technique, the risk of clinical administration of liposomes for intravenous administration to humans is also increased.

Disclosure of Invention

In view of the above-mentioned drawbacks and deficiencies of the prior art, the present invention provides an Apelin liposome with high encapsulation efficiency and long half-life, and a simple, environment-friendly and easily-industrialized method for preparing the Apelin liposome.

The present invention provides the following scheme:

an Apelin liposome is characterized by comprising Apelin, phosphatidylglycerol, phosphatidylcholine, cholesterol and phosphatidylethanolamine-polyethylene glycol derivatives, wherein the molar ratio of Apelin to phosphatidylglycerol is 1: 2-1: 10.

Preferably, the molar ratio of Apelin to phosphatidylglycerol is 1: 4-1: 10.

Preferably, the molar ratio of Apelin to phosphatidylcholine is 1: 2-1: 10.

Preferably, the molar ratio of Apelin to cholesterol is 1: 2-1: 16.

Preferably, the molar ratio of Apelin to the phosphatidylethanolamine-polyethylene glycol derivative is 1: 0.2-1: 3.

Preferably Apelin is one selected from the group consisting of [ Pyrl ] -Apelin-13, Apelin-17, Apelin-36, Apelin-55 and pharmaceutically acceptable salts thereof;

the phosphatidyl glycerol is selected from one of distearoyl phosphatidyl glycerol, dioleoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol, dilauroyl phosphatidyl glycerol, dimyristoyl phosphatidyl glycerol, erucyl phosphatidyl glycerol and 1-palmitoyl-2-oleoyl phosphatidyl glycerol;

the phosphatidylcholine is selected from one of soybean lecithin, yolk lecithin, hydrogenated soybean lecithin, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dilauroyl phosphatidylcholine, dioleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine and dianeoyl phosphatidylcholine;

the phosphatidylethanolamine-polyethylene glycol derivative is any one selected from the group consisting of distearoylphosphatidylethanolamine-polyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol 3400, distearoylphosphatidylethanolamine-polyethylene glycol 5000, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, dipalmitoylphosphatidylethanolamine-polyethylene glycol 5000, and dioleoylphosphatidylethanolamine-polyethylene glycol 2000.

Furthermore, Apelin is preferably [ Pyr1] -Apelin-13 or a pharmaceutically acceptable salt thereof.

In addition, the Apelin liposome can also comprise a freeze-drying protective agent, and the freeze-drying protective agent accounts for 0-30% (g/mL) of the weight-volume ratio of the liquid liposome. The freeze-drying protective agent is at least one selected from sucrose, lactose, trehalose and mannitol.

In addition, the invention also provides a preparation method of the Apelin liposome, which is characterized by comprising the following steps:

(1) preparing an organic phase, namely weighing Apelin, phosphatidylcholine, phosphatidylglycerol, cholesterol and phosphatidylethanolamine-polyethylene glycol derivatives in a container to ensure that the molar ratio of Apelin to phosphatidylglycerol is 1: 2-1: 10, and then adding a proper amount of organic solvent to completely dissolve the Apelin and the phosphatidylglycerol to form a homogeneous solution;

(2) preparing a water phase, namely weighing a freeze-drying protective agent, and completely dissolving the freeze-drying protective agent with a proper amount of sterile water for injection under the condition of a water bath at 50-75 ℃ to prepare the water phase;

(3) preparing a liposome primary product, namely mixing the organic phase obtained in the step (1) and the water phase obtained in the step (2) under the water bath condition of 50-75 ℃, and stirring for 10-30 minutes under the condition of 100-500 revolutions per minute to obtain the liposome primary product;

(4) controlling the particle size of the liposome, namely placing the liposome primary product obtained in the step (3) into a homogenizer, and circulating for 1-10 times under the homogenizing pressure condition of 600-2000 Bar to obtain a final liquid liposome preparation, wherein the average particle size of the liposome is 50-500 nm;

(5) and (4) freeze-drying the liposome, namely removing a heat source from the liquid liposome preparation obtained in the step (4), sterilizing, subpackaging and freeze-drying to obtain liposome freeze-dried powder.

The Apelin liposome with high-efficiency encapsulation and long half-life can be obtained by the method, and the preparation method of the Apelin liposome is simple, environment-friendly and easy to industrialize.

Drawings

FIG. 1 shows the plasma concentration time curves of [ Pyr1] -Apelin-13 solution and [ Pyr1] -Apelin-13 liposome of the invention in rats.

Detailed Description

In order to improve the Apelin entrapment rate of the liposome, prolong the half-life of Apelin in a living body and solve the defects of the existing physical encapsulation technology, the inventor of the invention carries out a great deal of research, prepares Apelin into a PEG liposome by using various methods based on the classical formulation of the liposome (the membrane material comprises phosphatidylcholine, cholesterol and phosphatidylethanolamine-polyethylene glycol derivatives), respectively uses the conventional methods such as a film dispersion method, a reverse phase evaporation method, an ethanol injection method, an ammonium sulfate gradient method, a pH gradient method, a calcium acetate gradient method and the like to prepare the PEG modified Apelin liposome, and has the result similar to the PEG modified Apelin liposome disclosed in US2016/0058705A1, the entrapment rate of the obtained liposome is about 30 percent, and the high-efficiency entrapment of Apelin cannot be realized.

It is well known to those skilled in the art that the use of methylene chloride and/or chloroform is inevitable in the preparation of liposomes by the thin film dispersion method; ether and/or methanol are/is used in the reverse phase evaporation method, and the organic solvents are easy to cause harm to human bodies and environment; the preparation methods of the ammonium sulfate gradient method, the calcium acetate gradient method, the pH gradient method and the like relate to the establishment of an ammonium gradient, a calcium acetate gradient and a pH gradient, a large amount of dialysis and/or ion exchange is required in the process, and the defects of complex and complicated operation, high production cost, poor industrial production reproducibility and the like exist; according to the classical formulation of the liposome (the membrane material is composed of phosphatidylcholine, cholesterol, phosphatidylethanolamine-polyethylene glycol derivatives), the ethanol injection method which can realize industrial production is adopted, and the prepared liposome has low entrapment rate and can not meet the clinical medication requirements.

After intensive research, the inventors of the present invention unexpectedly found that: the Apelin liposome with the entrapment rate of more than 90 percent can be obtained by a simple ethanol injection method when a specific amount of phosphatidylglycerol is added into the conventional liposome formula (the membrane material comprises phosphatidylcholine, cholesterol and phosphatidylethanolamine-polyethylene glycol derivatives), namely the liposome membrane material comprises the phosphatidylglycerol, the phosphatidylcholine, the cholesterol and the phosphatidylethanolamine-polyethylene glycol derivatives, and the molar ratio of Apelin to the phosphatidylglycerol is 1: 2-1: 10.

The Apelin liposome of the present invention contains Apelin as a pharmaceutically active ingredient and a liposome membrane material. The concentration of Apelin is 0.005-10 mg/mL, preferably 1mg/mL, the liposome membrane material comprises phosphatidyl glycerol, phosphatidyl choline, cholesterol and phosphatidyl ethanolamine-polyethylene glycol derivatives, the molar ratio of Apelin to phosphatidyl glycerol is 1: 2-1: 10, preferably 1: 4-1: 10, so that the high-efficiency encapsulation of Apelin can be realized, and the encapsulation efficiency is more than 90%. The liposome membrane materials such as phosphatidylcholine, cholesterol, phosphatidylethanolamine-polyethylene glycol derivatives and the like can be used according to the commonly adopted proportion in the classical formula of the liposome, and specifically, in the invention, the molar ratio of Apelin to phosphatidylcholine is 1: 2-1: 10, preferably 1: 3-1: 8, and further preferably 1: 4-1: 7; the molar ratio of Apelin to cholesterol is 1: 2-1: 16, preferably 1: 2-1: 8, and further preferably 1: 3-1: 6; the molar ratio of Apelin to the phosphatidylethanolamine-polyethylene glycol derivative is 1: 0.2-1: 3, preferably 1: 0.2-1: 1, and more preferably 1: 0.4-1: 0.8.

The present invention will be described in further detail below based on examples and test examples. In the present invention, all the devices and materials are commercially available or commonly used in the industry. The following test examples and examples are only for specifically describing the present invention, and the present invention is not limited thereto.

The abbreviations for the respective components used in the specification are shown in table 1 below.

TABLE 1

Apelin types and amino acid sequences thereof used in the present invention are as described in "European Journal of Pharmacology 2015, 763: 149- "and 159", as shown in Table 2 below. These apelins are all available from shishi biotechnology (shanghai) ltd.

Example 1

Prescription:

[Pyrl]-Apelin-13	0.058mmol
		DSPG	0.116mmol
DSPC	0.116mmol
		CH	0.116mmol
DSPE-mPEG2000	0.012mmol
		sucrose	10g
Trehalose	5.5g
		Sterilized water for injection	Adding to 100mL

The process comprises the following steps:

(1) preparing an organic phase, namely weighing Apelin, phosphatidylcholine, phosphatidylglycerol, cholesterol and phosphatidylethanolamine-polyethylene glycol derivatives in the formula amount in a container, and adding a proper amount of organic solvent to completely dissolve the Apelin, the phosphatidylcholine, the phosphatidylglycerol, the cholesterol and the phosphatidylethanolamine-polyethylene glycol derivatives, wherein the system presents a homogeneous solution, and the organic solvent is selected from ethanol or ethanol/water mixed solution;

(2) preparing a water phase, namely weighing the freeze-drying protective agent according to the formula amount, and completely dissolving the freeze-drying protective agent by using a proper amount of sterilized injection water under the water bath condition of 50-75 ℃ to prepare the water phase;

(3) preparing a liposome primary product, namely mixing the organic phase obtained in the step (1) and the water phase obtained in the step (2) under the water bath condition of 50-75 ℃, and stirring for 10-30 minutes under the condition of 100-500 revolutions per minute to obtain the liposome primary product;

(4) controlling the particle size of the liposome, namely placing the liposome primary product obtained in the step (3) into a homogenizer, and circulating for 1-10 times under the homogenizing pressure condition of 600-2000 Bar to obtain a final liquid liposome preparation, wherein the average particle size of the liposome is 50-500 nm;

(5) and (4) freeze-drying the liposome, namely removing a heat source from the liquid liposome preparation obtained in the step (4), sterilizing, subpackaging and freeze-drying to obtain liposome freeze-dried powder.

Redissolving the obtained liposome freeze-dried preparation to 1mg/mL by using 5% glucose injection to obtain Apelin liposome solution, adding 0.5mL of the solution to the upper layer of an ultrafiltration centrifugal tube (30K daltons), then placing the centrifugal tube into a centrifugal machine, centrifuging at 6000rpm for 30min, collecting lower layer liquid, and measuring the content A of the lower layer free drug by using High Performance Liquid Chromatography (HPLC); the resulting liposomes were disrupted with a breaker and the total drug content B in the liposome formulation was determined by HPLC. The encapsulation efficiency of the liposome was measured by the following formula, and the measurement result was 95%.

The encapsulation efficiency of the liposome (E%) (1-A/B) × 100%

Examples 2 to 45

Examples 2 to 45 were prepared according to the recipes shown in tables 3 to 5 below by the same preparation method as in example 1 to obtain Apelin liposomes. The encapsulation efficiency was measured by the same measurement method as in example 1, and the results are shown in tables 3 to 5.

From the above examples 1 to 45, it is understood that Apelin liposomes having an entrapment rate of 90% or more can be obtained by the present invention.

Test examples

Test example one, Apelin liposome without phosphatidylglycerol

1. Preparation of Apelin liposome without phosphatidylglycerol

Apelin liposomes containing no phosphatidylglycerol were prepared according to the conventional formulation of conventional liposomes by the following formulations 1 to 10 shown in table 6 and the preparation method of example 1.

TABLE 6

2. Measurement of Liposome encapsulation efficiency

Apelin liposomes prepared according to recipes 1 to 10 were measured by the same encapsulation efficiency measuring method as in example 1, and the measurement results are shown in Table 7.

TABLE 7

Prescription	Prescription 1	Prescription 2	Prescription 3	Prescription 4	Prescription 5	Prescription 6	Prescription 7	Prescription 8	Prescription 9	Prescription 10
											E％	30	26	18	23	32	19	25	28	30	27

The experimental data in tables 6 and 7 show that the encapsulation efficiency of Apelin liposome prepared by the conventional classical liposome prescription without phosphatidylglycerol is between 20% and 30%, the high encapsulation efficiency of Apelin is not realized, and the basic requirements of the encapsulation efficiency in the clinical application of liposome preparation are not met.

Test example II Effect of the amount of phosphatidylglycerol on Apelin Liposome encapsulation efficiency

1. Preparation of Apelin liposome containing phosphatidylglycerol

Apelin liposomes were prepared according to the recipes 11 to 20 shown in table 8 below and the preparation method of example 1 above.

TABLE 8

2. Determination of Apelin liposome encapsulation efficiency

Encapsulation efficiency was measured in the same manner as in example 1 for Apelin liposomes prepared according to the above recipes 11 to 20, and the results are shown in table 9.

TABLE 9

Prescription

Prescription 11

Prescription 12

Prescription 13

Prescription 14

Prescription 15

Prescription 16

Prescription 17

Prescription 18

Prescription 19

Prescription 20

E％

35

40

52

91

98

99

99

99

NA

NA

NA: this indicates that the liposome was not successfully prepared and thus the encapsulation efficiency could not be determined.

Comparing the prescriptions 1-10 (especially prescription 2) with the prescriptions 11-18, it can be seen that the encapsulation efficiency of Apelin liposome increases by adding phosphatidylglycerol to the prescription of Apelin conventional long-circulating liposome, and when the molar ratio of Apelin to phosphatidylglycerol is within a specific range, namely 1: 2-1: within 10, Apelin liposome with an entrapment rate of more than 90% can be obtained.

Test example III Effect of Apelin types on Liposome encapsulation efficiency

1. Preparation of liposomes of Apelin: drug-loaded liposomes containing different Apelin types were prepared according to the recipes 21-25 in table 10 and the preparation method described in example 1 above.

Watch 10

2. Determination of Apelin liposome encapsulation efficiency

Encapsulation efficiency was measured in the same manner as in example 1 for Apelin liposomes prepared according to the above formulations 21 to 25, and the results are shown in table 11 below.

TABLE 11

Prescription composition	Prescription 21	Prescription 22	Prescription 23	Prescription 24	Prescription 25
						Encapsulation efficiency (E%)	99	97	99	98	99

As is clear from the data in tables 10 and 11, liposomes having high encapsulation efficiency can be prepared according to the specific composition of the present invention when the pharmaceutically active ingredients are [ Pyrl ] -Apelin-13, Apelin-17, Apelin-36 and Apelin-55.

Test example four Effect of phosphatidylglycerol species on Apelin Liposome encapsulation efficiency

1. Preparation of Apelin liposomes

Apelin-containing liposomes were prepared using different kinds of phosphatidylglycerols according to the recipes 26 to 32 in Table 12 and the preparation method described in example 1 above.

TABLE 12

2. Determination of Apelin liposome encapsulation efficiency

Encapsulation efficiency was measured in the same manner as in example 1 for Apelin liposomes prepared according to the above recipes 26 to 32, and the results are shown in table 13.

Watch 13

Prescription	Prescription 26	Prescription 27	Prescription 28	Prescription 29	Prescription 30	Prescription 31	Prescription 32
								Encapsulation efficiency (%)	99	96	98	97	98	99	98

The experimental data show that the encapsulation efficiency of the Apelin liposome is over 90% no matter what kind of phosphatidylglycerol is used, and the efficient encapsulation of the Apelin is realized as long as the molar ratio of the phosphatidylglycerol to the Apelin is within the range of the invention.

Fifth test example examination of the biological half-life of Apelin liposomes in rats

The in vivo biological half-life period of rats of the Apelin liposome preparation is examined by taking a self-made [ Pyr1] -Apelin-13 solution as a control.

1. Preparation of drug administration test article

(1) Preparation of [ Pyrl ] -Apelin-13 solution

Prescription:

prescription composition	Dosage of prescription
		[Pyrl]-Apelin-13	0.058mmol
5% glucose solution	Adding to 100mL

The process comprises the following steps:

weighing the prescription amount of Pyrl]Apelin-13, completely dissolving the raw materials by using a 5% glucose solution under the condition of stirring at room temperature, then fixing the volume to 100mL by using the 5% glucose solution, and filtering the solution through a 0.22 mu m microporous filter membrane to obtain the self-made [ Pyr1]]-Apelin-13 solution, [ Pyr1]Apelin-13 specification 1mg mL^-1。

(2) [ Pyr1] -Apelin-13 liposome preparation

The [ Pyr1] -Apelin-13 liposome is prepared according to the formula 16, and the entrapment rate is up to 99%. The [ Pyrl ] -Apelin-13 specification was 1mg/mL and reconstituted with 5% glucose solution prior to administration.

2. Investigation of biological half-life of different [ Pyr1] -Apelin-13 preparations in rats

(1) Laboratory animal information

SPF grade SD rats, 12 males, weighing 190g to 210g, were provided by Schbefu (Beijing) Biotechnology Inc.

(2) Dosing regimen and plasma sample collection processing

The rats were randomly divided into 2 groups (n ═ 6) of a home-made [ Pyr1] -Apelin-13 solution group and a [ Pyrl ] -Apelin-13 liposome group, and the rats were fixed and administered through the tail vein. After administration, 0.2mL of whole blood was collected via jugular vein cannula at different time points, and placed in a 1.5mL EDTA-2K anticoagulation centrifuge tube, after blood collection, 0.2mL of physiological saline was supplemented from the cannula, and the tube was sealed with 50 μ L of heparin sodium solution (50IU/mL) for smooth collection at the next time point. The blood samples were centrifuged at 4000g for 5 minutes at 4℃, plasma was separated, a protein blood sample was precipitated, and LC-MS/MS was used to determine the concentration of the test substance in rat plasma after administration of the test substance and to calculate the biological half-life.

Blood sampling points:

a) the [ Pyrl ] -Apelin-13 solution group is used for collecting blood samples 1min, 3min, 5min, 10min, 15min, 30min, 1h and 2h after administration;

b) the [ Pyr1] -Apelin-13 liposome group is used for collecting blood samples 1min, 15min, 30min, 1h, 4h, 8h, 24h, 48h, 72h and 96h after administration;

(3) results of the experiment

The time-dependent change curve of the blood concentration of the [ Pyr1] -Apelin-13 solution and liposome preparation is shown in fig. 1, and the biological half-life in rats is shown in table 14 below.

TABLE 14

Prescription	[Pyr1]-Apelin-13 injection	[Pyr1]-Apelin-13 liposomes
			Half-life period (h)	0.016	9.5

The experimental data show that the [ Pyrl ] -Apelin-13 injection has extremely short biological half-life in rats, and is only about 0.016h (about 1 min); the [ Pyr1] -Apelin-13 liposome of the invention has obviously prolonged biological half-life in rats in vivo, which reaches 9.5 h. The above results show that: compared with [ Pyr1] -Apelin-13 injection, the Apelin liposome of the invention obviously prolongs the half life of the medicine, and the half life is about 590 times of that of Apelin solution.

In conclusion, the Apelin liposome provided by the invention can realize high-efficiency encapsulation of Apelin, the encapsulation rate is more than 90%, the half-life of Apelin in a living body is remarkably prolonged, and the biological half-life of the Apelin liposome provided by the invention is about 590 times of that of an Apelin solution.

The foregoing is intended to further illustrate the invention and not to limit the scope of the invention. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims (5)

1. An Apelin liposome, which is characterized by comprising Apelin, phosphatidylglycerol, phosphatidylcholine, cholesterol, phosphatidylethanolamine-polyethylene glycol derivative and a freeze-drying protective agent, and the Apelin liposome is characterized in that

The molar ratio of Apelin to phosphatidylglycerol is 1: 4-1: 10; the molar ratio of Apelin to phosphatidylcholine is 1: 2-1: 10; the molar ratio of Apelin to cholesterol is 1: 2-1: 16; the molar ratio of Apelin to the phosphatidylethanolamine-polyethylene glycol derivative is 1: 0.2-1: 3; and is

The freeze-drying protective agent accounts for 0-30% g/mL of the weight volume ratio of the liquid liposome.

2. The Apelin liposome of claim 1, wherein,

apelin is any one selected from [ Pyr1] -Apelin-13, Apelin-17, Apelin-36 and Apelin-55;

the phosphatidyl glycerol is selected from one of distearoyl phosphatidyl glycerol, dioleoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol, dilauroyl phosphatidyl glycerol, dimyristoyl phosphatidyl glycerol, erucyl phosphatidyl glycerol and 1-palmitoyl-2-oleoyl phosphatidyl glycerol;

the phosphatidylcholine is selected from one of soybean lecithin, yolk lecithin, hydrogenated soybean lecithin, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dilauroyl phosphatidylcholine, dioleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine and dianeoyl phosphatidylcholine;

the phosphatidylethanolamine-polyethylene glycol derivative is any one selected from the group consisting of distearoylphosphatidylethanolamine-polyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol 3400, distearoylphosphatidylethanolamine-polyethylene glycol 5000, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000, dipalmitoylphosphatidylethanolamine-polyethylene glycol 5000, and dioleoylphosphatidylethanolamine-polyethylene glycol 2000.

3. The Apelin liposome of claim 2, wherein Apelin is [ Pyr1] -Apelin-13.

4. The Apelin liposome of claim 1, wherein the lyoprotectant is at least one selected from the group consisting of sucrose, lactose, trehalose, and mannitol.

The preparation method of the Apelin liposome is characterized by comprising the following steps:

(1) preparing an organic phase, namely weighing Apelin, phosphatidylcholine, phosphatidylglycerol, cholesterol and phosphatidylethanolamine-polyethylene glycol derivatives into a container, wherein the molar ratio of Apelin to phosphatidylglycerol is 1: 4-1: 10, the molar ratio of Apelin to phosphatidylcholine is 1: 2-1: 10, the molar ratio of Apelin to cholesterol is 1: 2-1: 16, and the molar ratio of Apelin to phosphatidylethanolamine-polyethylene glycol derivatives is 1: 0.2-1: 3, adding a proper amount of organic solvent to completely dissolve the Apelin and phosphatidylethanolamine-polyethylene glycol derivatives, wherein the organic solvent is selected from ethanol or ethanol/water mixed solution;

(2) preparing a water phase, namely weighing a freeze-drying protective agent, and completely dissolving the freeze-drying protective agent with a proper amount of sterile water for injection under the water bath condition of 50-75 ℃ to prepare the water phase;

(3) preparing a primary liposome product, namely mixing the organic phase obtained in the step (1) and the water phase obtained in the step (2) under the water bath condition of 50-75 ℃, and stirring for 10-30 minutes under the condition of 100-500 revolutions per minute to obtain the primary liposome product;

(4) controlling the particle size of the liposome, namely placing the liposome primary product obtained in the step (3) into a homogenizer, and circulating for 1-10 times under the homogenizing pressure condition of 600-2000 Bar to obtain a final liquid liposome preparation, wherein the average particle size of the liposome is 50-500 nm;

(5) and (4) freeze-drying the liposome, namely removing a heat source from the liquid liposome preparation obtained in the step (4), sterilizing, subpackaging and freeze-drying to obtain liposome freeze-dried powder.

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