CN110339166A - A kind of Liraglutide multivesicular liposome and its preparation method and application - Google Patents
A kind of Liraglutide multivesicular liposome and its preparation method and application Download PDFInfo
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- CN110339166A CN110339166A CN201810300287.8A CN201810300287A CN110339166A CN 110339166 A CN110339166 A CN 110339166A CN 201810300287 A CN201810300287 A CN 201810300287A CN 110339166 A CN110339166 A CN 110339166A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/26—Glucagons
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
Abstract
The present invention relates to field of medicaments more particularly to a kind of Liraglutide multivesicular liposome and preparation method thereof.It include: Liraglutide, membrane material, osmotic pressure regulator, stabilizer in Liraglutide multivesicular liposome provided by the invention.Liraglutide multivesicular liposome stability prepared by the present invention is good, and entrapment efficiency is high, and drugloading rate is big, and rate of releasing drug is slowly steady, no phenomenon of burst release, and significantly improves the bioavilability of drug.To improve curative effect, reduce drug dose correlation toxic side effect, drug cost is reduced, there is larger application value.Experiment shows, nearly 432 hours of sustainable constant speed release medicine outside Via Liposomes provided by the invention, it can provide stable blood concentration in vivo, the internal residence time extends compared with its injection compared to significant, the characteristics of pharmacokinetics of apparent sustained release preparation is presented, it can provide normal steady blood glucose level, for hypoglycemic effect up to 312 hours, the relative bioavailability with injection was 661%.
Description
Technical field
The present invention relates to field of medicaments more particularly to a kind of Liraglutide multivesicular liposome and its preparation method and application.
Background technique
Diabetes have become the third-largest killer for endangering human life, are global epidemic diseases.Estimate according to WHO,
Quantity to the year two thousand fifty, whole world diabetic is up to 300,000,000.Diabetes are one group with defect of insulin secretion and/or pancreas
The metabolic disease that island element effect the shown hyperglycemia of obstacle is characterized.The state of chronic hyperglycemia and long-term complications of diabetes
Significant correlation, i.e., damage, dysfunction and the functional failure of numerous organs, especially kidney, eye, nerve, heart and blood vessel etc.,
Serious person can cause to be water, the acute complications such as electrolyte disturbance and acid-base imbalance ketoacidosis and Hyperosmotic coma.Cause
This, research and development efficient long-acting novel diabetes therapeutic agent has important social effect and economic value.
Liraglutide (Liraglutide) is Ucagon/glp-1 (7~37) class of danish novo nordisk exploitation
Ratified to list in the U.S. by FDA on January 25th, 2010 as the new drug for the treatment of diabetes B like object.Liraglutide and people
The natural GLP-1 of body has 97% homology, and structure has only done the modification of following two parts: i.e. 34 bad ammonia to GLP-1
Acid is replaced by arginine, and the palmitinic acid side chain of 16 carbon of glutamate-induced is increased on 26 lysines.This structural modification
So that Liraglutide is remained the bioactivity of GLP-1, in turn avoid being degraded by DPP-4 enzyme, with traditional regular iletin
It compares, Liraglutide has long-term effect.
Liraglutide has following pharmacological action: (1) concentration of glucose dependence insulin secretion accelerating；(2) inhibit postprandial
The secretion of glucagon reduces the release of glycogen；(3) enhance the sensibility of insulin；(4) slow down the emptying of stomach；(5) inhibit
Appetite loses weight.(6) beta Cell of islet is repaired；(7) protection is cardiovascular.
Liraglutide insulin secretion accelerating and glucagon suppression secretion have blood sugar concentration dependence, therefore work as blood
When sugar level is normal, hypoglycemia can't be caused by persistently giving Liraglutide, it can be seen that Liraglutide can not only make patient
Blood glucose maintains normal level, and can repair beta Cell of islet to a certain degree, also has certain curative effect for diabetic complication, is
Current advanced Remedies for diabetes.
The commercially available Liraglutide injection of current Novo Nordisk Co., Ltd's research and developmentIt is daily for subcutaneous administrations
Primary injection is injected, which has been found have good effect in terms of improving glycemic control and losing weight.
It is this for diabetes to need long-term treatment although Liraglutide has longer Half-life in vivo (12-14 hours), control blood
The disease of sugar level, Liraglutide injection, which still has, need to reduce administration number of times, and the improvement for improving patient to drug compliance needs
It asks.
For this purpose, people, which begin one's study, develops its slow release long-acting form of administration to reduce the injection frequency of Liraglutide.It is grinding
The long-acting dosage form in stage be mainly Liraglutide and PLGA, PLA etc. synthesize high molecular microball preparation (CN102085355A,
CN104382860A), however, the generally existing burst effect of polymer microsphere formulation (Sheikh Hasan, A., et al.,
Reduction of the in vivo burst release of insulin-loaded
microparticles.Journal ofDrug Delivery Science and Technology,2015.30,Part B:
P.486-493. it) and in prolonged stay body can generate inflammatory reaction, lead to organism immune response (Anderson, J.M.and
M.S.Shive,Biodegradation and biocompatibility of PLA and PLGA
Microspheres.Advanced Drug Delivery Reviews, 2012.64, Supplement (0): p.72-82.),
In addition, the problems such as acidic environment that polymer degradation generates can promote the denaturation of polypeptide protein class drug, activity to decline (Jiskoot,
W.,et al.,Protein Instability and Immunogenicity:Roadblocks to Clinical
Application of Injectable Protein Delivery Systems for Sustained
Release.Journal of Pharmaceutical Sciences,2012.101(3):p.946-954)。
Multivesicular liposome (Multivesicular liposome, MVLs) is by multiple non-concentric pharmaceutical aqueous solution folliculus
Bubble composition, these vesicas are separated by continuous non-concentric lipoids bilayer, packet with higher for water soluble drug
Envelope rate.Especially multivesicular liposome is to discharge drug by the vesica of rupture, and complete vesica still maintains original state.More capsule rouge
The topological structure of this non-concentric of plastid makes it form " storage cavern " in injection site, continuous with lipoids bilayer
Degradation, is encapsulated in drug therein and gradually discharges, generate good slow release effect, while in turn avoiding burst effect, can also lead to
Adjustment preparation prescription and technological parameter are crossed, obtains the Drug controlled release time from several days to the effect of several weeks.MVLs is as drug
Transmission system is ratified by FDA, morphine multivesicular liposome (DepoMorphinTM) listed in 2004 in the U.S..
In preparation process, the composition and preparation process of multivesicular liposome to entrapment efficiency, release time, particularly
The physical stability of multivesicular liposome has a significant impact.It is heavy that multivesicular liposome is easy to appear since partial size is larger, in storage process
It drops and assembles, used organic solvent in preparation process, easily occur deactivation prob etc. to protein and peptide drugs, therefore improve more
The aggregation stability and medicine stability of capsule liposome are still to develop the urgent problem to be solved of multivesicular liposome preparation.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that providing a kind of Liraglutide multivesicular liposome and its preparation
Methods and applications, Liraglutide multivesicular liposome provided by the invention have good slow release and long-term effect, and have good
Stability and bioavilability.
Liraglutide multivesicular liposome provided by the invention includes following mass parts:
A kind of Liraglutide multivesicular liposome provided by the invention is used as base by the phosphatide etc. for selecting high-biocompatibility
This membrane material is detained it and is not likely to produce inflammatory reaction in vivo.It obtained liposome encapsulation rate with higher and stable releases
Medicine rate, successfully avoids burst effect.Also, multivesicular liposome provided by the invention can also avoid Liraglutide easy in vivo
The problem of degrading extends the circulation time of drug in vivo, achieves the effect for significantly improving bioavilability.
In the present invention, Liraglutide multivesicular liposome includes following mass parts:
In the present invention, membrane material includes phosphatide, cholesterol and triglycerides, the mass ratio of phosphatide, cholesterol and triglycerides
For (46.8~342.3): (28.0~100.5): (8.6~191.0).
In the present invention, phosphatide is selected from lecithin (EPC), soybean lecithin (SPC), cephalin, dioleyl phosphatidyl choline
(DOPC), hydrogenated soya phosphatide (HSPC), Distearoyl Phosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC),
Dimyristoyl phosphatidyl choline (DMPC), Dioleoyl Phosphatidylcholine (DOPC), distearoylphosphatidylglycerol (DSPG), two
Palmityl phosphatidyl glycerol (DPPG), GLYCEROL,DIMYRISTOYL PHOSPHATIDYL (DMPG), dioleoylphosphatidylglycerol (DOPG) or phosphorus
Acyl ethanol amine (DOPE).
In some embodiments, phosphatide is selected from soybean lecithin, hydrogenated soya phosphatide, Dioleoyl Phosphatidylcholine, yolk ovum
Phosphatide, Dioleoyl Phosphatidylcholine or dipalmitoylphosphatidylglycerol.
DPPG, DSPG are the substance for increasing film negative electrical charge, can be to increase the stability of liposome.
Triglycerides is the important component that multivesicular liposome is formed, and plays a part of to support its structure in multivesicular liposome.
The triglycerides can be natural and synthesis.Heretofore described triglycerides is selected from olein, three
At least one of glycerol caprylate, soybean oil, preferably olein.
The present invention is provided in the membrane material of Liraglutide multivesicular liposome, and the molar percentage of triglycerides is 5%~50%.
The present invention is provided in the membrane material of Liraglutide multivesicular liposome, and the mass ratio of phosphatide and cholesterol is (0.5~20):
1。
In the present invention, the mass ratio of Liraglutide and membrane material is 1:(1~100).
In some embodiments, the mass ratio of Liraglutide and membrane material is 1:(1.44~92.35).
In the present invention, osmotic pressure regulator is selected from lysine, histidine, sucrose, glucose, dextran, glycerol, sweet dew
Alcohol or sorbierite.
In some embodiments, osmotic pressure regulator is selected from histidine, lysine, glucose or sucrose.
Liraglutide multivesicular liposome provided by the invention includes stabilizer, and heretofore described " stabilizer " refers to can
The internal external stability of drug is improved, acceptable auxiliary material in the pharmacy of drug Half-life in vivo is extended.
In the present invention, stabilizer is selected from human serum albumin, ox blood albumin, collagen, gelatin or gelatin hydrolysate.
In some embodiments, stabilizer is selected from human serum albumin, ox in Liraglutide multivesicular liposome provided by the invention
Blood albumin, gelatin hydrolysate or gelatin.
In the present invention, Liraglutide and stabilizer and mass ratio be 1:(0.3~2.5).
In some embodiments, Liraglutide and stabilizer and mass ratio be 1:(0.34~2.42).
In some embodiments, phosphatide is soybean lecithin；Triglycerides is olein；Wherein, soybean lecithin
The mass ratio of rouge, cholesterol and triglycerides is 201.1:90.2:125.
In this embodiment, Liraglutide and the mass ratio of membrane material are 1:41.62.
Preferably, stabilizer is human serum albumin；The mass ratio of Liraglutide and stabilizer is 1:1.6.
Preferably, osmotic pressure regulator is the mixture of sucrose and lysine, wherein the mass ratio of lysine and sucrose
For 15:1；The mass ratio of Liraglutide and osmotic pressure regulator is 1:67.2.
In some embodiments, phosphatide is hydrogenated soy phosphatidyl choline；Triglycerides is olein；In membrane material, hydrogen
The mass ratio for changing soybean lecithin, cholesterol and triglycerides is 203:96:121.
In this embodiment, Liraglutide and the mass ratio of membrane material are 1:42.1.
Preferably, stabilizer is human serum albumin；The mass ratio of Liraglutide and stabilizer is 1:2.
Preferably, osmotic pressure regulator is the mixture of sucrose and lysine, wherein the mass ratio of sucrose and lysine
For 15:1；The mass ratio of Liraglutide and osmotic pressure regulator is 1:67.2.
In some embodiments, phosphatide is Dioleoyl Phosphatidylcholine；Triglycerides is olein；In membrane material,
The mass ratio of Dioleoyl Phosphatidylcholine, cholesterol and triglycerides is 196.8:99.4:126.1.
In this embodiment, Liraglutide and the mass ratio of membrane material are 1:42.2.
Preferably, stabilizer is human serum albumin；The mass ratio of Liraglutide and stabilizer is 1:1.8.
Preferably, osmotic pressure regulator is the mixture of sucrose and lysine, wherein the mass ratio of sucrose and lysine
For 17.4:1；The mass ratio of Liraglutide and osmotic pressure regulator is 1:180.4.
In some embodiments, phosphatide is soybean lecithin；Triglycerides is olein；In membrane material, soybean lecithin,
The mass ratio of cholesterol and triglycerides is 202.1:98:122.6.
In this embodiment, Liraglutide and the mass ratio of membrane material are 1:7.7.
Preferably, stabilizer is human serum albumin；The mass ratio of Liraglutide and stabilizer is 1:1.6.
Preferably, osmotic pressure regulator is the mixture of sucrose and lysine, wherein the mass ratio of sucrose and lysine
For 15:1；The mass ratio of Liraglutide and osmotic pressure regulator is 1:12.2.
In some embodiments, phosphatide is soybean lecithin；Triglycerides is olein；Membrane material includes soybean phosphorus
Rouge, cholesterol, triglycerides and DPPG, wherein soybean lecithin, cholesterol, triglycerides and DPPG mass ratio be 210.5:
95:128.5:44.2.
In this embodiment, Liraglutide and the mass ratio of membrane material are 1:7.4.
Preferably, stabilizer is human serum albumin；The mass ratio of Liraglutide and stabilizer is 1:2.3.
Preferably, osmotic pressure regulator is the mixture of sucrose and lysine, wherein the mass ratio of sucrose and lysine
For 12.5:1；The mass ratio of Liraglutide and osmotic pressure regulator is 1:26.3.
In some embodiments, phosphatide is soybean lecithin；Triglycerides is olein；In membrane material, soybean lecithin,
The mass ratio of cholesterol and triglycerides is 199:90.5:187.
In this embodiment, Liraglutide and the mass ratio of membrane material are 1:42.2.
Preferably, stabilizer is ox blood albumin；The mass ratio of Liraglutide and stabilizer is 1:2.1.
Preferably, osmotic pressure regulator is the mixture of sucrose and histidine, preferably, osmotic pressure regulator is Portugal
The mixture of grape sugar, sucrose and histidine, wherein the mass ratio of glucose, sucrose and histidine is 100:980:112；Li La
The mass ratio of Shandong peptide and osmotic pressure regulator is 1:105.5.
In some embodiments, phosphatide is soybean lecithin；Triglycerides is olein；In membrane material, soybean lecithin,
The mass ratio of cholesterol and triglycerides is 260.3:100.5:191.
In this embodiment, Liraglutide and the mass ratio of membrane material are 1:30.3.
Preferably, stabilizer is gelatin；The mass ratio of Liraglutide and stabilizer is 1:2.4.
Preferably, osmotic pressure regulator is the mixture of sucrose and histidine, wherein the mass ratio of sucrose and histidine
For 12.3:1；The mass ratio of Liraglutide and osmotic pressure regulator is 1:158.0.
In some embodiments, phosphatide is soybean lecithin；Triglycerides is olein；In membrane material, soybean lecithin,
The mass ratio of cholesterol and triglycerides is 220:88.5:126.
In this embodiment, Liraglutide and the mass ratio of membrane material are 1:14.3.
Preferably, stabilizer is human serum albumin；The mass ratio of Liraglutide and stabilizer is 1:2.2.
Preferably, osmotic pressure regulator is the mixture of sucrose and histidine, wherein the mass ratio of sucrose and histidine
For 16.3:1；The mass ratio of Liraglutide and osmotic pressure regulator is 1:34.2.
In some embodiments, phosphatide is soybean lecithin；Triglycerides is olein；In membrane material, soybean lecithin,
The mass ratio of cholesterol and triglycerides is 220:95:156.
In this embodiment, Liraglutide and the mass ratio of membrane material are 1:92.4.
Preferably, stabilizer is ox blood albumin；The mass ratio of Liraglutide and stabilizer is 1:2.4.
Preferably, osmotic pressure regulator is the mixture of sucrose and histidine, wherein the mass ratio of sucrose and histidine
For 13.1:1；The mass ratio of Liraglutide and osmotic pressure regulator is 1:265.8.
In some embodiments, phosphatide is soybean lecithin；Membrane material includes soybean lecithin, cholesterol, tricaprylin, oil
Acid；Wherein, soybean lecithin, cholesterol, tricaprylin, oleic acid mass ratio be 220:76.5:122.3:25.6.
In this embodiment, Liraglutide and the mass ratio of membrane material are 1:31.3.
Preferably, stabilizer is human serum albumin；The mass ratio of Liraglutide and stabilizer is 1:2.1.
Preferably, osmotic pressure regulator is the mixture of sucrose and histidine, wherein the mass ratio of sucrose and histidine
For 18:1；The mass ratio of Liraglutide and osmotic pressure regulator is 1:93.7.
In some embodiments, phosphatide is soybean lecithin；In membrane material, soybean lecithin, cholesterol and olein
Mass ratio is 220:76.5:55.3.
In this embodiment, Liraglutide and the mass ratio of membrane material are 1:6.5.
Preferably, stabilizer is gelatin hydrolysate；The mass ratio of Liraglutide and stabilizer is 1:1.9.
Preferably, osmotic pressure regulator is the mixture of sucrose and histidine, wherein the mass ratio of sucrose and histidine
For 13.3:1；The mass ratio of Liraglutide and osmotic pressure regulator is 1:22.3.
In some embodiments, phosphatide DOPC；Membrane material includes DOPC, DPPG, cholesterol, triglycerides；Wherein,
DOPC, DPPG, cholesterol, triglycerides mass ratio be 36.6:10.2:28:8.6.
In this embodiment, Liraglutide and the mass ratio of membrane material are 1:1.4.
Preferably, stabilizer is human serum albumin；The mass ratio of Liraglutide and stabilizer is 1:0.34.
Preferably, osmotic pressure regulator is the mixture of sucrose and histidine, wherein the mass ratio of sucrose and histidine
For 15:1；The mass ratio of Liraglutide and osmotic pressure regulator is 1:23.1.
In some embodiments, phosphatide is egg yolk lecithin；In membrane material, egg yolk lecithin, cholesterol and triglycerides
Mass ratio is 183.6:70.7:61.1.
In this embodiment, Liraglutide and the mass ratio of membrane material are 1:7.0.
Preferably, stabilizer is human serum albumin；The mass ratio of Liraglutide and stabilizer is 1:2.0.
Preferably, osmotic pressure regulator is the mixture of sucrose and histidine, wherein the mass ratio of sucrose and histidine
For 13.3:1；The mass ratio of Liraglutide and osmotic pressure regulator is 1:26.9.
It is good that experiment shows that the Liraglutide multivesicular liposome prepared using prescription provided by the invention and preparation method is had
Good stability and bioavilability, particle size is suitable for, drugloading rate and encapsulation rate are all higher.Further, it is possible to improve drug
Residence time in vivo, bioavilability improve nearly 6 times compared with normal injection.And, it was also found that some bad in experimentation
The shape that prescription or inappropriate preparation method will lead to liposome will appear variation, cannot form complete liposome sometimes,
Sometimes many oil droplets are had.
Liraglutide multivesicular liposome prepared by the present invention is multivesicular liposome.
The preparation method of Liraglutide multivesicular liposome provided by the invention includes:
Step 1: membrane material is dissolved in organic solvent as oily phase；Stabilizer, osmotic pressure regulator and Liraglutide are dissolved in
Water is as inner aqueous phase；Osmotic pressure regulator is dissolved in water as outer aqueous phase；
Step 2: inner aqueous phase being made mutually to form W/O level-one cream with oily；
Step 3: the level-one cream and outer aqueous phase being made to form W/O/W emulsion；
Step 4: Liraglutide multivesicular liposome is made in dry emulsion.
In the present invention, organic solvent is selected from ether, methylene chloride, chloroform, ethyl acetate or hexamethylene in step 1.
In some embodiments, the organic solvent in step 1 is selected from ether, chloroform or hexamethylene.
In some embodiments, the concentration of membrane material is 10mg/ml~400mg/ml in oily phase.
In some embodiments, phospholipid concentration is 5~100mg/ml in oily phase, and cholesterol concentration is 3~50mg/ml, sweet
Oily three ester concentrations are 1~95mg/ml.
In the present invention, inner aqueous phase the preparation method comprises the following steps: by stabilizer stirring and dissolving Yu Shuizhong, osmotic pressure regulator, which is added, to be made
Dissolution, Liraglutide, which is added, to be made to dissolve.
In some embodiments, the concentration of stabilizer is 5~100mg/ml in inner aqueous phase；The concentration of Liraglutide be 1~
100mg/ml；The mass fraction of osmotic pressure regulator is 4%~10%.
In some embodiments, the mass fraction of osmotic pressure regulator is 4%~10% in outer aqueous phase.
Preferably, osmotic pressure regulator is glucose or sucrose, mass fraction 7% in inner aqueous phase；It is seeped in outer aqueous phase
Pressure regulator is the mixture of sucrose and histidine or sucrose and lysine thoroughly, and wherein the mass fraction of sucrose is 7%；Lysine
Or the concentration of histidine is 2mg/mL~8mg/mL.
In the present invention, the volume ratio of inner aqueous phase and oily phase is 1:(0.8~10).The method of preparation level-one cream is in step 2
Inner aqueous phase is added dropwise in oily phase, the method for emulsification uses the ordinary skill in the art, can use mechanical stirring, vortex, surpass
Adoptable method is emulsified in the pharmacies such as sound, high speed disperser, high pressure homogenizer, microjet high pressure homogenizer.
In the present invention, the volume ratio of level-one cream and outer aqueous phase is 1:(1~4).The method of emulsion is prepared in step 3 are as follows: outer
Water phase is added dropwise to outer aqueous phase under agitation, by level-one emulsion, and the method for emulsification uses the ordinary skill in the art, can be used
Selected from the methods of mechanical stirring, ultrasonic emulsification, mechanical high-speed emulsification pretreatment, high-pressure homogeneous.
In the present invention, dry emulsion is the process for removing organic solvent in emulsion, wherein the method for removing organic solvent
For the Conventional solvents minimizing technology in field, organic solvent is evaporated as normal pressure volatilization, air-flow brush, is dried under reduced pressure or is spray-dried
Deng, preferred method be first with inert gas flow drying, remaining organic solvent is further removed with Rotary Evaporators again.
In the present invention, washing is further included the steps that after step 4.
The washing is by washing the non-encapsulated free drug of removing with isotonic solution.Wherein, it is washed with isotonic solution
The method method that can be this field routine, isotonic solution can be used as cleaning solution, the side filtered using centrifugation, dialysis or film
Method removes the solute in free drug therein and outer aqueous phase, then with isotonic solution suspension to required drug concentration
Liraglutide multivesicular liposome.In the present invention, " isotonic solution " refers to that osmotic pressure is equal to blood plasma or subcutaneous, intramuscular injection
The aqueous solution of tolerable osmotic pressure, preferably sodium chloride or sucrose or glucose.
Liraglutide multivesicular liposome provided by the invention can be used in the drug of preparation treatment and/or prevention diabetes.
The present invention also provides the drugs of a kind for the treatment of and/or prevention diabetes including Li Lalu provided by the invention
Peptide multivesicular liposome.
The method provided by the invention for preparing liposome is also applied for glucagon-like peptide (GLP-1) class antidiabetic drug
Exenatide (Exenatide).
The dosage form of drug provided by the present invention for treating and/or preventing diabetes is injection.Its administration mode is
Subcutaneous injection, intramuscular injection, epidural injection or intrathecal injection, preferably subcutaneous injection and intramuscular injection.
According to the present invention, the active pharmaceutical ingredient Liraglutide is compound Liraglutide, with its source and preparation
Method is unrelated, can be genetic recombination source, is also possible to chemical synthesis source or semi-synthetic source.It is implemented all in this hair
Within bright protection scope.
It include: Liraglutide, membrane material, osmotic pressure regulator, stabilization in Liraglutide multivesicular liposome provided by the invention
Agent.Compared with the prior art, remarkable result of the invention is as follows:
(1) the Liraglutide multivesicular liposome prepared by the present invention has good stability, and entrapment efficiency is high, and drugloading rate is big,
Rate of releasing drug is slowly steady, no phenomenon of burst release, especially successfully avoids the incident internal drug drop of Liraglutide injection
Solution problem；Experiment shows that the partial size of liposome provided by the invention is mostly 5~30 μm；Encapsulation rate is not less than 75%, and drugloading rate is not
Lower than 2%；Its releasing effect stabilization, sustainable drug release 168-336 hours or more.
(2) liposome provided by the invention can reduce effect of the vivo protein degrading enzyme to Liraglutide, improve medicine
The internal external stability of object, meanwhile, liposome provided by the invention also slows down the release and absorption of drug, extends drug body
Interior circulation time.So Liraglutide multivesicular liposome prepared by the present invention significantly improves the bioavilability of drug.From
And improve curative effect, reduce drug dose correlation toxic side effect, drug cost is reduced, there is larger application value.It is real
It tests and shows that liposome rat skin lower injection administration blood concentration provided by the invention is stablized, sustained release is small up to 336 in vivo
When, the internal residence time significantly extends (respectively 163.4 hours and 13.9 hours) compared with Liraglutide injection, presents apparent
The characteristics of pharmacokinetics of sustained release preparation, the relative bioavailability with injection are 661%.
(3) Liraglutide multivesicular liposome provided by the present invention, drug release rate are slow, it is possible to provide long-acting hypoglycemic is made
With there is more preferably hypoglycemic effect, greatly reduce the administration number of times and frequency of patient, improve the administration compliance of patient.
Detailed description of the invention
Fig. 1 shows the optical microscope picture of Liraglutide multivesicular liposome prepared by the embodiment of the present invention 1；
Fig. 2 shows the optical microscope picture of Liraglutide multivesicular liposome prepared by the embodiment of the present invention 2；
Fig. 3 shows the optical microscope picture of Liraglutide multivesicular liposome prepared by the embodiment of the present invention 5；
Fig. 4 shows the In-vitro release curves of Liraglutide multivesicular liposome prepared by the embodiment of the present invention 1；
Fig. 5 shows the In-vitro release curves of Liraglutide multivesicular liposome prepared by comparative example 1 of the present invention；
Fig. 6 shows after the administration of Liraglutide multivesicular liposome diabetes rat prepared by the embodiment of the present invention 1 the 1st day to the
15 days blood sugar concentration-time graphs, ordinate its be blood sugar concentration (mmol/L), abscissa its be the time (h)；
Fig. 7 shows that comparative example 1 prepares the 1st day to the 7th day after Liraglutide multivesicular liposome diabetes rat is administered blood glucose
Concentration-time graph, ordinate are blood sugar concentration (mmol/L), and abscissa is the time (h)；
Fig. 8 shows that blood sugar concentration-time graph after commercially available Liraglutide injection diabetes rat administration, ordinate are
Blood sugar concentration (mmol/L), abscissa are the time (h)；
Liraglutide multivesicular liposome diabetes rat subcutaneous administrations Drug-time curve prepared by Fig. 9 embodiment 1；Blood
Concentration (pmol/l), abscissa are the time (h)；
Liraglutide multivesicular liposome diabetes rat subcutaneous administrations Drug-time curve prepared by Figure 10 comparative example 1；Blood
Concentration (pmol/l), abscissa are the time (h)；
The commercially available Liraglutide injection diabetes rat subcutaneous administrations Drug-time curve of Figure 11；Blood concentration (pmol/
L), abscissa is the time (h).
Specific embodiment
The present invention provides a kind of Liraglutide multivesicular liposomes and preparation method thereof, and those skilled in the art can use for reference
Present disclosure is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this field skill
It is it will be apparent that they are considered as being included in the present invention for art personnel.Method of the invention and application by compared with
Good embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to methods herein
It is modified or appropriate changes and combinations with application, carrys out implementation and application the technology of the present invention.
Reagent that the present invention uses, drug, instrument are all common commercially available product, can all be bought in market.
Wherein to the method for Liraglutide multivesicular liposome (Lrg-MVLs) quality testing are as follows:
Encapsulate the measurement of dose: precision measures Lrg-MVLs suspension 1ml and sets in 4ml centrifuge tube, 3500rpm centrifugation
10min discards supernatant liquid, and lower sediment is dispersed to 1ml with the PBS washing of pH 7.4 afterwards three times, and precision measures 0.2ml and sets 10ml
Measuring bottle is settled to scale to be acidified isopropanol (containing 1% hydrochloric acid), shakes up, HPLC method measures the drug of the intracapsular encapsulating of Lrg-MVLs
Concentration CIt is intracapsular, computational envelope dose MCapsuleIt is interior
Total dose measurement: precision measures Lrg-MVLs suspension 0.2ml and sets 10ml measuring bottle, to be acidified isopropanol (containing 1% salt
Acid) it is settled to scale, it shakes up, HPLC method measures the drug concentration C of the intracapsular encapsulating of Lrg-MVLsAlways, calculate total dose MAlways
Encapsulation rate (EE) is calculated as follows:
EE%=MIt is intracapsular/MAlways×100
Drugloading rate is calculated according to the following formula:
(DL): DL%=MIt is intracapsular/m
In formula: m is lipid total amount
The measuring method of relative bioavailability
Relative bioavailability is biology obtained from comparing degree of absorption and speed between same drug difference preparation
Availability.Relative bioavailability is calculated as follows:
In formula: A, B respectively represents example 1 group and Liraglutide commercial preparation group, and Dose indicates dosage
Liraglutide assay HPLC method:
Chromatographic condition: chromatographic column: C18Column (250mm × 4.6mm)；Mobile phase: acetonitrile-water (60:40) contains 0.1% trifluoro
Acetic acid；Flow velocity: 1mlmin-1；Column temperature: 30 DEG C；Detection wavelength: 290nm；Sample volume: 20 μ l.
The measurement of form and granularity: in 400 times of optical microphotographs its form under the microscope；Take sample with physiological saline for dispersion
After medium, utilize laser diffraction particle size instrument (LS230 laser particle analyzer analyzer, Beckman Coulter company, the U.S.)
Measure its partial size and size distribution.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1
Precision weigh Liraglutide 10.0mg be dissolved in 2ml containing 0.8% human serum albumin and 7% aqueous sucrose solution in formed
Inner aqueous phase；Precision weighs soybean lecithin 201.1mg, cholesterol 90.2mg, olein 125.0mg, is dissolved in 5ml ether shape
At oily phase, above-mentioned inner aqueous phase is added in oily phase, vortex 3min forms w/o type emulsion.The emulsion is instilled into 6mg/ml lysine
7% aqueous sucrose solution 7ml in, 40 DEG C persistently stirring 5min form W/O/W type emulsion, while being blown into nitrogen, make organic solvent
Naturally it volatilizees, after the volatilization of most of solvent, it is more to get Liraglutide sustained release to move it to revolving instrument removing residual organic solvent
Capsule liposome, measuring encapsulation rate is 87.5%, drugloading rate 2.3%, and average grain diameter is 12.7 μm (Fig. 1).
Embodiment 2
Precision weighs Liraglutide 10.0mg and is dissolved in 2ml containing being formed in 1% human serum albumin and 7% aqueous sucrose solution
Inner aqueous phase；Precision weighs hydrogenated soya phosphatide 203.0mg, cholesterol 96.6mg, and olein 121.0mg is dissolved in 5ml chlorine
Oily phase is formed in imitative, above-mentioned inner aqueous phase is added in oily phase, and the dispersion that is vortexed forms w/o type emulsion.The emulsion is instilled and contains 6mg/
In 7% aqueous sucrose solution 7ml of ml lysine, 40 DEG C are persistently stirred 10min and form W/O/W type emulsion, and continuing to stir at low speed makes
Organic solvent volatilizees naturally, after the volatilization of most of solvent, moves it to revolving instrument and removes residual organic solvent and both get profit La Lu
Peptide is sustained multivesicular liposome, and measuring encapsulation rate is 74.4%, drugloading rate 2.3%, and average grain diameter is 22.0 μm (Fig. 2).
Embodiment 3
Precision weigh Liraglutide 10.0mg be dissolved in 2ml contain 0.9% human serum albumin and 7% aqueous sucrose solution in shape
At inner aqueous phase；Precision weighs Dioleoyl Phosphatidylcholine 196.8mg, cholesterol 99.4mg, olein 126.1mg, molten
Oily phase is formed in 6ml chloroform, above-mentioned inner aqueous phase is added in oily phase, and the dispersion 5min that is vortexed forms w/o type emulsion.In 1000r/
Under min stirring, in 8% aqueous sucrose solution 20ml of the lysine containing 5mg/ml which is instilled, 40 DEG C are persistently stirred 10min
W/O/W type emulsion is formed, being blown into nitrogen makes organic solvent volatilize naturally, when having sedimentation phenomenon to occur, moves it to revolving instrument
It removes residual organic solvent and had both obtained Liraglutide sustained release multivesicular liposome, measuring encapsulation rate is 88.0%, drugloading rate 2.4%,
Average grain diameter is 11.6 μm.
Embodiment 4
Precision weighs Liraglutide 55.1mg and is dissolved in 2ml containing shape in 4.3% human serum albumin and 7% aqueous sucrose solution
At inner aqueous phase；Precision weighs soybean lecithin 202.1mg, cholesterol 98.0mg, and olein 122.6mg is dissolved in 5ml ether
Oily phase is formed, above-mentioned inner aqueous phase is added in oily phase, water bath sonicator 3min forms w/o type emulsion.The emulsion is instilled and contains 6mg/
In 7% aqueous sucrose solution 7ml of ml lysine, 40 DEG C are persistently stirred 5min and form W/O/W type emulsion, while being blown into nitrogen, are made
Organic solvent volatilizees naturally, when having sedimentation phenomenon to occur, moves it to revolving instrument and removes residual organic solvent and both get profit La Lu
Peptide is sustained multivesicular liposome, and measuring encapsulation rate is 84.5%, drugloading rate 11.5%, and average grain diameter is 12.4 μm.
Embodiment 5
Precision weighs Liraglutide 64.6mg, be dissolved in 1.5ml containing 10% human serum albumin and 7% sucrose solution in formed
Inner aqueous phase；Precision weighs soybean lecithin 210.5mg, cholesterol 95.0mg, and three oleic 128.5mg, DPPG 44.2mg are dissolved in
6ml chloroform: ether (volume ratio 1:1) forms oily phase, and above-mentioned inner aqueous phase is added in oily phase, and quick vortex 5min forms w/o type
Emulsion.The emulsion is instilled in 7% aqueous sucrose solution 21ml of the lysine containing 6mg/ml, 40 DEG C are persistently stirred 10min and form W/
O/W type emulsion, being blown into nitrogen makes organic solvent volatilize, and when having sedimentation phenomenon to occur, moves it to revolving instrument removing and remains
Solvent had both obtained Liraglutide and has been sustained multivesicular liposome, and measuring encapsulation rate is 91.3%, drugloading rate 11.9, and average grain diameter is
16.2 μm (Fig. 3).
Embodiment 6
Precision weigh Liraglutide 11.3mg be dissolved in 2ml containing 1.2% ox blood albumin and 5% glucose solution in formed
Inner aqueous phase；Precision weighs soybean lecithin 199.0mg, cholesterol 90.5mg, olein 187.0mg, is dissolved in 2ml ether shape
At oily phase, above-mentioned inner aqueous phase is added in oily phase, water bath sonicator forms w/o type emulsion.The emulsion is instilled into the ammonia of group containing 8mg/ml
In the water-soluble 14ml of 7% sucrose of acid, persistently stirs 5min and form W/O/W type emulsion, 40 DEG C stir at low speed down, while being blown into nitrogen
Gas makes organic solvent volatilize naturally, when having sedimentation phenomenon to occur, moves it to revolving instrument removing residual organic solvent and both got profit
Shandong peptide is drawn to be sustained multivesicular liposome, measuring encapsulation rate is 80.0%, drugloading rate 2.3%, and average grain diameter is 11.3 μm.
Embodiment 7
Precision weigh Liraglutide 18.2mg be dissolved in 2ml containing 2.2% gelatin (250Bloom Type B) and 7% sucrose it is molten
Inner aqueous phase is formed in liquid；Precision weighs soybean lecithin 260.3mg, cholesterol 100.5mg, and triglycerides 191.0mg is dissolved in 16ml
Ether forms oily phase, and above-mentioned inner aqueous phase is added in oily phase, and ice-bath ultrasonic 3min forms w/o type emulsion.The emulsion is instilled
In 7% aqueous sucrose solution 36ml of 6mg/ml histidine, 10 DEG C of ice baths persistently stir 8min and form W/O/W type emulsion, are blown into nitrogen
Gas makes organic solvent volatilize naturally, when having sedimentation phenomenon to occur, moves it to revolving instrument removing residual organic solvent and both got profit
Shandong peptide is drawn to be sustained multivesicular liposome, measuring encapsulation rate is 86.6%, drugloading rate 3.2%, and average grain diameter is 11.0 μm.
Embodiment 8
Precision weighs Liraglutide 30.4mg and is dissolved in 2ml containing being formed in 3.4% human serum albumin and 7% sucrose solution
Inner aqueous phase；Precision weighs soybean lecithin 220.0mg, cholesterol 88.5mg, and olein 126.0mg is dissolved in 4ml hexamethylene
The oily phase of middle formation, above-mentioned inner aqueous phase is added in oily phase, ice-bath ultrasonic 3min forms w/o type emulsion.Emulsion instillation is contained
In 7% aqueous sucrose solution of 6mg/ml histidine (phase volume ratio of colostrum and outer aqueous phase is 1:2), 45 DEG C continue stirring until to be formed
After W/O/W type emulsion, it is blown into nitrogen, organic solvent is made to volatilize naturally, when having sedimentation phenomenon to occur, revolving instrument is moved it to and removes
Residual organic solvent is gone both to obtain Liraglutide sustained release multivesicular liposome, measuring encapsulation rate is 77.7%, and drugloading rate 6.6% is put down
Equal partial size is 25.5 μm.
Embodiment 9
Precision weighs Liraglutide 5.1mg and is dissolved in 2ml containing shape in 0.125% ox blood albumin and 7% sucrose solution
At inner aqueous phase；Precision weighs soybean lecithin 220.0mg, cholesterol 95mg, and olein 156.0mg is dissolved in 6ml ether
Oily phase is formed, above-mentioned inner aqueous phase is added in oily phase, ice-bath ultrasonic 3min forms w/o type emulsion.Emulsion instillation is contained for 40 DEG C
In 7% aqueous sucrose solution of 6mg/ml histidine (phase volume ratio of colostrum and outer aqueous phase is 1:2), continue stirring until to form W/O/
W type emulsion, stirs at low speed, while being blown into nitrogen, and organic solvent is made to volatilize naturally, when having sedimentation phenomenon to occur, moves it to
It rotates instrument removing residual organic solvent and had both obtained Liraglutide sustained release multivesicular liposome, measuring encapsulation rate is 79.9%, and drugloading rate is
1.1%, average grain diameter is 10.5 μm.
Embodiment 10
Precision weigh Liraglutide 14.2mg be dissolved in 2ml containing 1.5% human serum albumin and 7% sucrose solution in formed in
Water phase；Precision weighs soybean lecithin 220.0mg, cholesterol 76.5mg, oleic acid 25.6mg, and tricaprylin 122.3mg is dissolved in
Oily phase is formed in 5ml ether, above-mentioned inner aqueous phase is added in oily phase, and vortex 5min forms w/o type emulsion.Emulsion instillation is contained
In 7% aqueous sucrose solution of 6mg/ml histidine (phase volume ratio of colostrum and outer aqueous phase is 1:2), 40 DEG C continue stirring until to be formed
W/O/W type emulsion, 600r/min is stirred at low speed down, while being blown into nitrogen, and organic solvent is made to volatilize naturally, and sedimentation phenomenon to be had goes out
Now, it moves it to revolving instrument removing residual organic solvent and had both obtained Liraglutide sustained release multivesicular liposome, measuring encapsulation rate is
88.1%, drugloading rate 3.1%, average grain diameter is 13.9 μm.
Embodiment 11
Precision weigh Liraglutide 54.0mg be dissolved in 2ml containing 5% gelatin hydrolysate and 7% sucrose solution in formed in water
Phase；Precision weighs soybean lecithin 220.0mg, cholesterol 76.5mg, triglycerides 55.3mg, is dissolved in 5ml ether and forms oily phase,
Above-mentioned inner aqueous phase is added in oily phase, ice-bath ultrasonic 5min forms w/o type emulsion.The emulsion is instilled into the histidine containing 6mg/ml
In 7% aqueous sucrose solution (phase volume ratio of colostrum and outer aqueous phase is 1:2), continue stirring until to form W/O/W type at a temperature of 40 DEG C
After emulsion, it is blown into nitrogen, organic solvent is made to volatilize naturally, when having sedimentation phenomenon to occur, revolving instrument is moved it to and removes residual
Organic solvent had both obtained Liraglutide and has been sustained multivesicular liposome, and measuring encapsulation rate is 86.8%, drugloading rate 13.3%, average grain diameter
It is 15.5 μm.
Embodiment 12
Precision weigh Liraglutide 58.1mg be dissolved in 2ml containing 1% human serum albumin and 7% sucrose solution in formed in water
Phase；Precision weighs DOPC 36.6mg (Lipoid), DPPG 10.2mg, cholesterol 28.0mg, and triglycerides 8.6mg is dissolved in 5ml
Chloroform: oily phase is formed in ether in the mixed solvent (volume ratio 1:1), above-mentioned inner aqueous phase is added in oily phase, vortex 5min is formed
W/o type emulsion.The emulsion is instilled into (the phase volume ratio of colostrum and outer aqueous phase in 7% aqueous sucrose solution of the histidine containing 6mg/ml
To continue stirring until to form W/O/W type emulsion under 40 DEG C of constant temperature of 1:2), it is blown into nitrogen under stirring, organic solvent is made to volatilize naturally,
When thering is sedimentation phenomenon to occur, moves it to revolving instrument removing residual organic solvent and had both obtained Liraglutide sustained release multivesicular liposome,
Measuring encapsulation rate is 82.3%, drugloading rate 41.2%, and average grain diameter is 19.7 μm.
Embodiment 13
Precision weigh Liraglutide 44.8mg be dissolved in 2ml containing 4.5% human serum albumin and 7% sucrose solution in formed in
Water phase；Precision weighs egg yolk lecithin 183.6mg (Lipoid), cholesterol 70.7mg, and olein 61.1mg is dissolved in
Oily phase is formed in 5ml ether in the mixed solvent (volume ratio 0.3:1), above-mentioned inner aqueous phase is added in oily phase, vortex 5min is formed
W/o type emulsion.The emulsion is instilled into (the phase volume ratio of colostrum and outer aqueous phase in 7% aqueous sucrose solution of the histidine containing 6mg/ml
For 1:2), continues stirring until to form W/O/W type emulsion under 40 DEG C of constant temperature, be blown into nitrogen, organic solvent is made to volatilize naturally, it is heavy wait have
When drop phenomenon occurs, moves it to revolving instrument removing residual organic solvent and both obtained Liraglutide sustained release multivesicular liposome, measure packet
Envelope rate is 86.0%, drugloading rate 12.5%, and average grain diameter is 18.3 μm.
Comparative example 1 (is free of stabilizer)
Precision weigh Liraglutide 10.0mg be dissolved in 2ml contain and 7% aqueous sucrose solution in form inner aqueous phase；Precision weighs
Soybean lecithin 201.1mg, cholesterol 100.2mg, olein 125.0mg are dissolved in 5ml ether and form oily phase, will be above-mentioned
Inner aqueous phase is added in oily phase, and vortex 3min forms w/o type emulsion.The emulsion is instilled to 7% sucrose water of the lysine containing 6mg/ml
5min is persistently stirred in solution 7ml, at a temperature of 40 DEG C and forms W/O/W type emulsion, is blown into nitrogen, organic solvent is made to volatilize naturally,
After the volatilization of most of solvent, revolving instrument removing residual organic solvent is moved it to get Liraglutide and is sustained multivesicular liposome,
Measuring encapsulation rate is 73%, drugloading rate 2.3%.
The release experiment of 14 Liraglutide multivesicular liposome of embodiment
It is molten that precision measures Lrg-MVL suspension 0.5ml, the PBS for being separately added into pH7.4 prepared by embodiment 1 and comparative example 1
Test tube is jumped a queue and is placed in 37 DEG C of constant temperature to 5ml by liquid, in the shaking table of 100rpm, is sampled respectively at different time points, sample in
5000rpm is centrifuged 5min and removes supernatant, repeats above operation again twice, and acidification isopropanol (containing 1% hydrochloric acid) is added in precipitating
Dissolution, HPLC method measure its content, calculate the drug that each sample point is trapped in MVLs, are with zero moment encapsulating dose
100%, the remaining percentage of each sample point is calculated, to obtain release percentage, draws the cumulative release percentage of Lrg-MVLs
Number curve.
The release profiles that embodiment 1 prepares liposome are as shown in Figure 4, the results showed that the more capsules of Liraglutide of the present invention
Slowly, sustained release sustainable nearly 432 hours, slow release effect was significant and without burst effect for liposome medicament release.Other are implemented
The releasing effect of liposome made from example is similarly.The release profiles that comparative example prepares liposome are as shown in Figure 5, the results showed that
Liraglutide multivesicular liposome drug release prepared by comparative example 1 continues 216 hours, and slow release effect is weaker than the preparation of embodiment 1
Multivesicular liposome.
The pharmacodynamics and Pharmacokinetic experiments of 15 Liraglutide multivesicular liposome of embodiment
The preparation of 1.1 diabetes rat models
Male rat is taken, fasting overnight is weighed after 12 hours, new with physiological saline with the dosage tail vein injection of 45mg/kg
The alloxan solution of the 1% of fresh preparation, 2ml is given in every stomach-filling after 30min, and 50% glucose solution normally raises 72h
Posterior orbit takes blood, detects its blood sugar concentration using Glucose Oxidase kit, and being greater than 11.1mmol/L person is successful glycosuria
Disease model rat.
1.2 medication
The successful diabetes rat of modeling is divided into tri- groups of A, B, C, is administered as follows
A group rat the nape of the neck subcutaneously disposably gives the suspension of 7mg/kg (in terms of Liraglutide) Liraglutide multivesicular liposome
Liquid (Liraglutide multivesicular liposome is prepared by the embodiment of the present invention 1~13, is resuspended with physiological saline), respectively at 0,24,96,
168, when 240,312,336,360h, blood 0.5ml is taken to be placed in the EP pipe for being coated with heparin, through 3500r/min centrifugation 10min separation
Blood plasma, -20 DEG C of freezen protectives are to be measured.
B group rat the nape of the neck subcutaneously disposably give same dosage Liraglutide injection (Novo Nordisk),
Respectively in 0,2,4,8,12,24,30 and 36h, blood 0.5ml is taken to be placed in the EP pipe for being coated with heparin.It is centrifuged through 3500r/min
10min separated plasma, -20 DEG C of freezen protectives are to be measured.
C group rat the nape of the neck subcutaneously disposably gives 5mg/kg Liraglutide multivesicular liposome suspension, and (Liraglutide is more
Capsule liposome is prepared by comparative example 1 of the present invention, is resuspended with physiological saline), when 0,24,48,72,96,120,168h, take
Blood 0.5ml is placed in the EP pipe for being coated with heparin.
The measurement of 1.3 blood glucose and blood concentration
Method: utilizing Glucose Oxidase kit (biotech inc Zhong Shanbeikong), using light splitting light
The OD value that reaction solution is measured under 505 wavelength of degree method, is calculated the blood sugar concentration of every rat of corresponding moment, is tried using people with GLP-1
Agent box (Shanghai Hu Ding Biotechnology Co., Ltd) measures corresponding blood concentration.
1.4 pharmacodynamic result
Rat skin lower injection gives the blood sugar detections of Liraglutide different dosage forms, and the results are shown in Table 1,
Table 1: the blood sugar concentration after rat administration
The result shows that single injection Liraglutide multivesicular liposome provided by the invention can provide just at least 312 hours
Chang Pingwen blood glucose level, hypoglycemic effect was up to 312 hours.However, the drop blood of the Liraglutide injection (commercially available) of same dose
Sugar effect only maintains 24 hours.It proves that Liraglutide multivesicular liposome has significant slow release effect, Li Lalu can be greatly reduced
The administration number of times of peptide improves the compliance of patient.Single injection Liraglutide multivesicular liposome (without stabilizer) can only mention
For 120 hours blood sugar reducing functions, long-acting was greater than injection, but not as good as the Liraglutide multivesicular liposome containing stabilizer, and two
Person has significant difference (p < 0.05).
1.5 Pharmacokinetic Results
Subcutaneous rat distinguishes single injection Liraglutide multivesicular liposome (present invention preparation) injection (commercially available), comparison system
The blood concentration of agent such as table 2.
Table 2: blood concentration after rat administration
3 pharmacokinetic parameters of table
Note, * show P < 0.05
The result shows that Liraglutide multivesicular liposome blood concentration prepared by the present invention is stablized compared with injection, release
Up to 360h.The internal residence time significantly extends (respectively 163.4 hours and 13.9 hours), and apparent sustained release preparation is presented
Characteristics of pharmacokinetics, the relative bioavailability with injection are 661.6%.Illustrate that Liraglutide multivesicular liposome significantly improves
Internal medicine stability, bioavilability significantly improve, and greatly reduce drug cost.The common more capsules of single injection Liraglutide
Liposome (preparation of comparative example 1) the internal residence time is 74.9 hours, is greater than injection, but is substantially less than (p < 0.05) containing stabilization
The Liraglutide multivesicular liposome of agent.
The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (11)
1. a kind of Liraglutide multivesicular liposome, which is characterized in that be made by the raw material of following quality point:
2. Liraglutide multivesicular liposome according to claim 1, which is characterized in that the membrane material includes that phosphatide, gallbladder are solid
The mass ratio of pure and mild triglycerides, the phosphatide, cholesterol and triglycerides is (46.8~342.3): (28.0~100.5):
(8.6~191.0).
3. Liraglutide multivesicular liposome according to claim 1 or 2, which is characterized in that the phosphatide be selected from lecithin,
Soybean lecithin, cephalin, dioleyl phosphatidyl choline, hydrogenated soya phosphatide, Distearoyl Phosphatidylcholine, two palmityl phosphorus
Phosphatidylcholine, dimyristoyl phosphatidyl choline, Dioleoyl Phosphatidylcholine, distearoylphosphatidylglycerol, two palmityl phosphorus
At least one of phosphatidyl glycerol, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, dioleoylphosphatidylglycerol or phosphatidyl-ethanolamine.
4. Liraglutide multivesicular liposome according to claim 1, which is characterized in that the triglycerides is selected from three oleic acid
At least one of glyceride, tricaprylin or soybean oil.
5. Liraglutide multivesicular liposome according to claim 1, which is characterized in that the osmotic pressure regulator, which is selected from, to be relied
At least one of propylhomoserin, histidine, sucrose, glucose, dextran, glycerol, mannitol or sorbierite.
6. Liraglutide multivesicular liposome according to claim 1, which is characterized in that the stabilizer is selected from the white egg of people's blood
At least one of white, ox blood albumin, collagen, gelatin or gelatin hydrolysate.
7. the preparation method of any one of the claim 1~6 Liraglutide multivesicular liposome characterized by comprising
Step 1: membrane material is dissolved in organic solvent as oily phase；Stabilizer, osmotic pressure regulator and Liraglutide are dissolved in water to make
For inner aqueous phase；Osmotic pressure regulator is dissolved in water as outer aqueous phase；
Step 2: inner aqueous phase being made mutually to form level-one cream with oily；
Step 3: the level-one cream and outer aqueous phase being made to form emulsion；
Step 4: Liraglutide multivesicular liposome is made in the organic solvent in dry emulsion.
8. preparation method according to claim 7, which is characterized in that organic solvent described in step 1 is selected from ether, dichloro
Methane, chloroform, ethyl acetate or hexamethylene.
9. preparation method according to claim 7, which is characterized in that the volume ratio of the inner aqueous phase and the oily phase is 1:
(0.8~10)；The volume ratio of the level-one cream and the outer aqueous phase is 1:(1~4).
10. any one of the claim 1~6 Liraglutide multivesicular liposome is in preparation treatment and/or the medicine of prevention diabetes
Application in object.
11. the drug of a kind for the treatment of and/or prevention diabetes, which is characterized in that described in any item including claim 1~6
Liraglutide multivesicular liposome.
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| WO2020127476A1 (en) | 2018-12-19 | 2020-06-25 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
| WO2020257260A1 (en) * | 2019-06-17 | 2020-12-24 | Massachusetts Institute Of Technology | Formulation of peptide loaded liposomes and related applications |
| WO2021123228A1 (en) | 2019-12-18 | 2021-06-24 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
| CN114344280A (en) * | 2022-01-10 | 2022-04-15 | 益奇健康科技（上海）有限公司 | Preparation method of vitamin and/or mineral microcapsule |
| CN114452258A (en) * | 2022-03-09 | 2022-05-10 | 成都大学 | Liraglutide liposome preparation and preparation method and application thereof |
| WO2023041588A1 (en) | 2021-09-14 | 2023-03-23 | Advapharm Gmbh | Novel lipopeptide formulation |
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| WO2020127476A1 (en) | 2018-12-19 | 2020-06-25 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
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| WO2021123228A1 (en) | 2019-12-18 | 2021-06-24 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
| WO2023041588A1 (en) | 2021-09-14 | 2023-03-23 | Advapharm Gmbh | Novel lipopeptide formulation |
| CN114344280A (en) * | 2022-01-10 | 2022-04-15 | 益奇健康科技（上海）有限公司 | Preparation method of vitamin and/or mineral microcapsule |
| CN114452258A (en) * | 2022-03-09 | 2022-05-10 | 成都大学 | Liraglutide liposome preparation and preparation method and application thereof |
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