CN110339166B - Liraglutide multivesicular liposome and preparation method and application thereof - Google Patents
Liraglutide multivesicular liposome and preparation method and application thereof Download PDFInfo
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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 invention relates to the field of medicines, and in particular relates to a liraglutide multivesicular liposome and a preparation method thereof. The liraglutide multivesicular liposome provided by the invention comprises: liraglutide, membrane material, osmotic pressure regulator and stabilizer. The liraglutide multivesicular liposome prepared by the invention has the advantages of good stability, high drug encapsulation rate, large drug-loading rate, slow and stable drug release rate, no burst release phenomenon and remarkably improved bioavailability of the drug. Thereby improving the curative effect, reducing the toxic and side effects related to the drug dosage, reducing the drug cost and having larger application value. Experiments show that the liposome provided by the invention can release drugs in vitro at a sustained constant speed for nearly 432 hours, can provide stable blood concentration in vivo, has remarkably prolonged in vivo retention time compared with the injection, presents obvious pharmacokinetic characteristics of a sustained release preparation, can provide normal and stable blood sugar level, has the blood sugar reducing effect of 312 hours, and has relative bioavailability of 661 percent compared with the injection.
Description
Technical Field
The invention relates to the field of medicines, and particularly relates to a liraglutide multivesicular liposome and a preparation method and application thereof.
Background
Diabetes has become the third leading killer to harm human life and is a global epidemic. According to WHO's estimation, the number of diabetic patients worldwide will reach 3 hundred million by 2050. Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia due to defects in insulin secretion and/or impaired insulin action. The chronic hyperglycemic state of diabetes is significantly associated with long-term complications, namely damage, dysfunction and failure of numerous organs, particularly kidneys, eyes, nerves, heart and blood vessels, and the severe can cause acute complications of ketoacidosis and hyperosmolar coma, such as water, electrolyte disorders and acid-base balance disorders. Therefore, the research and development of the novel efficient long-acting diabetes treatment drug has important social significance and economic value.
Liraglutide (Liraglutide) is a glucagon GLP-1 (7-37) analogue developed by Danish Novonide, and is approved by FDA to be marketed in the United states on 25/1 of 2010 as a new drug for treating type 2 diabetes. The liraglutide has 97 percent of homology with the natural GLP-1 of a human body, and the structure of the liraglutide only modifies the GLP-1 by two parts as follows: namely, the 34 th lysine is replaced by arginine, and the 26 th lysine is added with a glutamic acid mediated palmitic acid side chain with 16 carbons. The structural modification not only enables the liraglutide to retain the bioactivity of GLP-1, but also avoids the degradation by DPP-4 enzyme, and compared with the traditional insulin injection, the liraglutide has long-acting property.
Liraglutide has the following pharmacological actions: (1) glucose concentration-dependent insulinotropic secretion; (2) inhibiting secretion of glucagon after meal, and reducing release of glycogen; (3) enhancing insulin sensitivity; (4) slowing gastric emptying; (5) suppressing appetite and reducing weight. (6) Repairing islet beta cells; (7) protecting cardiovascular system.
The effects of liraglutide in promoting insulin secretion and inhibiting glucagon secretion have blood sugar concentration dependency, so that when the blood sugar level is normal, the continuous administration of liraglutide does not cause hypoglycemia, so that the liraglutide can not only maintain the normal blood sugar level of a patient, but also repair islet beta cells to a certain degree, has a certain curative effect on diabetic complications, and is a currently advanced diabetes treatment drug.
Currently marketed liraglutide injection developed by Novonide
The injection solution is administered by subcutaneous injection once daily, and has been proved to have good effects in improving blood sugar control and reducing body weight. Although liraglutide has a long half-life (12-14 hours) in vivo, for diabetes mellitus, a disease requiring long-term treatment and blood sugar level control, the liraglutide injection still has the need of reducing the administration frequency and improving the administration compliance of patients.
Therefore, in order to reduce the injection frequency of liraglutide, research and development of sustained-release long-acting administration forms of liraglutide are carried out. The long-acting dosage forms in the research stage are mainly the microsphere formulations of liraglutide and synthetic macromolecules such as PLGA, PLA, etc. (CN102085355A, CN104382860A), however, the polymeric microsphere formulations generally have burst effect (Shekh Hasan, A., et al, Reduction of the in vivo viral burst release of insulin-loaded microorganisms. journal of Drug Delivery and Science, 2015.30, part: p.486-493., and long-term retention in vivo may produce inflammatory reaction, cause body immune reaction (Anderson, J.M.and M.S.Shield, Biodegradation and biocompatibility of PLGA and PLGA. advanced Drug Delivery review, 2012.64, Supplement (0): p.72-82, and polymer degradation promoting Protein production, environmental activity of Protein, biological degradation, biological activity of Protein, biological degradation, issue of Protein, 2012.101(3): p.946-954).
Multivesicular liposomes (MVLs) are composed of a plurality of non-concentric vesicles of aqueous drug solution, which are separated by continuous non-concentric lipid bilayer, and have high encapsulation efficiency for water-soluble drugs. In particular, multivesicular liposomes release drug through ruptured vesicles, while intact vesicles remain intact. The multivesicular liposome, which is a non-concentric topological structure, forms a reservoir at an injection site, and the medicament encapsulated in the multivesicular liposome is gradually released along with the continuous degradation of a lipid bilayer to generate good effectThe sustained-release effect of the composition can avoid burst effect, and the effect of controlling the release time of the medicament from several days to several weeks can be obtained by adjusting the preparation prescription and the process parameters. MVLs have been approved by FDA as drug delivery systems, morphine multivesicular liposomes (Depo morphine)TM) Has been marketed in 2004 in the united states.
In the preparation process, the prescription composition and the preparation process of the multivesicular liposome have obvious influence on the drug encapsulation efficiency and the release time, particularly the physical stability of the multivesicular liposome. The multivesicular liposome has large particle size, is easy to precipitate and aggregate in the storage process, uses an organic solvent in the preparation process, and has inactivation problem and the like on protein polypeptide drugs, so that the improvement of the aggregation stability and the drug stability of the multivesicular liposome is still the problem to be solved urgently for developing multivesicular liposome preparations.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a liraglutide multivesicular liposome, and a preparation method and an application thereof.
The liraglutide multivesicular liposome provided by the invention comprises the following components in parts by mass:
according to the liraglutide multivesicular liposome provided by the invention, phospholipid with high biocompatibility and the like are selected as basic membrane materials, so that the liraglutide multivesicular liposome is retained in a body and is difficult to generate inflammatory reaction. The prepared liposome has higher entrapment rate and stable drug release rate, and successfully avoids burst effect. In addition, the multivesicular liposome provided by the invention can also avoid the problem that liraglutide is easy to degrade in vivo, prolongs the circulation time of the medicament in vivo and achieves the effect of remarkably improving the bioavailability.
In the invention, the liraglutide multivesicular liposome comprises the following components in parts by mass:
in the invention, the membrane material comprises phospholipid, cholesterol and triglyceride, wherein the mass ratio of the phospholipid to the cholesterol to the triglyceride is (46.8-342.3): (28.0-100.5): (8.6-191.0).
In the present invention, the phospholipid is selected from lecithin (EPC), Soybean Phospholipid (SPC), cephalin, Dioleoylphosphatidylcholine (DOPC), Hydrogenated Soybean Phospholipid (HSPC), Distearoylphosphatidylcholine (DSPC), Dipalmitoylphosphatidylcholine (DPPC), Dimyristoylphosphatidylcholine (DMPC), Dioleoylphosphatidylcholine (DOPC), Distearoylphosphatidylglycerol (DSPG), Dipalmitoylphosphatidylglycerol (DPPG), Dimyristoylphosphatidylglycerol (DMPG), Dioleoylphosphatidylglycerol (DOPG) or phosphatidylethanolamine (DOPE).
In some embodiments, the phospholipid is selected from the group consisting of soybean phospholipid, hydrogenated soybean phospholipid, dioleoylphosphatidylcholine, egg yolk lecithin, dioleoylphosphatidylcholine, and dipalmitoylphosphatidylglycerol.
DPPG and DSPG are substances for increasing negative membrane charge, and can increase the stability of liposome.
Triglycerides are important components of multivesicular liposome formation, where they serve to support their structure. The triglycerides may be natural and synthetic. The triglyceride in the invention is selected from at least one of triolein, tricaprylin and soybean oil, and preferably triolein.
The invention provides a film material of liraglutide multivesicular liposome, wherein the molar percentage of triglyceride is 5-50%.
In the membrane material of the liraglutide multivesicular liposome, the mass ratio of phospholipid to cholesterol is (0.5-20): 1.
in the invention, the mass ratio of the liraglutide to the membrane material is 1: (1-100).
In some embodiments, the mass ratio of liraglutide to membrane material is 1: (1.44 to 92.35).
In the present invention, the osmotic pressure regulator is selected from lysine, histidine, sucrose, glucose, dextran, glycerol, mannitol or sorbitol.
In some embodiments, the osmolality adjusting agent is selected from histidine, lysine, glucose or sucrose.
The liraglutide multivesicular liposome provided by the invention comprises a stabilizer, wherein the stabilizer is a pharmaceutically acceptable auxiliary material capable of improving the in vivo and in vitro stability of a medicament and prolonging the in vivo half-life period of the medicament.
In the present invention, the stabilizer is selected from human blood albumin, bovine blood albumin, collagen, gelatin or hydrolyzed gelatin.
In some embodiments, the stabilizing agent in the liraglutide multivesicular liposome provided by the invention is selected from human blood albumin, bovine blood albumin, hydrolyzed gelatin or gelatin.
In the invention, the mass ratio of the liraglutide to the stabilizer is 1: (0.3-2.5).
In some embodiments, the mass ratio of liraglutide to stabilizer is 1: (0.34-2.42).
In some embodiments, the phospholipid is soy lecithin; the triglyceride is triolein; wherein the mass ratio of the soybean lecithin to the cholesterol to the triglyceride is 201.1:90.2: 125.
In this example, the mass ratio of liraglutide to membrane material was 1: 41.62.
Preferably, the stabilizer is human blood albumin; the mass ratio of the liraglutide to the stabilizer is 1: 1.6.
Preferably, the osmotic pressure regulator is a mixture of sucrose and lysine, wherein the mass ratio of lysine to sucrose is 15: 1; the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 67.2.
In some embodiments, the phospholipid is hydrogenated soy phospholipid; the triglyceride is triolein; the mass ratio of the hydrogenated soybean phospholipids to the cholesterol to the triglyceride in the membrane material is 203:96: 121.
In this example, the mass ratio of liraglutide to membrane material was 1: 42.1.
Preferably, the stabilizer is human blood albumin; the mass ratio of the liraglutide to the stabilizer is 1:2.
Preferably, the osmotic pressure regulator is a mixture of sucrose and lysine, wherein the mass ratio of the sucrose to the lysine is 15: 1; the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 67.2.
In some embodiments, the phospholipid is dioleoylphosphatidylcholine; the triglyceride is triolein; in the membrane material, the mass ratio of the dioleoylphosphatidylcholine to the cholesterol to the triglyceride is 196.8:99.4: 126.1.
In this example, the mass ratio of liraglutide to membrane material was 1: 42.2.
Preferably, the stabilizer is human blood albumin; the mass ratio of the liraglutide to the stabilizer is 1: 1.8.
Preferably, the osmotic pressure regulator is a mixture of sucrose and lysine, wherein the mass ratio of the sucrose to the lysine is 17.4: 1; the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 180.4.
In some embodiments, the phospholipid is a soy phospholipid; the triglyceride is triolein; the mass ratio of the soybean phospholipids to the cholesterol to the triglyceride in the membrane material is 202.1:98: 122.6.
In this example, the mass ratio of liraglutide to membrane material was 1: 7.7.
Preferably, the stabilizer is human blood albumin; the mass ratio of the liraglutide to the stabilizer is 1: 1.6.
Preferably, the osmotic pressure regulator is a mixture of sucrose and lysine, wherein the mass ratio of the sucrose to the lysine is 15: 1; the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 12.2.
In some embodiments, the phospholipid is a soy phospholipid; the triglyceride is triolein; the membrane material comprises soybean phospholipid, cholesterol, triglyceride and DPPG, wherein the mass ratio of the soybean phospholipid to the cholesterol to the triglyceride to the DPPG is 210.5:95: 128.5: 44.2.
in this example, the mass ratio of liraglutide to membrane material was 1: 7.4.
Preferably, the stabilizer is human blood albumin; the mass ratio of the liraglutide to the stabilizer is 1: 2.3.
Preferably, the osmotic pressure regulator is a mixture of sucrose and lysine, wherein the mass ratio of the sucrose to the lysine is 12.5: 1; the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 26.3.
In some embodiments, the phospholipid is a soy phospholipid; the triglyceride is triolein; the mass ratio of the soybean phospholipids, the cholesterol and the triglyceride in the membrane material is 199:90.5: 187.
In this example, the mass ratio of liraglutide to membrane material was 1: 42.2.
Preferably, the stabilizer is bovine serum albumin; the mass ratio of the liraglutide to the stabilizer is 1: 2.1.
Preferably, the osmotic pressure regulator is a mixture of sucrose and histidine, and preferably, the osmotic pressure regulator is a mixture of glucose, sucrose and histidine, wherein the mass ratio of the glucose, the sucrose and the histidine is 100:980: 112; the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 105.5.
In some embodiments, the phospholipid is a soy phospholipid; the triglyceride is triolein; the mass ratio of the soybean phospholipid, the cholesterol and the triglyceride in the membrane material is 260.3:100.5: 191.
In this example, the mass ratio of liraglutide to membrane material was 1: 30.3.
Preferably, the stabilizer is gelatin; the mass ratio of the liraglutide to the stabilizer is 1: 2.4.
Preferably, the osmotic pressure regulator is a mixture of sucrose and histidine, wherein the mass ratio of sucrose to histidine is 12.3: 1; the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 158.0.
In some embodiments, the phospholipid is a soy phospholipid; the triglyceride is triolein; the mass ratio of the soybean phospholipids, the cholesterol and the triglyceride in the membrane material is 220:88.5: 126.
In this example, the mass ratio of liraglutide to membrane material was 1: 14.3.
Preferably, the stabilizer is human blood albumin; the mass ratio of the liraglutide to the stabilizer is 1: 2.2.
Preferably, the osmotic pressure regulator is a mixture of sucrose and histidine, wherein the mass ratio of sucrose to histidine is 16.3: 1; the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 34.2.
In some embodiments, the phospholipid is a soy phospholipid; the triglyceride is triolein; the mass ratio of the soybean phospholipids, the cholesterol and the triglyceride in the membrane material is 220:95: 156.
In this example, the mass ratio of liraglutide to membrane material was 1: 92.4.
Preferably, the stabilizer is bovine serum albumin; the mass ratio of the liraglutide to the stabilizer is 1: 2.4.
Preferably, the osmotic pressure regulator is a mixture of sucrose and histidine, wherein the mass ratio of sucrose to histidine is 13.1: 1; the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 265.8.
In some embodiments, the phospholipid is a soy phospholipid; the membrane material comprises soybean lecithin, cholesterol, tricaprylin and oleic acid; wherein the mass ratio of the soybean phospholipid to the cholesterol to the tricaprylin to the oleic acid is 220:76.5:122.3: 25.6.
In this example, the mass ratio of liraglutide to membrane material was 1: 31.3.
Preferably, the stabilizer is human blood albumin; the mass ratio of the liraglutide to the stabilizer is 1: 2.1.
Preferably, the osmotic pressure regulator is a mixture of sucrose and histidine, wherein the mass ratio of the sucrose to the histidine is 18: 1; the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 93.7.
In some embodiments, the phospholipid is a soy phospholipid; in the membrane material, the mass ratio of the soybean lecithin, the cholesterol and the triolein is 220:76.5: 55.3.
In this example, the mass ratio of liraglutide to membrane material was 1: 6.5.
Preferably, the stabilizer is hydrolyzed gelatin; the mass ratio of the liraglutide to the stabilizer is 1: 1.9.
Preferably, the osmotic pressure regulator is a mixture of sucrose and histidine, wherein the mass ratio of sucrose to histidine is 13.3: 1; the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 22.3.
in some embodiments, the phospholipid is DOPC; the membrane material comprises DOPC, DPPG, cholesterol and triglyceride; wherein the mass ratio of DOPC, DPPG, cholesterol and triglyceride is 36.6:10.2:28: 8.6.
In this example, the mass ratio of liraglutide to membrane material was 1: 1.4.
Preferably, the stabilizer is human blood albumin; the mass ratio of the liraglutide to the stabilizer is 1: 0.34.
Preferably, the osmotic pressure regulator is a mixture of sucrose and histidine, wherein the mass ratio of the sucrose to the histidine is 15: 1; the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 23.1.
In some embodiments, the phospholipid is egg yolk lecithin; in the membrane material, the mass ratio of the egg yolk lecithin to the cholesterol to the triglyceride is 183.6:70.7: 61.1.
In this example, the mass ratio of liraglutide to membrane material was 1: 7.0.
Preferably, the stabilizer is human blood albumin; the mass ratio of the liraglutide to the stabilizer is 1: 2.0.
Preferably, the osmotic pressure regulator is a mixture of sucrose and histidine, wherein the mass ratio of sucrose to histidine is 13.3: 1; the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 26.9.
Experiments show that the liraglutide multivesicular liposome prepared by the formula and the preparation method provided by the invention has good stability and bioavailability, proper particle size, high drug loading rate and high entrapment rate. In addition, the retention time of the medicine in the body can be improved, and the bioavailability is improved by about 6 times compared with that of the common injection. In the experimental process, some bad formulations or improper preparation methods can cause the shape of the liposome to change, sometimes the complete liposome cannot be formed, and sometimes a plurality of oil drops exist.
The liraglutide multivesicular liposome prepared by the invention is a multivesicular liposome.
The preparation method of the liraglutide multivesicular liposome provided by the invention comprises the following steps:
step 1: dissolving the membrane material in an organic solvent to be used as an oil phase; dissolving a stabilizer, an osmotic pressure regulator and liraglutide in water to serve as an internal water phase; dissolving osmotic pressure regulator in water as external water phase;
step 2: forming W/O first-stage emulsion by the internal water phase and the oil phase;
and step 3: forming W/O/W multiple emulsion by the primary emulsion and the external water phase;
and 4, step 4: drying and re-emulsifying to obtain the liraglutide multivesicular liposome.
In the invention, the organic solvent in the step 1 is selected from diethyl ether, dichloromethane, chloroform, ethyl acetate or cyclohexane.
In some embodiments, the organic solvent in step 1 is selected from diethyl ether, chloroform or cyclohexane.
In some embodiments, the concentration of the film material in the oil phase is from 10mg/ml to 400 mg/ml.
In some embodiments, the oil phase has a phospholipid concentration of 5-100 mg/ml, a cholesterol concentration of 3-50 mg/ml, and a triglyceride concentration of 1-95 mg/ml.
In the invention, the preparation method of the internal water phase comprises the following steps: the stabilizer is stirred and dissolved in water, the osmotic pressure regulator is added for dissolution, and the liraglutide is added for dissolution.
In some embodiments, the concentration of the stabilizer in the internal aqueous phase is 5-100 mg/ml; the concentration of the liraglutide is 1-100 mg/ml; the osmotic pressure regulator accounts for 4 to 10 percent by mass.
In some embodiments, the osmotic pressure regulator is present in the external aqueous phase in a mass fraction of between 4% and 10%.
Preferably, the osmotic pressure regulator in the internal water phase is glucose or sucrose, and the mass fraction is 7%; the osmotic pressure regulator in the external water phase is a mixture of sucrose and histidine or sucrose and lysine, wherein the mass fraction of the sucrose is 7%; the concentration of lysine or histidine is 2 mg/mL-8 mg/mL.
In the present invention, the volume ratio of the internal water phase to the oil phase is 1: (0.8-10). The method for preparing the first-stage emulsion in the step 2 is to drop the inner water phase into the oil phase, and the emulsification method adopts the conventional technology in the field, and can adopt methods which can be adopted in pharmaceutics such as mechanical stirring, vortex, ultrasound, a high-speed dispersion machine, a high-pressure homogenizer, a micro-jet high-pressure homogenizer and the like to carry out emulsification.
In the invention, the volume ratio of the primary milk to the external water phase is 1: (1-4). The method for preparing the multiple emulsion in the step 3 comprises the following steps: the first-stage emulsion is added dropwise into the external water phase under stirring, and the emulsifying method adopts conventional techniques in the field, and can adopt methods selected from mechanical stirring, ultrasonic emulsification, mechanical high-speed shearing emulsification, high-pressure homogenization and the like.
In the present invention, drying the double emulsion is a process for removing the organic solvent in the double emulsion, wherein the method for removing the organic solvent is a conventional solvent removal method in the field, such as normal pressure volatilization, air flow blowing evaporation of the organic solvent, reduced pressure drying or spray drying, and the like, and the preferred method is first blowing dry with inert gas flow, and then further removing the residual organic solvent by using a rotary evaporator.
In the present invention, step 4 is followed by a washing step.
The washing is to remove unencapsulated free drug by washing with an isotonic solution. The washing method with the isotonic solution can be a conventional method in the field, and the isotonic solution can be used as a washing solution, free medicines and solutes in an external water phase are removed by centrifugation, dialysis or membrane filtration, and then the liraglutide multivesicular liposome is suspended to a required medicine concentration by the isotonic solution. In the present invention, the term "isotonic solution" means an osmotic pressure equal to that tolerated by plasma or subcutaneous, intramuscular injection, preferably an aqueous solution of sodium chloride or sucrose or glucose.
The liraglutide multivesicular liposome provided by the invention can be used for preparing medicines for treating and/or preventing diabetes.
The invention also provides a medicament for treating and/or preventing diabetes, which comprises the liraglutide multivesicular liposome provided by the invention.
The method for preparing the liposome is also suitable for glucagon-like peptide (GLP-1) hypoglycemic drugs including Exenatide (Exenatide).
The medicament for treating and/or preventing diabetes provided by the invention is in an injection form. The administration mode is subcutaneous injection, intramuscular injection, epidural injection or intrathecal injection, and subcutaneous injection and intramuscular injection are preferred.
According to the invention, the active pharmaceutical ingredient liraglutide is a compound liraglutide, which is irrelevant to the source and the preparation method thereof, and can be a gene recombination source, a chemical synthesis source or a semi-synthesis source. The implementation of which is within the scope of the present invention.
The liraglutide multivesicular liposome provided by the invention comprises: liraglutide, membrane material, osmotic pressure regulator and stabilizer. Compared with the prior art, the invention has the following remarkable effects:
(1) the liraglutide multivesicular liposome prepared by the invention has good stability, high drug encapsulation rate, large drug loading capacity, slow and stable drug release rate and no burst release phenomenon, and particularly successfully avoids the problem of easy in-vivo drug degradation of liraglutide injection; experiments show that the particle size of the liposome provided by the invention is 5-30 μm; the encapsulation rate is not lower than 75 percent, and the drug loading rate is not lower than 2 percent; the release effect is stable, and the drug can be continuously released for more than 168-336 hours.
(2) The liposome provided by the invention can reduce the effect of in vivo protein degrading enzyme on liraglutide, improves the in vivo and in vitro stability of the medicament, and simultaneously slows down the release and absorption of the medicament and prolongs the in vivo circulation time of the medicament. Therefore, the liraglutide multivesicular liposome prepared by the invention obviously improves the bioavailability of the medicament. Thereby improving the curative effect, reducing the toxic and side effects related to the drug dosage, reducing the drug cost and having larger application value. Experiments show that the liposome provided by the invention is stable in plasma concentration after subcutaneous injection administration of rats, sustained drug release in vivo reaches 336 hours, the in vivo retention time is remarkably prolonged (163.4 hours and 13.9 hours respectively) compared with that of a liraglutide injection, obvious pharmacokinetic characteristics of a sustained release preparation are presented, and the relative bioavailability with the injection is 661%.
(3) The liraglutide multivesicular liposome provided by the invention has slow drug release speed, can provide long-acting hypoglycemic effect, has better hypoglycemic effect, greatly reduces the administration times and frequency of patients and improves the administration compliance of the patients.
Drawings
FIG. 1 shows an optical microscope photograph of liraglutide multivesicular liposomes prepared in example 1 of the present invention;
FIG. 2 shows an optical microscope photograph of liraglutide multivesicular liposomes prepared in example 2 of the present invention;
FIG. 3 shows an optical microscope photograph of liraglutide multivesicular liposomes prepared in example 5 of the present invention;
FIG. 4 shows the in vitro release profile of liraglutide multivesicular liposomes prepared in example 1 of the present invention;
FIG. 5 shows the in vitro release profile of liraglutide multivesicular liposomes prepared according to comparative example 1 of the present invention;
FIG. 6 shows the blood glucose concentration-time curves on day 1 to day 15 after administration of liraglutide multivesicular liposomes diabetic rats prepared in example 1 of the present invention, the ordinate being the blood glucose concentration (mmol/L) and the abscissa being the time (h);
FIG. 7 shows the blood glucose concentration-time curves from day 1 to day 7 after administration of liraglutide multivesicular liposomes to diabetic rats prepared in comparative example 1, with blood glucose concentration (mmol/L) on the ordinate and time (h) on the abscissa;
FIG. 8 shows the blood glucose concentration-time curve after administration to commercially available Liraglutide injection diabetic rats, with blood glucose concentration (mmol/L) on the ordinate and time (h) on the abscissa;
FIG. 9 is a curve of the injection of the liraglutide multivesicular liposome prepared in example 1 into a diabetic rat; plasma concentration (pmol/l) with time (h) on the abscissa;
FIG. 10 is a curve of the injection of liraglutide multivesicular liposomes prepared in comparative example 1 into diabetic rats subcutaneously; plasma concentration (pmol/l) with time (h) on the abscissa;
FIG. 11 is a graph showing the subcutaneous injection of commercially available liraglutide injection diabetic rats; plasma concentration (pmol/l) and time (h) on the abscissa.
Detailed Description
The invention provides a liraglutide multivesicular liposome and a preparation method thereof, and a person skilled in the art can realize the liraglutide multivesicular liposome by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The reagents, medicines and instruments adopted by the invention are all common products on the market and can be purchased on the market.
The method for detecting the quality of the liraglutide multivesicular liposomes (Lrg-MVLs) comprises the following steps:
determination of the amount of drug encapsulated: precisely measuring 1ml of Lrg-MVLs suspension, placing the Lrg-MVLs suspension in a 4ml centrifuge tube, centrifuging the Lrg-MVLs suspension for 10min at 3500rpm, removing supernatant, washing the lower-layer precipitate with PBS (pH 7.4) for three times, dispersing the precipitate to 1ml, precisely measuring 0.2ml, placing the precipitate in a 10ml measuring flask, metering the volume to scale with acidified isopropanol (containing 1% hydrochloric acid), shaking up, and measuring the concentration C of the drug encapsulated in the Lrg-MVLs by an HPLC methodIn the capsuleCalculating the amount of drug M encapsulatedIn the capsule
And (3) total drug quantity determination: precisely measuring 0.2ml of Lrg-MVLs suspension, placing the Lrg-MVLs suspension in a 10ml measuring flask, fixing the volume to scale by using acidified isopropanol (containing 1% hydrochloric acid), shaking up, and measuring the concentration C of the drug encapsulated in the Lrg-MVLs by using an HPLC methodGeneral assemblyCalculating the total dose MGeneral assembly
The Encapsulation Efficiency (EE) was calculated as follows:
EE％＝Min the capsule/MGeneral assembly×100
Drug loading was calculated as follows:
(DL):DL％＝Min the capsule/m
In the formula: m is the total amount of lipids
Method for determining relative bioavailability
Relative phase pairBioavailability is the bioavailability obtained by comparing the extent and rate of absorption between different formulations of the same drug. The relative bioavailability was calculated as follows:
in the formula: A. b represents the group of example 1 and the group of commercially available formulations of liraglutide, respectively, and Dose is indicated by Dose
Liraglutide assay HPLC method:
chromatographic conditions are as follows: a chromatographic column: c18Columns (250 mm. times.4.6 mm); mobile phase: acetonitrile-water (60: 40) containing 0.1% trifluoroacetic acid; flow rate: 1 ml. min-1(ii) a Column temperature: 30 ℃; detection wavelength: 290 nm; sample introduction amount: 20 μ l.
Determination of morphology and particle size: observing the morphology under a 400-fold optical microscope; after a sample was diluted with physiological saline as a dispersion medium, the particle size and particle size distribution thereof were measured by a laser scattering particle sizer (LS230 laser particle sizer analyzer, Beckman Coulter, usa).
The invention is further illustrated by the following examples:
example 1
Precisely weighing 10.0mg of liraglutide, and dissolving the liraglutide in 2ml of aqueous solution containing 0.8% of human serum albumin and 7% of sucrose to form an internal water phase; precisely weighing 201.1mg of soybean phospholipid, 90.2mg of cholesterol and 125.0mg of triolein, dissolving in 5ml of diethyl ether to form an oil phase, adding the internal water phase into the oil phase, and vortexing for 3min to form a W/O type emulsion. Dripping the emulsion into 7ml of 7% sucrose aqueous solution of 6mg/ml lysine, continuously stirring for 5min at 40 ℃ to form W/O/W type multiple emulsion, blowing nitrogen gas to naturally volatilize the organic solvent, transferring the emulsion to a rotary evaporator to remove residual organic solvent after most of the solvent is volatilized to obtain the liraglutide sustained-release multivesicular liposome, wherein the encapsulation rate is 87.5%, the drug-loading rate is 2.3%, and the average particle size is 12.7 mu m (shown in figure 1).
Example 2
Precisely weighing 10.0mg of liraglutide, and dissolving the liraglutide in 2ml of aqueous solution containing 1% of human serum albumin and 7% of sucrose to form an internal water phase; precisely weighing 203.0mg of hydrogenated soybean phospholipid, 96.6mg of cholesterol and 121.0mg of triolein, dissolving in 5ml of chloroform to form an oil phase, adding the internal water phase into the oil phase, and performing vortex dispersion to form a W/O type emulsion. Dripping the emulsion into 7ml of 7% sucrose aqueous solution containing 6mg/ml lysine, continuously stirring at 40 ℃ for 10min to form W/O/W type multiple emulsion, continuously stirring at low speed to naturally volatilize the organic solvent, after most of the solvent is volatilized, transferring the emulsion to a rotary evaporator to remove residual organic solvent to obtain the liraglutide sustained-release multivesicular liposome, wherein the encapsulation rate is 74.4%, the drug-loading rate is 2.3%, and the average particle size is 22.0 μm (shown in figure 2).
Example 3
Precisely weighing 10.0mg of liraglutide, and dissolving the liraglutide in 2ml of aqueous solution containing 0.9% of human serum albumin and 7% of sucrose to form an internal water phase; precisely weighing dioleoylphosphatidylcholine 196.8mg, cholesterol 99.4mg and triolein 126.1mg, dissolving in 6ml chloroform to form oil phase, adding the above internal water phase into the oil phase, and vortex dispersing for 5min to obtain W/O type emulsion. Dropping the emulsion into 20ml of 8% sucrose aqueous solution containing 5mg/ml lysine under stirring at 1000r/min, continuously stirring for 10min at 40 ℃ to form W/O/W type multiple emulsion, blowing nitrogen to naturally volatilize the organic solvent, and when a sedimentation phenomenon occurs, transferring the emulsion to a rotary evaporator to remove the residual organic solvent to obtain the liraglutide sustained-release multivesicular liposome, wherein the encapsulation rate is 88.0%, the drug-loading rate is 2.4%, and the average particle size is 11.6 mu m.
Example 4
Accurately weighing 55.1mg of liraglutide, dissolving in 2ml of aqueous solution containing 4.3% of human serum albumin and 7% of sucrose to form an internal water phase; precisely weighing 202.1mg of soybean phospholipid, 98.0mg of cholesterol and 122.6mg of triolein, dissolving in 5ml of diethyl ether to form an oil phase, adding the internal water phase into the oil phase, and performing ultrasonic treatment in water bath for 3min to form a W/O type emulsion. Dripping the emulsion into 7ml of 7% sucrose aqueous solution containing 6mg/ml lysine, continuously stirring for 5min at 40 ℃ to form W/O/W type multiple emulsion, blowing nitrogen gas to naturally volatilize the organic solvent, and when a sedimentation phenomenon occurs, transferring the emulsion to a rotary evaporator to remove residual organic solvent to obtain the liraglutide sustained-release multivesicular liposome, wherein the encapsulation rate is 84.5%, the drug-loading rate is 11.5%, and the average particle size is 12.4 mu m.
Example 5
Accurately weighing 64.6mg of liraglutide, and dissolving the liraglutide in 1.5ml of a solution containing 10% of human serum albumin and 7% of sucrose to form an internal water phase; precisely weighing 210.5mg of soybean phospholipid, 95.0mg of cholesterol, 128.5mg of glycerol trioleate and 44.2mg of DPPG dissolved in 6ml of chloroform to ether (volume ratio is 1:1) to form an oil phase, adding the internal water phase into the oil phase, and quickly swirling for 5min to form a W/O type emulsion. Dripping the emulsion into 21ml of 7% sucrose aqueous solution containing 6mg/ml lysine, continuously stirring at 40 deg.C for 10min to form W/O/W type multiple emulsion, blowing nitrogen gas to volatilize organic solvent, removing residual organic solvent when precipitation occurs to obtain liraglutide sustained release multivesicular liposome with entrapment rate of 91.3%, drug-loading rate of 11.9, and average particle diameter of 16.2 μm (figure 3).
Example 6
Precisely weighing 11.3mg of liraglutide, and dissolving the liraglutide in 2ml of a solution containing 1.2% of bovine serum albumin and 5% of glucose to form an internal water phase; precisely weighing 199.0mg of soybean phospholipid, 90.5mg of cholesterol and 187.0mg of triolein, dissolving in 2ml of diethyl ether to form an oil phase, adding the internal water phase into the oil phase, and performing ultrasonic treatment in a water bath to form a W/O type emulsion. Dripping the emulsion into 14ml of 7% sucrose solution containing 8mg/ml histidine, continuously stirring for 5min to form W/O/W type multiple emulsion, blowing nitrogen gas at 40 ℃ under low-speed stirring to naturally volatilize the organic solvent, and when a sedimentation phenomenon occurs, transferring the emulsion to a rotary evaporator to remove the residual organic solvent to obtain the liraglutide sustained-release multivesicular liposome, wherein the encapsulation rate is 80.0%, the drug-loading rate is 2.3%, and the average particle size is 11.3 mu m.
Example 7
Accurately weighing liraglutide 18.2mg, dissolving in 2ml of 2.2% gelatin (250Bloom type B) and 7% sucrose solution to form an inner water phase; accurately weighing soybean phospholipid 260.3mg, cholesterol 100.5mg and triglyceride 191.0mg, dissolving in 16ml diethyl ether to form oil phase, adding the above internal water phase into the oil phase, and performing ultrasonic treatment in ice bath for 3min to form W/O type emulsion. Dripping the emulsion into 36ml of 7% sucrose aqueous solution of 6mg/ml histidine, continuously stirring for 8min at 10 ℃ in an ice bath to form W/O/W type multiple emulsion, blowing nitrogen to naturally volatilize the organic solvent, and when a sedimentation phenomenon appears, transferring the emulsion to a rotary evaporator to remove the residual organic solvent to obtain the liraglutide sustained-release multivesicular liposome, wherein the encapsulation rate is 86.6%, the drug-loading rate is 3.2%, and the average particle size is 11.0 mu m.
Example 8
Precisely weighing 30.4mg of liraglutide, dissolving in 2ml of a solution containing 3.4% of human serum albumin and 7% of sucrose to form an internal water phase; precisely weighing 220.0mg of soybean phospholipid, 88.5mg of cholesterol and 126.0mg of triolein, dissolving in 4ml of cyclohexane to form an oil phase, adding the internal water phase into the oil phase, and performing ultrasonic treatment in an ice bath for 3min to form a W/O type emulsion. Dripping the emulsion into 7% sucrose aqueous solution containing 6mg/ml histidine (the phase volume ratio of primary emulsion to external water phase is 1:2), continuously stirring at 45 deg.C to form W/O/W type multiple emulsion, blowing nitrogen gas to make organic solvent naturally volatilize, removing residual organic solvent when precipitation occurs, and obtaining liraglutide sustained-release multivesicular liposome with entrapment rate of 77.7%, drug-loading rate of 6.6%, and average particle size of 25.5 μm.
Example 9
Precisely weighing liraglutide 5.1mg, and dissolving in 2ml solution containing bovine blood albumin 0.125% and sucrose 7% to form an internal water phase; precisely weighing 220.0mg of soybean phospholipid, 95mg of cholesterol and 156.0mg of triolein, dissolving in 6ml of diethyl ether to form an oil phase, adding the internal water phase into the oil phase, and performing ultrasonic treatment in ice bath for 3min to form a W/O type emulsion. Dripping the emulsion into 7% sucrose aqueous solution containing 6mg/ml histidine at 40 deg.C (the phase volume ratio of primary emulsion to external water phase is 1:2), continuously stirring to form W/O/W type multiple emulsion, stirring at low speed, blowing nitrogen gas to volatilize organic solvent naturally, removing residual organic solvent when precipitation occurs, and transferring to rotary evaporation apparatus to obtain liraglutide sustained-release multivesicular liposome with entrapment rate of 79.9%, drug loading rate of 1.1%, and average particle diameter of 10.5 μm.
Example 10
Accurately weighing 14.2mg of liraglutide, dissolving in 2ml of a solution containing 1.5% of human serum albumin and 7% of sucrose to form an internal water phase; precisely weighing 220.0mg of soybean phospholipid, 76.5mg of cholesterol, 25.6mg of oleic acid and 122.3mg of tricaprylin, dissolving in 5ml of diethyl ether to form an oil phase, adding the internal water phase into the oil phase, and vortexing for 5min to form a W/O type emulsion. Dripping the emulsion into 7% sucrose aqueous solution containing 6mg/ml histidine (the phase volume ratio of primary emulsion to external water phase is 1:2), continuously stirring at 40 deg.C to form W/O/W type multiple emulsion, stirring at low speed of 600r/min while blowing nitrogen gas to naturally volatilize organic solvent, removing residual organic solvent when precipitation occurs, and transferring to rotary evaporator to obtain liraglutide sustained-release multivesicular liposome with entrapment rate of 88.1%, drug-loading rate of 3.1%, and average particle diameter of 13.9 μm.
Example 11
Accurately weighing 54.0mg of liraglutide, and dissolving in 2ml of a solution containing 5% hydrolyzed gelatin and 7% sucrose to form an internal water phase; precisely weighing soybean phospholipid 220.0mg, cholesterol 76.5mg and triglyceride 55.3mg, dissolving in 5ml diethyl ether to form oil phase, adding the above internal water phase into the oil phase, and performing ultrasonic treatment in ice bath for 5min to form W/O type emulsion. Dripping the emulsion into 7% sucrose aqueous solution containing 6mg/ml histidine (the phase volume ratio of primary emulsion to external aqueous phase is 1:2), continuously stirring at 40 deg.C to form W/O/W type multiple emulsion, blowing nitrogen gas to volatilize organic solvent naturally, removing residual organic solvent when precipitation occurs, and obtaining liraglutide sustained-release multivesicular liposome with entrapment rate of 86.8%, drug-loading rate of 13.3%, and average particle diameter of 15.5 μm.
Example 12
Accurately weighing 58.1mg of liraglutide, dissolving in 2ml of a solution containing 1% of human serum albumin and 7% of sucrose to form an internal water phase; precisely weighing 36.6mg (lipoid) of DOPC, 10.2mg of DPPG, 28.0mg of cholesterol and 8.6mg of triglyceride, dissolving in 5ml of chloroform-diethyl ether mixed solvent (volume ratio is 1:1) to form an oil phase, adding the internal water phase into the oil phase, and vortexing for 5min to form a W/O type emulsion. The emulsion is dropped into 7 percent sucrose aqueous solution containing 6mg/ml histidine (the phase volume ratio of the primary emulsion to the external aqueous phase is 1:2), the mixture is continuously stirred at the constant temperature of 40 ℃ to form W/O/W type multiple emulsion, nitrogen is blown in under the stirring to enable the organic solvent to be naturally volatilized, when the sedimentation phenomenon occurs, the mixture is moved to a rotary evaporation instrument to remove the residual organic solvent, and the liraglutide sustained-release multivesicular liposome is obtained, wherein the entrapment rate is 82.3 percent, the drug-loading rate is 41.2 percent, and the average particle size is 19.7 mu m.
Example 13
Precisely weighing 44.8mg of liraglutide, dissolving in 2ml of a solution containing 4.5% of human serum albumin and 7% of sucrose to form an internal water phase; 183.6mg of egg yolk lecithin (lipoid), 70.7mg of cholesterol and 61.1mg of triolein are precisely weighed, dissolved in 5ml of diethyl ether mixed solvent (volume ratio is 0.3:1) to form an oil phase, the internal water phase is added into the oil phase, and the mixture is vortexed for 5min to form a W/O type emulsion. Dripping the emulsion into 7% sucrose aqueous solution containing 6mg/ml histidine (the phase volume ratio of primary emulsion to external water phase is 1:2), continuously stirring at constant temperature of 40 ℃ to form W/O/W type multiple emulsion, blowing nitrogen gas to enable organic solvent to be naturally volatilized, when a sedimentation phenomenon occurs, moving the emulsion to a rotary evaporator to remove residual organic solvent to obtain the liraglutide sustained-release multivesicular liposome, wherein the entrapment rate is 86.0%, the drug loading rate is 12.5%, and the average particle size is 18.3 mu m.
Comparative example 1 (without stabilizer)
Precisely weighing 10.0mg of liraglutide, and dissolving the liraglutide in 2ml of sucrose aqueous solution containing 7 percent of the liraglutide to form an internal water phase; precisely weighing 201.1mg of soybean phospholipid, 100.2mg of cholesterol and 125.0mg of triolein, dissolving in 5ml of diethyl ether to form an oil phase, adding the internal water phase into the oil phase, and vortexing for 3min to form a W/O type emulsion. Dripping the emulsion into 7ml of 7% sucrose aqueous solution containing 6mg/ml lysine, continuously stirring for 5min at 40 ℃ to form W/O/W type multiple emulsion, blowing nitrogen to naturally volatilize the organic solvent, transferring the emulsion to a rotary evaporator to remove residual organic solvent after most of the solvent is volatilized to obtain the liraglutide sustained-release multivesicular liposome, wherein the encapsulation rate is 73% and the drug-loading rate is 2.3%.
Example 14 Release assay for Liraglutide multivesicular liposomes
Precisely measuring 0.5ml of the Lrg-MVL suspension prepared in example 1 and comparative example 1, adding a PBS solution with pH7.4 to 5ml, plugging a test tube, placing the test tube in a shaking table with constant temperature of 37 ℃ and 100rpm, sampling at different time points, centrifuging the sample at 5000rpm for 5min to remove supernatant, repeating the operation for two more times, adding acidified isopropanol (containing 1% hydrochloric acid) into the precipitate for dissolution, measuring the content by an HPLC method, calculating the drug retained in the MVLs at each sampling point, taking the encapsulated drug amount at zero time as 100%, calculating the residual percentage of each sampling point, thus obtaining the release percentage, and drawing a cumulative release percentage curve of the Lrg-MVLs.
The release curve of the liposome prepared in example 1 is shown in fig. 4, and the result shows that the liraglutide multivesicular liposome drug of the present invention is slowly released, the sustained release can be continued for almost 432 hours, the sustained release effect is significant, and no burst release effect exists. The release effect of the liposomes prepared in the other examples was similar. The release curve of the liposome prepared in comparative example is shown in fig. 5, and the result shows that the drug release of the liraglutide multivesicular liposome prepared in comparative example 1 lasts for 216 hours, and the slow release effect is weaker than that of the multivesicular liposome prepared in example 1.
Example 15 pharmacodynamic and pharmacokinetic experiments with liraglutide multivesicular liposomes
1.1 preparation of diabetic rat model
Taking male rats, fasting for 12 hours overnight, weighing, injecting a 1% alloxan solution freshly prepared by physiological saline into caudal vein at a dose of 45mg/kg, feeding 2ml of a 50% glucose solution into each gavage after 30min, taking blood from eye sockets after normally feeding for 72 hours, and detecting the blood glucose concentration by adopting a glucose oxidase kit, wherein the rats with the blood glucose concentration of more than 11.1mmol/L are successful diabetes model rats.
1.2 methods of administration
The successfully molded diabetic rats were divided into A, B, C groups and administered as follows
Group A rats were subcutaneously administered 7mg/kg (based on liraglutide) of liraglutide multivesicular liposome suspension (liraglutide multivesicular liposome prepared in examples 1-13 of the present invention, resuspended in physiological saline) at one time in the neck and back, 0.5ml of blood was taken and placed in an EP tube coated with heparin at 0, 24, 96, 168, 240, 312, 336, and 360 hours, and plasma was separated by centrifugation at 3500r/min for 10min, and frozen at-20 ℃ for storage and testing.
Group B rats are given the same dose of liraglutide injection(s) subcutaneously at the neck and back in one time (
Noh and nord), 0.5ml of blood was taken at 0, 2, 4, 8, 12, 24, 30 and 36h, respectively, into EP tubes coated with heparin. Centrifuging at 3500r/min for 10min to separate plasma, and freezing at-20 deg.C for testing.
Group C rats were administered a single subcutaneous dose of liraglutide multivesicular liposome suspension (liraglutide multivesicular liposomes prepared according to the present invention in comparative example 1, resuspended in physiological saline) at 5mg/kg at the back of the neck, and 0.5ml of blood was taken at 0, 24, 48, 72, 96, 120, 168 hours and placed in an EP tube coated with heparin.
1.3 measurement of blood glucose and blood drug levels
The method comprises the following steps: the OD value of the reaction solution was measured at 505 wavelengths by spectrophotometry using a glucose oxidase kit (North China Biotechnology Ltd.) in Zhongshan, and the blood glucose concentration of each rat at the corresponding time was calculated, and the corresponding blood concentration was measured using a GLP-1 kit (Shanghai Huding Biotechnology Ltd.).
1.4 pharmacodynamic results
The results of blood glucose measurements of rats given different formulations of liraglutide by subcutaneous injection are shown in table 1,
table 1: blood glucose concentration after dosing in rats
The results show that the liraglutide multivesicular liposome provided by the invention can provide normal and stable blood sugar level within at least 312 hours through single injection, and the blood sugar reducing effect can reach 312 hours. However, the hypoglycemic effect of the same dose of liraglutide injection (commercially available) was maintained for only 24 hours. The liraglutide multivesicular liposome is proved to have obvious slow release effect, the administration times of the liraglutide can be greatly reduced, and the compliance of patients is improved. The single injection of liraglutide multivesicular liposome (without stabilizer) can only provide 120 hours of hypoglycemic effect, the long-acting effect of the liraglutide multivesicular liposome is greater than that of injection, but the long-acting effect of the liraglutide multivesicular liposome is less than that of the liraglutide multivesicular liposome containing the stabilizer, and the two have obvious difference (p is less than 0.05).
1.5 pharmacokinetic results
The injection (commercially available) of liraglutide multivesicular liposome (prepared according to the invention) was injected into rats subcutaneously in a single dose, and the blood concentrations of the comparative formulations are shown in table 2.
Table 2: blood concentration after administration to rats
TABLE 3 pharmacokinetic parameters
Note,. indicates P <0.05
The result shows that compared with the injection, the liraglutide multivesicular liposome prepared by the invention has stable blood concentration and the release time is 360 h. The in vivo retention time is obviously prolonged (163.4 hours and 13.9 hours respectively), and the pharmacokinetics characteristics of the sustained-release preparation are obvious, and the relative bioavailability with the injection is 661.6%. The liraglutide multivesicular liposome is proved to remarkably improve the stability of the medicament in vivo, remarkably improve the bioavailability and greatly reduce the medicament cost. The single injection liraglutide ordinary multivesicular liposomes (prepared in comparative example 1) had an in vivo retention time of 74.9 hours, which was greater than that of the injections, but significantly lower (p <0.05) than that of the liraglutide multivesicular liposomes containing a stabilizer.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (8)
1. The liraglutide multivesicular liposome is characterized by being prepared from the following raw materials in parts by mass:
1 part of liraglutide;
1-100 parts of a membrane material;
10-300 parts of an osmotic pressure regulator;
0.3-2.5 parts of a stabilizer;
the stabilizer is human blood albumin, bovine blood albumin or hydrolyzed gelatin;
the membrane material comprises phospholipid, cholesterol and triglyceride, wherein the mass ratio of the phospholipid to the cholesterol to the triglyceride is (46.8-342.3): (28.0-100.5): (8.6-191.0);
wherein the phospholipid is at least one of soybean phospholipid, lecithin, hydrogenated soybean phospholipid, dipalmitoyl phosphatidyl glycerol and dioleoyl phosphatidyl choline, and the triglyceride is triolein or tricaprylin;
the osmotic pressure regulator is at least one selected from lysine, histidine, glucose and sucrose.
2. The liraglutide multivesicular liposome of claim 1, wherein the liraglutide multivesicular liposome comprises the following components in parts by mass:
1 part of liraglutide;
1.44-92.35 parts of a membrane material;
12.2-265.8 parts of osmotic pressure regulator;
0.34-2.42 parts of a stabilizer.
3. The liraglutide multivesicular liposome of claim 1, wherein the liposome comprises a liposome-like structure,
the phospholipid is soybean lecithin, the triglyceride is glycerol trioleate, the stabilizer is human blood albumin, and the osmotic pressure regulator is a mixture of sucrose and lysine; wherein the mass ratio of the liraglutide to the membrane material is 1:41.62, the mass ratio of the liraglutide to the stabilizing agent is 1:1.6, the mass ratio of the liraglutide to the osmotic pressure regulator is 1:67.2, and the mass ratio of the soybean lecithin, the cholesterol and the triglyceride in the membrane material is 201.1:90.2: 125; the mass ratio of the sucrose to the lysine in the osmotic pressure regulator is 15: 1;
or the phospholipid is hydrogenated soybean phospholipid, the triglyceride is triolein, the stabilizer is human blood albumin, and the osmotic pressure regulator is a mixture of sucrose and lysine; wherein the mass ratio of the liraglutide to the membrane material is 1:42.1, the mass ratio of the liraglutide to the stabilizing agent is 1:2, the mass ratio of the liraglutide to the osmotic pressure regulator is 1:67.2, and the mass ratio of the hydrogenated soybean lecithin, the cholesterol and the triglyceride in the membrane material is 203:96: 121; the mass ratio of the sucrose to the lysine in the osmotic pressure regulator is 15: 1;
or the phospholipid is dioleoyl phosphatidylcholine, the triglyceride is triolein, the stabilizer is human blood albumin, and the osmotic pressure regulator is a mixture of sucrose and lysine; wherein the mass ratio of the liraglutide to the membrane material is 1:42.2, the mass ratio of the liraglutide to the stabilizing agent is 1:1.8, the mass ratio of the liraglutide to the osmotic pressure regulator is 1:180.4, and the mass ratio of the dioleoylphosphatidylcholine, the cholesterol and the triglyceride in the membrane material is 196.8:99.4: 126.1; the mass ratio of the sucrose to the lysine in the osmotic pressure regulator is 17.4: 1;
or the phospholipid is soybean phospholipid, the triglyceride is triolein, the stabilizer is human blood albumin, and the osmotic pressure regulator is a mixture of sucrose and lysine; wherein the mass ratio of the liraglutide to the membrane material is 1:7.7, the mass ratio of the liraglutide to the stabilizing agent is 1:1.6, the mass ratio of the liraglutide to the osmotic pressure regulator is 1:12.2, and the mass ratio of the soybean lecithin, the cholesterol and the triglyceride in the membrane material is 202.1:98: 122.6; the mass ratio of the sucrose to the lysine in the osmotic pressure regulator is 15: 1;
or the phospholipid is soybean phospholipid, the triglyceride is triolein, the stabilizer is human blood albumin, and the osmotic pressure regulator is a mixture of sucrose and lysine; the mass ratio of the liraglutide to the membrane material is 1:7.4, the mass ratio of the liraglutide to the stabilizing agent is 1:2.3, and the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 26.3; the membrane material comprises soybean phospholipid, cholesterol, triglyceride and DPPG, and the mass ratio of the soybean phospholipid to the cholesterol to the triglyceride to the DPPG in the membrane material is 210.5:95: 128.5: 44.2, the mass ratio of the sucrose to the lysine in the osmotic pressure regulator is 12.5: 1;
or the phospholipid is soybean phospholipid, the triglyceride is triolein, the stabilizer is bovine serum albumin, and the osmotic pressure regulator is a mixture of glucose, sucrose and histidine; the mass ratio of the liraglutide to the membrane material is 1:42.2, the mass ratio of the liraglutide to the stabilizing agent is 1:2.1, the mass ratio of the liraglutide to the osmotic pressure regulator is 1:105.5, the mass ratio of the soybean lecithin, the cholesterol and the triglyceride in the membrane material is 199:90.5:187, and the mass ratio of the glucose, the sucrose and the histidine in the osmotic pressure regulator is 100:98.0: 112;
or the phospholipid is soybean phospholipid, the triglyceride is triolein, the stabilizer is human serum albumin, and the osmotic pressure regulator is a mixture of sucrose and histidine; the mass ratio of the liraglutide to the membrane material is 1:14.3, the mass ratio of the liraglutide to the stabilizing agent is 1:2.2, the mass ratio of the liraglutide to the osmotic pressure regulator is 1:34.2, the mass ratio of the soybean lecithin, the cholesterol and the triglyceride in the membrane material is 220:88.5:126, and the mass ratio of the sucrose to the histidine in the osmotic pressure regulator is 16.3: 1;
or the phospholipid is soybean phospholipid, and the triglyceride is glycerol trioleate; the stabilizer is bovine blood albumin, and the osmotic pressure regulator is a mixture of sucrose and histidine; the mass ratio of liraglutide to membrane material is 1:92.4, the mass ratio of liraglutide to stabilizer is 1:2.4, the mass ratio of liraglutide to osmotic pressure regulator is 1:265.8, the mass ratio of soybean lecithin, cholesterol and triglyceride in the membrane material is 220:95:156, the mass ratio of sucrose to histidine in the osmotic pressure regulator is 13.1: 1;
or the phospholipid is soybean phospholipid, the membrane material comprises soybean phospholipid, cholesterol, tricaprylin and oleic acid, the stabilizer is human serum albumin, and the osmotic pressure regulator is a mixture of sucrose and histidine; the mass ratio of the liraglutide to the membrane material is 1:31.3, the mass ratio of the liraglutide to the stabilizing agent is 1:2.1, the mass ratio of the liraglutide to the osmotic pressure regulator is 1:93.7, the mass ratio of soybean lecithin, cholesterol, tricaprylin and oleic acid in the membrane material is 220:76.5:122.3:25.6, and the mass ratio of sucrose to histidine in the osmotic pressure regulator is 18: 1;
or the phospholipid is soybean phospholipid, the stabilizing agent is hydrolyzed gelatin, and the osmotic pressure regulator is a mixture of sucrose and histidine; the mass ratio of the liraglutide to the membrane material is 1:6.5, the mass ratio of the liraglutide to the stabilizing agent is 1:1.9, and the mass ratio of the liraglutide to the osmotic pressure regulator is 1: 22.3, the mass ratio of the soybean lecithin, the cholesterol and the triolein in the membrane material is 220:76.5:55.3, and the mass ratio of the sucrose to the histidine in the osmotic pressure regulator is 13.3: 1;
or the phospholipid is DOPC and DPPG, the stabilizer is human blood albumin, and the osmotic pressure regulator is a mixture of sucrose and histidine; the mass ratio of the liraglutide to the membrane material is 1:1.4, the mass ratio of the liraglutide to the stabilizing agent is 1:0.34, the mass ratio of the liraglutide to the osmotic pressure regulator is 1:23.1, the mass ratio of DOPC, DPPG, cholesterol and triglyceride in the membrane material is 36.6:10.2:28:8.6, and the mass ratio of sucrose to histidine in the osmotic pressure regulator is 15: 1;
or the phospholipid is yolk lecithin, the stabilizer is human blood albumin, the osmotic pressure regulator is a mixture of sucrose and histidine, the mass ratio of liraglutide to the membrane material is 1:7.0, the mass ratio of liraglutide to the osmotic pressure regulator is 1:26.9, the mass ratio of liraglutide to the stabilizer is 1:2.0, the mass ratio of yolk lecithin, cholesterol and triglyceride in the membrane material is 183.6:70.7:61.1, and the mass ratio of sucrose to histidine in the osmotic pressure regulator is 13.3: 1.
4. A method for preparing liraglutide multivesicular liposomes according to any one of claims 1 to 3, comprising:
step 1: dissolving the membrane material in an organic solvent to be used as an oil phase; dissolving a stabilizing agent, a partial osmotic pressure regulator and liraglutide in water to serve as an internal water phase; dissolving another part of osmotic pressure regulator in water to serve as an external water phase;
step 2: forming the inner water phase and the oil phase into first-stage emulsion;
and step 3: allowing the primary milk to form a multiple emulsion with the external water phase;
and 4, step 4: drying the organic solvent in the re-emulsion to prepare the liraglutide multivesicular liposome.
5. The method according to claim 4, wherein the organic solvent in step 1 is selected from diethyl ether, dichloromethane, chloroform, ethyl acetate and cyclohexane.
6. The method according to claim 4, wherein the volume ratio of the internal aqueous phase to the oil phase is 1: (0.8-10); the volume ratio of the first-stage milk to the external water phase is 1: (1-4).
7. Use of the liraglutide multivesicular liposome of any one of claims 1 to 3 for the preparation of a medicament for the treatment and/or prevention of diabetes.
8. A medicament for treating and/or preventing diabetes, comprising the liraglutide multivesicular liposome according to any one of claims 1 to 3.
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| 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 |
| 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 |
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