Injectable sustained release preparation and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses an injectable sustained-release preparation and a preparation method thereof.The injectable sustained-release preparation comprises an active drug and amphiphilic phospholipid, and the preparation method of the injectable sustained-release preparation comprises the following steps that active drug colloidal particles are prepared through a nanoparticle preparation method; active drug colloidal particles are dispersed in an organic solvent in which amphiphilic phospholipid is dissolved and mixed uniformly, and the mass ratio of the active drug to the amphiphilic phospholipid is (10: 0.05)-(10: 25); and removing the organic solvent, and dispersing in oil for injection to obtain the injectable sustained release preparation. The particle size of the injectable sustained-release preparation is 20-500 nm, the in-vitro release period varies from one day to one month, no obvious burst release phenomenon exists in in-vitro release, adverse reactions caused by too high blood concentration can be relieved, toxic and side effects can be reduced, and the treatment effect can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an injectable sustained-release preparation and a preparation method thereof. Background Art

[0002] Nanoformulations refer to a new type of drug preparation with a particle size ranging from 1 to 1000 nm, which is mainly made by combining drugs into nanocarriers by physical embedding or chemical bonding. By changing the composition, stability, responsiveness and surface charge of the carrier, the in vivo pharmacokinetic behavior of nanopharmaceutical preparations can be regulated to achieve the research goal of sustained and controlled release. In addition, compared with other types of preparations, nanoformulations can improve drug delivery and drug stability, increase drug safety, and are therefore often used to improve drug tissue distribution and thereby increase drug bioavailability. They are currently commonly used in the fields of anti-tumor drugs, antiviral drugs, polypeptide protein drugs, nucleic acid drugs, and the like.

[0003] Peptide protein drugs are biological macromolecules composed of amino acids connected by peptide bonds. Due to their high activity and low toxicity, they have achieved great success in the treatment of cancer, diabetes, genetic diseases and inflammation in recent years. However, the pharmacological activity of such drugs is closely related to their complex spatial conformation, which is easily damaged by the external environment, resulting in extremely low bioavailability. At the same time, they have the disadvantages of short biological half-life, poor stability, and easy decomposition by enzymes in the body, which restricts their clinical development.

[0004] For example, patent CN03110974.8 discloses a polypeptide protein oral nanoparticle preparation, which is made of polypeptide protein drugs and polymers or amphiphilic compounds as coating materials. The coating materials are used to reduce the burst release of nanoparticles, improve the stability of insulin to various proteases in the gastrointestinal tract, and improve its bioadhesion, thereby promoting its gastrointestinal absorption. However, the drug release of this preparation is completed within one day, and the gastrointestinal environment will quickly destroy the drug structure, and the bioavailability of polypeptide protein drugs is still low.

[0005] Patent CN202011514187.9 discloses a polypeptide nanoparticle, polypeptide nanoparticle microneedle and preparation method thereof for treating diabetes. This patent uses the naturally formed cavities in the preparation process of polymer microneedles to load freeze-dried powder of exenatide nanoparticles to prepare dry powder microneedles that can be used for the treatment of type 2 diabetes, which can effectively reduce the risk of the drug causing strong inflammation and immune response. However, the preparation process of this preparation involves the preparation of nanoparticles and polymer microneedles, which is cumbersome, has high production costs, and brings more uncertainties to the final microneedles.

[0006] Patent CN201410584447.8 discloses a novel insulin-phospholipid-chitosan self-assembled microparticle carrier and its preparation, in which insulin and phospholipid are formed into a complex powder, which is then dissolved and self-assembled with chitosan. This method has a complex prescription and a short sustained release time.

[0007] Therefore, there is a need for an injectable sustained-release preparation that can prolong the release time of polypeptide protein drugs. Summary of the invention

[0008] The purpose of the present invention is to solve the deficiencies in the prior art and provide an injectable sustained-release preparation and a preparation method thereof, so as to provide a method for prolonging the release time of polypeptide protein drugs.

[0009] In order to achieve the above object, the present invention is implemented by the following technical scheme: an injectable sustained-release preparation, comprising an active drug and an amphiphilic phospholipid, and the preparation method of the injectable sustained-release preparation is as follows:

[0010] (1) preparing active drug colloidal particles using a nanoparticle preparation method;

[0011] (2) dispersing the active drug colloid particles in an organic solvent containing an amphiphilic phospholipid and mixing them uniformly, wherein the mass ratio of the active drug to the amphiphilic phospholipid is 10:0.05-10:25;

[0012] (3) removing the organic solvent and dispersing the mixture in oil for injection to obtain the injectable sustained-release preparation.

[0013] Preferably, the active drug component is a water-soluble polypeptide or protein substance.

[0014] Preferably, the active drug is selected from one or more of insulin, exenatide, liraglutide, dulaglutide, lixisenatide, benaglutide, loxenatide, semaglutide, telpotide, semaglutide, albiglutide, retaglutide, svetidine, mashidu peptide, bovanglutide, ibenatide, goserelin, leuprorelin, triptorelin, buserelin, gonadorelin, nafarelin, histrelin, abarelix, cetrorelix, degarelix, ganirelix, lanreotide, octreotide, pasireotide, and lutetoxide octreotide.

[0015] Preferably, the amphiphilic phospholipid is selected from one or more of (2,3-dioleoyl-propyl)-trimethylammonium-chloride (DOTAP), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), dierucoylphosphatidylcholine (DEPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidic acid (DOPA), dipalmitoylphosphatidic acid (DPPA), distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), and dioleoylphosphatidylglycerol (DOPG).

[0016] Preferably, the nanoparticle preparation method in step (1) includes nanoprecipitation, emulsification, spray drying and ball milling.

[0017] Nanoprecipitation method: 1. Dissolve the active drug in a good solvent; 2. Add a poor solvent to precipitate the active drug to form colloidal particles;

[0018] Ball milling method: Weigh the active drug, add organic solvent and ball milling beads, adjust the grinding speed and time, and use a ball mill to prepare drug colloidal particles.

[0019] Preferably, the organic solvent in step (2) is selected from one or more of methanol, ethanol, benzyl alcohol, dichloromethane, chloroform, acetone, ethyl acetate, ethyl benzoate, and benzyl benzoate.

[0020] Preferably, the injection oil in step (3) is selected from one or more of soybean oil, sesame oil, olive oil, tea oil, castor oil, peanut oil, polyoxyethylene castor oil, medium chain triglycerides, and tricaprylin.

[0021] The present invention has the following beneficial effects: (1) The particle size of the injectable sustained-release preparation provided by the present invention is 20-500 nm, the in vitro release period ranges from one day to one month, and there is no obvious burst release phenomenon in the in vitro release, which can reduce the adverse reactions caused by excessive blood drug concentration, reduce toxic side effects, and improve the therapeutic effect.

[0022] (2) The injectable sustained-release preparation provided by the present invention has a clear prescription composition, a simple preparation process, and is easy to produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the in vitro release curve of the insulin sustained-release preparation prepared in Example 2;

[0024] Figure 2 This is the in vitro release curve of the insulin sustained-release preparation prepared in Example 4;

[0025] Figure 3The in vitro release curve of the leuprorelin sustained-release preparation prepared in Example 6;

[0026] Figure 4 This is the in vitro release curve of the leuprorelin sustained-release preparation prepared in Example 8;

[0027] Figure 5 This is the in vitro release curve of the liraglutide sustained-release preparation prepared in Example 10;

[0028] Figure 6 This is the in vitro release curve of the liraglutide sustained-release preparation prepared in Example 11. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0030] Example 1: An injectable sustained-release preparation, the specific preparation steps are as follows:

[0031] Dissolve insulin in 0.012M hydrochloric acid aqueous solution to prepare a 20mg / mL insulin aqueous solution. Under magnetic stirring at 1200rpm, add 4500μL acetone and then add 15mL dimethyl carbonate. After vortexing, centrifuge at 4000rpm for 5min, discard the supernatant, add 1mL ethyl acetate and disperse evenly by ultrasound to obtain 10mg / mL insulin colloid particles. Take 200μL insulin colloid particles, add 100μL 10mg / mL DOTAP, ultrasonicate with probe, centrifuge at 15000rpm for 5min, and discard the supernatant. Add 500μL medium-chain triglycerides and ultrasonicate with probe to obtain a sustained-release preparation.

[0032] Example 2: An injectable sustained-release preparation, the specific preparation steps are as follows:

[0033] Insulin was dissolved in 0.012M hydrochloric acid aqueous solution to prepare a 20mg / mL insulin aqueous solution. Under magnetic stirring at 1200rpm, 4500μL acetone was added dropwise, and then 15mL dimethyl carbonate was added. After vortexing, centrifuged at 4000rpm for 5min, the supernatant was discarded, and 1mL dichloromethane was added for ultrasonic dispersion to obtain 10mg / mL insulin colloid particles. Take 200μL insulin colloid particles, add 100μL 0.1mg / mL DOTAP, probe ultrasonic, centrifuge at 15000rpm for 5min, and discard the supernatant. Add 500μL medium-chain triglycerides, probe ultrasonic uniformly, and a sustained-release preparation was obtained. The preparation was released for a total of 9 days in vitro, with a cumulative release of 88.13%.

[0034] Example 3: An injectable sustained-release preparation, the specific preparation steps are as follows:

[0035] Dissolve insulin in 0.012M hydrochloric acid aqueous solution to prepare a 20mg / mL insulin aqueous solution. Under magnetic stirring at 1200rpm, add 4500μL acetone and then 15mL dimethyl carbonate. After vortexing, centrifuge at 4000rpm for 5min, discard the supernatant, add 1mL ethyl acetate and disperse evenly by ultrasound to obtain 10mg / mL insulin colloid particles. Take 200μL insulin colloid particles, add 100μL 1mg / mL DOPC, ultrasonicate with probe, centrifuge at 15000rpm for 5min, and discard the supernatant. Add 500μL medium-chain triglycerides and ultrasonicate with probe to obtain a sustained-release preparation.

[0036] Example 4: An injectable sustained-release preparation, the specific preparation steps are as follows:

[0037] Insulin was dissolved in 0.012M hydrochloric acid aqueous solution to prepare a 20mg / mL insulin aqueous solution. Under magnetic stirring at 1200rpm, 4500μL acetone was added dropwise, and then 15mL dimethyl carbonate was added. After vortexing, centrifuged at 4000rpm for 5min, the supernatant was discarded, and 1mL dichloromethane was added for ultrasonic dispersion to obtain 10mg / mL insulin colloid particles. Take 200μL insulin colloid particles, add 100μL 0.1mg / mL DOPA, probe ultrasonic, centrifuge at 15000rpm for 5min, and discard the supernatant. Add 500μL medium-chain triglycerides, probe ultrasonic uniformly, and a sustained-release preparation was obtained. The preparation was released for a total of 9 days in vitro, with a cumulative release of 89.73%.

[0038] Example 5: An injectable sustained-release preparation, the specific preparation steps are as follows:

[0039] Dissolve insulin in 0.012M hydrochloric acid aqueous solution to prepare a 20mg / mL insulin aqueous solution. Under magnetic stirring at 1200rpm, add 4500μL acetone and then 15mL dimethyl carbonate. After vortexing, centrifuge at 4000rpm for 5min, discard the supernatant, add 1mL ethyl acetate and disperse evenly by ultrasound to obtain 10mg / mL insulin colloid particles. Take 200μL insulin colloid particles, add 100μL 10mg / mL DOPA, ultrasonicate with a probe, centrifuge at 15000rpm for 5min, and discard the supernatant. Add 500μL medium-chain triglycerides and ultrasonicate with a probe to obtain a sustained-release preparation.

[0040] Example 6: An injectable sustained-release preparation, the specific preparation steps are as follows:

[0041] Weigh leuprorelin to prepare a 50 mg / mL leuprorelin aqueous solution. Pipette 50 μL of the solution into a centrifuge tube, add 1 mL of acetonitrile, and repeat the above operation four times. Transfer all of it to a 50 mL centrifuge tube and add 12.6 mL of dimethyl carbonate. After vortexing, centrifuge at 4000 rpm for 5 min, discard the supernatant, add 1 mL of dichloromethane and ultrasonically disperse evenly to obtain 10 mg / mL leuprorelin colloidal particles. Take 200 μL of leuprorelin colloidal particles, add 100 μL of 5 mg / mL DEPC, ultrasonicate with a probe, centrifuge at 15000 rpm for 5 min, and discard the supernatant. Add 500 μL of medium-chain triglycerides, ultrasonicate with a probe evenly, and a sustained-release preparation is obtained. The preparation was released for a total of 20 days in vitro, with a cumulative release of 35.64%.

[0042] Example 7: An injectable sustained-release preparation, the specific preparation steps are as follows:

[0043] Weigh leuprorelin to prepare a 50 mg / mL leuprorelin aqueous solution. Pipette 50 μL of the solution into a centrifuge tube, add 1 mL of acetonitrile, and repeat the above operation four times. Transfer all of it to a 50 mL centrifuge tube and add 12.6 mL of dimethyl carbonate. After vortexing, centrifuge at 4000 rpm for 5 min, discard the supernatant, add 1 mL of ethyl benzoate and ultrasonically disperse evenly to obtain 10 mg / mL leuprorelin colloidal particles. Take 200 μL of leuprorelin colloidal particles, add 100 μL of 10 mg / mL DOTAP, ultrasonicate with a probe, centrifuge at 15000 rpm for 5 min, and discard the supernatant. Add 500 μL of sesame oil and ultrasonicate with a probe to obtain a sustained-release preparation.

[0044] Example 8: An injectable sustained-release preparation, the specific preparation steps are as follows:

[0045] Weigh leuprorelin to prepare a 50 mg / mL leuprorelin aqueous solution. Pipette 50 μL of the solution into a centrifuge tube, add 1 mL of acetonitrile, and repeat the above operation four times. Transfer all of it to a 50 mL centrifuge tube and add 12.6 mL of dimethyl carbonate. After vortexing, centrifuge at 4000 rpm for 5 min, discard the supernatant, add 1 mL of dichloromethane and ultrasonically disperse evenly to obtain 10 mg / mL leuprorelin colloidal particles. Take 200 μL of leuprorelin colloidal particles, add 100 μL of 2 mg / mL DPPC, ultrasonicate with a probe, centrifuge at 15000 rpm for 5 min, and discard the supernatant. Add 500 μL of olive oil and ultrasonicate with a probe to obtain a sustained-release preparation. The preparation was released for a total of 30 days in vitro, with a cumulative release of 28.58%.

[0046] Example 9: An injectable sustained-release preparation, the specific preparation steps are as follows:

[0047] Weigh leuprorelin to prepare a 50 mg / mL leuprorelin aqueous solution. Pipette 50 μL of the solution into a centrifuge tube, add 1 mL of acetonitrile, and repeat the above operation four times. Transfer all of it to a 50 mL centrifuge tube and add 12.6 mL of dimethyl carbonate. After vortexing, centrifuge at 4000 rpm for 5 min, discard the supernatant, add 1 mL of ethyl acetate and ultrasonically disperse evenly to obtain 10 mg / mL leuprorelin colloidal particles. Take 200 μL of leuprorelin colloidal particles, add 100 μL of 10 mg / mL DOPC, ultrasonicate with a probe, centrifuge at 15000 rpm for 5 min, and discard the supernatant. Add 500 μL of medium-chain triglycerides, ultrasonicate with a probe evenly, and a sustained-release preparation is obtained.

[0048] Example 10: An injectable sustained-release preparation, the specific preparation steps are as follows:

[0049] Liraglutide was dissolved in 0.012M hydrochloric acid aqueous solution to prepare a 20mg / mL liraglutide aqueous solution. Under magnetic stirring at 1200rpm, 4500μL acetone was added dropwise, and then 15mL dimethyl carbonate was added. After vortexing, centrifuged at 4000rpm for 5min, the supernatant was discarded, and 1mL dichloromethane was added for ultrasonic dispersion to obtain 10mg / mL liraglutide colloidal particles. Take 200μL liraglutide colloidal particles, add 100μL 10mg / mL DPPG, probe ultrasonic, centrifuge at 15000rpm for 5min, and discard the supernatant. Add 500μL medium-chain triglycerides, probe ultrasonic uniformly, and a sustained-release preparation is prepared. The preparation was released for a total of 26 days in vitro, with a cumulative release of 88.10%.

[0050] Example 11: An injectable sustained-release preparation, the specific preparation steps are as follows:

[0051] Liraglutide was dissolved in 0.012M hydrochloric acid aqueous solution to prepare a 20mg / mL liraglutide aqueous solution. Under magnetic stirring at 1200rpm, 4500μL acetone was added dropwise, and then 15mL dimethyl carbonate was added. After vortexing, centrifuged at 4000rpm for 5min, the supernatant was discarded, and 1mL dichloromethane was added for ultrasonic dispersion to obtain 10mg / mL liraglutide colloidal particles. Take 200μL liraglutide colloidal particles, add 100μL 2mg / mL DOTAP, probe ultrasonic, centrifuge at 15000rpm for 5min, and discard the supernatant. Add 500μL olive oil and probe ultrasonic uniformly to obtain a sustained-release preparation. The preparation was released for a total of 7 days in vitro, with a cumulative release of 32.12%.

[0052] The above shows and describes the basic principles, main features and advantages of the present invention. However, the above is only a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other implementation methods derived by any technician in the field without departing from the technical solution of the present invention should be included in the patent scope of the present invention.

Claims

1. An injectable sustained-release preparation, characterized in that: The injectable sustained-release preparation comprises an active drug and an amphiphilic phospholipid, and the preparation method of the injectable sustained-release preparation is as follows: (1) preparing active drug colloidal particles using a nanoparticle preparation method; (2) dispersing the active drug colloid particles in an organic solvent containing an amphiphilic phospholipid and mixing them uniformly, wherein the mass ratio of the active drug to the amphiphilic phospholipid is 10:0.05-10:25; (3) removing the organic solvent and dispersing the mixture in oil for injection to obtain the injectable sustained-release preparation.

2. The injectable sustained-release preparation according to claim 1, characterized in that: The active drug component is a water-soluble polypeptide or protein substance.

3. The injectable sustained-release preparation according to claim 1, characterized in that: The active drug is selected from one or more of insulin, exenatide, liraglutide, dulaglutide, lixisenatide, benaglutide, loxenatide, semaglutide, telpotide, semaglutide, albiglutide, retaglutide, svetidine, mashidu peptide, bovanglutide, ibenatide, goserelin, leuprorelin, triptorelin, buserelin, gonadorelin, nafarelin, histrelin, abarelix, cetrorelix, degarelix, ganirelix, lanreotide, octreotide, pasireotide, and lutetoxide octreotide.

4. The injectable sustained-release preparation according to claim 1, characterized in that: The amphiphilic phospholipid is selected from one or more of (2,3-dioleoyl-propyl)-trimethylammonium-chloride, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, hydrogenated soybean phosphatidylcholine, dierucoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoyl lecithin, dioleoylphosphatidylethanolamine, dioleoylphosphatidic acid, dipalmitoylphosphatidic acid, distearoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol.

5. The injectable sustained-release preparation according to claim 1, characterized in that: The nanoparticle preparation method in step (1) includes nanoprecipitation method, emulsification method, spray drying method and ball milling method.

6. The injectable sustained-release preparation according to claim 1, characterized in that: The organic solvent in step (2) is selected from one or more of methanol, ethanol, benzyl alcohol, dichloromethane, chloroform, acetone, ethyl acetate, ethyl benzoate, and benzyl benzoate.

7. The injectable sustained-release preparation according to claim 1, characterized in that: The injection oil described in step (3) is selected from one or more of soybean oil, sesame oil, olive oil, tea oil, castor oil, peanut oil, polyoxyethylene castor oil, medium chain triglycerides, and tricaprylin.

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