An injectable sustained release formulation and a method for preparing the same

Abstract

The application belongs to the technical field of biological medicine, and discloses an injectable sustained-release preparation and a preparation method thereof, which comprises an active drug and an amphiphilic phospholipid; the preparation method of the injectable sustained-release preparation is as follows: active drug colloidal particles are prepared by using a nanoparticle preparation method; the active drug colloidal particles are dispersed in an organic solvent in which the amphiphilic phospholipid is dissolved and are uniformly mixed, wherein the mass ratio of the active drug to the amphiphilic phospholipid is 10:0.05-10:25; the organic solvent is removed, and the active drug colloidal particles are dispersed in an injection oil to obtain the injectable sustained-release preparation; the injectable sustained-release preparation provided by the application has a particle size of 20-500 nm, an in-vitro release period of one day to one month, and no obvious burst release phenomenon in in-vitro release, so that the adverse reactions caused by excessively high blood drug concentration can be reduced, the toxic side effects can be reduced, and the treatment effect can be improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an injectable sustained-release formulation and its preparation method. Background Technology

[0002] Nanoparticles refer to a new type of drug formulation with particle sizes ranging from 1 to 1000 nm. They are primarily manufactured by physically encapsulating or chemically bonding drugs to nanocarriers. By altering the composition, stability, responsiveness, and surface charge of the carrier, the in vivo pharmacokinetic behavior of nanoparticles can be regulated to achieve the research goals of sustained-release and controlled-release formulations. Furthermore, compared to other formulation types, nanoparticles can improve drug delivery and stability, and increase drug safety. Therefore, they are often used to improve drug distribution in tissues, thereby enhancing bioavailability. Currently, they are commonly used in the fields of antitumor drugs, antiviral drugs, peptide / protein drugs, and nucleic acid drugs.

[0003] Polypeptide-protein drugs are biological macromolecules composed of amino acids linked by peptide bonds. Due to their high activity and low toxicity, they have achieved great success in recent years in the treatment of cancer, diabetes, genetic diseases, and inflammation. However, the pharmacological activity of these drugs is closely related to their complex spatial conformation, which is highly susceptible to degradation by the external environment, resulting in extremely low bioavailability. They also suffer from drawbacks such as short biological half-life, poor stability, and susceptibility to degradation by enzymes in the body, which limits their clinical development.

[0004] For example, patent CN03110974.8 discloses an oral nanoparticle formulation of polypeptides and proteins. This formulation is made from a polypeptide / protein drug and a polymer or amphiphilic compound as a coating material. The coating material reduces the burst release of the nanoparticles, improves the stability of insulin against various gastrointestinal proteases, and enhances its bioadhesion, thereby promoting its absorption in the gastrointestinal tract. However, this formulation releases the drug completely within a day, and the gastrointestinal environment rapidly destroys the drug structure, resulting in relatively low bioavailability of the polypeptide / protein drug.

[0005] Patent CN202011514187.9 discloses polypeptide nanoparticles for treating diabetes, polypeptide nanoparticle microneedles, and their preparation method. This patent utilizes naturally formed cavities during the polymer microneedle preparation process to load exenatide nanoparticle lyophilized powder, preparing dry powder microneedles suitable for treating type 2 diabetes, effectively reducing the risk of the drug causing strong inflammatory and immune responses. However, the preparation process involves both nanoparticle and polymer microneedle preparation, which is complex, costly, and introduces considerable uncertainty into the final microneedles.

[0006] Patent CN201410584447.8 discloses a novel insulin-phospholipid-chitosan self-assembled microparticle carrier and its formulation, which forms a complex powder of insulin and phospholipid together, then dissolves and self-assembles with chitosan. This method has a complex formulation and a short sustained-release time.

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

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide an injectable sustained-release formulation and its preparation method, thereby providing a way to prolong the release time of polypeptide protein drugs.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution: an injectable sustained-release formulation comprising an active drug and an amphiphilic phospholipid, wherein the preparation method of the injectable sustained-release formulation is as follows:

[0010] (1) Prepare active drug colloidal particles using nanoparticle preparation methods;

[0011] (2) Disperse the colloidal particles of the active drug in an organic solvent containing amphiphilic phospholipids and mix them evenly, wherein the mass ratio of the active drug to the amphiphilic phospholipids is 10:0.05-10:25.

[0012] (3) Remove the organic solvent and disperse it in the injection oil to obtain the injectable sustained-release formulation.

[0013] Preferably, the active pharmaceutical ingredient is a water-soluble polypeptide or protein.

[0014] Preferably, the active drug is selected from one or more of insulin, exenatide, liraglutide, dulaglutide, liximabide, benaglutide, loxenatide, smegglutide, telpogglutide, semaglutide, abirate, retaglutide, svidiopeptide, mastolide, bofanogglutide, ibenaglutide, goserelin, leuprorelin, triptorelin, buserrelin, gonadorelin, nafarelin, histaminerelin, abarelix, cetrorelin, degarelix, ganirelinix, lanreitide, octreotide, parreitide, and lutetium oxyoctreotide.

[0015] Preferably, the amphiphilic phospholipid is selected from one or more of (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), dioleoylphosphatidylcholine (DOPC), distearylphosphatidylcholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), disqualylphosphatidylcholine (DEPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoyllecithin (DMPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidic acid (DOPA), dipalmitoylphosphatidic acid (DPPA), distearylphosphatidylglycerol (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 cause the active drug to precipitate and form colloidal particles;

[0018] Ball milling method: Weigh the active drug, add organic solvent and milling beads, adjust the milling 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 oil for injection 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 tricaprylic acid glycerides.

[0021] The present invention has the following beneficial effects: (1) The injectable sustained-release formulation provided by the present invention has a particle size of 20-500nm, and the in vitro release cycle varies from one day to one month. There is no obvious burst release phenomenon in vitro, 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 formulation provided by the present invention has a clear formulation composition, a simple preparation process, and is easy to produce. Attached Figure Description

[0023] Figure 1 The in vitro release curve of the insulin sustained-release formulation prepared in Example 2;

[0024] Figure 2 The in vitro release curve of the insulin sustained-release formulation prepared in Example 4;

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

[0026] Figure 4 The in vitro release curve of the leuprorelin sustained-release formulation prepared in Example 8;

[0027] Figure 5 The in vitro release curve of the liraglutide sustained-release formulation prepared in Example 10;

[0028] Figure 6 The in vitro release curve of the liraglutide sustained-release formulation prepared in Example 11 is shown. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

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

[0031] Insulin was dissolved in 0.012M hydrochloric acid aqueous solution to prepare a 20 mg / mL insulin aqueous solution. Under magnetic stirring at 1200 rpm, 4500 μL of acetone was added dropwise, followed by 15 mL of dimethyl carbonate. After vortexing, the mixture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and 1 mL of ethyl acetate was added. The mixture was then ultrasonically dispersed to obtain 10 mg / mL insulin colloidal particles. 200 μL of the insulin colloidal particles were taken, and 100 μL of 10 mg / mL DOTAP was added. The mixture was ultrasonically dispersed at 15000 rpm for 5 min, and the supernatant was discarded. 500 μL of medium-chain triglycerides was added, and the mixture was ultrasonically dispersed to obtain the sustained-release formulation.

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

[0033] Insulin was dissolved in 0.012M hydrochloric acid aqueous solution to prepare a 20 mg / mL insulin aqueous solution. Under magnetic stirring at 1200 rpm, 4500 μL of acetone was added dropwise, followed by 15 mL of dimethyl carbonate. After vortexing, the mixture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and 1 mL of dichloromethane was added for ultrasonic dispersion to obtain 10 mg / mL insulin colloidal particles. 200 μL of insulin colloidal particles were taken, and 100 μL of 0.1 mg / mL DOTAP was added. The mixture was ultrasonicated with a probe and centrifuged at 15000 rpm for 5 min, and the supernatant was discarded. 500 μL of medium-chain triglycerides was added, and the mixture was ultrasonicated with a probe for homogenization to obtain the sustained-release formulation. This formulation released insulin for a total of 9 days in vitro, with a cumulative release of 88.13%.

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

[0035] Insulin was dissolved in 0.012M hydrochloric acid aqueous solution to prepare a 20 mg / mL insulin aqueous solution. Under magnetic stirring at 1200 rpm, 4500 μL of acetone was added dropwise, followed by 15 mL of dimethyl carbonate. After vortexing, the mixture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and 1 mL of ethyl acetate was added. The mixture was then ultrasonically dispersed to obtain 10 mg / mL insulin colloidal particles. 200 μL of the insulin colloidal particles were taken, and 100 μL of 1 mg / mL DOPC was added. The mixture was ultrasonically dispersed at 15000 rpm for 5 min, and the supernatant was discarded. 500 μL of medium-chain triglycerides was added, and the mixture was ultrasonically dispersed to obtain the sustained-release formulation.

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

[0037] Insulin was dissolved in 0.012M hydrochloric acid aqueous solution to prepare a 20 mg / mL insulin aqueous solution. Under magnetic stirring at 1200 rpm, 4500 μL of acetone was added dropwise, followed by 15 mL of dimethyl carbonate. After vortexing, the mixture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and 1 mL of dichloromethane was added for ultrasonic dispersion to obtain 10 mg / mL insulin colloidal particles. 200 μL of insulin colloidal particles were taken, and 100 μL of 0.1 mg / mL DOPA was added. The mixture was ultrasonicated with a probe and centrifuged at 15000 rpm for 5 min, and the supernatant was discarded. 500 μL of medium-chain triglycerides was added, and the mixture was ultrasonicated with a probe for homogenization to obtain the sustained-release formulation. This formulation released insulin for a total of 9 days in vitro, with a cumulative release of 89.73%.

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

[0039] Insulin was dissolved in 0.012M hydrochloric acid aqueous solution to prepare a 20 mg / mL insulin aqueous solution. Under magnetic stirring at 1200 rpm, 4500 μL of acetone was added dropwise, followed by 15 mL of dimethyl carbonate. After vortexing, the mixture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and 1 mL of ethyl acetate was added. The mixture was then ultrasonically dispersed to obtain 10 mg / mL insulin colloidal particles. 200 μL of the insulin colloidal particles were taken, and 100 μL of 10 mg / mL DOPA was added. The mixture was ultrasonically dispersed at 15000 rpm for 5 min, and the supernatant was discarded. 500 μL of medium-chain triglycerides was added, and the mixture was ultrasonically dispersed to obtain the sustained-release formulation.

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

[0041] Weigh out leuprorelin to prepare a 50 mg / mL leuprorelin aqueous solution. Transfer 50 μL of this solution to a centrifuge tube, add 1 mL of acetonitrile, and repeat the above operation four times. Transfer the entire solution to a 50 mL centrifuge tube and add 12.6 mL of dimethyl carbonate. Vortex, centrifuge at 4000 rpm for 5 min, discard the supernatant, add 1 mL of dichloromethane, and sonicate to disperse evenly, obtaining 10 mg / mL leuprorelin colloidal particles. Take 200 μL of leuprorelin colloidal particles, add 100 μL of 5 mg / mL DEPC, sonicate with a probe, centrifuge at 15000 rpm for 5 min, and discard the supernatant. Add 500 μL of medium-chain triglycerides, and sonicate evenly with a probe to obtain the sustained-release formulation. This formulation released for a total of 20 days in vitro, with a cumulative release of 35.64%.

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

[0043] Weigh out leuprorelin to prepare a 50 mg / mL leuprorelin aqueous solution. Transfer 50 μL of this solution to a centrifuge tube, add 1 mL of acetonitrile, and repeat the above operation four times. Transfer the entire solution to a 50 mL centrifuge tube and add 12.6 mL of dimethyl carbonate. Vortex, centrifuge at 4000 rpm for 5 min, discard the supernatant, add 1 mL of ethyl benzoate, and sonicate to disperse evenly, obtaining 10 mg / mL leuprorelin colloidal particles. Take 200 μL of leuprorelin colloidal particles, add 100 μL of 10 mg / mL DOTAP, sonicate with a probe, centrifuge at 15000 rpm for 5 min, and discard the supernatant. Add 500 μL of sesame oil, sonicate with a probe to homogenize, and the sustained-release formulation is obtained.

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

[0045] Prepare a 50 mg / mL leuprorelin aqueous solution. Transfer 50 μL of this solution to a centrifuge tube, add 1 mL of acetonitrile, and repeat the above operation four times. Transfer the entire solution to a 50 mL centrifuge tube and add 12.6 mL of dimethyl carbonate. Vortex, centrifuge at 4000 rpm for 5 min, discard the supernatant, add 1 mL of dichloromethane, and sonicate to disperse evenly, obtaining 10 mg / mL leuprorelin colloidal particles. Take 200 μL of leuprorelin colloidal particles, add 100 μL of 2 mg / mL DPPC, sonicate with a probe, centrifuge at 15000 rpm for 5 min, and discard the supernatant. Add 500 μL of olive oil, sonicate with a probe to homogenize, thus obtaining the sustained-release formulation. This formulation released for a total of 30 days in vitro, with a cumulative release of 28.58%.

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

[0047] Weigh out leuprorelin to prepare a 50 mg / mL leuprorelin aqueous solution. Transfer 50 μL of this solution to a centrifuge tube, add 1 mL of acetonitrile, and repeat the above operation four times. Transfer the entire solution to a 50 mL centrifuge tube and add 12.6 mL of dimethyl carbonate. Vortex, centrifuge at 4000 rpm for 5 min, discard the supernatant, add 1 mL of ethyl acetate, and sonicate to disperse evenly, obtaining 10 mg / mL leuprorelin colloidal particles. Take 200 μL of leuprorelin colloidal particles, add 100 μL of 10 mg / mL DOPC, sonicate with a probe, centrifuge at 15000 rpm for 5 min, and discard the supernatant. Add 500 μL of medium-chain triglycerides, sonicate with a probe to homogenize, and the sustained-release formulation is obtained.

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

[0049] Liraglutide was dissolved in 0.012M hydrochloric acid aqueous solution to prepare a 20 mg / mL liraglutide aqueous solution. Under magnetic stirring at 1200 rpm, 4500 μL of acetone was added dropwise, followed by 15 mL of dimethyl carbonate. After vortexing, the mixture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and 1 mL of dichloromethane was added for ultrasonic dispersion to obtain 10 mg / mL liraglutide colloidal particles. 200 μL of liraglutide colloidal particles were taken, and 100 μL of 10 mg / mL DPPG was added. The mixture was ultrasonicated with a probe and centrifuged at 15000 rpm for 5 min, and the supernatant was discarded. 500 μL of medium-chain triglycerides was added, and the mixture was ultrasonicated with a probe to obtain the sustained-release formulation. This formulation released 88.10% of its target amount over 26 days in vitro.

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

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

[0052] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. However, the above description is merely a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments derived by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. An injectable sustained-release formulation, characterized in that, The formulation comprises an active pharmaceutical ingredient and an amphiphilic phospholipid, wherein the active pharmaceutical ingredient is insulin and the amphiphilic phospholipid is (2,3-dioleoyl-propyl)-trimethylammonium chloride. The preparation method of the injectable sustained-release formulation is as follows: (1) Prepare colloidal particles of active drug using a nanoparticle preparation method; the nanoparticle preparation method adopts the nanoprecipitation method, and the specific steps are: dissolve the active drug in a good solvent; add a poor solvent to precipitate the active drug to form colloidal particles; (2) Disperse the colloidal particles of the active drug in an organic solvent containing amphiphilic phospholipids and mix well, wherein the mass ratio of the active drug to the amphiphilic phospholipids is 10:0.05; (3) Remove the organic solvent and disperse it in the oil for injection to obtain the injectable sustained-release formulation, wherein the oil for injection is a medium-chain triglyceride.

2. The injectable sustained-release formulation 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.

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