Papaverine hydrochloride sustained release microsphere for injection and preparation method thereof

Abstract

The invention discloses papaverine hydrochloride sustained-release microspheres for injection and a preparation method of the papaverine hydrochloride sustained-release microspheres, and relates to the technical field of pharmaceutical preparations. The sustained-release microsphere comprises an active component, a composite carrier, a composite stabilizer and a freeze-drying protective agent. The composite carrier is a compound formed by grafting a polylactic acid-glycolic acid copolymer and hyaluronic acid, the composite stabilizer is a mixture of an antioxidant and an amino acid compound, the freeze-drying protective agent is a mixture of polyol and saccharides, and the surface of the microsphere is provided with a hydrophilic-hydrophobic gradient coating composed of a hydrophobic layer, an amphiphilic layer and a hydrophilic layer. The preparation method comprises the steps of primary emulsion preparation, multiple emulsion preparation, step-by-step curing, surface modification and freeze-drying treatment, and adopts the processes of gradient emulsification, step-by-step curing, layer-by-layer grafting, gradient cooling and segmented sublimation. According to the technical scheme, the drug encapsulation efficiency and the drug loading capacity are effectively improved, the burst release rate is reduced, long-acting stable slow release is realized, the storage stability and the biocompatibility are enhanced, and a safe and effective drug administration choice is provided for treatment of ischemic diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a sustained-release microsphere of papaverine hydrochloride for injection and its preparation method. Background Technology

[0002] Papaverine hydrochloride is a commonly used vasodilator in clinical practice, primarily used to treat acute ischemic cerebrovascular diseases and peripheral artery occlusion. It works by relaxing vascular smooth muscle and improving blood circulation in ischemic tissues. Currently, the main clinically available papaverine hydrochloride preparations are injectable and oral formulations. Injectable formulations have a short half-life (only 1-2 hours), requiring repeated administration 3-4 times daily, which not only severely impacts patient adherence but also increases the risk of adverse reactions due to significant fluctuations in blood drug concentration. Oral formulations are greatly affected by the gastrointestinal environment, have low bioavailability, and exhibit a significant first-pass effect, failing to rapidly meet the treatment needs of acute conditions.

[0003] To address the shortcomings of conventional formulations, the industry has gradually begun research and development of sustained-release formulations of papaverine hydrochloride, attempting to extend the dosing cycle through liposomes, nanoparticles, or conventional sustained-release microspheres. However, existing sustained-release formulations consistently face a core technical challenge: papaverine hydrochloride is highly lipid-soluble, and when using conventional single carrier materials and preparation processes, the drug is prone to migration and leakage at the oil-water phase interface, making it difficult to improve encapsulation efficiency and drug loading. At the same time, there is a significant burst-release effect, making it impossible to achieve long-term stable sustained release. Furthermore, some formulations lack biocompatibility, easily causing local inflammation after injection or being rapidly cleared by the reticuloendothelial system, making it difficult to simultaneously meet the comprehensive clinical requirements for efficacy stability, drug safety, and long-term efficacy.

[0004] In view of the above, this application is hereby submitted. Summary of the Invention

[0005] The purpose of this invention is to provide a sustained-release microsphere for injection of papaverine hydrochloride and its preparation method, so as to solve the problems mentioned in the background art.

[0006] To address the aforementioned technical problems, the present invention provides sustained-release microspheres of papaverine hydrochloride for injection, comprising an active ingredient, a composite carrier, a composite stabilizer, and a lyophilization protectant. The components are proportioned by weight as follows: 10-40 parts of the active ingredient papaverine hydrochloride, 50-80 parts of the composite carrier, 1-5 parts of the composite stabilizer, and 10-30 parts of the lyophilization protectant. The composite carrier is a complex formed by grafting polylactic acid-glycolic acid copolymer with hyaluronic acid. The composite stabilizer is a mixture of antioxidants and amino acid compounds. The lyophilization protectant is a mixture of polyols and sugars. The surface of the sustained-release microspheres has a hydrophilic-hydrophobic gradient coating, which consists of a hydrophobic layer, an amphiphilic layer, and a hydrophilic layer from the inside out. This achieves long-term sustained release of papaverine hydrochloride, reduces burst release rate, improves microsphere storage stability and biocompatibility, and provides excellent reconstitution properties, meeting the requirements for injection administration.

[0007] Furthermore, the components are formulated in the following proportions by weight: 30 to 35 parts papaverine hydrochloride, 60 to 70 parts composite carrier, 2 to 3 parts composite stabilizer, and 15 to 20 parts lyophilization protectant; this further optimizes the balance between drug loading and encapsulation efficiency, while improving the stability of the sustained-release period and adapting to the routine clinical dosage requirements.

[0008] Furthermore, the grafting mass ratio of polylactic acid-glycolic acid copolymer to hyaluronic acid in the composite carrier is 5:1 to 10:1; the composite stabilizer is a mixture of α-tocopherol and L-arginine, with a mass ratio of 1:2 to 1:4; and the lyophilization protectant is a mixture of mannitol and trehalose, with a mass ratio of 2:1 to 4:1. This significantly improves the antioxidant properties and crystal stability of the drug, reduces the drug degradation rate during storage, and optimizes the structural integrity of the microspheres after lyophilization.

[0009] A method for preparing papaverine hydrochloride sustained-release microspheres for injection includes the following steps:

[0010] S1 Preparation of primary emulsion: Papaverine hydrochloride and composite carrier are mixed evenly, added to the oil phase and emulsified under low shear to obtain primary emulsion; S11 Preparation of secondary emulsion: Primary emulsion is added to the aqueous phase containing emulsifier and emulsified under high shear to obtain secondary emulsion;

[0011] S2 stepwise curing: First, the composite emulsion is pre-cured at room temperature and pressure, and then the temperature is increased and the pressure is reduced for final curing to obtain the microsphere precursor;

[0012] S3 Surface Modification: A hydrophilic-hydrophobic gradient coating was constructed on the surface of the microsphere precursor using a layer-by-layer grafting method to obtain modified microspheres;

[0013] S4 freeze-drying process: A freeze-drying protectant is added to the modified microspheres, and after gradient cooling and segmented sublimation freeze-drying, papaverine hydrochloride sustained-release microspheres for injection are obtained; the uniformity of microsphere particle size is controlled by gradient emulsification, the organic solvent residue is reduced by step-by-step solidification, the surface gradient modification prolongs the in vivo circulation time, and the gradient freeze-drying improves the resolubility of microspheres, making them suitable for industrial production.

[0014] Furthermore, in S1, the low-shear emulsification rotation speed is 400 to 600 rpm, and the emulsification time is 8 to 12 minutes; in S11, the emulsifier is a mixture of Tween and poloxamer, and the high-shear emulsification rotation speed is 2,500 to 3,500 rpm, and the emulsification time is 3 to 7 minutes; this further improves the stability of the primary and secondary emulsions, avoids drug migration, increases the encapsulation rate, and ensures uniform microsphere particle size distribution.

[0015] Furthermore, in S2, the pre-curing temperature is 25 to 35 degrees Celsius, and the curing time is 1 to 3 hours; the final curing temperature is 40 to 60 degrees Celsius, the pressure is -0.05 to -0.07 MPa, and the curing time is 3 to 5 hours; this achieves precise control of the microsphere porosity, avoids burst release effects, and completely removes residual organic solvents, thereby improving the safety of the formulation.

[0016] Furthermore, in S3, the hydrophobic layer is polycaprolactone, the amphiphilic layer is polylactic-glycolic acid copolymer, and the hydrophilic layer is polyethylene glycol; the total thickness of the gradient coating is forty to one hundred and ten nanometers; this ensures the stability of the hydrophilic and hydrophobic gradient on the surface of the microspheres, effectively preventing rapid clearance by the reticuloendothelial system in vivo, prolonging blood circulation time, and enhancing the potential for targeted enrichment.

[0017] Furthermore, in S4, the gradient cooling step is as follows: first, maintain the temperature at -15 to -25 degrees Celsius for one to three hours, then lower the temperature to -35 to -45 degrees Celsius and maintain it for three to five hours, and finally lower the temperature to -55 to -65 degrees Celsius and maintain it for five to seven hours; the segmented sublimation is carried out under a vacuum of -0.07 to -0.09 MPa, first sublimating below 0 degrees Celsius for six to ten hours, and then raising the temperature to 15 to 25 degrees Celsius for sublimation for three to five hours; this avoids the microspheres from breaking and collapsing during the freeze-drying process, ensures the integrity of the microsphere structure after freeze-drying, results in a short reconstitution time and a clear solution, and improves the convenience of clinical use.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. The composite carrier is made of polylactic acid-glycolic acid copolymer grafted with hyaluronic acid, breaking the limitations of single carriers, reducing drug migration and leakage, and significantly improving encapsulation efficiency and drug loading. The composite stabilizer combines antioxidants and amino acid compounds to achieve dual functions of anti-oxidation and crystal form stabilization, effectively inhibiting drug degradation, significantly improving storage stability, and avoiding the single-function defects of single stabilizers.

[0020] 2. The gradient emulsification-stepwise curing process breaks through the conventional understanding of single emulsification and one-time curing. By using differentiated shear strength and stepwise curing operations, it ensures the uniformity of microsphere size, precisely controls porosity, reduces organic solvent residue, and completely solves the burst release effect. Surface hydrophilic-hydrophobic gradient modification constructs a three-layer structure, overcoming the functional contradictions of single modification, prolonging in vivo circulation time, reducing local inflammatory responses, and improving biocompatibility.

[0021] 3. The composite lyophilization protectant combines polyols and sugars to exert a synergistic protective effect, preventing microsphere lyophilization breakage and ensuring structural integrity and excellent reconstitution properties. These unique technologies work in close synergy to achieve long-lasting, stable, and sustained release, extending the dosing cycle, reducing the risk of adverse reactions, and making it suitable for industrial production. This provides a safer and more effective dosing option for the treatment of ischemic diseases and offers innovative ideas for the development of sustained-release formulations of lipid-soluble drugs. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating the preparation method of papaverine hydrochloride sustained-release microspheres for injection. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Please see Figure 1 This invention provides a technical solution: papaverine hydrochloride sustained-release microspheres for injection and their preparation method. Example 1 is the best example. Through five core technical means: polylactic acid-glycolic acid copolymer (PLGA) grafted composite carrier with hyaluronic acid, α-tocopherol and L-arginine composite stabilizer, gradient emulsification-stepwise curing preparation process, surface hydrophilicity-hydrophobicity gradient modification technology, and mannitol and trehalose composite freeze-drying protectant system, a whole-chain innovative solution of "carrier-stabilizer-process-modification-protection" is formed.

[0025] Comparative Examples 2 through 7 each eliminated one core technology, while the remaining raw material ratios, preparation steps, and testing methods were strictly consistent with Example 1, constructing a single-variable comparison model: Comparative Example 2 eliminated the composite carrier and used a single PLGA carrier; Comparative Example 3 eliminated the gradient emulsification process and used a single high-shear emulsification; Comparative Example 4 eliminated the stepwise curing process and used conventional one-time curing; Comparative Example 5 eliminated the surface hydrophilic-hydrophobic gradient modification and used a single PEG hydrophilic modification; Comparative Example 6 eliminated the composite freeze-drying protectant and used a single mannitol protectant; Comparative Example 7 eliminated the composite stabilizer and did not add any stabilizer.

[0026] The raw materials include: papaverine hydrochloride (purity 99.5%); polylactic acid-glycolic acid copolymer (PLGA, lactate-glycolic acid molar ratio 50:50, molecular weight 20kDa); hyaluronic acid (HA, molecular weight 10kDa); α-tocopherol (purity 99.0%); L-arginine (purity 99.5%); mannitol (injection grade); trehalose (injection grade); polycaprolactone (PCL, molecular weight 15kDa); polyethylene glycol (PEG, molecular weight 2kDa); Tween 80 (injection grade); poloxamer 188 (injection grade); ethyl acetate (chromatographic grade); carbodiimide (EDC, purity 99.0%); N,N-dimethylformamide (DMF, analytical grade); anhydrous ethanol (analytical grade); and deionized water (self-made, conductivity <10μS / cm).

[0027] The equipment includes: low-shear emulsifier; high-shear emulsifier; vacuum drying oven; freeze dryer; laser particle size analyzer; high-performance liquid chromatograph; Zeta potentiometer; scanning electron microscope; gas chromatograph; analytical balance; constant temperature water bath; three-necked flask; rotary evaporator; vacuum filtration device; agate mortar and pestle; ultrasonic cleaner.

[0028] Example 1 is the preferred embodiment, and specifically includes:

[0029] Preparation of the composite carrier (PLGA-HA graft): Weigh 60g of PLGA and 6g of hyaluronic acid, place them in a 500mL three-necked flask, add 200mL of N,N-dimethylformamide, heat to 60℃, set the stirring speed to 300r / min, and keep warm for 2 hours to dissolve, forming a homogeneous and transparent mixed solution. Weigh 3g of carbodiimide and slowly add it to the above mixed solution, maintain the stirring conditions at 60℃ and 300r / min, and continue the reaction for 4 hours. Through the condensation reaction mediated by carbodiimide, the carboxyl group in the PLGA molecule and the hydroxyl group in the hyaluronic acid molecule are grafted, forming a PLGA-HA covalently bonded composite carrier.

[0030] After the reaction was complete, the reaction solution was slowly added dropwise to 500 mL of deionized water at a rate of 5 mL / min, while stirring (150 rpm). After the addition was complete, stirring continued for 30 minutes, and the mixture was allowed to stand for 12 hours, resulting in the precipitation of a white flocculent precipitate. The precipitate was collected by vacuum filtration and washed three times with 200 mL of deionized water each time, until the pH of the washing solution stabilized at 6.8-7.2 (neutral). The washed precipitate was transferred to a vacuum drying oven at 50 °C and a vacuum of -0.09 MPa for 12 hours to obtain a white powdery PLGA-HA composite carrier, which was then sealed and stored in a desiccator for later use.

[0031] Preparation of the composite stabilizer (α-tocopherol + L-arginine): Weigh 1.5g of α-tocopherol and 4.5g of L-arginine, place them in a 50mL beaker, add 20mL of anhydrous ethanol, set the stirring speed to 200r / min, and dissolve at room temperature (25℃) for 1 hour to form a clear and transparent mixed solution. Transfer the solution to a rotary evaporator, set the temperature to 40℃ and the vacuum degree to -0.08MPa, and rotary evaporate for 30 minutes to remove the ethanol, obtaining a pale yellow, loose solid composite stabilizer. Place the solid product in an agate mortar, grind it into a fine powder, pass it through a 100-mesh sieve, and seal it for later use.

[0032] Preparation of colostrum: Weigh 35g of papaverine hydrochloride, 70g of the PLGA-HA composite carrier prepared above, and 3g of composite stabilizer, place them in a 250mL beaker, mix them manually with a glass stir bar until uniform, add 80mL of ethyl acetate (oil phase), transfer to a low-shear emulsifier, set the speed to 500r / min and the emulsification time to 10 minutes, observe the state of the emulsion every 2 minutes during the process to ensure that there is no layering or clumping, and finally obtain a uniform and stable water-in-oil (W1 / O) type colostrum. The emulsion is milky white and semi-transparent with uniform viscosity.

[0033] Preparation of the double emulsion: Weigh 2 g of Tween 80 and 4 g of Poloxamer 188, add 200 mL of deionized water (aqueous phase), set the stirring speed to 200 r / min, and dissolve at room temperature for 1 hour to obtain a clear and transparent aqueous solution. Slowly add the prepared primary emulsion to the aqueous solution at a rate of 3 mL / min while stirring (100 r / min). After all the emulsion has been added, transfer it to a high-shear emulsifier, set the speed to 3000 r / min and the emulsification time to 5 minutes, and break the primary emulsion droplets to the target particle size to obtain a uniform and fine oil-in-water-in-oil (W1 / O / W2) double emulsion. The emulsion is milky white and opaque, with no visible particles.

[0034] Stepwise curing: The re-emulsion was transferred to a 500mL three-necked flask and placed in a constant-temperature water bath. Under constant temperature and atmospheric pressure, and with a stirring speed of 100 rpm, pre-curing was performed for 2 hours to slowly evaporate some of the ethyl acetate, allowing the microspheres to initially form and preventing excessive porosity due to rapid solvent evaporation. After pre-curing, the temperature of the constant-temperature water bath was raised to 50℃, the vacuum was adjusted to -0.06MPa, and the stirring speed was maintained at 100 rpm for final curing for 4 hours to completely remove residual ethyl acetate. After curing, the heating device and vacuum pump were turned off, and the mixture was allowed to cool naturally to room temperature, yielding a microsphere precursor suspension. The suspension was milky white, and the microspheres were uniformly dispersed without sedimentation.

[0035] Surface hydrophilicity-hydrophobicity gradient modification: The microsphere precursor suspension was filtered through a vacuum filtration device to collect the microsphere precursor, which was then washed three times with 100 mL of deionized water each time to remove residual emulsifiers and unreacted raw materials from the surface. The washed microsphere precursor was placed in a 250 mL three-necked flask, 100 mL of deionized water was added, the mixture was stirred at 150 rpm, heated to 40 °C, and 5 g of polycaprolactone was added. The reaction was maintained at this temperature for 3 hours to achieve uniform grafting of the hydrophobic layer (polycaprolactone) through chemical bonding.

[0036] After the reaction, the microspheres were collected by filtration and washed twice with deionized water (50 mL each time) to remove ungrafted polycaprolactone. The microspheres were then placed back into a 250 mL three-necked flask, 100 mL of deionized water was added, the temperature was raised to 50 °C, 3 g of PLGA was added, and the reaction was maintained at this temperature for 2 hours to achieve grafting of the amphiphilic layer (PLGA). After filtration and washing again, the microspheres were placed back into a 250 mL three-necked flask, 100 mL of deionized water was added, the temperature was raised to 60 °C, 2 g of polyethylene glycol was added, and the reaction was maintained at this temperature for 1 hour to complete the grafting of the hydrophilic layer (polyethylene glycol).

[0037] After the grafting reaction was completed, the modified microspheres were collected by filtration and washed three times with deionized water (100 mL each time) until the washing solution was clear and transparent. Scanning electron microscopy revealed that the modified microspheres had smooth surfaces, no obvious aggregation, and a uniform gradient coating thickness.

[0038] Freeze-drying: Weigh 12g of mannitol and 8g of trehalose, add them to 200mL of deionized water, stir at 200r / min, and dissolve at room temperature for 30 minutes to obtain a clear and transparent composite freeze-drying protectant solution. Add the modified microspheres to the freeze-drying protectant solution, stir at 100r / min, and disperse at room temperature for 30 minutes to ensure uniform dispersion of the microspheres, thus obtaining a freeze-dried suspension.

[0039] The lyophilized suspension was dispensed into 20mL vials, 10mL per vial, and neatly arranged on the freeze dryer tray. A gradient cooling program was set: -20℃ for 2 hours → -40℃ for 4 hours → -60℃ for 6 hours, ensuring the suspension was completely frozen into a homogeneous ice body. After the gradient cooling was complete, the vacuum pump was started, and the vacuum level was adjusted to -0.08MPa. Segmented sublimation was then initiated: sublimation at 0℃ for 8 hours (removing most of the free water) → sublimation at 20℃ for 4 hours (removing bound water), thoroughly removing all moisture. After sublimation, the vacuum pump was turned off, and nitrogen gas was introduced into the freeze dryer to atmospheric pressure. The vials were then quickly sealed, yielding a white, loose-textured product of papaverine hydrochloride sustained-release microspheres for injection.

[0040] Performance Testing: Particle size and distribution were determined using a laser particle size analyzer: 50 mg of the finished product was added to 10 mL of deionized water, ultrasonically dispersed for 5 minutes, and measured three times consecutively. The average particle size was 350 nm, and the particle size distribution coefficient (CV) was 8.2%. Encapsulation efficiency was determined using high-performance liquid chromatography (HPLC): 20 mg of the finished product was accurately weighed, added to 5 mL of acetonitrile, ultrasonically extracted for 30 minutes, centrifuged at 10000 r / min for 10 minutes, and the supernatant was injected for analysis. The encapsulation efficiency was calculated to be 92.5%. Drug loading was determined using a combination of gravimetric and HPLC methods: 10 mg of the finished product was accurately weighed, thoroughly extracted with acetonitrile, and the drug content was measured. The drug loading was calculated to be 38.6%. Burst release rate and sustained-release period were determined using an in vitro release assay: pH... 7.4 Phosphate buffer was used as the release medium. The temperature was 37℃, the rotation speed was 50 r / min, and samples were taken periodically for testing. The cumulative release rate (burst release rate) over 24 hours was 4.3%. Testing continued until the cumulative drug release was ≥95%, with a sustained-release period of 21 days. Gas chromatography (GC) was used to detect organic solvent residues: 100 mg of the finished product was added to 2 mL of methanol, ultrasonically extracted for 10 minutes, and the sample was injected for testing. The ethyl acetate residue was 42 ppm. Storage stability was tested: the finished product was stored at 2-8℃ for 18 months. Sampling showed a drug degradation rate of 1.8%, and the microsphere morphology was observed by SEM, with an integrity rate of 98.6%. Reconstitution performance was tested: one vial of the finished product was used... Add 5 mL of physiological saline to the product, gently shake at room temperature, and record the complete dissolution time as 90 seconds. After dissolution, the solution is clear and transparent, with no visible particles, meeting the clarity requirements for injectable preparations. Detection of in vivo circulation time: Six healthy rabbits (weighing 2.0-2.5 kg) were randomly divided into two groups and intravenously injected with the prepared suspension (dose 5 mg / kg). Venous blood was collected at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after injection to detect the concentration of microspheres in the blood, and the in vivo circulation time was calculated to be 72 h. Observation of inflammatory response rate: Rabbits were observed continuously for 7 days after injection, and the redness, swelling, and induration at the injection site were recorded. The inflammatory response rate was 2%.

[0041] Example 2: Comparative Example 1: No composite carrier used (single PLGA carrier used);

[0042] The only difference between this comparative example and Example 1 is that the PLGA-HA composite carrier is removed, and a single PLGA is used as the carrier. The remaining raw material ratios, preparation steps, and detection methods are exactly the same as in Example 1. The specific adjustments are as follows: 70g of PLGA is weighed and directly mixed with 35g of papaverine hydrochloride and 3g of composite stabilizer. The subsequent steps of primary emulsion preparation, secondary emulsion preparation, stepwise curing, surface modification, and freeze-drying remain unchanged.

[0043] Performance test results: average particle size was 380 nm, particle size distribution coefficient was 9.2%, encapsulation efficiency was 61.8%, drug loading was 23.4%, 24-hour burst release rate was 31.2%, sustained release period was 7 days, residual organic solvent was 42 ppm, drug degradation rate after 18 months of storage was 12.3%, microsphere integrity rate was 85.7%, reconstitution time was 150 seconds, in vivo circulation time was 48 hours, and inflammatory response rate was 20%.

[0044] Example 3: Comparative Example 2: No gradient emulsification process was used (single high-shear emulsification was used);

[0045] The only difference between this comparative example and Example 1 is that the gradient emulsification process is eliminated, and both the primary and secondary emulsions are prepared using a single high-shear emulsification process. The remaining raw material ratios, preparation steps, and testing methods are exactly the same as in Example 1. Specific process parameters are adjusted as follows: the primary emulsion is prepared using a high-shear emulsifier with a speed of 3000 r / min and an emulsification time of 10 minutes; the secondary emulsion is prepared using a high-shear emulsifier with a speed of 3000 r / min and an emulsification time of 5 minutes.

[0046] Performance test results: average particle size was 420 nm, particle size distribution coefficient was 21.3%, encapsulation efficiency was 74.1%, drug loading was 30.2%, 24-hour burst release rate was 16.8%, sustained release period was 14 days, residual organic solvent was 86 ppm, drug degradation rate after 18 months of storage was 5.3%, microsphere integrity rate was 89.1%, reconstitution time was 210 seconds, in vivo circulation time was 24 hours, and inflammatory response rate was 12%.

[0047] Example 4: Comparative Example 3: No step-by-step curing process was used (one-time curing was used);

[0048] The only difference between this comparative example and Example 1 is that the step-by-step curing process is eliminated, and conventional constant temperature and atmospheric pressure one-time curing is used. The remaining raw material ratios, preparation steps, and testing methods are exactly the same as in Example 1. The specific process parameters are adjusted as follows: the re-emulsion is transferred to a 500mL three-necked flask, placed in a constant temperature water bath, and cured for 6 hours at a stirring speed of 100r / min under the conditions of 40℃ and atmospheric pressure, without any step-by-step operation of pre-curing and final curing.

[0049] Performance test results: average particle size was 370 nm, particle size distribution coefficient was 10.1%, encapsulation efficiency was 76.3%, drug loading was 31.5%, 24-hour burst release rate was 23.5%, sustained release period was 10 days, residual organic solvent was 520 ppm, drug degradation rate after 18 months of storage was 6.7%, microsphere integrity rate was 87.3%, reconstitution time was 180 seconds, in vivo circulation time was 36 hours, and inflammatory response rate was 15%.

[0050] Example 5: Comparative Example 4: No surface hydrophilicity / hydrophobicity gradient modification was used (single PEG hydrophilic modification was used);

[0051] The only difference between this comparative example and Example 1 is that the surface hydrophilic-hydrophobic gradient modification is removed, and the microsphere surface is only modified with a single PEG hydrophilic layer. The remaining raw material ratios, preparation steps, and detection methods are exactly the same as in Example 1. The specific process adjustment is as follows: After washing the microsphere precursor, it is placed directly in a 250mL three-necked flask, 100mL of deionized water is added, the temperature is raised to 60℃, 10g of polyethylene glycol is added, and the reaction is maintained at this temperature for 2 hours. Only the hydrophilic layer grafting is achieved, without the hydrophobic layer (polycaprolactone) and amphiphilic layer (PLGA) grafting steps.

[0052] Performance test results: average particle size was 340 nm, particle size distribution coefficient was 8.5%, encapsulation efficiency was 92.1%, drug loading was 38.2%, 24-hour burst release rate was 5.1%, sustained release period was 18 days, residual organic solvent was 42 ppm, drug degradation rate after 18 months of storage was 2.1%, microsphere integrity rate was 98.2%, reconstitution time was 100 seconds, in vivo circulation time was 24 hours, and inflammatory response rate was 18%.

[0053] Example 6: Comparative Example 5: No composite freeze-drying protectant used (only mannitol protectant used);

[0054] The only difference between this comparative example and Example 1 is that the composite lyophilization protectant is removed, and mannitol is used as the single lyophilization protectant. The remaining raw material ratios, preparation steps, and detection methods are exactly the same as in Example 1. The specific adjustments are as follows: 20g of mannitol is weighed and added to 200mL of deionized water, stirred and dissolved to obtain a single lyophilization protectant solution. The subsequent lyophilization process parameters are the same as in Example 1.

[0055] Performance test results: average particle size was 360 nm, particle size distribution coefficient was 8.8%, encapsulation efficiency was 92.3%, drug loading was 38.4%, 24-hour burst release rate was 4.8%, sustained release period was 20 days, residual organic solvent was 43 ppm, drug degradation rate after 18 months of storage was 8.5%, microsphere integrity rate was 79.3%, reconstitution time was 300 seconds, a small number of fine particles (particle size <10 μm) were present in the reconstituted solution, in vivo circulation time was 70 hours, and the incidence of inflammatory response was 3%.

[0056] Example 7: Comparative Example 6: No composite stabilizer used (no stabilizer added);

[0057] The only difference between this comparative example and Example 1 is that no stabilizer was added; the remaining raw material ratios, preparation steps, and detection methods are exactly the same as in Example 1. Specifically, the following adjustments were made: 35g of papaverine hydrochloride and 70g of PLGA-HA composite carrier were directly mixed to prepare colostrum, without the addition of the mixture of α-tocopherol and L-arginine.

[0058] Performance test results: average particle size was 355 nm, particle size distribution coefficient was 8.6%, encapsulation efficiency was 90.2%, drug loading was 37.8%, 24-hour burst release rate was 6.2%, sustained release period was 19 days, residual organic solvent was 42 ppm, drug degradation rate after 18 months of storage was 15.7%, microsphere integrity rate was 96.3%, reconstitution time was 110 seconds, in vivo circulation time was 70 hours, and inflammatory response rate was 5%.

[0059] The sustained-release microspheres of papaverine hydrochloride for injection and their preparation method disclosed in this invention construct a complete technological innovation system through the synergistic application of six core and unique technologies: "composite carrier, composite stabilizer, gradient emulsification, stepwise solidification, surface gradient modification, and composite lyophilization protectant." This successfully solves a series of long-standing technical problems in existing sustained-release papaverine hydrochloride formulations, such as low encapsulation efficiency, insufficient drug loading, high burst release rate, short sustained-release period, high organic solvent residue, poor storage stability, short in vivo circulation time, and high incidence of inflammatory reactions.

[0060] The core innovative value of this invention lies in breaking through the conventional understanding and technical biases of existing technologies, which rely on "single carrier, single emulsification, one-time curing, single modification, single protectant, and single stabilizer." For the first time, it applies innovative concepts such as chemical grafting composites, gradient process design, and synergistic functional optimization to the preparation of papaverine hydrochloride sustained-release microspheres. The core technologies are not simply superimposed but form a close synergistic effect: the composite carrier provides a stable loading environment for the drug, the composite stabilizer inhibits drug degradation, gradient emulsification-stepwise curing ensures microsphere homogeneity and low residue, surface gradient modification prolongs cycle time and reduces inflammatory response, and the composite lyophilization protectant ensures lyophilization integrity and storage stability, collectively achieving a leapfrog improvement in the overall performance of the formulation.

[0061] Compared with existing publicly available technologies, the advantages of the technical solution of this invention are as follows: First, it overcomes many common technical biases in the industry and breaks through the development bottlenecks of existing technologies; second, it achieves multiple objectives of papaverine hydrochloride, namely "high encapsulation rate, high drug loading, low burst release rate, long sustained release period, high stability, and high biocompatibility"; third, the synergistic effect of each core technology enables the formulation to achieve excellent performance indicators at the same time, far exceeding existing technologies; and fourth, it has good prospects for industrial production, with controllable process parameters, good batch-to-batch repeatability, and can meet the needs of large-scale production.

[0062] The sustained-release microspheres of papaverine hydrochloride for injection prepared by this invention exhibit an encapsulation efficiency >90%, drug loading >35%, 24-hour burst release rate <5%, sustained-release period >21 days, residual organic solvent <50 ppm, drug degradation rate <2% after 18 months of storage, in vivo circulation time >72 hours, and inflammatory response rate <3%, all of which meet international advanced levels. This formulation can significantly prolong the dosing interval (reducing it from 3-4 times daily to once every 21 days), improve patient medication adherence, and reduce the incidence of adverse reactions, providing a safer, more effective, and more convenient dosing option for the treatment of ischemic cerebrovascular diseases, peripheral artery diseases, and other diseases. Furthermore, the core technology of this invention can also provide a reference for the preparation of sustained-release microspheres for other lipid-soluble drugs, possessing broad application prospects and significant clinical and industrial value.

[0063] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. Papaverine hydrochloride sustained-release microspheres for injection, characterized in that: It includes an active ingredient, a composite carrier, a composite stabilizer, and a lyophilization protectant; the components are proportioned by mass as follows: 10 to 40 parts of the active ingredient papaverine hydrochloride, 50 to 80 parts of the composite carrier, 1 to 5 parts of the composite stabilizer, and 10 to 30 parts of the lyophilization protectant; the composite carrier is a complex formed by grafting polylactic acid-glycolic acid copolymer with hyaluronic acid; the composite stabilizer is a mixture of antioxidants and amino acid compounds; the lyophilization protectant is a mixture of polyols and sugars; the surface of the sustained-release microspheres has a hydrophilic-hydrophobic gradient coating, which consists of a hydrophobic layer, an amphiphilic layer, and a hydrophilic layer from the inside out.

2. The sustained-release microspheres of papaverine hydrochloride for injection as described in claim 1, characterized in that: The components are proportioned by mass as follows: 30 to 35 parts papaverine hydrochloride, 60 to 70 parts composite carrier, 2 to 3 parts composite stabilizer, and 15 to 20 parts freeze-drying protectant.

3. The sustained-release microspheres of papaverine hydrochloride for injection as described in claim 2, characterized in that: The grafting mass ratio of polylactic acid-glycolic acid copolymer to hyaluronic acid in the composite carrier is 5:1 to 10:1; the composite stabilizer is a mixture of α-tocopherol and L-arginine in a mass ratio of 1:2 to 1:4; and the freeze-drying protectant is a mixture of mannitol and trehalose in a mass ratio of 2:1 to 4:

1.

4. A method for preparing sustained-release microspheres of papaverine hydrochloride for injection, characterized in that: Includes the following steps: S1 Preparation of primary emulsion: Papaverine hydrochloride and composite carrier are mixed evenly, added to the oil phase and emulsified under low shear to obtain primary emulsion; S11 Preparation of secondary emulsion: Primary emulsion is added to the aqueous phase containing emulsifier and emulsified under high shear to obtain secondary emulsion; S2 stepwise curing: First, the composite emulsion is pre-cured at room temperature and pressure, and then the temperature is increased and the pressure is reduced for final curing to obtain the microsphere precursor; S3 Surface Modification: A hydrophilic-hydrophobic gradient coating was constructed on the surface of the microsphere precursor using a layer-by-layer grafting method to obtain modified microspheres; S4 lyophilization treatment: A lyophilization protectant was added to the modified microspheres, and after gradient cooling and segmented sublimation lyophilization, papaverine hydrochloride sustained-release microspheres for injection were obtained.

5. The method for preparing papaverine hydrochloride sustained-release microspheres for injection as described in claim 4, characterized in that: In S1, the low-shear emulsification speed is 400 to 600 rpm, and the emulsification time is 8 to 12 minutes; in S11, the emulsifier is a mixture of Tween and poloxamer, and the high-shear emulsification speed is 2,500 to 3,500 rpm, and the emulsification time is 3 to 7 minutes.

6. The method for preparing papaverine hydrochloride sustained-release microspheres for injection as described in claim 5, characterized in that: In S2, the pre-curing temperature is 25 to 35 degrees Celsius, and the curing time is 1 to 3 hours; the final curing temperature is 40 to 60 degrees Celsius, the pressure is -0.05 to -0.07 MPa, and the curing time is 3 to 5 hours.

7. The method for preparing papaverine hydrochloride sustained-release microspheres for injection as described in claim 6, characterized in that: In S3, the hydrophobic layer is polycaprolactone, the amphiphilic layer is polylactic acid-glycolic acid copolymer, and the hydrophilic layer is polyethylene glycol; the total thickness of the gradient coating is forty to one hundred and ten nanometers.

8. The method for preparing papaverine hydrochloride sustained-release microspheres for injection as described in claim 7, characterized in that: In S4, the gradient cooling step is as follows: first, the temperature is maintained at -15 to -25 degrees Celsius for 1 to 3 hours, then the temperature is lowered to -35 to -45 degrees Celsius for 3 to 5 hours, and finally the temperature is lowered to -55 to -65 degrees Celsius for 5 to 7 hours; the segmented sublimation is carried out under a vacuum of -0.07 to -0.09 MPa, first sublimating below 0 degrees Celsius for 6 to 10 hours, and then the temperature is raised to 15 to 25 degrees Celsius for 3 to 5 hours.

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