Bupivacaine sustained-release microspheres, and preparation method and application thereof

Abstract

This invention provides bupivacaine microspheres with controllable in vitro release and long-lasting analgesia. The microspheres use lactide-glycolic acid copolymer (PLGA) as the carrier material and bupivacaine as the drug; they have high drug loading, encapsulation efficiency and good sustained-release effect.

Description

[0001] This application claims priority to an earlier application filed by the applicant with the China National Intellectual Property Administration on December 2, 2024, with patent application number 202411763456.3, entitled "A Bupivacaine Sustained-Release Microsphere and Its Preparation Method and Application". The entire contents of the earlier application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of pharmaceutical formulation technology, and relates to a bupivacaine sustained-release microsphere, its preparation method, and its application. Background Technology

[0003] Postoperative pain, also known as analgesia, is the pain felt by patients at or in the surgical site after surgery. It is a type of acute pain, primarily caused by the extreme trauma and / or organ damage resulting from the surgery. The peak pain period is generally 3-5 days post-surgery. If not controlled in the initial stage, it can easily develop into chronic postoperative pain (CPSP), severely impacting patients' quality of life and increasing morbidity and mortality. Currently used analgesics such as opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) often cause side effects such as nausea, vomiting, and gastrointestinal reactions, and do not provide targeted pain relief. Therefore, other long-acting anesthetics are needed for better analgesia.

[0004] Bupivacaine (BUP) is a commonly used amide-type local anesthetic in clinical practice, characterized by its strong anesthetic potency, long duration of action, and significant dissociation of sensory and motor blockade. BUP is widely used in spinal anesthesia, epidural anesthesia, brachial plexus blocks, and the treatment of postoperative anesthesia pain, as well as some long-term, severe, and cancer pain. Its chemical name is 1... n-Butyl 2 (2, 6) (dimethylaminoformyl)piperidine, with the molecular formula C 18 H 28 N2O. Bupivacaine has low solubility in water but is readily soluble in organic solvents such as methanol, ethanol, and dichloromethane. Clinically, bupivacaine hydrochloride is commonly used; it is a white crystalline powder, odorless, and bitter. Although bupivacaine hydrochloride is a long-acting local anesthetic, its onset time is only 3-6 hours, and its effect is dose-dependent. While frequent administration of small doses can prolong the duration of action, it increases the risk of drug-induced toxicity, such as neurotoxicity and cardiovascular toxicity. Therefore, developing a slow-release, long-acting bupivacaine formulation can effectively improve postoperative analgesia and address problems in chronic pain management.

[0005] Bupivacaine liposome injection, developed by Pacira Pharmaceuticals in the United States, was the first long-acting analgesic for direct injection at the surgical site. Approved for marketing in the US in 2011 under the brand name Exparal, it is approved for local postoperative wound infiltration analgesia after hemorrhoidectomy and bunionectomy. Multi-capsule liposomes encapsulating bupivacaine, produced using DepoFoam technology, can achieve analgesia for up to 72 hours in vivo, but literature review and animal studies have shown that it is only effective for about 24 hours. Due to the large injection volume of liposomes (specifications: 266mg / 20ml and 133mg / 10ml) and the cumbersome administration method (requiring separate injections into the surrounding tissue and subcutaneous tissue), Exparal's analgesic effect is not satisfactory for early postoperative periods or surgeries with severe postoperative pain.

[0006] Microsphere formulations are tiny spherical particle dispersion systems in which drugs are dispersed in a polymer matrix, typically with a particle size ranging from 1 to 250 μm. They represent a promising field for sustained-release and controlled-release formulations. The carrier materials used to prepare microspheres usually possess good biocompatibility and biodegradability. By adjusting the microsphere formulation and preparation process, the drug release rate and onset time can be controlled, thereby reducing dosing frequency and improving patient compliance. However, microsphere formulations still have some drawbacks, such as low drug loading, difficulty in controlling the release cycle, and insufficient duration of action. Controlling the release cycle of analgesic microspheres is crucial. Clinically, analgesia often needs to last 3 to 7 days postoperatively. During this period, controlling the slow release of anesthetic analgesics can help patients avoid postoperative pain and reduce the side effects of frequent dosing. Due to the aforementioned drawbacks, there are currently no commercially available analgesic microsphere formulations on the market.

[0007] Patent document CN113440496A discloses a method for preparing microspheres loaded with anesthetic and analgesic drugs, as well as the products and uses thereof. The method involves dissolving the hydrochloride salt of analgesic drugs in the inner aqueous phase using a W / O / W double emulsion method to prepare microspheres. However, if bupivacaine hydrochloride is selected, its high solubility in an aqueous physiological environment may lead to drug leakage, making it difficult to effectively control drug release. To prevent drug leakage, this method involves adding additives to both the inner aqueous and oil phases, while simultaneously adjusting the pH to dissolve the drug, followed by membrane treatment. This formulation and process are overly complex, increasing the difficulty of industrialization, and the drug release is too rapid, making it difficult to maintain the analgesic effect. Patent document CN116763741A discloses a bupivacaine microsphere, its preparation method, and its application. This method uses free bupivacaine as the encapsulating drug to increase the drug loading capacity. Bupivacaine microspheres are prepared using the O / W monoemulsion method. By adding poloxamer 188 as a pore-forming agent to the oil phase, the in vitro release rate is adjusted, which helps the microspheres to release rapidly in the early stage. However, the drug loading capacity of this method is too high, resulting in excessive burst release in vitro. The in vitro release exceeds 40% within 1 hour, and the drug effect in rats can only be maintained for 10 hours, which is insufficient to achieve the treatment time required for postoperative analgesia. Patent document CN106344521A discloses the preparation and application of biodegradable bupivacaine microspheres with high drug loading. It uses PLLA as a carrier material and prepares bupivacaine microspheres by O / W single emulsion method. By selecting high viscosity materials and adding Tween-80 to the aqueous phase, the microspheres are round and smooth with a drug loading of not less than 60%. However, it still has defects such as too rapid in vitro release in the early stage and insufficient release in the later stage due to the high molecular weight of PLLA, requiring a higher dose to maintain the efficacy. Summary of the Invention

[0008] To improve the above-mentioned technical problems, the present invention provides bupivacaine microspheres, comprising bupivacaine (BUP), lactide-glycolic acid copolymer (PLGA), surfactant and osmotic pressure regulator; wherein the weight ratio of BUP to PLGA is 10:90-40:60.

[0009] According to an embodiment of the present invention, the bupivacaine microspheres further include water.

[0010] According to an embodiment of the present invention, the surfactant is polyvinyl alcohol (PVA), such as PVA1788.

[0011] According to an embodiment of the present invention, the osmotic pressure regulator is selected from one or more of sodium chloride, potassium chloride, mannitol, sucrose, glucose and sodium citrate.

[0012] According to an embodiment of the present invention, the osmotic pressure regulator is sodium chloride.

[0013] According to an embodiment of the present invention, the weight-average molecular weight (Mw) of the PLGA is 5 kDa - 50 kDa, more preferably 10 kDa - 30 kDa, for example 16 kDa, 20 kDa, 26 kDa.

[0014] According to an embodiment of the present invention, the ratio of PLGA lactide to glycolide is 85:15-50:50, for example 75:25.

[0015] According to an embodiment of the present invention, the intrinsic viscosity of the PLGA is 0.10 dL / g to 0.30 dL / g, more preferably 0.12 dL / g to 0.20 dL / g, for example 0.15 dL / g, 0.21 dL / g, or 0.23 dL / g.

[0016] According to an embodiment of the present invention, the PLGA is purchased from Evonik Specialty Chemicals (Shanghai) Co., Ltd., and the model is 50:50 2A, 50:50 2.5A, 75:25 2CA; more preferably 75:25 2CA.

[0017] According to an embodiment of the present invention, the weight ratio of the BUP to the PLGA is 20:80-30:70.

[0018] According to an embodiment of the present invention, the total weight of BUP and PLGA, the weight of surfactant and the weight of osmotic pressure regulator are in the ratio of 1:(1-6):(2.1-2.7), for example 1:(2-5):(2.2-2.6), 1:(3-4):(2.3-2.5), such as 6.65:20:16.

[0019] According to an embodiment of the present invention, the particle size distribution d(50) of the bupivacaine microspheres is 20μm-50μm, for example 30μm-45μm, such as 33.1μm, 35.8μm, 36.4μm, 37.9μm, 44.4μm.

[0020] According to an embodiment of the present invention, the drug loading of the bupivacaine microspheres is 0.1%-40%, for example 10%-30%, such as 17.2%, 20.0%, 23.1%, 26.0%, 27.1%, or 28.1%.

[0021] According to an embodiment of the present invention, the encapsulation efficiency of the bupivacaine microspheres is 50%-98%, for example 70%-95%, such as 77.0%, 86%, 86.7%, 90.3%, or 93.7%.

[0022] This invention also provides a method for preparing the bupivacaine microspheres using a double emulsion-solvent evaporation method, comprising the following steps: A lactide-glycolic acid copolymer (PLGA) and bupivacaine (BUP) were dissolved in dichloromethane (DCM) to prepare an oil phase; a surfactant and an osmotic pressure regulator were dissolved in water to prepare an external aqueous phase; the oil phase, internal aqueous phase and external aqueous phase were combined to form a W / O / W type emulsion, and the emulsion was cured to obtain the bupivacaine microspheres.

[0023] According to an embodiment of the present invention, the ratio of the total mass of the BUP and PLGA to the volume of dichloromethane is 0.25-0.5 g / mL, for example 0.5 g / mL.

[0024] According to an embodiment of the present invention, the internal aqueous phase includes water.

[0025] According to an embodiment of the present invention, the volume ratio of the inner aqueous phase, dichloromethane and the outer aqueous phase is (0.01-1):1:(10-1000), for example (0.05-0.3):1:(100-300), such as 0.11:1:150.

[0026] According to an embodiment of the present invention, the concentration of the surfactant in the external aqueous phase is 0.2 g / mL to 4 g / mL, more preferably 0.5 g / mL to 2 g / mL, for example 1 g / mL.

[0027] According to an embodiment of the present invention, the concentration of the osmotic pressure regulator in the external aqueous phase is 0.7 g / mL - 0.9 g / mL, more preferably 0.75 g / mL - 0.85 g / mL, such as 0.8 g / mL. According to an embodiment of the present invention, the osmotic pressure regulator is sodium chloride. It is understood that, in embodiments of the present invention, 0.7 g / mL - 0.9 g / mL of sodium chloride can achieve technical effects equivalent to 0.8 g / mL of sodium chloride.

[0028] According to an embodiment of the present invention, the curing is first low-temperature curing followed by high-temperature curing. The low-temperature curing temperature can be 0℃-12℃, for example 4℃-10℃, and the low-temperature curing time can be 0.1h-2h, for example 0.5h, 1h, or 1.5h. The high-temperature curing temperature can be 25℃-35℃, for example 30℃, and the high-temperature curing time can be 1h-10h, for example 2h, 4h, 6h, or 8h.

[0029] According to an embodiment of the present invention, the method includes the following steps: (1) The oil phase was prepared by dissolving lactide-glycolic acid copolymer (PLGA) and bupivacaine (BUP) in dichloromethane (DCM); the external aqueous phase was prepared by dissolving surfactant and osmotic pressure regulator in water. (2) The aqueous phase and the oil phase are mixed and sheared to prepare the primary emulsion; (3) The colostrum is mixed with the external aqueous phase and sheared to prepare a compound emulsion; (4) The re-emulsion is solidified to prepare the bupivacaine microspheres.

[0030] According to an embodiment of the present invention, the initial emulsion shearing speed in step (2) is 5000rpm-20000rpm, more preferably 8000rpm-12000rpm; According to an embodiment of the present invention, in step (3), the volume ratio of the primary emulsion to the external aqueous phase is 1:100-1:150, preferably 1:100-1:120, such as 1:110; According to an embodiment of the present invention, the re-emulsification shearing speed in step (3) is 1200rpm-5000rpm, preferably 1800rpm-4000rpm, more preferably 2500rpm-3500rpm, such as 3000rpm; A bupivacaine microsphere comprises bupivacaine (BUP), lactide-glycolic acid copolymer (PLGA), and a surfactant; the bupivacaine microsphere does not include an osmotic pressure regulator; the weight ratio of BUP to PLGA is 10:90-40:60.

[0031] According to an embodiment of the present invention, the bupivacaine microspheres further include water.

[0032] According to an embodiment of the present invention, the surfactant is polyvinyl alcohol (PVA), such as PVA1788.

[0033] According to an embodiment of the present invention, the weight-average molecular weight (Mw) of the PLGA is 5 kDa - 50 kDa, more preferably 10 kDa - 30 kDa, for example 16 kDa, 20 kDa, 26 kDa.

[0034] According to an embodiment of the present invention, the ratio of PLGA lactide to glycolide is 85:15-50:50, for example 75:25.

[0035] According to an embodiment of the present invention, the intrinsic viscosity of the PLGA is 0.10~0.30 dL / g, more preferably 0.12 dL / g~0.20 dL / g, for example 0.15 dL / g, 0.21 dL / g, 0.23 dL / g.

[0036] According to an embodiment of the present invention, the PLGA is purchased from Evonik Specialty Chemicals (Shanghai) Co., Ltd., and the model is 50:50 2A, 50:50 2.5A, 75:25 2CA; more preferably 75:25 2CA.

[0037] According to an embodiment of the present invention, the weight ratio of the BUP to the PLGA is 20:80-30:70.

[0038] According to an embodiment of the present invention, the total weight ratio of BUP and PLGA to the surfactant weight is 1:(1-6), for example 1:(2-5), 1:(3-4), such as 6.65:20.

[0039] According to an embodiment of the present invention, the particle size distribution d(50) of the bupivacaine microspheres is 20μm-50μm, for example 30μm-45μm, such as 37.2μm, 38.5μm, 43.2μm.

[0040] According to an embodiment of the present invention, the drug loading of the bupivacaine microspheres is 0.1%-30%, for example 10%-25%, such as 13.0%, 18.2%, or 21.3%.

[0041] According to an embodiment of the present invention, the encapsulation efficiency of the bupivacaine microspheres is 50%-98%, for example 60%-75%, such as 60.7%, 65.0%, or 71.0%.

[0042] This invention also provides a method for preparing the bupivacaine microspheres, comprising the following steps: A lactide-glycolic acid copolymer (PLGA) and bupivacaine (BUP) were dissolved in a solvent to prepare an oil phase; a surfactant was dissolved in water to prepare an aqueous phase; the oil phase and the aqueous phase were combined to form an O / W emulsion, and the emulsion was cured to obtain the bupivacaine microspheres.

[0043] According to an embodiment of the present invention, the solvent is selected from one or more of dichloromethane and n-heptane.

[0044] According to an embodiment of the present invention, the solvent is selected from dichloromethane or a mixture of dichloromethane and n-heptane.

[0045] According to an embodiment of the present invention, the ratio of the total mass of the BUP and PLGA to the volume of the solvent is 0.25 g / mL to 0.5 g / mL, for example, 0.5 g / mL.

[0046] According to an embodiment of the present invention, the volume ratio of the solvent to the aqueous phase is 1:(10-1000), for example 1:(100-300), such as 1:140 or 1:150.

[0047] According to an embodiment of the present invention, the concentration of the surfactant in the aqueous phase is 0.2 g / mL to 4 g / mL, more preferably 0.5 g / mL to 2 g / mL, for example 1 g / mL.

[0048] According to an embodiment of the present invention, the shearing speed in step (2) is 1200rpm-5000rpm, preferably 1800rpm-4000rpm, more preferably 2500rpm-3500rpm, such as 3000rpm.

[0049] According to an embodiment of the present invention, the curing is first low-temperature curing followed by high-temperature curing. The low-temperature curing temperature can be 0℃-12℃, for example 4℃-10℃, and the low-temperature curing time can be 0.1h-2h, for example 0.5h, 1h, or 1.5h. The high-temperature curing temperature can be 25℃-35℃, for example 30℃, and the high-temperature curing time can be 1h-10h, for example 2h, 4h, 6h, or 8h.

[0050] According to an embodiment of the present invention, the method includes the following steps: (1) Prepare an oil phase by dissolving lactide-glycolic acid copolymer (PLGA) and bupivacaine (BUP) in a solvent; prepare an aqueous phase by dissolving a surfactant in water; (2) The oil phase and the aqueous phase are mixed and sheared to prepare an emulsion; (3) The emulsion is solidified to prepare the bupivacaine microspheres.

[0051] According to an embodiment of the present invention, the shearing speed in step (2) is 1200rpm-5000rpm, preferably 1800rpm-4000rpm, more preferably 2500rpm-3500rpm, such as 3000rpm.

[0052] The present invention also provides bupivacaine microspheres prepared by the method described above.

[0053] The present invention also provides a pharmaceutical composition comprising the bupivacaine microspheres.

[0054] The present invention also provides a bupivacaine formulation comprising the bupivacaine microspheres.

[0055] According to an embodiment of the present invention, the drug is formulated as a lyophilized powder for injection.

[0056] The present invention also provides the use of the bupivacaine microspheres or the pharmaceutical composition in the preparation of a drug.

[0057] According to an embodiment of the present invention, the drug is an anesthetic or analgesic, such as a drug for postoperative analgesia or relief of local pain.

[0058] According to an embodiment of the present invention, the drug is a sustained-release analgesic.

[0059] The pharmacokinetic (PK) test results of the bupivacaine microspheres described in this invention in rats showed that the blood concentration Cmax of bupivacaine could reach more than 200 ng / mL, and the blood concentration was maintained at 50 ng / mL within 72 hours, ensuring the analgesic effect. The in vivo efficacy test of the bupivacaine microspheres described in this invention showed that the analgesic duration could be maintained for 72h-120h; This invention also provides a method for preparing bupivacaine formulations, comprising the following steps: after sterilization and filtration of each phase solution, aseptic production, freeze-drying, sieving, transfer, powder dispensing, and capping are carried out on a sterile production line that has undergone sterility validation. Before administration, the sterile powder is dispersed in a sterile solvent, and after uniform dispersion, it is administered by local injection for postoperative analgesia or relief of local pain.

[0060] Beneficial effects (1) The present invention provides a bupivacaine microsphere with controllable in vitro release and long-lasting analgesia, wherein the microsphere uses lactide-glycolic acid copolymer (PLGA) as a carrier material and bupivacaine as a drug; (2) The present invention uses the W / O / W type double emulsion solvent evaporation method to prepare the bupivacaine microspheres, controls the drug loading of the microspheres to be 0.1%-40%, the encapsulation rate to be more than 75%, and adds NaCl to the external aqueous phase to adjust the osmotic pressure of the external aqueous phase, increase the microsphere pores, regulate the in vitro release rate, and make the drug be released completely at a uniform rate within 3-5 days. (3) The bupivacaine microspheres prepared by the O / W type single emulsion method of the present invention have high drug loading and encapsulation efficiency, and can play a certain sustained release effect.

[0061] (4) The analgesic effect of the present invention can be maintained for 72h-120h after in vivo administration, reducing the number of times bupivacaine is administered and the toxic side effects, and improving patient compliance with medication.

[0062] (5) The preparation process of the bupivacaine microspheres described in this invention is relatively simple, which reduces the difficulty of industrialization of microsphere formulation process. Attached Figure Description

[0063] Figure 1 These are microscope images of bupivacaine microspheres from Examples 1, 2, 3, and 4.

[0064] Figure 2 Microscopic images of bupivacaine microspheres in Examples 5, 6, 7, and 9.

[0065] Figure 3 The images shown are SEM images of the bupivacaine microspheres in Examples 3 and 4.

[0066] Figure 4 The images show the in vitro release curves of bupivacaine microspheres in Examples 1, 2, and 4.

[0067] Figure 5 The images show the in vitro release curves of bupivacaine microspheres in Examples 3, 4, 5, and 6.

[0068] Figure 6 The images show the in vitro release curves of bupivacaine microspheres in Examples 3, 7, 8, and 9.

[0069] Figure 7 The data are the drug-time curves after administration of bupivacaine microspheres to rats in Examples 1, 4, and 5. Detailed Implementation

[0070] The technical solutions of this disclosure will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of this disclosure and should not be construed as limiting the scope of protection of this disclosure. All technologies implemented based on the above content of this disclosure are covered within the scope of protection intended by this disclosure.

[0071] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0072] Additional notes regarding the polymers involved in the examples: PLGA: The preferred PLGA molecular weight range is defined in the invention description. Each embodiment specifies the molecular weight and type of PLGA used, manufactured by Evonik Specialty Chemicals (Shanghai) Co., Ltd. PVA: Model number 1788, molecular weight 130, no special specifications from the manufacturer.

[0073] DM represents particle size distribution d(50); DL represents drug loading; EE represents encapsulation efficiency.

[0074] Example 1 PLGA (LA:GA=75:25, 2CA, IV=0.15 dL / g, Mw=16 kDa) was used as the carrier, with a theoretical drug loading of 30%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 30:70, and the oil-water volume ratio is 1:133. The formulation composition is shown in Table 1.

[0075] Preparation method: Weigh 1.995g BUP and 4.655g PLGA and dissolve them in 13.3mL dichloromethane as the oil phase. Use 2000mL of 1% PVA (model 1788, molecular weight = 130) aqueous solution as the aqueous phase. Under shear conditions, add the oil phase to the aqueous phase at a uniform rate using a syringe. Emulsify at 3000rpm for 3min to form an O / W type emulsion. Stir and solidify at 4-12℃ for 0.5h, then heat to 30℃ for 4h. After solidification, wash the suspension multiple times with purified water and centrifuge to remove the supernatant to obtain bupivacaine microspheres. Transfer the microspheres to a vial for freeze-drying to obtain bupivacaine microsphere lyophilized powder. d(50) = 37.2μm, drug loading is 18.2%. In vitro, in pH 7.4 phosphate buffer, the cumulative release of bupivacaine in 2h is 9.9%, and the cumulative release of bupivacaine in 120h is 88.9%, indicating suitable sustained-release effect.

[0076] Example 2 PLGA (LA:GA=75:25, 2CA, IV=0.15 dL / g, Mw=16 kDa) was used as the carrier, with a theoretical drug loading of 20%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 20:80, and the oil-water volume ratio is 1:133. The formulation composition is shown in Table 1.

[0077] Bupivacaine microspheres were prepared using the microsphere preparation method described in Example 1, with a d(50) = 38.5 μm and a drug loading of 13.0%. In vitro, the cumulative release of bupivacaine in pH 7.4 phosphate buffer was 7.1% in 2 hours and 89.7% in 120 hours, indicating a suitable sustained-release effect.

[0078] Example 3 PLGA (LA:GA=75:25, 2CA, IV=0.15 dL / g, Mw=16 kDa) was used as the carrier, with a theoretical drug loading of 30%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 30:70, and the volume ratio of colostrum to external aqueous phase is 1:110. The formulation composition is shown in Table 1.

[0079] Preparation method: Weigh 1.995g BUP and 4.655g PLGA and dissolve them in 13.3mL dichloromethane as the oil phase. Accurately measure 1.5mL purified water as the inner aqueous phase and 2000mL 1%PVA + 0.8%NaCl aqueous solution as the outer aqueous phase. Under shear conditions, inject the inner aqueous phase into the oil phase and shear at 12000rpm for 2min. Then, under shear conditions, rapidly inject the primary emulsion into the outer aqueous phase and shear emulsify at 3000rpm for 3min to form a W / O / W type emulsion. Stir and solidify at 4-12℃ for 0.5h, and then heat to 30℃ for 4h. After that, wash the suspension multiple times with purified water and centrifuge to remove the supernatant to obtain bupivacaine microspheres. Transfer the microspheres to a vial for freeze-drying to obtain bupivacaine microsphere lyophilized powder. d(50) = 36.4μm, drug loading is 26.0%. In vitro, the cumulative release of bupivacaine in pH 7.4 phosphate buffer was 6.0% over 2 hours and 88.0% over 120 hours.

[0080] Example 4 PLGA (LA:GA=75:25, 2CA, IV=0.15 dL / g, Mw=16 kDa) was used as the carrier, with a theoretical drug loading of 20%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 20:80, and the volume ratio of colostrum to external aqueous phase is 1:110. The formulation composition is shown in Table 1.

[0081] Bupivacaine microspheres were prepared using the microsphere preparation method described in Example 3, with a d(50) = 35.8 μm and a drug loading of 17.2%. In vitro, the cumulative release of bupivacaine in pH 7.4 phosphate buffer was 7.0% in 2 hours and 90.4% in 120 hours. The microspheres prepared by this method have a good sustained-release effect, and bupivacaine is fully released within 3-5 days, meeting the clinical needs for postoperative analgesia.

[0082] Therefore, it can be seen that the preparation method has a certain influence on the in vitro release behavior. Figure 4 It can be seen that the double breast method (W / O / W) provides more complete sustained release in the early stage and more ideal release behavior in the later stage compared with the single breast method (O / W), which is more in line with the needs of postoperative analgesia.

[0083] Table 1. Prescriptions and quality evaluations for Examples 1-4

[0084] Example 5 PLGA (LA:GA=50:50, 2.5A, IV=0.23 dL / g, Mw=26 kDa) was used as the carrier, with a theoretical drug loading of 30%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 30:70, and the volume ratio of colostrum to external aqueous phase is 1:110. The formulation composition is shown in Table 2.

[0085] Using the preparation method in Example 3, bupivacaine microspheres with a diameter (d50) of 33.1 μm and a drug loading of 27.1% were obtained. In vitro, in pH 7.4 phosphate buffer, the cumulative release of bupivacaine was 4.8% in 2 hours and 81.0% in 120 hours, indicating a suitable sustained-release effect.

[0086] Example 6 PLGA (LA:GA=50:50, 2A, IV=0.21 dL / g, Mw=20 kDa) was used as the carrier, with a theoretical drug loading of 30%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 30:70, and the volume ratio of colostrum to external aqueous phase is 1:110. The formulation composition is shown in Table 2.

[0087] Using the preparation method in Example 3, bupivacaine microspheres with a diameter (d(50)) of 37.9 μm and a drug loading of 28.1% were obtained. In vitro, in pH 7.4 phosphate buffer, the cumulative release of bupivacaine was 3.9% in 2 hours, 53.5% in 72 hours, and 85.8% in 120 hours. The bupivacaine prepared by this method showed good sustained-release effect.

[0088] As can be seen from Examples 3, 4, 5, and 6, the PLGA model has a certain influence on the in vitro release behavior of bupivacaine microspheres. Based on the premise of relatively fast release and higher release completeness within 120 hours, PLGA75:25-2CA is preferred as the polymer carrier for bupivacaine microspheres.

[0089] Table 2. Prescriptions and quality evaluations for Examples 5 and 6

[0090] Example 7 PLGA (LA:GA=75:25, 2CA, IV=0.15 dL / g, Mw=16 kDa) was used as the carrier, with a theoretical drug loading of 30%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 30:70, and the volume ratio of colostrum to external aqueous phase is 1:110. The formulation composition is shown in Table 3.

[0091] Using the preparation method in Example 3, with a double emulsion shearing speed of 1800 rpm, the bupivacaine microspheres obtained had a d(50) = 44.4 μm, a drug loading of 23.1%, and an encapsulation efficiency of 77.0%. In vitro, in pH 7.4 phosphate buffer, the cumulative release of bupivacaine was 6.3% after 2 hours and 90.3% after 120 hours, indicating that the bupivacaine prepared by this method had a good sustained-release effect.

[0092] Example 8 PLGA (LA:GA=75:25, 2CA, IV=0.15 dL / g, Mw=16 kDa) was used as the carrier, with a theoretical drug loading of 50%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 50:50, and the volume ratio of colostrum to external aqueous phase is 1:110. The formulation composition is shown in Table 3.

[0093] Using the preparation method in Example 3, bupivacaine microspheres with a diameter (d) of 22.4 μm and a drug loading of 41.1% were obtained, with an encapsulation efficiency of 82.2%. In vitro, the cumulative release of bupivacaine in pH 7.4 phosphate buffer was 60.5% over 2 hours. Bupivacaine microspheres prepared with this drug loading did not have a sustained-release effect.

[0094] Example 9 PLGA (LA:GA=50:50, 2.5A, IV=0.23 dL / g, Mw=16 kDa) was used as the carrier, with a theoretical drug loading of 50%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 50:50, and the volume ratio of colostrum to external aqueous phase is 1:110. The formulation composition is shown in Table 3.

[0095] Using the preparation method in Example 3, bupivacaine microspheres with a diameter (d50) of 29.1 μm, a drug loading of 43.6%, and an encapsulation efficiency of 87.2% were obtained. In vitro, bupivacaine was completely released within 8 hours in pH 7.4 phosphate buffer, indicating that the bupivacaine microspheres prepared with this drug loading did not have a sustained-release effect.

[0096] As can be seen from Examples 3, 7, 8 and 9, the theoretical drug loading has a significant impact on the microsphere release behavior. If the drug loading is too high, the sustained release effect cannot be achieved. As the re-emulsification shear speed increases, the microsphere particle size decreases and the release rate accelerates. Based on the angle of higher release completeness within 120 hours, a theoretical drug loading of 20%-30% and a shear speed of 3000 rpm are preferred.

[0097] Table 3. Prescriptions and quality evaluations for Examples 7-9

[0098] Example 10 PLGA (LA:GA=75:25, 2CA, IV=0.15 dL / g, Mw=16 kDa) was used as the carrier, with a theoretical drug loading of 30%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 30:70, and the volume ratio of colostrum to external aqueous phase is 1:110. The formulation composition is shown in Table 4.

[0099] Using the microsphere preparation method described in Example 3, the NaCl content in the external aqueous phase was changed to 1%. Bupivacaine microspheres were obtained with d(50) = 25.7 μm and a drug loading of 14.4%. In vitro, in pH 7.4 phosphate buffer, the cumulative release of bupivacaine was 45.8% in 2 hours and 88.9% in 120 hours. The microspheres prepared by this method do not have a sustained-release effect and do not meet the clinical postoperative analgesia requirements.

[0100] Example 11 PLGA (LA:GA=75:25, 2CA, IV=0.15 dL / g, Mw=16 kDa) was used as the carrier, with a theoretical drug loading of 30%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 30:70, and the volume ratio of colostrum to external aqueous phase is 1:110. The formulation composition is shown in Table 4.

[0101] Using the microsphere preparation method described in Example 3, the NaCl content in the external aqueous phase was changed to 0.5%. Bupivacaine microspheres were obtained with d(50) = 26.5 μm and a drug loading of 10.8%. In vitro, in pH 7.4 phosphate buffer, the cumulative release of bupivacaine was 74.3% in 2 hours and 87.3% in 120 hours. The microspheres prepared by this method do not have a sustained-release effect and do not meet the clinical postoperative analgesia requirements.

[0102] Example 12 PLGA (LA:GA=75:25, 2CA, IV=0.15 dL / g, Mw=16 kDa) was used as the carrier, with a theoretical drug loading of 30%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 30:70, and the volume ratio of colostrum to external aqueous phase is 1:110. The formulation composition is shown in Table 4.

[0103] Using the microsphere preparation method described in Example 3, without adding NaCl to the external aqueous phase, bupivacaine microspheres were obtained with a d(50) = 28.7 μm and a drug loading of 11.8%. In vitro, in pH 7.4 phosphate buffer, the cumulative release of bupivacaine was 76.8% over 2 hours and 96.1% over 120 hours. The microspheres prepared by this method do not exhibit sustained-release effects and do not meet the clinical requirements for postoperative analgesia.

[0104] Examples 3, 10, 11, and 12 show that the addition of NaCl to the external aqueous phase and its content significantly affect the release behavior of bupivacaine microspheres. The absence of NaCl in the external aqueous phase leads to rapid drug release and lacks a sustained-release effect; therefore, a NaCl content of 0.8% in the external aqueous phase yields better results.

[0105] Table 4. Prescriptions and quality evaluations for Examples 10-12

[0106] Example 13 PLGA (LA:GA=75:25, 2CA, IV=0.15 dL / g, Mw=16 kDa) was used as the carrier, with a theoretical drug loading of 30%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 30:70, and the volume ratio of colostrum to external aqueous phase is 1:110.

[0107] Preparation method: Weigh 1.995g BUP and 4.655g PLGA and dissolve them in a mixed solvent of 10.0mL dichloromethane and 3.3mL ethyl acetate as the oil phase. Accurately measure 1.5mL purified water as the inner aqueous phase and 2000mL 1%PVA + 0.8%NaCl aqueous solution as the outer aqueous phase. Under shear conditions, inject the inner aqueous phase into the oil phase and shear at 12000rpm for 2min. Then, under shear conditions, rapidly inject the primary emulsion into the outer aqueous phase and shear emulsify at 3000rpm for 3min to form a W / O / W type emulsion. Stir and solidify at 4-12℃ for 0.5h, and then heat to 30℃ for 4h. After that, wash the suspension multiple times with purified water and centrifuge to remove the supernatant to obtain bupivacaine microspheres. Transfer the microspheres to a vial for freeze-drying to obtain bupivacaine microsphere lyophilized powder. d(50) = 25.1μm, drug loading is 13.9%. In vitro, bupivacaine was released at pH 7.4 phosphate buffer for 2 hours, with a cumulative release of 72.1% and 84.6% within 120 hours, indicating no sustained-release effect.

[0108] Example 14 PLGA (LA:GA=75:25, 2CA, IV=0.15 dL / g, Mw=16 kDa) was used as the carrier, with a theoretical drug loading of 50%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 50:50, and the volume ratio of colostrum to aqueous phase is 1:110.

[0109] Preparation method: Weigh 3.325g BUP and 3.325g PLGA and dissolve them in a mixed solvent of 10.0mL dichloromethane and 3.3mL ethyl acetate as the oil phase, and 2000mL 1% PVA aqueous solution as the aqueous phase. Under shear conditions, the oil phase is added to the aqueous phase at a uniform rate using a syringe. High-speed shear emulsification is carried out at 1600rpm for 3min to form an O / W type emulsion. The emulsion is stirred and solidified at 4-12℃ for 0.5h, and then solidified at 30℃ for 4h. The suspension is washed multiple times with purified water and the supernatant is removed by centrifugation to obtain bupivacaine microspheres. The microspheres are transferred to vials for freeze-drying to obtain bupivacaine microsphere lyophilized powder. d(50) = 36.1μm, and the drug loading is 44.8%. In vitro, in pH 7.4 phosphate buffer, the cumulative release of bupivacaine in 2h is 74.3%, and the cumulative release of bupivacaine in 120h is 98.4%, which does not have a sustained-release effect.

[0110] Example 15 PLGA (LA:GA=75:25, 2CA, IV=0.15 dL / g, Mw=16 kDa) was used as the carrier, with a theoretical drug loading of 30%, that is, the mass ratio of bupivacaine to carrier material (BUP:PLGA) is 30:70, and the volume ratio of colostrum to aqueous phase is 1:110.

[0111] Preparation method: 1.995g BUP and 4.655g PLGA were dissolved in a mixed solvent of 13.3mL dichloromethane and 1mL n-heptane as the oil phase, and 2000mL 1% PVA aqueous solution as the aqueous phase. Under shear conditions, the oil phase was added to the aqueous phase at a uniform rate using a syringe. High-speed shear emulsification was carried out at 1800rpm for 3min to form an O / W type emulsion. The emulsion was stirred and solidified at 4-12℃ for 0.5h, and then solidified at 30℃ for 4h. The suspension was washed multiple times with purified water, and the supernatant was removed by centrifugation to obtain bupivacaine microspheres. The microspheres were transferred to vials for freeze-drying to obtain bupivacaine microsphere lyophilized powder. d(50) = 43.2μm, and the drug loading was 21.3%. In vitro, in pH 7.4 phosphate buffer, the cumulative release of bupivacaine in 2h was 26.6%, and the cumulative release of bupivacaine in 120h was 91.3%, which showed a certain sustained-release effect.

[0112] Table 5. Prescriptions and quality evaluations for Examples 13-15

[0113] Example 16 Pharmacokinetic studies in Examples 1, 4, and 5 Experimental animals and grouping: Nine qualified male SD rats were selected and randomly divided into three groups of three rats each: Example 1 group (drug loading 18.2%, 28 mg / kg), Example 4 group (drug loading 17.2%, 40 mg / kg), and Example 5 group (drug loading 27.1%, 40 mg / kg). The subcutaneous injection volume was 2.67 mL / kg. Blood samples were collected at 0.5, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 96, and 120 h after administration to determine the blood drug concentration by LC-MS. The drug-time curves are shown in the figure.

[0114] Experimental results: Figure 7 It can be seen that Example 5 showed improved sustained-release effect and encapsulation efficiency compared to Example 1, indicating that the microspheres prepared by the double emulsion method had better release and encapsulation effects of bupivacaine. However, the initial release was low, making it difficult to maintain the analgesic effect. The bupivacaine microspheres prepared in Example 4 could sustain release for 120 hours after administration to rats. max The blood drug concentration reached 220 ng / mL, and remained at around 50 ng / mL within 48 hours, indicating that the formulation process has a good sustained-release effect.

[0115] Table 6. Pharmacokinetic parameters of bupivacaine (n=3, x±s)

[0116] The above description provides an exemplary account of the implementation methods of the technical solution disclosed herein. It should be understood that the scope of protection of this disclosure is not limited to the above-described embodiments. Any modifications, equivalent substitutions, or improvements made by those skilled in the art within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this application.

Claims

1. A bupivacaine microsphere comprising bupivacaine (BUP), lactide-glycolic acid copolymer (PLGA), a surfactant, and an osmotic pressure regulator; wherein the weight ratio of BUP to PLGA is 10:90-40:

60.

2. The bupivacaine microspheres according to claim 1, characterized in that, The bupivacaine microspheres also include water; Preferably, the surfactant is polyvinyl alcohol (PVA), such as PVA1788; Preferably, the osmotic pressure regulator is selected from one or more of sodium chloride, potassium chloride, mannitol, sucrose, glucose, and sodium citrate; Preferably, the weight-average molecular weight (Mw) of the PLGA is 5 kDa - 50 kDa, more preferably 10 kDa - 30 kDa; Preferably, the ratio of PLGA lactide to glycolide is 85:15-50:50, for example 75:25; Preferably, the intrinsic viscosity of the PLGA is 0.10 dL / g to 0.30 dL / g, more preferably 0.12 dL / g to 0.20 dL / g; Preferably, the weight ratio of the BUP to the PLGA is 20:80-30:70; Preferably, the weight ratio of the total weight of BUP and PLGA, the weight of the surfactant, and the weight of the osmotic pressure regulator is 1:(1-6):(2.1-2.7), for example 1:(2-5):(2.2-2.6), 1:(3-4):(2.3-2.5), such as 6.65:20:16; Preferably, the particle size distribution d(50) of the bupivacaine microspheres is 20μm-50μm, for example 30μm-45μm; Preferably, the drug loading of the bupivacaine microspheres is 0.1%-40%, for example 10%-30%; Preferably, the encapsulation rate of the bupivacaine microspheres is 50%-98%, for example 70%-95%.

3. The method for preparing bupivacaine microspheres according to claim 1 or 2, comprising the following steps: A lactide-glycolic acid copolymer (PLGA) and bupivacaine (BUP) were dissolved in dichloromethane (DCM) to prepare an oil phase; a surfactant and an osmotic pressure regulator were dissolved in water to prepare an external aqueous phase; the oil phase, internal aqueous phase, and external aqueous phase were combined to form a W / O / W type emulsion, and the emulsion was cured to obtain the bupivacaine microspheres; Preferably, the ratio of the total mass of the BUP and PLGA to the volume of dichloromethane is 0.25 g / mL - 0.5 g / mL, for example, 0.5 g / mL; Preferably, the volume ratio of the inner aqueous phase, dichloromethane, and outer aqueous phase is (0.01-1):1:(10-1000), for example (0.05-0.3):1:(100-300), such as 0.11:1:150; Preferably, the concentration of the surfactant in the external aqueous phase is 0.2 g / mL - 4 g / mL, more preferably 0.5 g / mL - 2 g / mL, for example 1 g / mL; Preferably, the concentration of the osmotic pressure regulator in the external aqueous phase is 0.7 g / mL - 0.9 g / mL, more preferably 0.75 g / mL - 0.85 g / mL, such as 0.8 g / mL; Preferably, the curing is performed by first curing at a low temperature and then curing at a higher temperature. The temperature for low-temperature curing can be 0℃-12℃, for example, 4℃-10℃, and the curing time can be 0.1h-2h. The temperature for curing at a higher temperature can be 25℃-35℃, and the curing time can be 1h-10h. Preferably, the internal aqueous phase contains water.

4. The method according to claim 3, characterized in that... The method includes the following steps: (1) The oil phase was prepared by dissolving lactide-glycolic acid copolymer (PLGA) and bupivacaine (BUP) in dichloromethane (DCM); the external aqueous phase was prepared by dissolving surfactant and osmotic pressure regulator in water. (2) The aqueous phase and the oil phase are mixed and sheared to prepare the primary emulsion; (3) The colostrum is mixed with the external aqueous phase and sheared to prepare a compound emulsion; (4) The composite emulsion is solidified to prepare the bupivacaine microspheres; Preferably, the initial shearing speed in step (2) is 5000rpm-20000rpm, more preferably 8000rpm-12000rpm; Preferably, in step (3), the volume ratio of the colostrum to the external aqueous phase is 1:100-1:150, more preferably 1:100-1:120, such as 1:110; Preferably, the re-emulsification shearing speed in step (3) is 1200rpm-5000rpm, and more preferably 1800rpm-4000rpm.

5. A bupivacaine microsphere comprising bupivacaine (BUP), lactide-glycolic acid copolymer (PLGA), and a surfactant; wherein the bupivacaine microsphere does not include an osmotic pressure regulator; Preferably, the bupivacaine microspheres further include water; Preferably, the surfactant is polyvinyl alcohol (PVA), such as PVA1788; Preferably, the weight-average molecular weight (Mw) of the PLGA is 5 kDa - 50 kDa, more preferably 10 kDa - 30 kDa; Preferably, the ratio of PLGA lactide to glycolide is 85:15-50:50, for example 75:25; Preferably, the intrinsic viscosity of the PLGA is 0.10 dL / g to 0.30 dL / g, more preferably 0.12 dL / g to 0.20 dL / g; Preferably, the weight ratio of the BUP to the PLGA is 20:80-30:70; Preferably, the total weight ratio of BUP and PLGA to the surfactant weight is 1:(1-6), for example 1:(2-5), 1:(3-4), such as 6.65:20; Preferably, the particle size distribution d(50) of the bupivacaine microspheres is 20μm-50μm, for example 30μm-45μm; Preferably, the drug loading of the bupivacaine microspheres is 0.1%-30%, for example 10%-25%; Preferably, the encapsulation rate of the bupivacaine microspheres is 50%-98%, for example 60%-75%.

6. The method for preparing the bupivacaine microspheres according to claim 5, comprising the following steps: A lactide-glycolic acid copolymer (PLGA) and bupivacaine (BUP) were dissolved in a solvent to prepare an oil phase; a surfactant was dissolved in water to prepare an aqueous phase; the oil phase and the aqueous phase were combined to form an O / W emulsion, and the emulsion was cured to obtain the bupivacaine microspheres. Preferably, the solvent is selected from one or more of dichloromethane and n-heptane; Preferably, the ratio of the total mass of BUP and PLGA to the volume of solvent is 0.25 g / mL - 0.5 g / mL, for example, 0.5 g / mL; Preferably, the volume ratio of the solvent to the aqueous phase is 1:(10-1000), for example 1:(100-300); Preferably, the concentration of the surfactant in the aqueous phase is 0.2 g / mL to 4 g / mL, more preferably 0.5 g / mL to 2 g / mL, for example 1 g / mL; Preferably, the curing is performed by first curing at a low temperature and then curing at a higher temperature. The temperature for low-temperature curing can be 0℃-12℃, for example, 4℃-10℃, and the curing time can be 0.1h-2h. The temperature for curing at a higher temperature can be 25℃-35℃, and the curing time can be 1h-10h. Preferably, the preparation method includes the following steps: (1) Prepare an oil phase by dissolving lactide-glycolic acid copolymer (PLGA) and bupivacaine (BUP) in a solvent; prepare an aqueous phase by dissolving a surfactant in water; (2) The oil phase and the aqueous phase are mixed and sheared to prepare an emulsion; (3) The emulsion is solidified to prepare the bupivacaine microspheres; Preferably, the shearing speed in step (2) is 1200rpm-5000rpm, and more preferably 1800rpm-4000rpm.

7. Bupivacaine microspheres prepared by the method according to any one of claims 3, 4 and 6.

8. A pharmaceutical composition comprising bupivacaine microspheres as described in any one of claims 1, 2, and 5.

9. A bupivacaine formulation comprising the bupivacaine microspheres according to any one of claims 1, 2, and 5; Preferably, the drug is in the form of a lyophilized powder for injection.

10. The use of the bupivacaine microspheres according to any one of claims 1, 2 and 5 or the pharmaceutical composition according to claim 8 in the preparation of a medicament; Preferably, the drug is an anesthetic or analgesic, such as a postoperative analgesic or a drug for relieving local pain; Preferably, the drug is a sustained-release analgesic.

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