Sustained-release microsphere preparation containing dihydrocannabidiol di(hetero)arylate derivative, and preparation method and application thereof

Abstract

The application relates to the field of biological medicine, in particular to a sustained-release microsphere preparation containing di(hetero)aromatic formate derivatives of dihydrocannabidiol and a preparation method and application thereof. The preparation takes biodegradable polylactic acid-glycolic acid copolymer as a carrier, and encapsulates an active ingredient PSF1-5 in the microspheres. The application solves the problems that existing cancer pain treatment drugs need frequent administration and have obvious side effects. Pharmacodynamic studies show that in the mouse plantar cancer pain model established by the application, a single subcutaneous injection of the microsphere preparation of the application can continuously produce a significant analgesic effect for up to 14 days and above, and the analgesic effect appears in the early stage of administration and is independent of the antitumor effect. Safety evaluation shows that the active ingredient of the application has no mental dependence or physical dependence, and has no addiction risk. The sustained-release microsphere preparation of the application provides a new strategy for long-term, effective and safe management of cancer pain.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to sustained-release microsphere formulations containing dihydrocannabidiol di(hetero)carbamate derivatives, their preparation methods, and applications. Background Technology

[0002] Cancer pain is one of the most common complications in patients with malignant tumors, seriously affecting their quality of life, treatment adherence, and even survival prognosis. Its etiology is complex, involving multiple mechanisms such as direct compression and invasion of peripheral nerves by tumor tissue, local inflammatory response caused by tumor growth, and tissue destruction caused by tumor metastasis (such as bone metastasis). It often manifests as persistent, moderate to severe chronic pain.

[0003] For moderate to severe cancer pain, opioids (such as morphine and oxycodone) are the standard treatment choice, with definite analgesic effects. However, opioids generally suffer from rapid onset but short duration of action, requiring multiple daily doses to maintain effective blood drug concentrations. This not only inconveniences patients and caregivers, but more importantly, the dramatic fluctuations in blood drug concentrations (peak-trough effect) are a major cause of adverse drug reactions and unstable efficacy. Furthermore, long-term use of opioids also faces the risks of tolerance development (requiring continuously increased doses to achieve the same analgesic effect), drug dependence (addiction), and serious adverse reactions such as constipation, nausea, vomiting, and respiratory depression, significantly limiting their application in the long-term management of chronic pain.

[0004] The applicant has previously developed a series of dihydrocannabidiol (DHC) di(hetero)carbamate derivatives, namely PSF1-5, see patents CN116983265A (PSF2), CN116983266A (PSF4), CN116983267A (PSF3), CN116983263A (PSF1), and CN116983264A (PSF5). These patents confirm that PSF1-5, as a DHC di(hetero)carbamate derivative, has a protective effect against oxidative damage to nerve cells and can alleviate insulin resistance. However, the aforementioned prior art does not disclose the application of PSF1-5 in pain treatment, nor does it disclose whether it is addictive.

[0005] To overcome the limitations of short-acting analgesics, the development of long-acting sustained-release analgesics has become an important direction in drug research and development. An ideal long-acting formulation should be able to maintain a stable and effective blood drug concentration for a relatively long period of time (several days to several weeks) with a single dose, thereby achieving continuous pain control, improving patient compliance, and reducing side effects caused by frequent dosing and fluctuations in blood drug concentration, while also possessing good safety, especially no risk of addiction.

[0006] Polylactic acid-glycolic acid copolymer (PLGA) is a biodegradable polymer material approved by the U.S. Food and Drug Administration (FDA). Due to its excellent biocompatibility, biodegradability (ultimately metabolized into carbon dioxide and water in vivo), and adjustable degradation rate, PLGA has been widely used as a drug delivery carrier, successfully leading to the development of various marketed long-acting injectable formulations. Encapsulating drugs in PLGA microspheres and administering them subcutaneously or intramuscularly creates a "drug reservoir" at the injection site, achieving sustained release for weeks or even months through the slow degradation of the polymer and drug diffusion.

[0007] Dihydrocannabidiol (PSF1-5) di(hetero)carbamate derivatives are a class of novel compounds with potential analgesic activity. However, these compounds are generally poorly water-soluble, and if administered in their free form, they may suffer from short in vivo half-lives and low bioavailability, making it difficult to achieve a long-lasting effect. Developing them into stable, long-acting analgesics that meet clinical needs is a key problem that must be solved to advance them from candidate compounds to clinical application. Currently, there is no publicly available technology for preparing long-acting sustained-release microsphere formulations of the PSF1-5 series compounds for the treatment of cancer pain. Summary of the Invention

[0008] To address the shortcomings of the existing technology, the present invention aims to provide a novel long-acting analgesic formulation, which involves encapsulating dihydrocannabidiol di(hetero)carbamate derivatives (PSF1-5) with analgesic activity in biodegradable PLGA microspheres to prepare a subcutaneous sustained-release microsphere formulation, aiming to achieve long-term, stable control of cancer pain through a single dose. The present invention also provides a method for preparing this sustained-release microsphere formulation and its application in pain treatment.

[0009] This invention is achieved using the following technical solution: According to a first aspect of the present invention, a sustained-release microsphere formulation comprising a di(hetero)arcarbamate derivative of dihydrocannabidiol is provided. The core components of this formulation are a di(hetero)arcarbamate derivative of dihydrocannabidiol of general formula I as the active ingredient, and a biodegradable polymer as the sustained-release matrix material. This combination allows the active ingredient to be encapsulated within a polymer matrix, forming microspheres capable of slowly releasing the drug.

[0010] Specifically, the formulation comprises a di(hetero)carbamate derivative of dihydrocannabidiol or a pharmaceutically acceptable salt thereof as the active ingredient, and a biodegradable polymer as a carrier; The general structural formula of the di(hetero)arylformate derivative of dihydrocannabidiol is shown in Formula I below:

[0011] Formula I; Where R can be any of the following structures: (1); (2); (3); (4);

[0016] (5).

[0017] As a preferred embodiment, the biodegradable polymer is polylactic-co-glycolic acid copolymer (PLGA). The degradation rate of PLGA can be precisely controlled by changing the ratio of lactic acid (LA) and glycolic acid (GA), the molecular weight, and the terminal groups (such as ester-capped or carboxyl-capped), thereby allowing the design of microsphere formulations with specific release cycles according to the properties of different drugs and clinical needs.

[0018] The microspheres prepared by this invention possess specific physical properties. Preferably, the average particle size of the microspheres is controlled within the range of 50-100 μm. This particle size range has significant clinical implications: on the one hand, microspheres of this size are large enough that they are not easily phagocytosed by macrophages after subcutaneous injection, allowing them to remain stably at the injection site to form a drug reservoir and ensure long-lasting release; on the other hand, microspheres of this size are also small enough to be easily injected through conventional injection needles, exhibiting good injection performance and minimal irritation to local tissues.

[0019] Among the compounds involved in this invention, experimental data show that dihydrocannabidiimidazolium carbamate (PSF1) exhibits particularly superior analgesic activity. Therefore, in a preferred embodiment, the active ingredient is specifically PSF1.

[0020] According to a second aspect of the present invention, a method for preparing the above-mentioned sustained-release microsphere formulation is provided. This method typically employs a mature and stable emulsification-solvent evaporation method, specifically comprising the following steps: (a) dissolving the active ingredients (PSF1-5) and PLGA in a water-immiscible organic solvent (such as dichloromethane) to form a homogeneous oil phase; (b) dispersing the oil phase under high-speed shearing or stirring in an aqueous phase containing an emulsifier (such as polyvinyl alcohol, PVA) to form a stable oil-in-water (O / W) emulsion; (c) evaporating the organic solvent in the oil phase by stirring or vacuum evaporation, causing the PLGA to precipitate and solidify, encapsulating the drug within to form solid microspheres; (d) collecting the microspheres by centrifugation or filtration, and repeatedly washing them with deionized water to remove residual emulsifier, unencapsulated drug, and organic solvent, finally obtaining the final microsphere powder by freeze-drying or conventional drying. This method offers good process controllability and high repeatability, enabling the preparation of microspheres with narrow particle size distribution and high encapsulation efficiency, making it suitable for industrial production.

[0021] According to a third aspect of the invention, a pharmaceutical application of the above-mentioned sustained-release microsphere formulation is provided. Specifically, its application is in the preparation of a medicament for treating pain, particularly cancer pain. The key to this invention is that by formulating PSF1-5 into PLGA sustained-release microspheres, a long-term release of the drug in vivo is achieved. The preferred route of administration is subcutaneous injection. After a single subcutaneous injection, the formulation can form a drug reservoir at the injection site, continuously releasing an effective concentration of the drug for up to one week or even longer, thereby achieving long-term analgesia.

[0022] The long-acting analgesic effect of the formulation of this invention is based on two levels: PLGA microspheres, as drug carriers, primarily utilize an initial "burst release" and subsequent sustained release mechanism. The burst release mainly originates from the drug adhering to the microsphere surface, while sustained release is dominated by two synergistic processes: first, drug molecules diffuse through the porous network of the PLGA polymer matrix; second, PLGA itself undergoes ester bond hydrolysis under the influence of water and enzymes, resulting in polymer chain breakage and degradation, thereby exposing the encapsulated drug. By precisely controlling the properties of PLGA and the microsphere preparation process, the rates of these two processes can be modulated, achieving stable drug release within a predetermined timeframe (e.g., 7-14 days).

[0023] This invention, through rigorous animal behavioral experiments, confirms that the analgesic effect of PSF1-5 has an intrinsic pharmacological basis, and is not merely a byproduct of its antitumor effect. Experimental data show that in the early stages of administration (e.g., day 3) of high-dose PSF1 in tumor-bearing mice, before significant changes in the size of the plantar tumors, the mechanical and thermal pain thresholds were already significantly improved.

[0024] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention achieves sustained drug release by preparing PSF1-5 into PLGA sustained-release microspheres. Experimental results show that a single subcutaneous injection of the formulation of this invention can produce a significant analgesic effect lasting for 14 days or more in a mouse cancer pain model. Its analgesic level is comparable to the peak effect of daily morphine, and there is no late-stage drug efficacy decay. This avoids the inconvenience of multiple daily administrations of traditional analgesics, and improves the convenience of medication and patient compliance.

[0025] (2) By comparing the analgesic effect with the time progression of tumor volume change, this invention strongly demonstrates the direct analgesic effect of the active ingredient. Experimental data show that on the 3rd day after administration, the mechanical pain threshold of the Example 1 group was significantly increased, while the foot volume was not statistically different from that of the model control group. This phenomenon of "analgesia being faster than tumor inhibition" indicates that this invention produces significant analgesia in the early stages of administration, and that this early analgesic effect is not dependent on the reduction of tumor volume, but has a direct analgesic mechanism independent of anti-tumor effects.

[0026] (3) Through a systematic review of the PSF1-5 series compounds, this invention clarified that their analgesic activity gradient in a cancer pain model is: PSF1>PSF5>PSF2≈PSF3≈PSF4, and that high doses of PSF2 / 3 / 4 are comparable to the positive control drug CBD. This provides a clear basis for screening lead compounds for subsequent drug development, among which PSF1 shows the best potential.

[0027] (4) This invention also demonstrates, through conditional place preference (CPP) and withdrawal behavior experiments, that the active ingredient PSF1-5 does not have psychological or physical dependence. This excellent safety profile, especially its non-addictive nature, gives it a significant clinical advantage over opioids, which pose a serious risk of addiction, in the long-term management of chronic pain (especially cancer pain), and addresses a major clinical pain point related to opioid addiction. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] Experimental materials and conditions: Unless otherwise stated, the experimental materials and instruments used in the embodiments of this invention are as follows: Active drug: Bis(hetero)arylcarboxylate derivatives of dihydrocannabidiol (PSF1, PSF2, PSF3, PSF4, PSF5), purity ≥98%, provided by the Institute of Agro-Products Processing, Chinese Academy of Agricultural Sciences.

[0030] Main excipients: Poly(lactic-co-glycolic) acid copolymer (PLGA, molecular weight 10,000 - 30,000, ester-terminated, LA:GA = 50:50); Polyvinyl alcohol (PVA, injection grade); Mannitol (injection grade); Dichloromethane (analytical grade).

[0031] Control drugs: Morphine hydrochloride injection; Cannabidiol (CBD, purity ≥98%).

[0032] Experimental animals: SPF-grade female Kunming mice, 6 - 8 weeks old, weighing 18 - 22 g, purchased from Spf (Beijing) Biotechnology Co., Ltd. [Production license number: SCXK (Beijing) 2024 - 0001]. The animals were housed in an SPF-grade barrier environment at a temperature of 22 - 25 °C, with a 12 h light / dark cycle, and free access to food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee.

[0033] Cell line: Mouse hepatoma H22 cell line, purchased from the Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection.

[0034] Main instruments: High-speed shear emulsifier, ultrasonic cleaner, rotary evaporator, high-speed refrigerated centrifuge, vacuum freeze dryer, laser particle size analyzer, electronic vernier caliper, hot plate, VonFrey analgesimeter.

[0035] I. General preparation method of sustained-release microspheres The sustained-release microsphere preparation described in the present invention can be prepared by conventional methods in the art, preferably by the oil-in-water (O / W) emulsion-solvent evaporation method. A typical preparation process includes the following steps: (a) Preparation of the oil phase: Weigh accurately a certain mass of the active ingredient (such as one of PSF1 - 5) and the biodegradable polymer (such as PLGA), and their mass ratio is preferably 1:5 to 1:20, more preferably 1:10. Add them to an appropriate amount of organic solvent (such as dichloromethane, ethyl acetate, chloroform, etc., preferably dichloromethane), and dissolve them completely by stirring, ultrasonic treatment, etc. to obtain a clear oil-phase solution. The ultrasonic conditions can be, for example, treating at a power of 200 W for 15 minutes at room temperature.

[0036] (b) Emulsification: An aqueous solution containing an emulsifier is used as a continuous aqueous phase. The emulsifier may be selected from polyvinyl alcohol (PVA), poloxamer, polysorbate, etc., preferably injection-grade polyvinyl alcohol, and its concentration in the aqueous phase is preferably 0.1%-2.0% (w / v), more preferably 0.5% (w / v). The volume ratio of the oil phase to the aqueous phase is preferably 1:2 to 1:10, more preferably 1:4. Under low-temperature conditions (e.g., an ice bath at 0-4°C), the oil phase is slowly added to the rapidly stirred aqueous phase at a controlled rate (e.g., 1.0 mL / min) using a high-speed shearing or homogenizing device. The shearing speed can be adjusted according to the desired particle size, for example, 9000 r / min, and shearing is continued until a uniform O / W emulsion is formed.

[0037] (c) Solvent evaporation and curing: Remove the low-temperature conditions and continue stirring at room temperature (e.g., 20-25°C) at a low speed (e.g., 300 r / min) for several hours (e.g., 4 hours) to allow most of the organic solvent to evaporate. To completely remove residual solvent, rotary evaporation under reduced pressure can be used, for example, rotary evaporation at 40°C and -0.08 MPa for 15 minutes. After solvent evaporation, the polymer solidifies to form drug-loaded microspheres.

[0038] (d) Collection, washing, and drying of microspheres: The solidified microsphere suspension is collected by centrifugation (e.g., 8000 r / min, 10 min) or filtration. The collected microspheres are resuspended and centrifuged multiple times (e.g., 3 times) with purified or deionized water to remove residual emulsifiers and unencapsulated drugs. After washing, the microspheres can be added to a suitable cryoprotectant (e.g., mannitol, sucrose, trehalose, etc., preferably a 5% w / v mannitol solution) to disperse them evenly, and then freeze-dried to obtain loose lyophilized microsphere powder. The freeze-drying process may include pre-freezing (e.g., -45°C, 4 hours), primary drying (e.g., vacuum 10 Pa, -20°C, 24 hours), and secondary drying (e.g., vacuum 5 Pa, 10°C, 8 hours).

[0039] II. Physicochemical Properties Characterization of Microspheres (a) Particle size and distribution determination: Take an appropriate amount of freeze-dried microsphere powder, redissolve and disperse it with purified water or a suitable dispersion medium, and then use a laser particle size analyzer to determine its particle size and polydispersity index (PDI).

[0040] (b) Morphological observation: Take an appropriate amount of freeze-dried microsphere powder, fix it on conductive adhesive, and after gold sputtering, observe its surface morphology and spheric regularity using a scanning electron microscope (SEM).

[0041] (c) Encapsulation efficiency and drug loading determination: Accurately weigh a certain amount (W) 微球Drug-loaded microspheres were completely dissolved in an appropriate amount of an organic solvent capable of dissolving the polymer (such as dichloromethane) to break the emulsion. Then, an extraction solution immiscible with the organic solvent (such as water or buffer) was added to extract the drug, or the solvent was evaporated and the microspheres were dissolved in the mobile phase. The drug concentration (C) in the solution was determined using high-performance liquid chromatography (HPLC), and the actual mass of drug encapsulated in the microspheres (W) was calculated. 实际 =C×V). Based on the theoretical dosage during preparation (W_theoretical), calculate the encapsulation efficiency and drug loading using the following formula: Encapsulation efficiency (%) = (W 实际 / W 理论 ) × 100%; Drug loading (%) = (W 实际 / W 微球 ) × 100%; Exemplary HPLC detection conditions were as follows: a C18 column (4.6 mm × 250 mm, 5 μm), acetonitrile-water (70:30, v / v) as the mobile phase, a detection wavelength of 210 nm, a flow rate of 1.0 mL / min, and a column temperature of 30 °C. Under these conditions, the main peaks and impurity peaks were well separated.

[0042] III. Establishment of Cancer Pain Models and Methods for Efficacy Evaluation To verify the analgesic effect of the formulation of the present invention, a mouse foot transplant tumor cancer pain model recognized in the art was used.

[0043] (a) Cell Culture and Preparation: Mouse hepatocellular carcinoma H22 cell line was cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells in the logarithmic growth phase were amplified by intraperitoneal inoculation and ascites fluid extraction. Ascites fluid cells were collected, washed with sterile physiological saline, counted, and their viability was confirmed to be above 95% using trypan blue staining. Finally, the cells were resuspended in sterile physiological saline and the cell concentration was adjusted to 6 × 10⁶ cells / mL. 7 Quantity / mL, keep on ice for later use.

[0044] (b) Animal modeling: SPF-grade female Kunming mice (6-8 weeks old, 18-22g) were used, and the sole of their right hind paw was exposed. 20 μL of the above H22 cell suspension (containing 1.2 × 10⁻⁶ cells) was aspirated using a microsyringe. 6 (1 cell), injected subcutaneously into the center of the sole of the foot. The blank control group was injected with an equal volume of sterile saline.

[0045] (c) Model Successful Confirmation: Observe daily after model establishment and measure foot volume at specific time points (e.g., days 3, 5, and 7). Approximately 7 days after model establishment, compare behavioral indicators before and after model establishment. If the mechanical pain threshold and thermal pain threshold of tumor-bearing mice are significantly lower than those of the blank control group (P<0.05), and the foot volume is significantly increased, it indicates that the cancer pain model has been successfully established and can be used for subsequent pharmacodynamic evaluation.

[0046] (d) Analgesia behavioral testing: Mice need to acclimatize to the testing environment for at least 30 minutes before any behavioral tests.

[0047] Mechanical pain threshold (PWMT) determination: Von Frey fibers were used, and the "up-and-down" method was employed. Mice were placed in a test box on an elevated metal mesh. The right hind paw of the mouse was vertically stimulated from below using a series of Von Frey fibers with specific weights. The minimum weight that elicited rapid paw withdrawal, licking, or avoidance in the mouse was taken as its mechanical stimulation response threshold.

[0048] Pain Threshold at Heat (PWTL) Measurement: A hot plate apparatus was used, with the surface temperature of the instrument maintained at 55±0.5℃. Mice were placed on the hot plate, and the time from contact with the hot plate to the appearance of a pain response such as licking the hind paw or jumping was recorded; this is the heat withdrawal latency. To avoid tissue damage, a maximum transection time (e.g., 30 seconds) should be set.

[0049] Foot volume measurement: The thickness (t) and width (w) of the mouse's right hind foot were measured using electronic vernier calipers, and the result was calculated using the formula V = 0.52 × t × w. 2 Calculate the volume of the sole of the foot to monitor tumor growth.

[0050] (e) Statistical analysis: All experimental data are expressed as mean ± standard deviation (Mean ± SD). Statistical software such as SPSS was used for analysis. One-way ANOVA was used for comparisons between groups, and P < 0.05 was used as the criterion for statistical significance.

[0051] Example 1: Preparation of PSF1PLGA sustained-release microspheres In this embodiment, PSF1PLGA sustained-release microspheres were prepared using an oil-in-water (O / W) emulsification-solvent evaporation method.

[0052] (1) Preparation of oil phase: Accurately weigh 1.0 g of active drug PSF1 and 10.0 g of polylactic acid-glycolic acid copolymer (PLGA, molecular weight 10000-30000, ester-terminated, LA:GA=50:50), and place them in 100 mL of dichloromethane (analytical grade). At room temperature, use an ultrasonic cleaner at 200 W power for 15 minutes to sonicate until the drug and PLGA are completely dissolved, and a clear oil phase solution is obtained.

[0053] (2) Preparation of aqueous phase: Weigh injection grade polyvinyl alcohol (PVA), add purified water, heat to 40°C and stir at low speed to dissolve completely. After cooling to room temperature, make up to 400 mL of 0.5% (w / v) PVA aqueous solution with purified water as the continuous aqueous phase.

[0054] (3) Emulsification: Place the aqueous phase obtained in step (2) in an ice bath at 0°C. Start the high-speed shear emulsifier and set the shearing speed to 9000 r / min. Add the oil phase obtained in step (1) slowly and uniformly to the high-speed sheared aqueous phase at a constant rate of 1.0 mL / min using a peristaltic pump or syringe pump. After the oil phase is completely added, continue shearing under these conditions for 5 minutes to form a water-in-oil (O / W) type primary emulsion with uniform particle size distribution.

[0055] (4) Solidification and Washing: Remove the ice bath and transfer the colostrum to a stirring paddle. Stir continuously for 4 hours at 300 r / min (room temperature 25°C) to allow most of the dichloromethane in the oil phase to evaporate. Subsequently, transfer the microsphere suspension to a rotary evaporator and evaporate under reduced pressure at 40°C and -0.08 MPa for 15 minutes to completely remove residual organic solvents and solidify the microspheres. Centrifuge the solidified microsphere suspension at 8000 r / min and 4°C for 10 minutes, discard the supernatant, and collect the microsphere precipitate. Resuspend the precipitate in sterile deionized water and repeat this centrifugation and washing step 3 times to remove residual PVA and unencapsulated drug.

[0056] (5) Freeze-drying: Collect the microsphere precipitate after final washing and weigh its wet weight. Add a pre-prepared 5% (w / v) mannitol aqueous solution, sterilized by a 0.22 μm filter membrane, at a ratio of 1 g: 20 mL of wet weight of precipitate to freeze-drying protectant solution. Gently stir at 150 r / min for 10 minutes to ensure uniform dispersion of the microspheres in the freeze-drying protectant. Quantitatively dispense the microsphere suspension into sterile vials, place them in a vacuum freeze dryer, and freeze-dry according to the following procedure: first, pre-freeze at -45℃ for 4 hours; then perform a first sublimation drying under vacuum of 10 Pa and shelf temperature of -20℃ for 24 hours; finally, perform a second desorption drying under vacuum of 5 Pa and shelf temperature of 10℃ for 8 hours. After drying, seal the vials under sterile conditions to obtain the PSF1PLGA sustained-release microsphere freeze-dried formulation.

[0057] Examples 2-5: Preparation of PSF2, PSF3, PSF4, PSF5 PLGA sustained-release microspheres Equal molar amounts of PSF2 (Example 2), PSF3 (Example 3), PSF4 (Example 4), and PSF5 (Example 5) were used to replace the active drug PSF1 in Example 1, while the remaining excipients, dosages, and all preparation steps were exactly the same as those in Example 1. The lyophilized PLGA sustained-release microsphere preparations of the corresponding PSF2, PSF3, PSF4, and PSF5 were prepared respectively.

[0058] Control Example 1: Preparation of blank PLGA microspheres The same formulation and process as in Example 1 were used for preparation. The only difference was that no active drug (i.e., no PSF1) was added to the oil phase in step (1), and only 10.0 g of PLGA was dissolved in 100 mL of dichloromethane. The remaining steps were the same as those in Example 1, and finally, drug-free blank PLGA microspheres were prepared.

[0059] Control Example 2: Preparation of positive control drug (morphine hydrochloride) solution Take morphine hydrochloride injection and dilute it to the required concentration with sterile normal saline before administration to meet the intraperitoneal injection dosage of 3 mg / kg and ensure the injection volume is the same for each mouse. The solution was freshly prepared daily.

[0060] Control Example 3: Preparation of CBD control drug solution Take CBD raw material (purity ≥ 98%), dissolve it with an appropriate amount of pharmaceutical-grade co-solvent (such as a mixture of Tween-80 and ethanol), and then dilute it with sterile normal saline to the required concentration to meet the intraperitoneal injection dosage of 20 mg / kg and ensure the injection volume is the same for each mouse. The solution was freshly prepared daily.

[0061] Experimental Example 1: Physicochemical property characterization of the microsphere preparation of the present invention and pharmacodynamic evaluation in a mouse model of cancer pain 1. Experimental materials and instruments Experimental animals: SPF-grade female Kunming mice, 6 - 8 weeks old, weighing 18 - 22 g, purchased from Beijing Specif Biological Technology Co., Ltd., production license number: SCXK (Beijing) 2024 - 0001. The animals were housed in an SPF-grade animal room at a temperature of 22 - 25 °C, with a 12 h light / 12 h dark cycle, and free access to food and water. All animal experiment protocols were approved by the Experimental Animal Ethics Committee.

[0062] Cell line: Mouse hepatocarcinoma H22 cell line, purchased from the Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection.

[0063] Drugs and reagents: PSF1-5 (purity ≥98%, provided by the Institute of Agricultural Product Processing, Chinese Academy of Agricultural Sciences); PLGA (molecular weight 10,000-30,000, ester-terminated, LA:GA=50:50); polyvinyl alcohol (PVA, injection grade); mannitol (injection grade); dichloromethane (analytical grade); morphine hydrochloride injection (Shenyang No.1 Pharmaceutical Co., Ltd., Northeast Pharmaceutical Group, specification 1mL:10mg); Von Frey fiber kit; sodium pentobarbital; RPMI 1640 culture medium; fetal bovine serum.

[0064] Main instruments: High-speed shear emulsifier; ultrasonic cleaner; rotary evaporator; high-speed refrigerated centrifuge; vacuum freeze dryer; laser particle size analyzer; electronic vernier caliper (accuracy 0.01mm); hot plate apparatus; Von Frey analgesic device; CO2 incubator; clean bench.

[0065] 2. Physicochemical characterization of microsphere formulations (1) Particle size determination: Take a small amount of freeze-dried microsphere powder prepared in Examples 1-5, add an appropriate amount of purified water to disperse it fully, and use a laser particle size analyzer to determine the average particle size and polydispersity index (PDI) of the microspheres.

[0066] (2) Encapsulation efficiency determination: Accurately weigh an appropriate amount of the lyophilized microsphere powder prepared in Examples 1-5, add sufficient dichloromethane to completely dissolve and demulsify it. After centrifugation, take the supernatant and determine the content of PSF1-5 by high performance liquid chromatography (HPLC) to calculate the actual drug loading in the microspheres. Calculate the encapsulation efficiency according to the formula: Encapsulation efficiency (%) = (Actual drug loading in microspheres / Theoretical drug loading) × 100%.

[0067] Characterization results: The microspheres prepared in Examples 1-5 were all spherical in shape with smooth surfaces. The average particle size of the microspheres of each component was 65.3 ± 8.7 μm (range 62.8-68.1 μm for each component), and the polydispersity index (PDI) was less than 0.2, indicating a narrow particle size distribution. The average encapsulation efficiency was 84.6% ± 5.2% (range 82.1%-87.4% for each component, n=3). The above results indicate that the preparation process adopted in this invention is stable and reliable, and can obtain sustained-release microsphere formulations with uniform physicochemical properties that meet the requirements for subcutaneous injection.

[0068] 3. Establishment of an animal model of cancer pain (1) Preparation of H22 cells: The frozen H22 cell line was rapidly thawed in a 37℃ water bath and cultured in RPMI 1640 complete medium containing 10% fetal bovine serum and 1% penicillin antibiotics at 37℃ and 5% CO2. Logarithmic growth phase cells were collected and passaged in ascites fluid of 4-6 week old male Kunming mice via intraperitoneal inoculation. After approximately 7-10 days, ascites fluid was aseptically aspirated, and cells were collected by centrifugation. After washing twice with sterile physiological saline, cell counts were performed using a cell counting chamber, and cell viability was confirmed to be above 95% by trypan blue staining. Finally, the cell volume was adjusted to 6×10⁶ cells / mL with sterile physiological saline. 7 Quantity / mL, keep on ice for later use.

[0069] (2) Foot injection model: Take the above-mentioned SPF-grade female Kunming mice, fix their right hind limbs by hand to fully expose the soles of their feet. Disinfect the injection area with 75% alcohol swabs. Use a 30-gauge microsyringe to draw 20 μL of the above-mentioned H22 cell suspension (containing 1.2 × 10⁻⁶ cells). 6 (1 cell), injected subcutaneously from the center of the sole of the foot. The blank control mice were injected with the same volume of sterile saline at the same site.

[0070] (3) Model confirmation: Animal behavior was observed daily after modeling, and foot volume was measured on days 3, 5, and 7. If the foot volume of tumor-bearing mice increased significantly compared to before modeling by day 7 after modeling, and their mechanical pain threshold and thermal pain threshold decreased significantly compared to the blank control group (P<0.05), the cancer pain model was considered to have been successfully established and could be used for subsequent grouping and drug administration experiments.

[0071] 4. Animal grouping and administration Mice with successfully established models were randomly divided into 16 groups of 10 mice each using a random number table. The grouping and administration regimens are shown in Table A. The microsphere formulations of Examples 1-5 and Comparative Example 1 were resuspended in sterile saline to the required concentration before administration and administered via single subcutaneous injection. The drug solutions of Comparative Examples 2 and 3 were administered intraperitoneally once daily for 14 consecutive days. Animal grouping and administration regimens are shown in Table 1.

[0072] Table 1: Animal grouping and dosing regimen

[0073] Pharmacodynamic evaluation of cancer pain models: A cancer pain model was established in mice by inoculating the footpads of mice with H22 hepatocellular carcinoma cells. After successful modeling, mice were randomly divided into groups and administered either a single subcutaneous injection of the microsphere formulations of each example, or a blank microsphere formulation of Comparative Example 1, or daily intraperitoneal injections of Comparative Example 2 (morphine) or Comparative Example 3 (CBD). The mechanical pain threshold (PWMT), thermal pain threshold (PWTL), and footpad volume of the mice were measured at different time points after drug administration.

[0074] The effects on the mechanical pain threshold (PWMT) of each group of mice at different time points are shown in Table 2.

[0075] Table 2: Comparison of mechanical pain threshold (PWMT, g) at different time points in each group of mice (Mean±SD, n=10)

[0076] Table 2 shows that the mechanical pain threshold of the model control group (Comparative Example 1) remained low throughout the 14 days, demonstrating the stability and reliability of the cancer pain model. While the positive control group, morphine (Comparative Example 2), had a rapid onset of action, it required daily administration and showed significant efficacy decline after day 10. In contrast, Examples 1-5 of this invention all demonstrated long-acting analgesia after a single dose. Specifically, Example 1 (PSF 120 mg / kg) raised the pain threshold to near-normal levels by day 3 and maintained this level until day 14, exhibiting superior long-acting analgesia compared to morphine and CBD, which require frequent administration. This result indicates that the formulation of this invention overcomes the short duration of action of traditional analgesics.

[0077] The effects on the pain threshold (PWTL) of heat were shown in Table 3 for each group of mice at different time points.

[0078] Table 3: Comparison of thermal pain threshold (PWTL, s) at different time points in each group of mice (Mean±SD, n=10)

[0079] As shown in Table 3, the thermal pain threshold data are consistent with the mechanical pain threshold results, further verifying the analgesic activity of the formulation of this invention. The model group maintained a persistently low pain threshold, while Example 1 also exhibited a strong and sustained analgesic effect, with its thermal pain threshold on day 14 still comparable to the peak effect of morphine, further demonstrating its good long-lasting effect.

[0080] The effects on paw volume and the independence analysis of analgesic effect were shown in Table 4.

[0081] Table 4: Foot volume (mm²) of mice in each group at different time points 3 Compare (Mean ± SD, n = 10)

[0082] As can be seen from Table 4, it confirms the key to the direct analgesic effect of the present invention. The data shows that on the 3rd day after administration, there was no statistical difference in the paw volume of the group of Example 1 (PSF1 120 mg / kg) compared with the model control group. However, at the same time point, the pain threshold of this group had been significantly reversed. This time difference strongly proves that the analgesic effect of the preparation of the present invention does not stem from its inhibition of tumor growth, but from the direct pharmacological action of the active drug itself.

[0083] In summary, the present invention successfully prepared PLGA sustained-release microspheres containing di (hetero) aromatic acid ester derivatives (PSF1-5) of cannabidiol. The results of pharmacodynamic evaluation confirmed that after single subcutaneous injection, this microsphere preparation could produce an analgesic effect lasting at least 14 days in a mouse model of cancer pain. Among them, PSF1 had the most excellent activity, and its analgesic efficacy was comparable to that of morphine administered daily and was superior to CBD. Among PSF2, PSF3, and PSF4, high doses also showed significant long-term analgesic activity, comparable to the effect of CBD.

[0084] Experimental Example 2: Evaluation of non-addiction of the active ingredients PSF1-5 of the present invention To evaluate the drug addiction of the active ingredients PSF1-5 of the present invention, the present invention conducted a conditioned place preference (CPP) experiment to evaluate its psychological dependence and evaluated its physical dependence after withdrawal through an open field test (OFT) and an elevated plus maze test (EPM).

[0085] 1. Experimental materials and methods 1.1 Experimental animals SPF-grade male Kunming mice, 6-8 weeks old, weighing 25-30 g, were purchased from Beijing Spearf Bio-Technology Co., Ltd. [Production License No.: SCXK (Beijing) 2024-0001]. The animals were housed in an SPF-grade animal room at a temperature of 22-25 °C, with a 12 h light / 12 h dark cycle, and free access to food and water. All animal experiments followed the relevant regulations of the Experimental Animal Ethics Committee.

[0086] 1.2 Drugs and reagents Each component of PSF1-5 (purity ≥ 98%, provided by the Institute of Agro-Products Processing, Chinese Academy of Agricultural Sciences); morphine hydrochloride injection (commercial specification, positive control drug); normal saline; DMSO (dimethyl sulfoxide, pharmaceutical grade); 75% alcohol.

[0087] Before administration, each component of PSF1, PSF2, PSF3, PSF4, and PSF5 was dissolved in DMSO respectively to prepare stock solutions, and were diluted to the required concentrations with normal saline before use (the final concentration of DMSO ≤ 5%). Each component was administered by intraperitoneal injection.

[0088] 1.3 Main instruments Conditional location preference box, open field test box, elevated cross maze and VisuTrack animal behavior video analysis software (Shanghai Xinruan Information Technology Co., Ltd.).

[0089] 1.4 Experimental grouping and dosing regimen Mice were randomly divided into the following groups, with 10 mice in each group: First batch: blank control group (physiological saline), solvent control group (5% DMSO), positive control group (morphine 3mg / kg), PSF1 group (10mg / kg), PSF5 group (10mg / kg).

[0090] The second batch consisted of: blank control group (physiological saline), solvent control group (5% DMSO), positive control group (morphine 3 mg / kg), PSF2 group (20 mg / kg), PSF3 group (20 mg / kg), and PSF4 group (20 mg / kg).

[0091] All medications were administered via intraperitoneal injection.

[0092] 2. Experimental Procedure 2.1 Conditional Place Preference (CPP) Experiment The experiment consisted of three phases: pre-test (3 days), training (12 days, drug-environment pairing), and post-test (1 day). The changes in the time mice spent in the drug-accompanied box before and after training (CPP score) were compared among the groups.

[0093] The training phase lasted for 12 days (days 1 to 12). A partition was inserted, and training was alternated between medication-accompanied and non-medication-accompanied boxes, 6 times each. On medication-accompanied box training days (days 1, 3, 5, 7, 9, and 11): Mice in each drug-treated group were intraperitoneally injected with the corresponding drug 30 minutes before training (PSF1-5 groups and the solvent control group were injected with the corresponding drug or 5% DMSO saline solution; the positive control group was injected with morphine; and the blank control group was injected with saline solution). After 30 minutes, the mice were placed in the medication-accompanied box and left for 35 minutes before being removed. On non-medication-accompanied box training days (days 2, 4, 6, 8, 10, and 12): Mice in all groups were not given any medication and were directly placed in the non-medication-accompanied box, left for 35 minutes, and then removed.

[0094] 2.2 Withdrawal Behavioral Experiments On days 7 and 8 after CPP training, the open field test (OFT) and elevated cross maze test (EPM) were conducted to evaluate voluntary activities and anxiety-like behaviors after drug withdrawal.

[0095] OFT metrics: total distance traveled, number of times entering the central zone, time spent in the central zone, average speed, and number of supporting stands.

[0096] EPM metrics: number of times the arm is opened and the dwell time in the arm.

[0097] 2.3 Statistical Analysis Experimental data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons among multiple groups; LSD method was used for pairwise comparisons when variances were homogeneous, and Dunnett's T3 method was used when variances were unequal. Paired t-tests were used to compare pre-test and post-test CPP scores. A p-value < 0.05 was considered statistically significant. GraphPad Prism 9.0 was used as the statistical software.

[0098] 3. Experimental Results 3.1 Results of the CPP experiment (evaluation of psychological dependence) The results are shown in Table 5. The positive control group (morphine) mice showed a significantly higher post-test CPP score than the pre-test (P<0.001), indicating a clear conditional position preference. However, the post-test CPP scores of all PSF1-5 groups showed no significant difference compared to the pre-test (P>0.05), comparable to the blank control group and the solvent control group. These results indicate that none of the components of PSF1-5 induces psychological dependence.

[0099] Table 5: Comparison of CPP scores before and after the test in each group of mice (s, Mean±SD, n=10)

[0100] 3.2 Results of the withdrawal behavior experiment (evaluation of physical dependence) Open field test (OFT) results (Table 6): The total range of motion and central area activity of mice in the morphine withdrawal group were significantly reduced, while the number of times they supported themselves on their feet was significantly increased (P<0.05), exhibiting typical anxiety-like behavior. There were no significant differences in any behavioral indicators of mice in the PSF1-5 groups compared with the blank control group (P>0.05).

[0101] Table 6: Comparison of Open Field Test (OFT) Results among Different Groups of Mice (Mean±SD, n=10)

[0102] The results of the elevated cross maze (EPM) test (Table 7) showed that the morphine withdrawal group had significantly less time spent in the open arm and fewer entries than the blank control group (P<0.05). There were no significant differences between the PSF1-5 groups and the blank control group (P>0.05).

[0103] Table 7: Comparison of Elevated Cross Maze (EPM) results among different groups of mice (Mean±SD, n=10)

[0104] In summary, the active ingredients PSF1-5 involved in this invention do not induce psychological dependence in the conditional position preference test, do not cause anxiety-like behavior after withdrawal, and do not induce physical dependence. These results strongly demonstrate that the PSF1-5 series of compounds do not have addictive potential and possess the good safety profile required for use as a chronic pain treatment drug.

Claims

1. A sustained-release microsphere formulation comprising a di(hetero)arylcarbamate derivative of dihydrocannabidiol, characterized in that, The formulation comprises a di(hetero)carbamate derivative of dihydrocannabidiol or a pharmaceutically acceptable salt thereof as the active ingredient, and a biodegradable polymer as a carrier. The general structural formula of the di(hetero)arylformate derivative of dihydrocannabidiol is shown in Formula I below: Formula I; Where R can be any of the following structures: （1）； （2）； （3）； （4）； （5）。 2. The sustained-release microsphere formulation according to claim 1, characterized in that, The biodegradable polymer is a polylactic acid-glycolic acid copolymer.

3. The sustained-release microsphere formulation according to claim 1 or 2, characterized in that, The average particle size of the microspheres is 50-100 μm.

4. The sustained-release microsphere formulation according to claim 1, characterized in that, The dihydrocannabidiol di(hetero)carbamate derivative is dihydrocannabidiol diimidazocarbamate.

5. A method for preparing the sustained-release microsphere formulation according to any one of claims 1-4, characterized in that, Includes the following steps: (a) The active ingredient and the biodegradable polymer are dissolved in an organic solvent to obtain an oil phase; (b) Dispersing the oil phase in an aqueous phase containing an emulsifier to form an oil-in-water emulsion; (c) The organic solvent is evaporated to remove the microspheres, thereby solidifying them; (d) Collect, wash and dry the microspheres.

6. The method according to claim 5, characterized in that, The method is an emulsification-solvent evaporation method.

7. The method according to claim 5, characterized in that, The organic solvent mentioned in step (a) is dichloromethane, and the emulsifier mentioned in step (b) is polyvinyl alcohol.

8. The application of a sustained-release microsphere formulation according to any one of claims 1-4, characterized in that, Used in the preparation of drugs for treating pain.

9. The application according to claim 8, characterized in that, The pain described is cancer pain.

10. The application according to claim 8 or 9, characterized in that, The drug is administered via subcutaneous injection and a single dose can produce a long-lasting analgesic effect for 14 days or more.

Images