Long-acting injectable formulations of di(hetero)arylic acid ester derivatives of dihydrocannabidiol and their use in perioperative analgesia

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a long-acting injection preparation of di(hetero)aromatic formate derivative containing dihydrocannabidiol and application thereof in perioperative analgesia. The long-acting injection preparation of the application shows rapid onset and sustained analgesic effect for up to 14 days, and the analgesic effect is significantly better than that of commonly used clinical drugs ibuprofen and tramadol. The di(hetero)aromatic formate derivative of dihydrocannabidiol is first confirmed from the mechanism level in the application to effectively protect the mitochondrial function of the spinal cord dorsal horn neurons damaged due to surgical trauma and stabilize the mitochondrial membrane potential. This indicates that the application can not only relieve the pain symptoms, but also inhibit the occurrence of postoperative central sensitization from the source, thereby possibly effectively preventing the formation of postoperative chronic pain.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to long-acting injectable formulations containing dihydrocannabidiol di(hetero)carbamate derivatives and their application in perioperative analgesia. Background Technology

[0002] Perioperative pain is one of the most common complications of surgery. Tissue trauma and local inflammation caused by surgical incisions, and the resulting peripheral and central sensitization, not only cause severe pain for patients but also induce sympathetic nerve excitation, exacerbate inflammatory stress, and disrupt immune function, hindering wound healing and early postoperative recovery, increasing the difficulty of intraoperative anesthesia management, prolonging hospital stays, and seriously affecting patient prognosis. Of particular concern is postoperative pain sensitization—manifested as persistent postoperative pain, hyperalgesia, abnormal pain, and prolonged pain—which not only severely reduces patients' postoperative quality of life but is also a significant contributing factor to the development of chronic postoperative neuropathic pain.

[0003] Currently, perioperative and postoperative analgesia in clinical practice mainly relies on opioids and nonsteroidal anti-inflammatory drugs (NSAIDs). However, the clinical application of both classes of drugs has significant limitations. While opioids have a definite analgesic effect, long-term use of high doses can easily cause adverse reactions such as respiratory depression, nausea, vomiting, and gastrointestinal discomfort. They also pose risks of drug tolerance, dependence, and addiction, with particularly prominent safety concerns in high-risk patients such as the elderly and those with underlying cardiopulmonary diseases. NSAIDs, on the other hand, pose problems such as gastrointestinal damage, liver and kidney toxicity, and cardiovascular risks. Furthermore, the conventional formulations of these drugs generally suffer from short duration of action, unstable blood drug concentrations, and the need for frequent dosing, which not only increases the clinical workload but also makes it difficult to achieve stable and long-lasting analgesic effects. More importantly, existing drugs are mostly limited to symptomatic relief of overt pain and are unable to effectively inhibit the occurrence and development of central pain sensitization, thus failing to block the formation of chronic postoperative pain at the mechanistic level.

[0004] In recent years, the concept of multimodal analgesia has gradually become a consensus in perioperative pain management. This strategy combines analgesics with different mechanisms of action to synergistically enhance analgesic effects while reducing the exposure dose of a single drug, thereby lowering the risk of adverse reactions. Natural plant active monomers, with their advantages of low toxicity, long-lasting analgesia, and no drug resistance, have become an important research direction for novel analgesics. Meanwhile, long-acting microcrystalline suspension injections, as a novel drug delivery system, can effectively prolong the duration of drug action, reduce the frequency of administration, and maintain stable blood drug concentrations, making them a key research focus for postoperative long-acting analgesics.

[0005] The applicant has previously developed a series of dihydrocannabidiol 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 dihydrocannabidiol di(hetero)carbamate derivative, has a protective effect against oxidative damage to nerve cells and can alleviate insulin resistance.

[0006] PSF1-5 is a series of natural active components that have been proven by the applicant to possess good analgesic activity. However, there is still a lack of systematic formulation development and pharmacodynamic studies on PSF1-5. Its suitability for long-acting injectable formulations, its value in perioperative multimodal analgesia, and its intervention effect on the core mechanism of postoperative pain sensitization: mitochondrial functional damage of spinal dorsal horn neurons, all lack in-depth experimental verification.

[0007] Therefore, there is an urgent need in this field to develop a long-acting injectable formulation based on PSF1-5, to achieve stable and continuous in vivo drug release through optimized formulation process, and to systematically verify its application value in postoperative analgesia, perioperative multimodal adjuvant analgesia, and prevention of postoperative pain sensitization, so as to provide a safe, efficient and long-acting new drug option for clinical postoperative pain management.

[0008] Cannabidiol (CBD) is one of the most widely studied natural analgesic active ingredients. Existing studies have shown that CBD can reduce hyperalgesia and mechanical abnormal pain in various animal pain models. However, the limitations of CBD in postoperative analgesia are also prominent: (1) The duration of action is short. Literature reports that the analgesic effect of CBD in postoperative pain models is measured in hours to several days, which cannot achieve long-term coverage of the entire postoperative wound healing cycle; (2) There is serious instability in efficacy: the analgesic effect of CBD is significantly affected by sex and the hormonal cycle of female individuals. In some physiological stages (such as proestrus), there is even no anti-nociceptive response, suggesting that its analgesic effect is unpredictable and difficult to achieve standardized clinical administration; (3) Clinical evidence is limited and controversial. Some randomized controlled trials have shown no significant difference between CBD and placebo in postoperative pain control, and systematic reviews have pointed out that cannabinoid drugs have no clinically significant benefit for acute postoperative pain. The above limitations seriously restrict the clinical application of CBD in perioperative long-acting analgesia.

[0009] Under the same experimental conditions, the PSF1-5 series compounds of this invention all exhibited analgesic activity superior to or equivalent to CBD. Among them, PSF1 and PSF5 had significantly better analgesic effects than CBD, and had a longer duration of action (up to 14 days) and more stable efficacy. Summary of the Invention

[0010] This invention aims to address the problems of short duration of action, significant side effects, and ineffective prevention of chronic pain transformation in existing perioperative analgesics. It provides a safe, efficient, and long-acting PSF1-5 injection formulation and elucidates its application and mechanism in perioperative analgesia and prevention of central sensitization.

[0011] A first aspect of the present invention provides the use of dihydrocannabidiol di(hetero)carbamate derivatives or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating or preventing perioperative pain, wherein the general structural formula of the dihydrocannabidiol di(hetero)carbamate derivatives is shown in Formula I below:

[0012] Formula I; Where R can be any of the following structures: (1); (2); (3); (4);

[0017] (5).

[0018] Specifically, the di(hetero)carbamate derivatives of dihydrocannabidiol are any one of the following compounds:

[0019] PSF1: Dihydrocannabidiol diimidazocarbamate;

[0020] PSF2: Dihydrocannabidiol-2,6-dioxapiperazine carboxylate;

[0021] PSF3: dihydrocannabidioxanone dinicotinate;

[0022] PSF4: dihydrocannabidiol difuranose ester;

[0023] PSF5: Dihydrocannabidiol dibenzoate; all subsequent uses are abbreviated by number.

[0024] The drug is a long-acting injectable formulation.

[0025] The perioperative pain includes acute incision pain, postoperative inflammatory pain, or postoperative neuropathic pain.

[0026] The invention unexpectedly revealed that the application also includes the preparation of drugs for preventing or alleviating perioperative pain sensitization. This prevention or alleviation of perioperative pain sensitization is achieved by protecting the mitochondrial function of dorsal horn neurons in the spinal cord.

[0027] A second aspect of the invention provides a long-acting injectable formulation for treating or preventing perioperative pain, which is a microcrystalline suspension comprising a di(hetero)carbamate derivative of dihydrocannabidiol or a pharmaceutically acceptable salt thereof.

[0028] The microcrystalline suspension also contains mannitol and poloxamer 188.

[0029] Furthermore, the dihydrocannabidiol di(hetero)carbamate derivative or its pharmaceutically acceptable salt has a crystallite size D90 ≤ 10 μm in the suspension. Preferably, the crystallite D50 is 1.8~3.0 μm, D90 ≤ 8.0 μm, and the particle size distribution span is < 1.8.

[0030] Furthermore, long-acting formulations are either subcutaneous or intramuscular injection formulations.

[0031] The suspension preferably contains mannitol as a stabilizer and isotonic adjuster, and / or poloxamer 188 as a suspending agent and solubilizer. The formulation can be a ready-to-use microcrystalline suspension for injection, or a lyophilized powder for injection reconstituted immediately before use.

[0032] A third aspect of the present invention provides a method for preparing a long-acting injectable formulation for treating or preventing perioperative pain, comprising the following steps: (1) Micronize dihydrocannabidiol di(hetero)carbamate derivatives or pharmaceutically acceptable salts thereof; (2) Disperse the micronized compound in an aqueous medium containing a suspending agent and / or a stabilizer to form a coarse suspension; (3) The coarse suspension is homogenized under high pressure to obtain a microcrystalline suspension.

[0033] This method produces a microcrystalline suspension with uniform particle size and good stability through steps such as micronization and high-pressure homogenization.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) In a rat paw incision pain model, the long-acting injectable formulation of the present invention showed rapid onset and sustained analgesia for up to 14 days, and its analgesic efficacy was significantly better than that of commonly used clinical drugs ibuprofen and tramadol.

[0035] 2) This invention is the first to demonstrate at the mechanistic level that PSF1-5 can effectively protect the mitochondrial function of spinal cord dorsal horn neurons damaged by surgical trauma and stabilize mitochondrial membrane potential. This indicates that this invention can not only alleviate pain symptoms but also inhibit the occurrence of postoperative central sensitization at its source, thereby potentially effectively preventing the formation of postoperative chronic pain.

[0036] 3) Muscle irritation tests showed that no local irritation occurred after 7 consecutive days of intramuscular injection of this preparation, indicating good biocompatibility. More importantly, PSF1-5 are lipid-soluble components and do not rely on strong liver and kidney metabolism. The injection administration method of this invention also avoids the first-pass effect of the liver. Therefore, for special patient groups with liver and kidney dysfunction, this invention provides a safer analgesic option.

[0037] 4) This invention provides a complete and controllable process for preparing microcrystalline suspensions, solving the formulation problem of water-insoluble drugs. In vitro release experiments have demonstrated its excellent sustained-release properties, enabling stable and continuous drug release. Detailed Implementation

[0038] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0039] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0040] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0041] Example 1 Experimental drugs: The active pharmaceutical ingredient used in this study was PSF1-5, provided by the Institute of Agricultural Product Processing, Chinese Academy of Agricultural Sciences. The product specification is 10 g / bottle, and it is a yellow oily substance with a purity ≥98%, free of pyrogens and impurities, and exhibits good batch stability. Positive control drug: Tramadol (a commonly used postoperative analgesic in clinical practice). CBD reference standard: purity ≥99%, purchased externally.

[0042] CBD preparation method: Preparation of CBD nanocrystals by antisolvent precipitation method (1) Organic phase preparation: Accurately weigh 100 mg of CBD raw material, dissolve it in 4 mL of anhydrous ethanol, and stir magnetically (500 r / min, room temperature) until CBD is completely dissolved and the solution is clear and transparent.

[0043] (2) Preparation of aqueous phase: Accurately weigh 10 mg of bovine serum albumin (BSA), dissolve it in 20 mL of water for injection, and stir magnetically until completely dissolved to form the aqueous phase.

[0044] (3) Antisolvent precipitation (nanocrystalline precipitation): Under magnetic stirring (1000 r / min), the organic phase is slowly added to the aqueous phase (dropping rate approximately 0.5 mL / min). CBD becomes instantaneously supersaturated due to a sudden drop in solubility in the unsuitable solvent (water), precipitating out to form nanoscale drug crystals. High-speed stirring must be maintained throughout the dropping process to avoid excessive local supersaturation leading to uneven particle size.

[0045] (4) High-pressure homogenization and refinement: The above-mentioned nanocrystalline coarse suspension is transferred to a high-pressure homogenizer, the homogenization pressure is set to 800 bar, and the homogenization is repeated 10 times to further reduce the particle size and make its distribution more uniform.

[0046] (5) Freeze-drying (preparation of freeze-dried powder): The homogenized CBD nanocrystal suspension was dispensed into freeze-drying pans and freeze-dried using bovine serum albumin (BSA) as a freeze-drying protectant. Freeze-drying conditions: Pre-freezing temperature ≤ -40 ℃, pre-freezing time 4h; primary drying temperature -15 ℃, vacuum degree <20 Pa, drying time 24h; secondary drying temperature 20 ℃, drying time 8h.

[0047] (6) Reconstitution before use: Store the lyophilized powder in a sealed container away from light. Before use, add an appropriate amount of sterile water for injection to reconstitute the powder. Gently shake to restore it to a uniform nanocrystalline suspension. The obtained nanocrystals exhibit the following characteristics: particle size approximately 141.7 ± 1.5 nm, PDI 0.18 ± 0.01, and Zeta potential -25.73 mV. The average particle size after reconstitution is 139.1 ± 1.4 nm. In vivo studies have shown that the AUC of CBD nanocrystals injected intramuscularly is... 0-24h and C max Both were significantly higher than those administered orally.

[0048] The preparation method of PSF1-5 oil is described in the following patent examples: PSF1: CN116983263A, PSF2: CN116983265A, PSF3: CN116983267A, PSF4: CN116983266A, PSF5: CN116983264A.

[0049] Laboratory animals: Healthy male mice, weighing 20-22 g, provided by the Laboratory Animal Center, Animal License No.: SCXK (Guangdong) 2026-0044. The laboratory animals were housed in an environment with a temperature of 22-25 ℃ and humidity of 50%-60%, with a circadian rhythm of 12 h / 12 ​​h, and free access to food and water. Experiments were conducted after one week of acclimatization.

[0050] Preparation of long-acting intramuscular injection formulation of PSF1-5: (1) Raw material pretreatment PSF1-5 is a yellow, oily substance, insoluble in water, and is pretreated in a Class A clean area. Accurately weigh 40.0 g of PSF1-5 raw material (purity ≥98%) (based on pure drug quantity) and place it in a sterile, sealed container. Heat in a water bath to 40 °C to reduce the viscosity and increase the fluidity of the oily substance. Under this temperature-maintaining condition, magnetically stir at 300 r / min for 10 min to ensure uniformity of the material and prevent stratification or precipitation. This step eliminates the internal temperature gradient of the oily substance, ensuring uniformity in subsequent weighing and dispersion.

[0051] (2) Preparation of sterile mannitol blank suspension Accurately weigh 50.0 g of injection-grade mannitol and add it to 800 mL of fresh water for injection. Stir magnetically at 300 r / min for 15 min at room temperature (20–25 °C) until the mannitol is completely dissolved. Add 20 mL of 0.2 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (Na₂HPO₄-NaH₂PO₄) to bring the final buffer salt concentration in the system to approximately 10 mmol / L. After mixing thoroughly, first coarsely filter through a 0.45 μm filter membrane to remove particulates, then sterilize by filtration through a 0.22 μm sterile microporous filter membrane. Adjust the volume to 1000 mL to obtain a 5% (w / v) mannitol sterile blank suspension with a pH of approximately 5.5–6.0 and an osmotic pressure of approximately 285–295 mOsmol / kg. Mannitol can increase the viscosity of the dispersion medium, significantly slowing down the sedimentation and aggregation of water-insoluble PSF₁–₅ nanoparticles, and maintaining the stability of the suspension system.

[0052] (3) Initial mixing and dispersion (coarse emulsification) In a sterile, closed stirring system, take 400 mL of the above mannitol blank suspension and stir at 1000 r / min. Slowly add 40.0 g of the pretreated PSF1-5 oily substance dropwise or in a thin stream to the suspension (avoid adding it all at once to prevent oil droplet aggregation). After all the material is added, stir at 1500 r / min for 20 min to fully disperse the oily substance and form a pale yellow coarse emulsion / coarse suspension. At this time, the oil droplet / particle D50 is about 5~10 μm, and there is no obvious visible oil droplet stratification.

[0053] (4) High-pressure homogenization and refining The coarse dispersion was transferred to a high-pressure homogenizer for gradient homogenization and refinement. The first homogenization pressure was 500 bar, and the second homogenization pressure was 900 bar. The homogenization cycle was 10 times (oily substances are more difficult to refine than solids, so the number of cycles needs to be increased). The temperature was controlled by an ice-water bath throughout the homogenization process, and the system temperature was ≤15 ℃ (oily substances are more susceptible to viscosity changes due to heat, so temperature control needs to be more stringent). After homogenization, the particle size of PSF1-5 particles decreased significantly, and the final controlled D50 was 0.5~1.5 μm, D90 ≤3.0 μm, and particle size distribution range <2.0, forming a uniform particle size, stable suspension of nanoscale particles / droplets. The appearance was a pale yellow, uniform suspension, without stratification or flocculation.

[0054] (5) Adjust pH to constant volume After homogenization, sterile water for injection was added to the homogenized solution to bring the volume to 500 mL, so that the theoretical concentration of PSF1-5 was 80 mg / mL (based on pure PSF1-5, here 40.0 g pure oily substance ÷ 500 mL = 80 mg / mL, no purity conversion is needed). Under sterile conditions, the pH of the system was slowly adjusted to 5.8-6.5 using 0.1 mol / L sterile phosphoric acid or 0.1 mol / L sterile sodium hydroxide solution. In this example, 0.1 mol / L sterile sodium hydroxide solution was used to adjust the pH of the system to 6.2. The mixture was gently stirred at 150 r / min for 5 min to avoid local pH abrupt changes that could cause flocculation and sedimentation of PSF1-5 nanoparticles. After adjustment, samples were taken for retesting. The pH stabilized within the target range, and the particle size of the suspension showed no significant change.

[0055] (6) Aseptic dispensing Because the dispersed phase particle size in the PSF1-5 nano-suspension is close to or exceeds the pore size of conventional sterile filter membranes (0.22 μm), it cannot be sterilized by terminal filtration. Therefore, a sterile production process is used throughout the entire process to control the microbial load. The qualified suspension is quantitatively filled into sterile vials at 2.0 mL per vial under Class A laminar flow, sealed with butyl rubber stoppers and aluminum caps, thus obtaining the PSF1-5 nano-suspension injection.

[0056] 1. In vitro release rate study PSF1-5 nano-suspension injection and free PSF1-5 oily active pharmaceutical ingredient (directly dispersed in a release medium containing SDS) were used as research subjects. Since the release medium contains 0.5% SDS, the PSF1-5 oily substance can be rapidly emulsified and dispersed in the medium, resulting in a faster release rate. This was used as a control to evaluate the sustained-release characteristics of the formulation.

[0057] Three parallel samples (n=3) were set up for in vitro release experiments using the paddle method. The release medium was phosphate buffered saline (PBS, pH 7.4) containing 0.5% (w / v) sodium dodecyl sulfate (SDS), and the release medium temperature was 37 ℃ with a paddle rotation speed of 50 r / min. Samples were taken at 0 min, 20 min, 40 min, 60 min, 120 min, and 240 min to detect the cumulative drug release rate at each time point, and the in vitro drug release patterns of the two systems were compared and analyzed.

[0058] 2. Muscle stimulation test Sixty-six experimental mice were randomly divided into 11 groups of six each, including a negative control group, a PSF1-5 nano-suspension injection group, and a positive control group. All treatment groups received intramuscular injection of 0.2 mL daily for 7 consecutive days. Specific administration methods for the muscle stimulation test are shown in Table 1. After administration, the muscle tissue of the mice was dissected and observed. The muscle stimulation of each group was determined according to the muscle stimulation response grading standard (0-5 grades) to evaluate the local safety of the formulation. The stimulation grading standard was as follows: Grade 0: no significant change; Grade 1: mild congestion, ≤0.5×1.0 cm; Grade 2: moderate congestion, >0.5×1.0 cm; Grade 3: severe congestion with muscle degeneration; Grade 4: necrosis with brown degeneration; Grade 5: extensive muscle necrosis. Specific muscle stimulation response grading standards are shown in Table 2.

[0059] Table 1. Administration methods for muscle irritation tests

[0060] Table 2 Grading Criteria for Muscle Stimulation Response

[0061] 3. Analgesic efficacy test for neuropathic pain A mouse model of neuropathic pain was constructed, with 26 experimental groups, including a normal control group, a sham-operated control group, a neuropathic pain model control group, a positive control group (tramadol), a CBD intramuscular injection control group (to compare the efficacy differences between the components of PSF1-5 and the known natural analgesic active ingredient CBD, a CBD intramuscular injection control group of 60 mg / kg was added in this experiment), a PSF gavage control group (PSF1 was uniformly selected as the representative of the gavage control group, and PSF1 was used as the representative component. PSF1 raw material was dissolved in 0.5% CMC-Na solution to prepare a 30 mg / mL suspension, which was then administered by gavage. PSF1 was selected as the representative of the gavage control group because it had the highest oral bioavailability in previous preliminary experiments and was the most meaningful for comparison by gavage), and low, medium, medium-high, and high-dose intramuscular injection groups of PSF1-5. Each group was administered the drug according to the predetermined dosage, route, and cycle, once daily for 7 consecutive days. The mechanical pain threshold of mice was measured before administration and on days 1, 2, 5, and 7 after administration. Changes in pain threshold were used to evaluate the analgesic effects of different formulations and doses of PSF. The specific experimental design for neuropathological pain is shown in Table 3 below. Table 3 Experimental Design for Neuropathic Pain

[0062] Experimental results 1. Analysis of in vitro release rate results The in vitro release results of PSF1-5 nano-suspension injection and free PSF1-5 oily active pharmaceutical ingredient are shown in Tables 4 and 5. Overall, the free PSF1-5 oily active pharmaceutical ingredient showed the fastest release rate, with a cumulative release rate of 35%–39% at 20 min and almost complete release (98%–99%) at 240 min, exhibiting no sustained-release effect. Compared to the free PSF1-5 oily active pharmaceutical ingredient, the release rate of the PSF1-5 nano-suspension injection was significantly slower. The sustained-release effects of each component showed a clear gradient: PSF1 had the best sustained-release effect (88% release rate at 240 min), followed by PSF5 (91%). The sustained-release effects of PSF2 (95%), PSF3 (94%), and PSF4 (96%) were similar and all weaker than those of PSF1 and PSF5.

[0063] Table 4. External release rate of PSF1-5 nanosuspension injection liquid

[0064] Table 5. In vitro release rate of free PSF1-5 oily active pharmaceutical ingredient.

[0065] 2. Results of muscle stimulation test The results of the five PSF muscle stimulation responses are shown in Table 6 below. After 7 consecutive days of intramuscular administration, the mice in each group showed normal mental state and activity, and no abnormalities in eating or drinking. Dissection and observation of the muscle tissue revealed no abnormal changes such as congestion, edema, degeneration, or necrosis at the injection site in all treatment groups, consistent with the negative control group, and the stimulation response level was 0 in all groups. These results indicate that PSF1-5 nano-suspension injection has good safety for intramuscular injection, with no local irritation, meeting the safety requirements for injectable formulations, and can be used for long-acting intramuscular administration.

[0066] Table 6 Results of PSF1-5 muscle stimulation response

[0067] 3. Analysis of analgesic effect on neuropathic pain The results of mechanical pain threshold determination are shown in Table 7. Before administration, there was no significant difference in mechanical pain threshold among the model mice in each group; all were at a low pain threshold level, indicating successful establishment of the neuropathic pain model. The pain thresholds of the normal control group and the sham-operated control group remained stable throughout the process, without significant fluctuations; the pain threshold of the model control group remained at an extremely low level, showing no tendency to recover. After administration, the pain thresholds of the positive control group (tramadol) and the PSF1 gavage group increased, but the increase was limited, and the efficacy decreased in the later stages (pain thresholds were 6.7 and 6.3 on day 7, respectively). The pain threshold of the CBD intramuscular injection control group (60 mg / kg) increased significantly after administration, reaching a peak of 8.5 on day 5 and maintaining 7.9 on day 7, indicating that CBD has a clear analgesic effect in this model. All high-dose PSF intramuscular injection groups (60 mg / kg) showed significant analgesic effects; the pain threshold continued to increase with the duration of administration, reaching a peak on day 5 and maintaining a high analgesic level on day 7, demonstrating a clear long-term effect advantage. The high-dose PSF1 group showed the best analgesic effect, with the pain threshold rising to 7.5 on the first day of administration, reaching a peak of 9.5 on the fifth day, and remaining at 8.8 on the seventh day. This was significantly better than the positive control group, the CBD control group, and other PSF preparations (P < 0.05), demonstrating rapid onset and strong duration of analgesia. The high-dose PSF5 group was the second best, with a pain threshold of 8.4 on the seventh day, which was also significantly better than the CBD control group (P < 0.05). The pain thresholds of the high-dose PSF2, PSF3, and PSF4 groups on the seventh day were 7.9, 7.8, and 8.0, respectively, which were comparable to the CBD control group (7.9) with no significant difference (P > 0.05).

[0068] The above results indicate that the analgesic effects of each component of PSF1-5 and CBD exhibit a clear gradient: PSF1 > PSF5 > PSF2 ≈ PSF3 ≈ PSF4 ≈ CBD. Among them, the analgesic activities of PSF1 and PSF5 are significantly better than those of the known natural analgesic component CBD, while PSF2, PSF3, and PSF4 are comparable to CBD. Overall, the PSF1-5 series of compounds all have good potential for perioperative analgesia.

[0069] Table 7 Results of Mechanical Pain Threshold Measurement

[0070] Long-acting nanosuspension injections can effectively overcome the shortcomings of traditional analgesics, such as short duration of action, need for repeated administration, and large fluctuations in blood drug concentration, making them a key research direction for postoperative long-acting analgesics. The optimized preparation process of PSF1-5 nanosuspension injections developed in this invention is simple and stable, enabling the batch production of nanosuspension formulations with uniform particle size and stable drug release patterns, meeting the needs of large-scale production. In vitro release tests show that PSF1-5 nanosuspension injections have a significant sustained-release advantage compared to the active pharmaceutical ingredient, slowing down the drug release rate and achieving stable in vivo drug release. This avoids adverse reactions caused by excessively high instantaneous blood drug concentrations and effectively prolongs the duration of drug action, meeting the research and development needs for long-acting postoperative analgesia.

[0071] Drug safety and analgesic efficacy are the core criteria for evaluating the clinical application value of analgesic preparations. Muscle irritation tests confirmed that PSF1-5 nano-suspension injection showed no local irritation after continuous administration, and the muscle tissue remained normal. This indicates that the drug formulation and excipient system of the PSF1-5 preparation prepared in this study are safe and reliable, with no local toxicity, and meet the standards for injectable preparations. In vivo neuropathic pain efficacy tests further validated the results, showing a clear gradient in analgesic effects among the administration groups: the high-dose PSF1 intramuscular injection group (60 mg / kg) showed the best analgesic effect, followed by the high-dose PSF5 intramuscular injection group. The efficacy levels of the high-dose PSF2, PSF3, and PSF4 intramuscular injection groups were comparable to the CBD intramuscular injection control group (60 mg / kg). Furthermore, the analgesic effects of each PSF intramuscular injection group were significantly better than those of gavage administration and the positive control drug tramadol. Among them, the PSF1 preparation had the fastest onset of action and the best analgesic effect, demonstrating significant potential for clinical translation.

[0072] PSF1-5 is a lipid-soluble active ingredient, primarily metabolized via a lipid-soluble pathway. It does not rely on strong hepatic metabolism or renal clearance, and after administration, it is stably distributed and slowly eliminated through the bloodstream, significantly reducing the metabolic burden in individuals with hepatic or renal insufficiency. This invention utilizes intramuscular injection, avoiding first-pass metabolism in the liver and further enhancing the safety of the drug in individuals with impaired metabolic function. It provides a safer analgesic option for patients with hepatic or renal insufficiency, demonstrating significant clinical research value and application prospects.

[0073] This invention successfully optimized and prepared PSF1-5 nano-suspension injections with a stable and highly reproducible preparation process. In vitro release tests confirmed that the formulation possesses excellent sustained-release characteristics, enabling stable and continuous drug release. Muscle irritation tests demonstrated that the formulation is safe and non-irritating for local application. In vivo pharmacodynamic studies showed that the analgesic activity of the PSF1-5 formulation exhibited a clear gradient: PSF1 > PSF5 > PSF2 ≈ PSF3 ≈ PSF4 ≈ CBD. Among them, the analgesic effects of PSF1 and PSF5 were significantly better than those of the known natural analgesic component CBD and the clinically commonly used drug tramadol, while PSF2, PSF3, and PSF4 were comparable to CBD, all demonstrating good perioperative analgesic application value.

[0074] This formulation has analgesia as its core function. Relying on its lipid-soluble metabolic characteristics and intramuscular injection route, it can reduce the metabolic burden on the liver and kidneys, making it especially suitable for people with liver and kidney dysfunction. It has a clear clinical positioning and outstanding safety advantages.

[0075] In summary, PSF1-5 nano-suspension injection is safe, efficient, and has significant sustained-release properties, making it effective for postoperative analgesia intervention. It caters to the analgesic needs of both the general population and patients with hepatic and renal insufficiency. Among them, PSF1 is the optimal active component, followed by PSF5, while PSF2, PSF3, PSF4 are comparable to CBD. It has good research and development value and clinical application prospects, and can provide an important reference for the development of novel long-acting postoperative analgesics.

[0076] Example 2 Experimental drugs: The PSF1-5 nano-suspension injection used in this experiment was prepared according to the method described in Example 1. The formulation is a pale yellow uniform suspension with a particle size D50 of 0.5~1.5 μm and D90≤3.0 μm. It should be gently shaken to ensure uniformity before use.

[0077] Positive control drug: Ibuprofen injection (a commonly used postoperative analgesic in clinical practice). CBD reference standard: purity ≥99%, purchased externally. (CBD control group: CBD nanocrystal lyophilized powder prepared according to the CBD nanocrystal preparation method in Example 1 before use, and reconstituted to 60 mg / mL before use.) 1. Validation experiment on the long-acting analgesic effect of a rat model of incision pain 1) Experimental Objective The rat paw incision pain model is a classic animal model simulating postoperative surgical traumatic pain. It can accurately replicate the complete pathological process of acute postoperative pain, persistent inflammatory pain, and chronic pain sensitization during the wound healing period, closely matching the characteristics of perioperative postoperative pain in clinical practice. This experiment constructed a rat paw incision pain model, using ibuprofen, a commonly used clinical analgesic, as a positive control. It systematically evaluated the intervention effects of different doses of PSF1, PSF2, PSF3, PSF4, and PSF5 on short-term acute postoperative pain and long-term incision pain over 14 days. By detecting changes in the mechanical pain threshold of rats at different time points, the long-acting analgesic activity, dose-response relationship, and pharmacodynamic gradient of each component were clarified, verifying the application value of PSF1-5 for perioperative multimodal analgesia and reducing the clinical use of opioids.

[0078] 2) Laboratory animals and their housing conditions SPF-grade healthy male SD rats, aged 8-10 weeks and weighing 220-280 g, were selected. All rats were acclimatized for 3 days before the experiment. To eliminate the potential interference of female rat sex hormone cycle fluctuations on the baseline pain threshold and efficacy evaluation, and to ensure the stability and reproducibility of analgesic dose-response data, this experiment used the classic rat paw incision pain model and its mechanical pain threshold evaluation method (Von Frey ciliary test) established by Brennan et al. Male SD rats were used for pharmacodynamic evaluation. The housing environment was kept at a constant temperature of 22±2 ℃ and a relative humidity of 50%±5%, with a regular 12h / 12h circadian rhythm. Rats had free access to food and water. Cages were regularly disinfected, and the environment was kept clean and dry to avoid environmental stress interfering with the experimental results. The entire experiment strictly adhered to animal ethics guidelines, and the experimental procedures followed standardized animal experimental protocols. Each group consisted of 6-8 rats, and weight differences between groups were strictly controlled to ensure the objectivity and reliability of the experimental data.

[0079] 3) Modeling steps ① Anesthesia and fixation: After the rats were acclimatized, they were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). After the rats’ corneal reflex was completely lost, the muscles of the limbs were relaxed, and there was no stress response, they were fixed in a prone position on a sterile operating table, and the sole of the right hind paw was fully exposed and the skin was prepared and disinfected. ② Incision preparation: The skin of the right hind paw of the rat was repeatedly disinfected with povidone-iodine. Starting from the middle of the sole, a surgical incision of about 1 cm in length was prepared longitudinally, parallel to the direction of the phalanges. The incision depth reached the fascia layer. The operation was precise and gentle to avoid damage to blood vessels and nerves, reduce intraoperative bleeding, and avoid additional trauma that would interfere with the experimental results. ③ Wound management: Gently separate the subcutaneous tissue and fascia of the incision with sterile forceps, let it stand for 30 seconds to simulate the trauma of clinical surgery, and then use 4-0 sterile sutures to intermittently suture the incision with 2-3 stitches. After suturing, disinfect the incision and surrounding skin with povidone-iodine again to prevent postoperative wound infection. ④ Postoperative recovery: After the model is established, the rats are gently placed back into a clean cage to wake up naturally and have free access to food and water. The rats' mental state and wound healing are observed regularly every day. Any abnormalities such as oozing or redness are treated promptly. After 24 hours of modeling, once the rats are stable and the wounds are free of infection, the drug administration and indicator testing phase begins.

[0080] 4) Experimental grouping and dosing regimen The successfully modeled SD rats were randomly divided into 18 groups, with 6-8 rats in each group. The grouping and administration regimens are as follows (the PSF1-5 nano-suspension injection used in this experiment was prepared according to the method described in Example 1, and the administration route was subcutaneous injection): ① Model control group: Administered an equal volume of sterile saline subcutaneously once daily (qd). ② Positive control group: Administered 30 mg / kg ibuprofen injection subcutaneously, once daily; ③CBD control group: 60 mg / kg CBD nanosuspension injection (the concentration of CBD was 60 mg / mL) was administered subcutaneously, once a day (refer to the effective dose of CBD in the rat incisional pain model reported in the literature). ④ Low, medium and high dose groups of each PSF component: Three dose gradients of 10 mg / kg, 20 mg / kg and 40 mg / kg were set up respectively. Each component of PSF1, PSF2, PSF3, PSF4 and PSF5 were administered subcutaneously, once a day.

[0081] All groups were given their first dose 24 hours after model establishment, with a dosing cycle of 14 days. The doses were administered at fixed times each day, with standardized dosages and procedures to avoid operational errors.

[0082] 5) Detection indicators and time nodes The core detection indicator was the rat paw mechanical pain threshold, used to evaluate the rat's postoperative pain sensitivity. A higher mechanical pain threshold value indicates stronger pain tolerance and better analgesic effect of the drug. Measurements were performed at fixed time points before drug administration (24 h after modeling), 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h after drug administration, and 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days after drug administration. Mechanical pain threshold data for each group of rats were recorded throughout the process. The mean and standard deviation were calculated to analyze the short-term onset rate and long-term analgesic maintenance effect of the drug.

[0083] 2. Experimental Results 1) Analgesic effect of PSF1-5 components Table 8 Results of PSF1 mechanical pain threshold testing

[0084] Table 9 Results of PSF2 mechanical pain threshold testing

[0085] Table 10 Results of PSF3 mechanical pain threshold testing

[0086] Table 11 Results of PSF4 mechanical pain threshold detection

[0087] Table 12 Results of PSF5 mechanical pain threshold testing

[0088] The results of the comprehensive mechanical pain threshold test (Table 8-12) show that the mechanical pain threshold of all rats before drug administration remained stable at 2.0±0.5 after modeling, with no significant difference between groups. This indicates that the incision pain model was well-uniformed and can exclude the interference of differences in baseline pain on the experimental results. After drug administration, the pain threshold of the model control group remained at a low level of 2.1~4.0, with no obvious self-recovery trend, indicating that the postoperative pain status remained stable and suitable for drug efficacy evaluation.

[0089] All treatment groups showed clear analgesic effects. The positive control group (ibuprofen 30 mg / kg) had a rapid onset of action, with the pain threshold rising to 5.0±0.6 at 0.25 h after administration, peaking at 11.0-14.0 h, and then gradually decreasing, returning to 4.5±0.5 at 14 days, which was not significantly different from the model control group (4.0±0.6). The CBD control group (60 mg / kg) had a similar efficacy level to the high-dose PSF2, PSF3, and PSF4 groups, and the difference in pain threshold between the control group and the high-dose PSF2, PSF3, and PSF4 groups at each time point was within the range of 0.2-0.4, suggesting that the analgesic activity of CBD in this model was at the same level as that of PSF2, PSF3, and PSF4.

[0090] The analgesic effects of each PSF component showed a clear dose-dependent relationship; for the same component, the pain threshold increased synchronously at all time points with increasing dosage. Under the same dosage conditions, the analgesic activity of each component exhibited a stable gradient difference. Based on the pain threshold on day 7 of the high-dose group (40 mg / kg), the ranking was: PSF1 > PSF5 > PSF4 > PSF2 > PSF3 > CBD, and the overall efficacy ranking was PSF1 > PSF5 > PSF2 ≈ PSF3 ≈ PSF4 ≈ CBD.

[0091] Regarding long-term maintenance, the pain threshold in the high-dose PSF1 group remained at 5.0±0.5 at 14 days, significantly higher than that in the model control group (4.0±0.6), making it the only component that maintained a clear analgesic effect at 14 days. The pain threshold in the high-dose PSF5 group was 4.8±0.5 at 14 days, still superior to the model control group. However, the pain thresholds in the high-dose PSF2, PSF3, and PSF4 groups, as well as the CBD control group, had decreased to 4.1-4.3 at 14 days, showing no significant difference from the model control group (4.0±0.6), suggesting an analgesic duration of approximately 7-10 days. These results indicate that the analgesic effects of the PSF1-5 components and CBD exhibit a clear gradient, with PSF1 being the most active component, followed by PSF5. The levels of PSF2, PSF3, and PSF4 were comparable to those of CBD, all possessing good potential for perioperative analgesia and providing differentiated drug selection for multimodal analgesia in clinical practice.

[0092] Example 3 Experimental drugs: The PSF1-5 local infiltration injection used in this experiment was prepared according to the method described in Example 1. It was diluted with sterile water for injection to the target concentration before use and was prepared and used immediately.

[0093] Detection of changes in mitochondrial membrane potential in the dorsal horn of the spinal cord 1. Experimental Objective Mitochondrial membrane potential (MMP) is a core indicator for evaluating the integrity of neuronal mitochondrial function, cell viability, and the degree of central sensitization. Postoperative surgical trauma can induce mitochondrial functional damage in spinal dorsal horn neurons, manifested as decreased mitochondrial membrane potential, oxidative stress imbalance, and abnormal neuronal excitation, thereby mediating the occurrence and maintenance of postoperative pain sensitization. This experiment evaluated the repair effects of PSF1, PSF2, PSF3, PSF4, PSF5, and CBD on postoperative mitochondrial functional damage and their inhibitory effects on postoperative pain sensitization by detecting changes in mitochondrial membrane potential in the spinal dorsal horn after intervention with different PSF1-5 components and CBD, clarifying the differences in efficacy among the components and identifying the optimal active component.

[0094] 2. Laboratory animals SPF-grade healthy male mice were selected and acclimatized for 3 days. The ambient temperature was 22-25 ℃, the relative humidity was 50%±5%, the diurnal rhythm was regular, and the mice had free access to food and water. The experimental procedures were in accordance with animal ethics requirements.

[0095] 3. Experimental reagents and instruments Main reagents: JC-1 mitochondrial membrane potential detection kit, sterile PBS buffer, tissue lysis buffer, etc.; Main instruments: flow cytometer, high-speed refrigerated centrifuge, constant temperature water bath, precision pipette, sterile dissecting instruments, etc.

[0096] 4. Experimental Methods 1) Animal grouping and administration The experimental mice were randomly divided into the following groups (n=6~8): The blank control group, model control group, PSF1 group, PSF2 group, PSF3 group, PSF4 group, PSF5 group, and CBD group were treated with local infiltration intervention around the surgical incision using the corresponding PSF local infiltration injection. A postoperative pain sensitization model was established and corresponding drug intervention was given. Routine feeding intervention continued until the detection time point.

[0097] Blank control group: No treatment was given, and the animals were fed according to routine conditions; Model control group: A postoperative pain sensitization model was established (the surgical incision preparation steps were the same as those in Example 2, rat foot incision pain model. A longitudinal incision of about 0.5 cm in length and depth to the fascia layer was prepared on the right hind foot of the mouse. After suturing, the mouse was fed normally). An equal volume of sterile saline was injected locally around the surgical incision once a day. PSF1, PSF2, PSF3, PSF4, and PSF5 groups: After establishing the same postoperative pain sensitization model, the corresponding PSF components were injected locally around the surgical incision at a dose of 30 mg / kg (based on the dose-response relationship results of the neuropathic pain model in Example 1, 30 mg / kg (medium-high dose) is the dose at which each PSF component showed a clear and stable analgesic effect, so this dose was selected for verification of mitochondrial functional mechanism). The PSF components were prepared according to the method in Example 1, diluted with sterile water for injection to the target concentration before use, and then subjected to local infiltration intervention once a day.

[0098] CBD group: After establishing the same postoperative pain sensitization model, CBD local infiltration injection (dose of 60 mg / kg, referring to the preparation method of CBD nanocrystals in Example 1, reconstituted with sterile water for injection and diluted to the target concentration before use) was administered around the surgical incision, once a day.

[0099] Each group was given the first dose 24 hours after modeling, and the mice were sacrificed after 7 days of continuous administration for sample collection and testing.

[0100] 2) Preparation of spinal cord dorsal horn samples Mice were euthanized after the intervention, and the dorsal horn tissue of the mouse spinal cord was quickly isolated. The tissue was washed with sterile PBS buffer, and a single-cell suspension was prepared. After centrifugation, washing, and resuspending, the suspension was stained and incubated according to the instructions of the JC-1 mitochondrial membrane potential detection kit. The cells were then tested after the reaction was completed in the dark.

[0101] 3) Flow cytometry detection of MMPs Mitochondrial membrane potential parameters were detected using flow cytometry in each group. 50,000 cells were collected from each group for data analysis. The number, percentage, and median fluorescence value of cells in different regions (E1, E2, E3) were statistically analyzed to reflect the mitochondrial membrane potential level. High fluorescence intensity represented normal mitochondrial membrane potential and intact mitochondrial function; a higher proportion of low fluorescence indicated more severe mitochondrial damage, decreased membrane potential, and increased central neuronal damage and pain sensitization. Specifically, E1 represented early damage / mildly decreased membrane potential, E2 represented intermediate damage, and E3 represented late damage / apoptotic cells. Since all three regions represent varying degrees of mitochondrial functional impairment, the total percentage of E1+E2+E3 was used as the core quantitative indicator for evaluating the overall degree of mitochondrial damage. The lower this value, the more comprehensive the protection of mitochondrial function by the drug.

[0102] 5. Experimental Results Mitochondrial membrane potential directly reflects the degree of mitochondrial damage in dorsal horn neurons of the spinal cord and is a core indicator for assessing the degree of postoperative central pain sensitization. The proportion of cells in E1, E2, and E3 regions and their fluorescence intensity can directly reflect the functional state of neuronal mitochondria. A higher proportion of low-damage, high-fluorescence cells and a lower proportion of damaged cells indicate a stronger protective effect of the drug on mitochondrial function and a better inhibitory effect on postoperative pain sensitization. The flow cytometry results of mitochondrial membrane potential in PSF1-5 mouse dorsal horn neurons are shown in Tables 13-17 below. Table 13 Results of flow cytometry analysis of mitochondrial membrane potential in dorsal horn neurons of the spinal cord of PSF1 mice

[0103] Table 14 Results of Flow Cytometry Detection of Mitochondrial Membrane Potential in Dorsal Horn Neurons of Spinal Cord in PSF2 Group Mice

[0104] Table 15 Results of flow cytometry analysis of mitochondrial membrane potential in dorsal horn neurons of the spinal cord of PSF3 mice

[0105] Table 16 Results of Flow Cytometry Detection of Mitochondrial Membrane Potential in Dorsal Horn Neurons of Spinal Cord in PSF4 Group Mice

[0106] Table 17 Results of flow cytometry analysis of mitochondrial membrane potential in dorsal horn neurons of the spinal cord of PSF5 mice

[0107] Table 18 Results of Flow Cytometry Detection of Mitochondrial Membrane Potential in Dorsal Horn Neurons of the Spinal Cord of CBD Group Mice

[0108] Based on the combined test data of each group, the total damage rate of E1+E2+E3 in the blank control group was 0.52%, and the mitochondrial function was intact; the total damage rate in the model control group was 6.85%, which was significantly higher than that in the blank control group. This indicates that the surgical trauma successfully induced mitochondrial function damage in the dorsal horn neurons of the spinal cord, and the postoperative central pain sensitization model was successfully constructed.

[0109] After administration of PSF1-5 and CBD, the proportions of total E1+E2+E3 injury were as follows: PSF1 group 4.51%, PSF5 group 4.96%, PSF4 group 5.37%, CBD group 5.38%, PSF2 group 5.38%, and PSF3 group 5.39%, all significantly lower than the model control group (6.85%). This indicates that each PSF component and CBD can improve postoperative spinal cord dorsal horn mitochondrial injury to varying degrees, stabilize mitochondrial membrane potential, and inhibit postoperative central pain sensitization. The protective effects of each component on mitochondrial function showed a clear gradient: PSF1 (4.51%) > PSF5 (4.96%) > PSF4 (5.37%) ≈ CBD (5.38%) ≈ PSF2 (5.38%) ≈ PSF3 (5.39%). Among them, the PSF1 group had the lowest total proportion of mitochondrial damaged cells, the best mitochondrial membrane potential stability, and the best protective effect on neuronal mitochondrial function. It could effectively improve postoperative mitochondrial dysfunction and strongly inhibit postoperative pain sensitization from a mechanistic perspective. The protective effect of PSF5 was second only to PSF1 (4.96%), and the overall effect was good. The effects of PSF2, PSF3, PSF4 and CBD groups were similar (5.37%~5.39%), and the protective effect on mitochondria was weaker than that of PSF1 and PSF5. There was no significant difference in efficacy among the groups.

[0110] Postoperative damage to mitochondrial function in the dorsal horn of the spinal cord is the core pathological mechanism inducing postoperative analgesia and a key central cause of persistent postoperative pain and analgesia. This study validated the central analgesic and anti-sensitization mechanisms of the PSF1-5 series of active components. Results showed that locally infiltrated injections prepared from these components effectively targeted and protected the mitochondrial function of neurons in the dorsal horn of the spinal cord after surgery. The drugs can effectively alleviate central nervous system damage induced by surgical trauma by stabilizing mitochondrial membrane potential and improving mitochondrial injury, thus intervening in the occurrence and development of analgesia at the core pathological level and achieving the therapeutic effect of preventing and reducing postoperative hyperalgesia. Meanwhile, the experiment confirmed that there were significant differences in the central protective and anti-sensitization effects of the various components of PSF1-5. Among them, PSF1 had the most prominent protective effect on the mitochondrial function of spinal cord neurons (total damage rate of 4.51%, a decrease of 34.2% compared with the model control group), and could effectively block the progression of postoperative central sensitization. The efficacy of PSF5 was second best (total damage rate of 4.96%, a decrease of 27.6% compared with the model control group). The efficacy levels of PSF2, PSF3, PSF4 and CBD groups were generally similar (total damage rate of 5.37%~5.39%, a decrease of 21.3%~21.6% compared with the model control group), which was basically the same. This clarified the difference in the activity gradient of different components in central analgesia and inhibition of pain sensitization.

[0111] The results of this mechanism validation experiment clearly demonstrate that the PSF1-5 series compounds can effectively intervene in the pathological process of postoperative pain sensitization by protecting the function of mitochondria in the dorsal horn of the spinal cord and repairing central nervous system damage, exhibiting good postoperative central analgesia and anti-sensitization effects. Considering the differences in efficacy among the components, PSF1 and PSF5 are identified as the core active components for improving and preventing postoperative pain sensitization. Among them, PSF1 shows the best overall effect in mitochondrial protection and central anti-sensitization, exhibiting outstanding pharmacological activity and application potential. This indicates that PSF1 possesses excellent drug development characteristics and can be used as a core active ingredient in the preparation of intraoperative local infiltration drugs to specifically prevent and treat postoperative pain sensitization. It provides a new direction for natural drug development for long-acting perioperative analgesia and avoiding postoperative chronic pain, possessing extremely high clinical development value and translational application prospects.

[0112] Experimental Example: Evaluation of the non-addictive properties of the active ingredient PSF1-5 in this invention To evaluate the addictive properties of the active ingredient PSF1-5 of this invention, the Conditioned Place Preferred (CPP) test was conducted to assess its psychological dependence, and the Open Field Test (OFT) and the Elevated Cross Maze Test (EPM) were used to assess its physical dependence after withdrawal.

[0113] 1. Experimental Materials and Methods 1.1 Laboratory Animals Specific pathogen-free (SPF) male Kunming mice, aged 6-8 weeks, weighing 25-30 g, were purchased from SiBeiFu (Beijing) Biotechnology Co., Ltd. [Production License No.: SCXK (Jing) 2024-0001]. The animals were raised in an SPF-grade animal room with a temperature of 22-25°C, alternating 12 h light / 12 h dark cycle, and free access to food and water. All animal experiments were conducted in accordance with the relevant regulations of the Institutional Animal Care and Use Committee.

[0114] 1.2 Drugs and Reagents Each component of PSF1-5 (purity ≥98%, provided by the Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences); morphine hydrochloride injection (commercially available specification, positive control drug); normal saline; DMSO (dimethyl sulfoxide, pharmaceutical grade); 75% alcohol.

[0115] Before administration, each component of PSF1, PSF2, PSF3, PSF4 and PSF5 was dissolved in DMSO separately to prepare stock solutions respectively. Before use, the stock solutions were diluted to the required concentrations with normal saline respectively (final concentration of DMSO ≤5%). Each component was administered by intraperitoneal injection separately.

[0116] 1.3 Main Instruments Conditioned place preference box, open field test box, elevated plus maze and VisuTrack animal behavior video analysis software (Shanghai Xinruan Information Technology Co., Ltd.).

[0117] 1.4 Experimental Grouping and Administration Regimen The mice were randomly divided into the following groups with 10 mice in each group: First batch: blank control group (normal saline), solvent control group (5% DMSO), positive control group (morphine 3 mg / kg), PSF1 group (10 mg / kg), PSF5 group (10 mg / kg).

[0118] Second batch: blank control group (normal saline), solvent control group (5% DMSO), positive control group (morphine 3 mg / kg), PSF2 group (20 mg / kg), PSF3 group (20 mg / kg), PSF4 group (20 mg / kg).

[0119] All administrations were performed by intraperitoneal injection.

[0120] 2. Experimental Procedure 2.1 Conditioned Place Preference (CPP) Experiment The experiment was divided into three phases: pre-test (3 days), training (12 days, drug-environment pairing) and post-test (1 day). The change in residence time in the drug-paired compartment (CPP score) before and after training was compared among groups of mice.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] EPM metrics: number of times the arm is opened and the dwell time in the arm.

[0125] 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.

[0126] 3. Experimental Results 3.1 Results of the CPP experiment (evaluation of psychological dependence) The results are shown in Table 19. The positive control group (morphine) mice showed a significantly higher post-test CPP score than the pre-test score (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 scores (P>0.05), and were 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.

[0127] Table 19: Comparison of CPP scores before and after the test in each group of mice (s, Mean±SD, n=10)

[0128] 3.2 Results of the withdrawal behavior experiment (evaluation of physical dependence) Open field test (OFT) results (Table 20): 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).

[0129] Table 20: Comparison of Open Field Test (OFT) Results among Different Groups of Mice (Mean±SD, n=10)

[0130] The results of the elevated cross maze (EPM) test (Table 21) 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).

[0131] Table 21: Comparison of Elevated Cross Maze (EPM) results among different groups of mice (Mean±SD, n=10)

[0132] 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. Use of a dihydrocannabidiol di(hetero)carbamate derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of perioperative pain, characterized in that, 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 application according to claim 1, characterized in that, The drug is a long-acting injectable formulation.

3. The application according to claim 1, characterized in that, The perioperative pain includes acute incision pain, postoperative inflammatory pain, or postoperative neuropathic pain.

4. The application according to claim 1, characterized in that, The applications also include the preparation of drugs to prevent or reduce perioperative pain sensitization.

5. The application according to claim 4, characterized in that, The prevention or reduction of perioperative pain sensitization is achieved by protecting the mitochondrial function of the dorsal horn neurons of the spinal cord.

6. A long-acting injectable formulation for the treatment or prevention of perioperative pain, characterized in that, It is a microcrystalline suspension containing dihydrocannabidiol di(hetero)carbamate derivatives or pharmaceutically acceptable salts thereof, the general structural formula of which is shown in Formula I below: Formula I; Where R can be any of the following structures: （1）； （2）； （3）； （4）； （5）。 7. The long-acting injectable formulation for treating or preventing perioperative pain according to claim 6, characterized in that, The microcrystalline suspension also contains mannitol and poloxamer 188.

8. The long-acting injectable formulation for treating or preventing perioperative pain according to claim 6, characterized in that, The microcrystal size D90 of the dihydrocannabidiol di(hetero)carbamate derivative or its pharmaceutically acceptable salt in the suspension is ≤10 μm.

9. The long-acting injectable formulation for treating or preventing perioperative pain according to claim 6, characterized in that, Long-acting formulations are administered via subcutaneous or intramuscular injection.

10. A method for preparing a long-acting injectable formulation for treating or preventing perioperative pain as described in any one of claims 6 to 9, characterized in that, Includes the following steps: (1) Micronize dihydrocannabidiol di(hetero)carbamate derivatives or pharmaceutically acceptable salts thereof; (2) Disperse the micronized compound in an aqueous medium containing a suspending agent and / or a stabilizer to form a coarse suspension; (3) The coarse suspension is homogenized under high pressure to obtain a microcrystalline suspension.

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