A compound for use in the treatment of acute and chronic pain

Abstract

The present application relates to the use of Elamipretide in the preparation of prevention and / or treatment of postoperative or traumatic acute and chronic pain, inflammatory pain, neuropathic pain, cancer pain and the like, pharmacological experiments have shown that it has significant efficacy. The intervention target of Elamipretide is different from that of opioid drugs, which effectively avoids the toxic side effects such as drug addiction and hyperalgesia. The present application uses Elamipretide to intervene in drug-induced pain, which has the advantages of fast analgesic effect, strong analgesic effect equivalent to morphine, longer duration than morphine, safety, low toxicity, significant effect and easy popularization.

Description

[0001] This application claims priority to an earlier application filed on April 23, 2025, with the China National Intellectual Property Administration, patent application number 2025105170859, entitled "Use of Elamipretide in the preparation of a medicament for treating pain induced by surgical trauma, etc." The entire contents of the earlier application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of medicine, specifically relating to the use of Elamipretide in the preparation of medicaments for the prevention and / or treatment of pain induced by surgical trauma, etc. Background Technology

[0003] Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage. More than 320 million people worldwide undergo surgery each year, and acute postoperative pain is extremely common. Safe and effective treatment of acute pain is crucial to preventing increased morbidity, mortality, and the transition to chronic pain. Acute pain lasting longer than 3 months transforms into chronic pain, which affects approximately 30% of the global population. Pain not only leads to complications such as anxiety, depression, insomnia, and cognitive impairment, but can also cause patients to lose their ability to live independently and even develop suicidal tendencies, significantly increasing the socioeconomic burden.

[0004] The prevention and treatment of pain mainly rely on medication. Currently, opioids targeting central nervous system mechanisms are most commonly used to treat acute pain. However, the use of opioids in surgical patients is still limited by safety concerns and the risk of addiction. A careful balance needs to be struck between managing acute pain induced by surgical trauma and minimizing the risks of continued opioid use postoperatively (CHOU R, GORDON DB, DE LEON-CASASOLA OA, et al. Management of Postoperative Pain: A Clinical Practice Guideline From the American Pain Society, the American Society of Regional Anesthesia and Pain Medicine, and the American Society of Anesthesiologists' Committee on Regional Anesthesia, Executive Committee, and Administrative Council [J]. J Pain, 2016, 17(2):131-57.). In addition, commonly used drugs for prevention and treatment in clinical practice include antiepileptic drugs, tricyclic antidepressants, nonsteroidal anti-inflammatory drugs (NSAIDs), and other novel drugs. However, besides significant side effects, the effectiveness of these drugs is also significantly limited by the type of pain. For example, a recent meta-analysis of drug treatments showed that only 30-40% of patients with neuropathic pain responded adequately to clinically administered drugs compared to placebo (ATTAL N, BOUHASSIRA D, COLVIN L. Advances and challenges in neuropathic pain: a narrative review and future directions [J].Br J Anaesth, 2023, 131(1): 79-92.)(FINNERUP NB, HAROUTOUNIAN S, BARON R, et al. Neuropathic pain clinical trials: factors associated with decreases inestimated drug efficacy [J]. Pain, 2018, 159(11): 2339-46.). Therefore, innovative strategies are extremely important for the treatment of acute and chronic pain, including drug repositioning, finding new therapeutic targets, and rational combination therapy.Overall, although some progress has been made in the prevention and treatment of pain, its high incidence and low cure rate due to the lack of safe and effective treatments remain pressing problems that need to be addressed now and in the future. Currently available medications for treating pain induced by surgical trauma and other factors have significant adverse reactions, severely limiting their application. Therefore, there is an urgent need in this field for a treatment regimen with clear therapeutic efficacy and no significant adverse reactions for clinical use.

[0005] Cancer pain refers to pain caused by cancer, cancer-related lesions, and anti-cancer treatments, with 80% of such pain directly caused by cancer. Cancer pain is often chronic. Pain is a common symptom in cancer patients; approximately one-quarter of newly diagnosed cancer patients, one-third of cancer patients undergoing treatment, and three-quarters of patients with advanced cancer experience pain. Cancer pain patients may survive for months or years. Without proper pain management, patients will suffer from pain for a long time, significantly impacting their activities, sleep, mood, and overall quality of life. The tumor itself can compress surrounding nerves, blood vessels, and tissues, producing pain. In addition, tumor-produced cytokines and tumor necrosis factor can release pain mediators, and proteolytic activity can also produce sensitization and hyperalgesia. Therefore, this disease has characteristics of inflammatory pain and neuropathic pain, but it is fundamentally different from simple inflammatory pain and neuropathic pain. In the early stages of cancer development, before inflammation and nerve damage occur, patients already exhibit hyperalgesia, touch-evoked pain, and spontaneous pain.

[0006] Preclinical studies have shown that Elamipretide has protective and restorative effects in models of heart failure, neurodegeneration, ischemia-reperfusion injury, metabolic syndrome, and muscle atrophy and weakness. Meanwhile, multiple clinical trials, including PROGRESS-HF, TAZPOWER, MMPOWER-3, and ReCLAIM, have highlighted the significant therapeutic potential of Elamipretide. However, its application in the development of drugs for treating acute and chronic pain induced by surgical trauma, etc., is currently unknown. Summary of the Invention

[0007] In view of the deficiencies in the prior art and / or the needs in the art, one of the objects of the present invention is to provide the use of Elamipretide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of postoperative or post-traumatic pain, inflammatory pain, neuropathic pain or cancer pain.

[0008] Elamipretide, also known as SS-31, MTP-131, and Bendavia, has CAS number 736992-21-5 and molecular formula C. 32 H 49 N9O5, molecular weight 639.79.

[0009] According to embodiments of the present invention, the Elamipretide or a pharmaceutically acceptable salt thereof is used as the sole active ingredient or in combination with other therapeutic agents to prepare a medicament for treating postoperative or post-traumatic pain, inflammatory pain, neuropathic pain, or cancer pain.

[0010] According to an embodiment of the present invention, the postoperative or post-traumatic pain, inflammatory pain includes acute and chronic pain. According to an embodiment of the present invention, the postoperative or post-traumatic pain includes: acute and chronic pain after surgery or trauma and pain at the site of the primary disease. Postoperative pain is acute pain that occurs immediately after surgery, including somatic pain and visceral pain, and usually lasts no more than 7 days.

[0011] According to an embodiment of the present invention, the postoperative chronic pain refers to pain caused by surgery that occurs in or near the surgical site and lasts for more than 3 months.

[0012] According to an embodiment of the present invention, the postoperative chronic pain refers to persistent postoperative pain caused by a complex neurosensitization mechanism following the traction of the skin and muscle incision.

[0013] According to embodiments of the present invention, the inflammatory pain includes acute and chronic pain caused by inflammatory stimulation, specifically including ankylosing spondylitis pain, osteoarthritis pain, rheumatoid arthritis pain, traumatic arthritis pain, suppurative arthritis pain, gouty arthritis pain, tuberculous arthritis pain, neurogenic arthritis pain, or hemophilic arthritis pain.

[0014] According to an embodiment of the present invention, the neuropathic pain includes pain directly caused by damage or disease of the somatic sensory nervous system, specifically including pain caused by causes such as diabetes, herpes zoster, spinal cord injury, stroke, multiple sclerosis, HIV infection, and lumbar or cervical radiculopathy.

[0015] According to embodiments of the present invention, the cancer pain includes tumor pain, bone cancer pain, pain caused by infiltration or compression in bone, viscera, soft tissue or nerves during tumor progression, metastasis-induced pain, bone cancer pain caused by metastasis, and / or pain induced by metastatic disease in patients with breast cancer, prostate cancer or lung cancer. According to an embodiment of the present invention, the cancer pain is preferably caused by lung cancer-induced bone cancer.

[0016] According to an embodiment of the present invention, the cancer pain includes mild, moderate and severe pain.

[0017] According to embodiments of the present invention, pharmaceutically acceptable salts of Elamipretide include at least one of hydrochloride, hydrobromide, acetate, citrate, benzoate, succinate, octanoate, fumarate, lactate, oxalate, phthalate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, tartrate, maleate, or trifluoroacetate.

[0018] According to embodiments of the present invention, in the above applications, the drug further includes a pharmaceutically acceptable carrier and / or excipients, suitable for preparation into formulations for intravenous infusion, intravenous drip, subcutaneous administration, intradermal administration, intramuscular injection, oral spray, oral administration, etc.

[0019] According to embodiments of the present invention, in the above applications, the drug may be an oral preparation, an injectable preparation, or an enema liquid preparation. Oral preparations include tablets, capsules, pills, powders, granules, suspensions, drops, etc., and injectable preparations include injection solutions, powder injections, etc.; more preferably, the tablets include ordinary tablets, orally disintegrating tablets, dispersible tablets, or sustained-release tablets.

[0020] The present invention also provides the use of a pharmaceutical composition containing Elamipretide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of postoperative or post-traumatic pain, inflammatory pain, neuropathic pain or cancer pain.

[0021] The present invention also provides a pharmaceutical composition for treating and preventing and / or treating postoperative or post-traumatic pain, inflammatory pain, neuropathic pain or cancer pain, comprising Elamipretide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0022] The present invention also provides a pharmaceutical composition for acute and chronic pain with rapid onset of analgesia and a longer duration of analgesia than morphine, the pharmaceutical composition containing Elamipretide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and / or excipient, wherein the pain is selected from one or more of postoperative or post-traumatic pain, inflammatory pain, neuropathic pain or cancer pain. According to an embodiment of the present invention, the analgesic onset time is preferably 5 minutes, and the analgesic duration is 2-3 hours.

[0023] The present invention also provides a method for preventing and / or treating postoperative or post-traumatic pain, inflammatory pain, neuropathic pain, or cancer pain, the method comprising administering a therapeutically effective amount of Elamipretide or a pharmaceutically acceptable salt thereof to a subject in need.

[0024] According to an embodiment of the present invention, the method has the effect of rapid analgesia, an effect intensity equivalent to morphine, and an analgesic duration longer than morphine. The analgesic onset time is preferably 5 minutes, and the analgesic duration lasts for 2-3 hours.

[0025] According to embodiments of the present invention, the dosage of the Elamipretide or a pharmaceutically acceptable salt thereof is 0.5 mg / kg to 40 mg / kg, preferably 0.5 mg / kg to 5 mg / kg, 5 mg / kg to 10 mg / kg, 10 mg / kg to 20 mg / kg, 20 mg / kg to 30 mg / kg, 30 mg / kg to 40 mg / kg, for example, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, etc. The ED50 of intravenous administration of 5 mg / kg, 10 mg / kg, 20 mg / kg, and 40 mg / kg to PINC model mice was 0.5036 mg / kg. In different pain models, dosages of 5 mg / kg, 10 mg / kg, 20 mg / kg, and 40 mg / kg all showed significant efficacy. The clinical dosage range can be 0.05 mg / kg to 4.4 mg / kg, preferably 0.05 mg / kg to 2 mg / kg, 2 mg / kg to 3 mg / kg, 3 mg / kg to 4 mg / kg, for example, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, and 4.4 mg / kg.

[0026] According to an embodiment of the invention, the subject in need may be a mammal, such as a human.

[0027] Beneficial effects This invention utilizes Elamipretide for pharmacological intervention in pain, offering advantages such as rapid onset of analgesia (within 5 minutes), an efficacy equivalent to morphine, and a longer duration of action. It is also safe, low in toxicity, highly effective, and easy to promote. Experimental verification demonstrates that Elamipretide is significantly effective in treating pain caused by various factors, including acute postoperative pain, chronic postoperative pain, inflammatory pain, neuropathic pain, and cancer pain. In PINC model mice, the ED50 after intravenous administration was 0.5036 mg / kg. Elamipretide rapidly regulates intracellular calcium homeostasis, thereby directly reducing neuronal activation induced by noxious stimuli. We have demonstrated that in the PINC model, Elamipretide rapidly reduces spinal cord neuronal activation through non-mitochondrial protective mechanisms and restores intracellular calcium levels in HT22 neurons after stress injury. Clearance ability.

[0028] Furthermore, because Elamipretide targets a different area than opioids, it effectively avoids toxic side effects such as drug addiction or hyperalgesia. Our pharmacological experiments have shown that Elamipretide can rapidly and effectively improve various types of pain, and further confirm its safety. Attached Figure Description

[0029] Figure 1 The effects of intraperitoneal injection of three different mitochondrial protectants, Elamipretide, Mdivi-1, and P110, on SMIR-induced chronic postoperative pain in mice were investigated.

[0030] Figure 2 Intraperitoneal injection of Elamipretide significantly improved acute postoperative pain induced by plantar incision in mice.

[0031] Figure 3 Intraperitoneal injection of Elamipretide in mice does not produce toxic side effects similar to those of other drugs.

[0032] Figure 4 The onset and duration of intravenous administration of Elamipretide improved acute postoperative pain induced by plantar incision in mice.

[0033] Figure 5 It is the ED50 of Elamipretide administered intravenously to mice.

[0034] Figure 6 The mitochondrial structure of the spinal cord tissue of SMIR model mice on Day 1 showed no significant changes.

[0035] Figure 7The differentially expressed genes in the Day 1 spinal cord tissue transcriptomes of female and male SMIR model mice were enriched in the calcium signaling pathway.

[0036] Figure 8 Intravenous injection of Elamipretide rapidly reduced the activation of spinal cord neurons in the PINC model.

[0037] Figure 9 Elamipretide restored the function of HT22 neurons after oxidative stress injury. Clearance ability.

[0038] Figure 10 Intraperitoneal injection of Elamipretide significantly improved acute inflammatory pain induced by carrageenan foot injection in mice.

[0039] Figure 11 Intraperitoneal injection of different doses of Elamipretide significantly improved SMIR-induced chronic postoperative pain in mice.

[0040] Figure 12 Intraperitoneal injection of Elamipretide significantly improved SNI-induced neuropathic pain in mice.

[0041] Figure 13 Intraperitoneal injection of Elamipretide significantly improved bone cancer pain induced by Leiws cancer cells in mice.

[0042] Terminology Definition The term "postoperative or post-traumatic pain" in this article includes acute and chronic pain following surgery or trauma, as well as pain at the site of the primary disease. Postoperative pain is acute pain that occurs immediately after surgery, including somatic and visceral pain, and usually lasts no more than 7 days. If postoperative pain is not adequately controlled in its initial state and persists for more than 3 months, it develops into chronic postoperative pain (CPSP), which may also change in nature to neuropathic or mixed pain. It is mostly moderate pain, but can also be mild or severe, lasting for up to six months or even decades.

[0043] The term "inflammatory pain" in this article encompasses both acute and chronic pain caused by the activation of corresponding pain receptors by inflammatory mediators. Acute inflammation is a short-term response to injury or infection, while chronic inflammation is a persistent inflammatory state that can last for months or years, including pain from ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, traumatic arthritis, septic arthritis, gouty arthritis, tuberculous arthritis, neurogenic arthritis, or hemophilic arthritis.

[0044] The term "neuropathic pain" in this article refers to pain directly caused by damage or disease of the somatic sensory nervous system, including pain caused by causes such as diabetes, herpes zoster, spinal cord injury, stroke, multiple sclerosis, HIV infection, and lumbar or cervical radiculopathy.

[0045] The term "cancer pain" in this article includes tumor pain, bone cancer pain, pain caused by infiltration or pressure on bone, viscera, soft tissue or nerves during tumor progression, metastasis-induced pain, bone cancer pain caused by metastasis, and / or pain induced by metastatic disease in patients with breast cancer, prostate cancer or lung cancer. The term "pharmaceutically acceptable salt" in this article includes at least one of the following: hydrochloride, hydrobromide, acetate, citrate, benzoate, succinate, octanoate, fumarate, lactate, oxalate, phthalate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, tartrate, maleate, or trifluoroacetate. The term "subject" in this article refers to an animal, such as a mammal. Mammals include, for example, mice, rats, dogs, cats, pigs, sheep, horses, cattle, and humans.

[0046] The term "therapeutic effective amount" or "effective amount" herein refers to the amount of a compound disclosed and / or described herein that, when administered to a patient requiring this treatment, is sufficient to achieve the treatment as defined herein. A therapeutically effective amount of a compound may be an amount sufficient to treat pain. Therapeuticly effective amounts will vary depending on factors such as the subject being treated and their disease condition, the subject's weight and age, the severity of the disease condition, the specific compound, the dosing regimen to be followed, the timing of administration, and the method of administration, all of which can be readily determined by one of ordinary skill in the art.

[0047] The term “treatment” in this article includes one or more of the following: suppressing a disease or condition; slowing or halting the development of clinical symptoms of a disease or condition; and / or alleviating a disease or condition (i.e., causing relief or resolution of clinical symptoms), and both complete or partial reduction of clinical symptoms of a disease or condition.

[0048] The term "therapeutic effect" in this article refers to the effect resulting from treatment, which at the animal level manifests as alteration, usually reduction or improvement of symptoms of disease or disease condition, or cure of disease or disease condition. Detailed Implementation

[0049] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0050] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0051] The following describes the protective effect and safety verification examples of Elamipretide in pain induced by surgical trauma, etc., as described in this invention.

[0052] The following are related abbreviations: D322: Elamipretide saline solution; GBP: Gabapentin solution; MOP: Morphine solution; Car, carrageenan; BCP, bone cancer pain.

[0053] Example 1: Comparing the effects of intraperitoneal injection of three different mitochondrial protectants on SMIR-induced chronic postoperative pain in mice, only Elamipretide significantly improved SMIR-induced chronic postoperative pain in mice.

[0054] Experimental materials Three mitochondrial protectants: Elamipretide, Mdivi-1, and P110.

[0055] Elamipretide was purchased from Selleck (S9803) and prepared with physiological saline; Mdivi-1 was purchased from Selleck (S7162), and P110 was purchased from Selleck (S9887). Male C57BL / 6J mice aged 7-8 weeks were purchased from Beijing Vital River Pharmaceutical Co., Ltd., and were subjected to experiments after one week of acclimatization.

[0056] Experimental methods Animal grouping Sham+vehicle (n=8): Normal mice were injected intraperitoneally with the control solvent; SMIR+vehicle (n=8): Peritoneal control solvent for SMIR model mice; SMIR+D322 (n=8): SMIR model mice were intraperitoneally injected with 5 mg / kg Elamipretide; SMIR+Mdivi-1 (n=8): SMIR model mice were intraperitoneally injected with 25 mg / kg Midiv-1; SMIR+ P110 (n=8): SMIR model mice were intraperitoneally injected with 0.5 mg / kg P110.

[0057] Establishment of a mouse model of postoperative chronic pain (SMIR) The SMIR model was prepared by replicating the modeling method used by Flatters et al. in rats (FLATTERS SJL Characterization of a model of persistent postoperative pain evoked by skin / muscle incision and retraction (SMIR) [J]. Pain, 2008, 135(1-2): 119-30.). The specific procedure was as follows: Mice were anesthetized with 2% isoflurane and fixed in a supine position. Hair was removed from the surgical area in the middle of the right hind limb. The surgical area was repeatedly wiped with 75% alcohol to expose the saphenous vein. Subsequently, a skin incision of about 1 cm was made 3 mm medial to the saphenous vein to expose the superficial gracilis muscle. The superficial muscle was bluntly dissected until the white adductor fascia was seen. A self-made micro-retractor about 1 cm wide was then inserted. The skin / muscle was pulled apart to reveal the white adductor fascia, and the retraction was maintained for 1 hour. During the traction process, the saphenous nerve is stretched and displaced by the retractor, but because it is located on the surface of the muscle, it is not compressed by hard objects such as bones. The incision is kept moist with sterile saline gauze during traction, and the muscle / skin is sutured after 1 hour. At the same time, a warming blanket (45℃) is used to keep the area warm throughout the process.

[0058] After the surgery, the mice were placed on a warming blanket to allow them to recover from anesthesia. Once the mice were confirmed to be fully awake, they were returned to their cages. SMIR was administered daily at 8:00 AM starting one day before the SMIR model was established until day 21.

[0059] Mechanical pain threshold measurement First, prepare 7-8 week old male C57 mice and acclimate them in the mouse house for a week (lights on at 8 am and off at 8 pm). Then, the day before the experiment, randomly divide them into 7 groups (8 mice in each group). After grouping, place the mice on the pain test rack for more than 30 minutes to acclimate. This acclimatization process starts at 8:30 am every day and lasts for three days. On the fourth day, use Von Frey needle wire to detect the 50% paw withdrawal threshold of the right hind paw of the mice (i.e., baseline detection).

[0060] After the mice have fully adapted to the environment, use Von Frey silk (0.04, 0.07, 0.16, 0.4, 0.6, 1.0, 1.4, 2.0, 4.0 g) to vertically stimulate the test area on the sole of the hind paw for 3-5 seconds. The force should be such that the Von Frey silk bends slightly. The initial test intensity is 0.6 g. If a positive reaction occurs during the test (such as rapid retraction of the paw, shaking the paw, or licking the paw), switch to a lower-level Von Frey silk. If the test result is negative (no positive reaction behavior), switch to a higher-level Von Frey silk.

[0061] Vonfrey tests were performed on Days 1, 3, 7, 14, and 21 after SMIR modeling, and data were recorded and analyzed using the up-and-down method. Following the method described in the reference (CHAPLAN SR, BACH FW, POGREL JW, et al. Quantitative assessment of tactile allodynia in the rat paw [J]. J NeurosciMethods, 1994, 53(1): 55-63.), the 50% PWT value was calculated using Matlab software.

[0062] Experimental results like Figure 1 The SMIR surgery model significantly reduced the mechanical pain threshold in mice. After intraperitoneal injection of Elamipretide, the area under the curve of the mechanical pain threshold was significantly higher than that of the SMIR+vehicle group, indicating that intraperitoneal injection of Elamipretide can effectively relieve hyperalgesia caused by SMIR surgery. In contrast, the other two mitochondrial protectants, Mdivi-1 and P110, did not produce significant analgesic effects.

[0063] Example 2: Intraperitoneal injection of Elamipretide significantly improved acute postoperative pain induced by plantar incision (PINC) in mice. Experimental materials The Elamipretide used in this invention was purchased from Selleck (S9803) and prepared with physiological saline. Male C57BL / 6J mice aged 7-8 weeks were purchased from Beijing Vital River Pharmaceutical Co., Ltd., and were subjected to experiments after one week of acclimatization feeding. The morphine was supplied by Northeast Pharmaceutical Group Shenyang First Pharmaceutical Co., Ltd.

[0064] Experimental methods Animal grouping Sham+vehicle (n=8): Normal mice were injected intraperitoneally with physiological saline; PINC+vehicle (n=8): PINC model mice were injected intraperitoneally with physiological saline; PINC+ morphine (n=8): PINC model mice were intraperitoneally injected with 5 mg / kg morphine; PINC+D322-5 (n=8): PINC model mice were intraperitoneally injected with 5 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). PINC+D322-10 (n=8): PINC model mice were intraperitoneally injected with 10 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). PINC +D322-20 (n=8): PINC model mice were intraperitoneally injected with 20 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). PINC +D322-40 (n=8): PINC model mice were injected intraperitoneally with 40 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline).

[0065] Establishment of a mouse model of plantar incision pain (PINC) The specific procedures for establishing the mouse plantar incision pain model (PINC) were performed according to previous literature (BANIK RK, WOO YC, PARK SS, et al. Strain and sex influence on pain sensitivity after plantar incision in the mouse [J]. Anesthesiology, 2006, 105(6): 1246-53.). The specific procedures were as follows: Mice were general anesthetized with 2% isoflurane to ensure an appropriate depth of anesthesia (no reaction when the foot was clamped). Then, the right hind foot of the mouse was disinfected with alcohol and iodine. After disinfection, a 5mm incision was made in the center of the right hind foot with a No. 111 blade (made in Korea). The foot muscle was lifted with curved forceps, and the foot muscle was longitudinally cut 10 times with the blade (care should be taken to avoid nerve damage). The skin was then sutured with 7-0 nylon sutures, and the wound was disinfected with iodine and the blood was removed. Finally, the mouse was put back in its cage, and its body temperature was maintained with a warming blanket. The recovery was observed. Administered medication immediately after surgery, at 10 hours, and on Day 1.

[0066] Mechanical pain threshold measurement Using Von Frey wire, the 50% claw retraction threshold of mice was detected by the Up-Down method as an indicator of mechanical hyperalgesia in mice, the method being the same as that in (3) of the experimental method in Example 1. Von Frey detection was performed at 2h, 6h, 12h after PINC surgery and on Days 1, 2, 3, 5, and 7.

[0067] Experimental results like Figure 2The PINC surgical model significantly reduced the mechanical pain threshold in mice. After intraperitoneal injection of Elamipretide at doses of 10, 20, and 40 mg / kg / day, the area under the curve (AUC) for the mechanical pain threshold was significantly higher in the Elamipretide group than in the PINC+vel group. The analgesic effect of 10 mg / kg Elamipretide was comparable to that of 5 mg / kg morphine. This indicates that intraperitoneal injection of Elamipretide can effectively alleviate hyperalgesia caused by plantar incision surgery, and its analgesic effect increases with increasing dose.

[0068] Example 3: Intraperitoneal injection of Elamipretide in mice did not produce toxic side effects similar to those of other drugs. Experimental materials The Elamipretide used in this invention was purchased from Selleck (S9803) and prepared with physiological saline. Male C57BL / 6J mice aged 7-8 weeks were purchased from Beijing Vital River Pharmaceutical Co., Ltd., and were subjected to experiments after one week of acclimatization feeding. Experimental methods Animal grouping Sham +vel (n=8): Normal mice were injected intraperitoneally with physiological saline; PINC+vel (n=8): PINC model mice were injected intraperitoneally with physiological saline; PINC+MOP (n=8): PINC model mice were injected intraperitoneally with 5 mg / kg morphine; PINC+D322 (n=8): PINC model mice were intraperitoneally injected with 10 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). PINC model establishment The method is the same as that in (2) of the experimental method in Example 2. The drug is administered at fixed times starting one day before modeling, once a day.

[0069] Open field experiment Before the experiment, mice were placed in the experimental environment for 30 minutes to acclimatize and reduce pre-experimental stress. At the start of the experiment, the mice were gently placed in the center of the open field box, and the video tracking system was activated to record the mice's behavior. The experiment lasted for 5 minutes. The total distance traveled, time spent in the central area, number of times the mice stood up, number of fecal particles, grooming behavior, speed, and acceleration were observed and recorded. After the experiment, the mice were gently removed and returned to their cages. The open field box was cleaned in preparation for the next mouse's experiment.

[0070] Experimental results like Figure 3Small animal open field experiments showed that mice injected intraperitoneally with 5 mg / kg morphine exhibited significant anomalous excitation in the open field, while intraperitoneal injection of Elamipretide did not produce similar toxic side effects. Survival curve results demonstrated that continuous intraperitoneal injection of 10 mg / kg Elamipretide for 21 days did not lead to mouse death. This indicates that intraperitoneal injection of Elamipretide has a clear safety profile and does not produce toxic side effects similar to those of opioids.

[0071] Example 4: Onset and duration of intravenous administration of Elamipretide in improving acute postoperative pain induced by plantar incision in mice Experimental materials The Elamipretide used in this invention was purchased from Selleck (S9803) and prepared with physiological saline. Male C57BL / 6J mice aged 7-8 weeks were purchased from Beijing Vital River Pharmaceutical Co., Ltd., and were subjected to experiments after one week of acclimatization feeding. Experimental methods Animal grouping Sham +vel (n=6): Normal mice were injected with physiological saline via the tail vein; PINC+vel (n=6): PINC model mice were injected with saline via the tail vein; PINC+MOP (n=6): PINC model mice were injected via the tail vein with 5 mg / kg morphine; PINC+D322-5 (n=6): PINC model mice were injected with 5 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline) via the tail vein.

[0072] PINC model establishment The method is the same as that in (2) of the experimental method in Example 2. The drug was administered immediately after modeling.

[0073] Mechanical pain threshold measurement Using Von Frey wire, the 50% claw retraction threshold of mice was detected by the up-down method as an indicator of mechanical hyperalgesia in mice, the method being the same as that in (3) of the experimental method in Example 1. Von Frey detection was performed at 0.5, 1, 2, 3, 4, 5, and 12 hours after the drug was injected into the tail vein.

[0074] Catwalk gait detection: For three consecutive days at 9:00 AM, training was conducted in a red light environment (simulating darkness). Before training, the mice were transferred to the 343 red light experimental environment for 30 minutes to acclimatize. The mice were placed into the CatWalk track from one end. If the mice did not explore forward in the track for a long time, a certain stimulus was given from behind the mice until they entered the dark box. The training continued until the mice could continuously and without stopping across the track.

[0075] Experiments were conducted at 5 min, 0.5, 1, 2, 3, 4, 5, and 12 h after intravenous injection of the drug into mice. Mice were placed into a CatWalk track from one end in a red light environment (simulating darkness), and their gait was recorded using a camera system. After each experiment, the track was wiped with 75% alcohol to remove odor. Print intensities were analyzed and quantified using Noldus CatWalk XT software, and the maximum and average pressure differences between the left and right hind limbs were compared (GraphPad Prism 9.0, Two-way ANOVA).

[0076] Experimental results like Figure 4 Both Vonfrey and Catwalk gait tests showed that tail vein injection of Elamipretide effectively relieved acute pain in PINC model mice, and that 5 mg / kg of Elamipretide took effect 5 minutes after intravenous administration, with the analgesic effect lasting for 2-3 hours.

[0077] Example 5: Intravenous injection of Elamipretide ED50 in mice Experimental materials The Elamipretid used in this invention was purchased from Selleck (S9803) and prepared with physiological saline. Male C57BL / 6J mice aged 7-8 weeks were purchased from Beijing Vital River Pharmaceutical Co., Ltd., and were subjected to experiments after one week of acclimatization feeding. Experimental methods ED50 animal grouping: Sham+vel (n=6): Normal mice were injected with physiological saline via the tail vein; PINC+vel (n=6): PINC model mice were injected with saline via the tail vein; PINC+D322-0.5 (n=6): PINC model mice were injected via the tail vein with 0.5 mg / kg Elamipretide; PINC+D322-1 (n=6): PINC model mice were injected intravenously with 1 mg / kg Elamipretide; PINC+D322-2 (n=6): PINC model mice were injected via the tail vein with 2 mg / kg Elamipretide; PINC+D322-3 (n=6): PINC model mice were injected via the tail vein with 3 mg / kg Elamipretide; PINC+D322-4 (n=6): PINC model mice were injected via the tail vein with 4 mg / kg Elamipretide; PINC+D322-5 (n=6): PINC model mice were injected via the tail vein with 5 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline).

[0078] PINC model establishment The method is the same as that in (2) of the experimental method in Example 2. The drug was administered immediately after modeling.

[0079] Mechanical pain threshold measurement Using Von Frey wire, the 50% claw retraction threshold of mice was detected by the Up-Down method as an indicator of mechanical hyperalgesia in mice, the method being the same as that in (3) of the experimental method in Example 1. Von Frey detection was performed 1 hour after the drug was injected into the tail vein.

[0080] Catwalk gait detection The method is the same as that in (3) of the experimental method in Example 4. Catwalk gait detection was performed 1 hour after the drug was injected into the tail vein.

[0081] Experimental results like Figure 5 Vonfrey assays showed that low-dose Elamipretide treatment also improved hyperalgesia in PINC model mice, with an ED50 of only 0.5036 mg / kg.

[0082] Example 6: No significant changes in mitochondrial structure in Day 1 spinal cord tissue of SMIR model mice Experimental materials: Elamipretide used in this invention was purchased from Selleck (S9803) and prepared with physiological saline. Male C57BL / 6J mice aged 7-8 weeks were purchased from Beijing Vital River Pharmaceutical Co., Ltd., and were subjected to experiments after one week of acclimatization. 2.5% glutaraldehyde used for tissue fixation was purchased from Solarbio (P1126).

[0083] Experimental methods Animal grouping Sham (n=5): Normal mice; SMIR-Day1 (n=5): Day 1 after SMIR model mice were established.

[0084] Establishment of a mouse model of postoperative chronic pain (SMIR) The SMIR model was prepared by replicating the modeling method used by Flatters et al. in rats (FLATTERS SJL Characterization of a model of persistent postoperative pain evoked by skin / muscle incision and retraction (SMIR) [J]. Pain, 2008, 135(1-2): 119-30.). The specific procedure was as follows: Mice were anesthetized with 2% isoflurane and fixed in a supine position. Hair was removed from the surgical area in the middle of the right hind limb. The surgical area was repeatedly wiped with 75% alcohol to expose the saphenous vein. Subsequently, a skin incision of about 1 cm was made 3 mm medial to the saphenous vein to expose the superficial gracilis muscle. The superficial muscle was bluntly dissected until the white adductor fascia was seen. A self-made micro-retractor about 1 cm wide was then inserted. The skin / muscle was pulled apart to reveal the white adductor fascia, and the retraction was maintained for 1 hour. During the traction process, the saphenous nerve was stretched and displaced by the retractor, but because it was located on the surface of the muscle, it was not compressed by hard objects such as bones. The incision was kept moist with sterile saline gauze during traction, and the muscle and skin were sutured one hour later. A warming blanket (45℃) was used to maintain warmth throughout the procedure. After the surgery, the mouse was placed on the warming blanket to allow it to recover from anesthesia. Once the mouse was confirmed to be fully awake, it was returned to its cage.

[0085] Spinal cord sampling and fixation Mice were anesthetized by intraperitoneal injection of 40 mg / kg 1% sodium pentobarbital solution. After the pinprick reflex disappeared, the mice were fixed in a supine position, and the heart was exposed. The mice were perfused sequentially with pre-cooled PBS buffer and fixative (2.5% glutaraldehyde). After perfusion, the L4-L6 segments of the spinal cord were completely removed and quickly placed in 2.5% glutaraldehyde fixative, and fixed overnight at 4°C. The samples were then transported to a laboratory equipped with a transmission electron microscope using a transport box with dry ice for embedding, sectioning, refixation, and imaging.

[0086] Experimental results like Figure 6 The mitochondrial structure of the dorsal horn neurons in the L4-L6 segment of the spinal cord in the SMIR-Day1 group was not different from that in the sham group, indicating that no structural abnormalities or damage occurred in the mitochondria of the dorsal horn neurons of the spinal cord in the SMIR model mice on the first day after surgery.

[0087] Example 7: Differentially expressed genes in Day 1 spinal cord tissue transcriptomes of female and male SMIR model mice were enriched in the calcium signaling pathway. Experimental materials The 7-8 week old male and female C57BL / 6J mice used in this invention were purchased from Beijing Vital River Company and were subjected to experiments after one week of acclimatization feeding.

[0088] Animal grouping male Sham (n=3): normal male mice; male D1_SMIR (n=3): Male mice on day 1 after SMIR modeling; female Sham (n=3): normal female mice; female D1_SMIR (n=3): Female mice on day 1 after SMIR modeling.

[0089] SMIR Model Establishment The method is the same as the content of (2) in the experimental method of Example 1.

[0090] Spinal cord sampling and fixation Mice were anesthetized by intraperitoneal injection of 40 mg / kg 1% sodium pentobarbital solution. After the pinprick reflex disappeared, the mice were fixed in a supine position, and the heart was exposed. The mice were then perfused sequentially with pre-cooled PBS buffer. After perfusion, the L4-L6 segments of the spinal cord were completely removed and rapidly placed in liquid nitrogen for preservation.

[0091] Experimental results like Figure 7 Transcriptomic differentially expressed genes in spinal cord tissues of mice of different sexes were enriched in the calcium signaling pathway on the first day after SMIR surgery, suggesting that changes in the calcium signaling pathway occur in the early stages of postoperative pain and may play an important role in the chronic progression of pain.

[0092] Example 8: Intravenous injection of Elamipretide rapidly reduced the activation of spinal cord neurons in the PINC model.

[0093] Experimental materials Elamipretide used in this invention was purchased from Selleck (S9803) and prepared with physiological saline. Male C57BL / 6J mice aged 7-8 weeks were purchased from Beijing Vital River Pharmaceutical Co., Ltd., and were subjected to experiments after one week of acclimatization. 4% paraformaldehyde was purchased from Biosharp (BL539A), P-ERK antibody from Cell Signaling Technology (#4370), HUC / HUD antibody from Invitrogen (16A11), DAPI from Sigma (D9542), and Anti-Rb-555 and Anti-Ms-488 antibodies from Abcam (ab150078, ab150113).

[0094] Experimental methods Animal grouping Sham+vel (n=4): Normal mice were injected with saline via the tail vein. PINC+vel (n=4): PINC model mice were injected with saline via the tail vein; PINC+D322 (n=4): PINC model mice were injected via the tail vein with 10 mg / kg Elamipretide. PINC model establishment The method is the same as that in (2) of the experimental method in Example 2. The drug was administered immediately after modeling, and the sample was collected 30 min after modeling and drug administration.

[0095] Spinal cord sampling and fixation Mice were anesthetized by intraperitoneal injection of 40 mg / kg 1% sodium pentobarbital solution. After the pinprick reflex disappeared, the mice were fixed in a supine position, and the heart was exposed. The mice were perfused sequentially with pre-cooled PBS buffer and 4% paraformaldehyde. After perfusion, the spinal cord segments of L4-L6 were completely removed and quickly placed in 4% paraformaldehyde for fixation overnight at 4°C. Subsequently, the tissue was dehydrated using a sucrose gradient and frozen tissue sections were prepared.

[0096] Immunofluorescence staining After washing tissue sections with PBS, the sections were blocked and perforated at 37°C using 0.3% Triton X-100 (Vetec, WXBF3198V) + 5% BSA (Beyotime, ST023). Primary antibodies were prepared using blocking buffer: p-ERK (1:300) + HUC / HUD (1:300), and incubated overnight at 4°C. Secondary fluorescent antibodies were prepared using 3% BSA: Anti-Rb-555 (1:500) + Anti-Ms-488 (1:500), and incubated at room temperature for 2 hours. DAPI (1:2000) was prepared with PBS and incubated on a shaker at room temperature for 10 min. The sections were then mounted and photographed under an Olympus SpinSR 20x microscope in the dorsal horn of the spinal cord.

[0097] Experimental results like Figure 8 In animal models, 30 minutes after PINC administration, the number of neurons expressing p-ERK in the dorsal horn of the L4-L6 spinal cord of mice began to increase, suggesting that noxious stimuli activated neurons in the dorsal horn of the spinal cord. However, after intravenous injection of Elamipretide in PINC mice, the number of neurons decreased significantly, indicating that Elamipretide can reduce neuronal activation caused by noxious stimuli 30 minutes after administration.

[0098] Example 9: Elamipretide restored intracellular function in HT22 neurons after oxidative stress injury. Its ability to clear debris.

[0099] Experimental materials The HT22 cell line used in this invention was purchased from ATCC, and Fluo-4 AM was purchased from Yeasen (40704ES72).

[0100] Experimental methods Cell grouping Control: Normal HT22 cells (blank control); 300 μM HT22 cells damaged by oxidative stress for 1 hour; +D322: 10 μM D322 treatment for 2 hours, 300 μM HT22 cells damaged by oxidative stress for 1 hour; Cellular calcium ion imaging Cells were incubated with 4 μM Fluo-4 AM in HBSS buffer for 20 min in the dark. After washing three times with HBSS to remove extracellular dye, 1 mL of fresh HBSS was added, and the cells were incubated for 20 min in the dark for deloading. Basal signal detection was performed by HBSS perfusion for 1 min. External stimulation was then performed by adding 100 μM ATP to the perfusion cycle for 1 min, followed by HBSS elution for 3 min. Calcium ion imaging was performed before, during, and after ATP application in the culture dish. All imaging experiments were performed using a confocal microscope (Olympus, SpinSR, Japan) with a 20x magnification.

[0101] Experimental results like Figure 9 In HT22 mouse neurons, The HT22 cells in the group produced a more sustained calcium ion response to ATP stimulation, suggesting that the cells' calcium ion clearance capacity decreased after oxidative stress injury, while the use of Elamipretide restored the intracellular calcium ion buffering and clearance capacity of HT22 cells.

[0102] Example 10: Intraperitoneal injection of Elamipretide significantly improved acute postoperative pain induced by carrageenan (Car) in mice. Experimental materials Elamipretide used in this invention was purchased from Selleck (S9803) and prepared with physiological saline. Male C57BL / 6J mice aged 7-8 weeks were purchased from Beijing Vital River Pharmaceutical Co., Ltd., and were subjected to experiments after one week of acclimatization. Carrageenan was purchased from aladdin reagent (C121014). Experimental methods Animal grouping Sham+vehicle (n=8): Normal mice were injected intraperitoneally with physiological saline; Car+vehicle (n=8): Mice with acute inflammatory pain caused by carrageenan were injected intraperitoneally with saline. Carrageenan + morphine (n=8): Carrageenan acute inflammatory pain model mice were intraperitoneally injected with 5 mg / kg morphine; Car +D322-5 (n=8): Carrageenan acute inflammatory pain model mice were intraperitoneally injected with 5 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). Car +D322-10 (n=8): Carrageenan acute inflammatory pain model mice were intraperitoneally injected with 10 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). Car +D322-20 (n=8): Carrageenan acute inflammatory pain model mice were intraperitoneally injected with 20 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). Car +D322-40 (n=8): Carrageenan acute inflammatory pain model mice were intraperitoneally injected with 40 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline).

[0103] Establishment of a carrageenan acute inflammatory pain model The establishment of a mouse model of acute inflammatory pain caused by carrageenan injection into the foot was performed according to previous literature (DELFINIM-C, MANTILLERI A, GAILLARD S, et al. TAFA4, a chemokine-like protein, modulates injury-induced mechanical and chemical pain hypersensitivity in mice [J]. Cell Rep, 2013, 5(2): 378-88.). The specific procedure was as follows: Mice were general anesthetized with 2% isoflurane to ensure appropriate depth of anesthesia. The foot of the mouse was then disinfected with alcohol and iodine. After disinfection, 5 μL of 1% v / w (kg / L) carrageenan (Sigma-Aldrich, 22049-5G-F) was injected into the foot of the mouse using a microsyringe (parallel needle insertion). The injection was stopped for 3 minutes after injection, the needle was removed and disinfected with iodine, and the mouse was returned to its cage. The body temperature was maintained with a warming blanket, and the recovery was observed. The drug was administered immediately after surgery, at 10 h, and on Day 1.

[0104] Mechanical pain threshold measurement Using Von Frey silk, the 50% paw retraction threshold of mice was detected by the up-down method as an indicator of mechanical hyperalgesia in mice, the method being the same as that in (3) of the experimental method in Example 1. Von Frey detection was performed at 2h, 6h, 12h and Day 1, 2, 3, 5 and 7 after injection of carrageenan into the foot.

[0105] Experimental results like Figure 10Foot-plantar injection of carrageenan significantly reduced the mechanical pain threshold in mice. The area under the curve (AUC) for the mechanical pain threshold after intraperitoneal injection of Elamipretide at doses of 5, 10, 20, and 40 mg / kg / day was significantly higher in the Elamipretide group than in the Carrageenan + Vel group. The analgesic effect of 20 mg / kg Elamipretide was comparable to that of 5 mg / kg morphine. This indicates that intraperitoneal injection of Elamipretide can effectively relieve inflammatory pain induced by foot-plantar carrageenan injection, and its analgesic effect increases with increasing dose.

[0106] Example 11: Effect of intraperitoneal injection of different doses of Elamipretide on SMIR-induced chronic postoperative pain in mice.

[0107] Experimental materials Elamipretid used in this invention was purchased from Selleck (S9803) and prepared with physiological saline. Male C57BL / 6J mice aged 7-8 weeks were purchased from Beijing Vital River Pharmaceutical Co., Ltd., and were subjected to experiments after one week of acclimatization. GBP (Gabapentin) was purchased from MCE (HY-A0057).

[0108] Experimental methods Animal grouping Sham+vehicle (n=8): Normal mice were injected intraperitoneally with physiological saline; SMIR+vehicle (n=8): SMIR model mice were intraperitoneally injected with physiological saline; SMIR+GBP (n=8): SMIR model mice were intraperitoneally injected with 50 mg / kg Gabapentin (MCE, HY-A0057, dissolved in physiological saline). SMIR+D322-5 (n=8): SMIR model mice were intraperitoneally injected with 5 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). SMIR+D322-10 (n=8): SMIR model mice were intraperitoneally injected with 10 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). SMIR+D322-20 (n=8): SMIR model mice were intraperitoneally injected with 20 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). SMIR+D322-40 (n=8): SMIR model mice were intraperitoneally injected with 40 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline).

[0109] Establishment of a mouse model of postoperative chronic pain (SMIR) The method is the same as that in (2) of the experimental method in Example 1. Administer the drug at 8:00 a.m. every day starting 7 days after SMIR modeling (chronic phase).

[0110] Mechanical pain threshold measurement First, prepare 7-8 week old male C57 mice and acclimate them in the mouse house for a week (lights on at 8 am and off at 8 pm). Then, the day before the experiment, randomly divide them into 7 groups (8 mice in each group). After grouping, place the mice on the pain test rack for more than 30 minutes to acclimate. This acclimatization process starts at 8:30 am every day and lasts for three days. On the fourth day, use Von Frey needle wire to detect the 50% paw withdrawal threshold of the right hind paw of the mice (i.e., baseline detection).

[0111] After the mice have fully adapted to the environment, use Von Frey silk (0.04, 0.07, 0.16, 0.4, 0.6, 1.0, 1.4, 2.0, 4.0 g) to vertically stimulate the test area on the sole of the hind paw for 3-5 seconds. The force should be such that the Von Frey silk bends slightly. The initial test intensity is 0.6 g. If a positive reaction occurs during the test (such as rapid retraction of the paw, shaking the paw, or licking the paw), switch to a lower-level Von Frey silk. If the test result is negative (no positive reaction behavior), switch to a higher-level Von Frey silk.

[0112] Vonfrey tests were performed on Days 1, 3, 5, 7, 14, and 21 after SMIR modeling. Data were recorded and analyzed using the up-and-down method. Following the method described in the reference (CHAPLAN SR, BACH FW, POGREL JW, et al. Quantitative assessment of tactile allodynia in the rat paw [J]. J NeurosciMethods, 1994, 53(1): 55-63.), the 50% PWT value was calculated using Matlab software.

[0113] Experimental results like Figure 11The SMIR surgical model significantly reduced the mechanical pain threshold in mice. After intraperitoneal injection of Elamipretide at doses of 5, 10, 20, and 40 mg / kg / day, the area under the curve (AUC) for the mechanical pain threshold was significantly higher in the Elamipretide group than in the SMIR+vel group, indicating that even a lower dose could produce an effect similar to intraperitoneal injection of 50 mg / kg / day GBP. This suggests that intraperitoneal injection of 5, 10, 20, and 40 mg / kg / day of Elamipretide can effectively alleviate hyperalgesia induced by SMIR surgery, and its analgesic effect increases with increasing dose.

[0114] Example 12: Intraperitoneal injection of Elamipretide significantly improved SNI-induced neuropathic pain in mice. Experimental materials The Elamipretide used in this invention was purchased from Selleck (S9803) and prepared with physiological saline. Male C57BL / 6J mice aged 7-8 weeks were purchased from Beijing Vital River Pharmaceutical Co., Ltd., and were subjected to experiments after one week of acclimatization feeding. Experimental methods Animal grouping Sham+vehicle (n=7): Normal mice were injected intraperitoneally with physiological saline; SNI+vehicle (n=7): SNI neuropathic pain model mice were injected intraperitoneally with saline. SNI +GBP (n=7): SNI neuropathic pain model mice were intraperitoneally injected with 50 mg / kg Gabapentin (MCE, HY-A0057, dissolved in physiological saline); SNI +D322-5 (n=7): SNI neuropathic pain model mice were intraperitoneally injected with 5 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). SNI +D322-10 (n=7): SNI neuropathic pain model mice were intraperitoneally injected with 10 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). SNI +D322-20 (n=7): SNI neuropathic pain model mice were intraperitoneally injected with 20 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). SNI +D322-40 (n=7): SNI neuropathic pain model mice were intraperitoneally injected with 40 mg / kg Elamipretid (Selleck, S9803, dissolved in physiological saline).

[0115] Establishment of the SNI neuropathic pain model The establishment of the mouse SNI neuropathic pain model was performed according to the description in previous literature (XU Z, XIE W, FENG Y, et al. Positive interaction between GPER and β-alanine in the dorsalroot ganglion uncovers potential mechanisms: mediating continuous neuronalsensitization and neuroinflammation responses in neuropathic pain [J]. JNeuroinflammation, 2022, 19(1): 164.). The specific procedure was as follows: Mice were anesthetized with 2% isoflurane to ensure appropriate depth of anesthesia. After shaving the hair near the right hind limb of the mouse, the mouse was placed on a sterile towel in a left lateral decubitus position. The right hind leg was disinfected with an iodine swab. The skin of the right hind leg was grasped with small toothed forceps in the left hand, and the skin of the right thigh of the mouse was cut open with ophthalmic scissors in the right hand. The muscles were then gently and bluntly separated with forceps to expose the sciatic nerve bundle composed of the sural nerve, common peroneal nerve and tibial nerve. After exposing these nerves, the common peroneal and common tibial nerves were tightly ligated with non-absorbable 7-0 surgical sutures and then severed distally with microsurgical scissors, while the sural nerve remained intact. Finally, the skin was sutured with 6-0 nylon sutures and disinfected with povidone-iodine.

[0116] After the surgery, the mice were placed on a warming blanket to allow them to recover from anesthesia. Once the mice were confirmed to be fully awake, they were returned to their cages. Administration of medication began at 8:00 AM daily, 7 days after SNI modeling (chronic phase).

[0117] Mechanical pain threshold measurement Using Von Frey wire, the 50% claw retraction threshold of mice was detected by the up-down method as an indicator of mechanical hyperalgesia in mice, the method being the same as that in (3) of the experimental method in Example 1. Von Frey detection was performed on Day 1, 3, 5, 7, 14, and 21 after SNI surgery.

[0118] Experimental results like Figure 12SNI (surgical induction of pain) significantly reduced the mechanical pain threshold in mice. After intraperitoneal injection of Elamipretide at doses of 5, 10, 20, and 40 mg / kg / day, the area under the curve (AUC) for the mechanical pain threshold was significantly higher in the Elamipretide group than in the SNI+vel group. The analgesic effect of 20 mg / kg Elamipretide was comparable to that of 50 mg / kg GBP. This indicates that intraperitoneal injection of Elamipretide can effectively alleviate hyperalgesia induced by SNI surgery, and its analgesic effect increases with increasing dose.

[0119] Example 13: Intraperitoneal injection of Elamipretide significantly improved bone cancer pain (BCP) induced by Leiws cancer cells in mice. Experimental materials The Elamipretide used in this invention was purchased from Selleck (S9803) and prepared with physiological saline. Male C57BL / 6J mice aged 7-8 weeks were purchased from Beijing Vital River Pharmaceutical Co., Ltd., and were subjected to experiments after one week of acclimatization feeding. Experimental methods Animal grouping Sham+vehicle (n=7): Normal mice were injected intraperitoneally with physiological saline; BCP+vehicle (n=7): BCP bone cancer pain model mice were injected intraperitoneally with physiological saline; BCP+morphine (n=7): BCP bone cancer pain model mice were intraperitoneally injected with 10 mg / kg morphine; BCP+D322-5 (n=7): BCP bone cancer pain model mice were intraperitoneally injected with 5 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). BCP+D322-10 (n=7): BCP bone cancer pain model mice were intraperitoneally injected with 10 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). BCP+D322-20 (n=7): BCP bone cancer pain model mice were intraperitoneally injected with 20 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline). BCP+D322-40 (n=7): BCP bone cancer pain model mice were intraperitoneally injected with 40 mg / kg Elamipretide (Selleck, S9803, dissolved in physiological saline).

[0120] Establishment of a mouse model of bone cancer pain (BCP) induced by Leiws cancer cells The method for establishing a mouse model of bone cancer pain was performed according to previous literature (ZHANG Z, DENG M, HUANG J, et al. Microglial annexin A3 downregulation alleviates bone cancer-induced pain through inhibiting the Hif-1α / vascular endothelial growth factors ignaling pathway [J]. Pain, 2020, 161(12): 2750-62.), the specific procedure is as follows: After weighing the mice, they are anesthetized by inhalation under 2% isoflurane. The hair near the knee joint of the right hind limb of the mouse is shaved clean with a hair removal machine. The small animal is then covered with a warm blanket and a sterile towel. The mouse is placed on the sterile towel in a left lateral decubitus position. The right knee joint is disinfected with an iodine swab. The skin of the right knee joint is grasped with small toothed forceps in the left hand. The skin above the right knee joint cavity is made longitudinally with microscissors in the right hand. The joint is opened transversely with a scalpel to expose the distal femoral head. The ligament is only cut in the middle part of the knee joint, not completely cut. The ligaments on both sides are partially preserved, with the principle of exposing the area near the femoral head plateau and minimizing damage to muscles, blood vessels and nerves. The left hand stabilizes the mouse's right hind limb, maintaining the left femur perpendicular to the horizontal plane and the tibia and fibula parallel to the horizontal plane, forming a 90-degree vertical angle. Using a disposable skin prick needle (30G needle), a puncture is made in the depression in the middle of the femoral head plateau with the right hand, parallel to the long axis of the femur and perpendicular to the horizontal plane, into the medullary cavity, rotating and advancing simultaneously. After establishing the injection channel, a new disposable skin prick needle is pre-filled with a well-mixed suspension of mouse lung cancer cells (4 x 10^7 cells / ml) or PBS. A 25 μl microsyringe is attached to the needle, and then 5 μl of mouse lung cancer cell suspension (4 x 10^7 cells / ml) or PBS is drawn up. Using the microsyringe, 5 μl of PBS, containing or not containing mouse lung cancer cells (2 x 10^5 cells), is slowly injected into the medullary cavity of the femur through the aforementioned puncture. After injection, the needle is left in place for 10 minutes before being withdrawn. The injection site is then sealed with a small piece of gelatin sponge and thoroughly flushed with saline. The skin is sutured with No. 4 mousse sutures, and the wound is disinfected.

[0121] After the surgery, the mice were placed on a warming blanket to recover from anesthesia. Once the mice were confirmed to be fully awake, they were returned to their cages. Elamipretide was administered daily at 8:00 AM starting 7 days after the bone cancer pain model was established (chronic phase). Morphine was administered at 8:00 AM on Days 1, 3, 5, 7, 14, and 21.

[0122] Mechanical pain threshold measurement Using Von Frey wire, the 50% claw retraction threshold of mice was detected by the up-down method as an indicator of mechanical hyperalgesia in mice, the method being the same as that in (3) of the experimental method in Example 1. Von Frey detection was performed on Day 1, 3, 5, 7, 14, and 21 after bone cancer pain modeling.

[0123] Experimental results like Figure 13 The establishment of a Leiws cancer cell-induced bone cancer pain (BCP) model in mice significantly reduced the mechanical pain threshold. After intraperitoneal injection of Elamipretide at doses of 10, 20, and 40 mg / kg / day, the area under the curve (AUC) of the mechanical pain threshold was significantly higher in the Elamipretide group than in the BCP+vel group. The analgesic effect of 40 mg / kg Elamipretide was comparable to that of 10 mg / kg morphine. This indicates that intraperitoneal injection of Elamipretide can effectively alleviate Leiws cancer cell-induced bone cancer pain in mice, and its analgesic effect increases with increasing dose.

[0124] The exemplary embodiments of the present invention have been described above. However, the scope of protection of this application is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of an Elamipretide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of postoperative or post-traumatic pain, inflammatory pain, neuropathic pain or cancer pain.

2. The application according to claim 1, characterized in that... The postoperative or post-traumatic pain refers to acute postoperative or post-traumatic pain, chronic postoperative or post-traumatic pain, or pain at the site of the primary disease. Preferably, the postoperative or post-traumatic pain is acute pain that occurs immediately after surgery, including somatic pain or visceral pain, and usually lasts no more than 7 days. Preferably, the chronic postoperative pain refers to pain caused by surgery that occurs in or near the surgical area and lasts for more than 3 months. Or, the chronic postoperative pain refers to persistent postoperative pain caused by traction of the skin and muscle incision.

3. The application according to claim 1, characterized in that, The inflammatory pain includes acute and chronic pain caused by inflammatory stimuli, preferably including ankylosing spondylitis pain, osteoarthritis pain, rheumatoid arthritis pain, traumatic arthritis pain, suppurative arthritis pain, gouty arthritis pain, tuberculous arthritis pain, neurogenic arthritis pain, or hemophilic arthritis pain.

4. The application according to claim 1, characterized in that, The neuropathic pain includes pain directly caused by damage or disease of the somatic sensory nervous system, preferably including pain caused by causes such as diabetes, herpes zoster, spinal cord injury, stroke, multiple sclerosis, HIV infection, or lumbar or cervical radiculopathy.

5. The application according to claim 1, characterized in that, The cancer pain includes tumor pain, bone cancer pain, pain caused by infiltration or pressure on bone, viscera, soft tissue or nerves during tumor progression, metastasis-induced pain, bone cancer pain caused by metastasis, and / or pain induced by metastatic disease in patients with breast cancer, prostate cancer or lung cancer. Preferably, the cancer pain is bone cancer induced by lung cancer. More preferably, the cancer pain includes mild, moderate or severe pain.

6. The application according to any one of claims 1-5, characterized in that, The pharmaceutically acceptable salts include at least one of hydrochloride, hydrobromide, acetate, citrate, benzoate, succinate, octanoate, fumarate, lactate, oxalate, phthalate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, tartrate, maleate, or trifluoroacetate.

7. The use of a pharmaceutical composition containing Elamipretide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of postoperative or post-traumatic pain, inflammatory pain, neuropathic pain or cancer pain.

8. The application according to any one of claims 1-7, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or excipients; Alternatively, the drug may be formulated as a preparation for intravenous infusion, intravenous drip, subcutaneous administration, intradermal administration, intramuscular injection, oral spray, or oral administration.

9. The application according to any one of claims 1-7, characterized in that, The drug is an oral preparation, an injectable preparation, or an enema liquid preparation. Oral preparations include tablets, capsules, pills, powders, granules, suspensions, and drops. Injectable preparations include injection solutions and powder injections. More preferably, the tablets include ordinary tablets, orally disintegrating tablets, dispersible tablets, or sustained-release tablets.

10. A method for preventing and / or treating postoperative or post-traumatic pain, inflammatory pain, neuropathic pain, or cancer pain, characterized in that, The method includes administering a therapeutically effective amount of Elamipretide or a pharmaceutically acceptable salt thereof to a subject in need.

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