WO2022105665A1 - Compound for prevention and treatment of pain and inflammation and application thereof - Google Patents

Abstract

Disclosed in the present invention is an application of an Hv1 channel serving as a target to treat pain. In the present invention, after an Hv1 gene is specifically knocked out, inflammatory pain and neuropathic pain thresholds of a corresponding mouse are increased. In the present invention, a small molecule compound (an inhibitor) represented by formula (I) or formula (II) and designed on the basis of an Hv1 protein structure can remarkably relieve the inflammatory pain and neuropathic pain in mice, and the small molecule compound can remarkably reduce the content of inflammatory factors under the inflammatory pain, and shows the effects of blocking opioid analgesic tolerance and improving opioid-induced pain hypersensitivity, thereby providing the basis for the treatment of pain and diseases such as cerebral stroke, atherosclerosis, and Parkinson's disease. Further disclosed in the present invention that an Hv1 channel can be used as a new target of a drug for analgesia, treatment of opioid analgesic tolerance and hypersensitivity, and reduction of inflammatory effects.

Description

A compound for preventing and treating pain and inflammation and its application technical field

The invention belongs to the fields of biotechnology and medical technology, and relates to a compound with Hv1 inhibitory activity using Hv1 channel as a drug target for preventing and treating pain and inflammatory diseases and its application.

Background technique

The main physiological function of voltage-gated proton channel (Hv1) is to rapidly move intracellular protons to extracellular. Hv1 regulates intracellular pH, activates nicotinamide-adenine dinucleotide phosphate oxidase (NOX), and cooperates with NOX to generate oxygen free radicals (ROS) (Wu LJ.Voltage-gated proton channel Hv1 in microglia. Neuroscientist. 2014;20:599-609.). Therefore, Hv1 may be related to neurological diseases and cardiovascular and cerebrovascular diseases caused by oxidative damage, including stroke, Parkinson's disease, and atherosclerosis.

Stroke, also known as stroke, is a sudden, rapidly developing acute cerebral ischemia or cerebral hemorrhage disease, of which about 80% are ischemic strokes. Under cerebral ischemia, microglia are activated and intracellular subunits of NOX are transported to the cell membrane. Hv1 channels on microglia assist NOX to generate ROS, which in turn induces neuronal cell death and a portion of brain damage from ischemic stroke. Atherosclerosis (AS) is a common disease in middle-aged and elderly patients, and it is also the main cause of cardiovascular and cerebrovascular diseases (Pasquel FJ, Gregg EW, Ali MK. The evolving epidemiology of atherosclerotic cardiovascular disease in people with diabetes [J]. Endocrinol Metab Clin North Am, 2018;47:1-32.).

Atherosclerosis and its fatal complications, such as myocardial infarction or stroke, are the most common cause of morbidity or death in the world today. Atherosclerosis is accompanied by excess ROS. Under physiological conditions, the accumulation of intracellular H ⁺ and the depolarization of the cell membrane during ROS production limit the ROS production process, thereby avoiding oxidative stress. However, the sustained production of ROS during atherosclerosis suggests that the physiologically self-limited ROS generation process is disrupted and that there may be an unknown mechanism that supports continued ROS generation. Hv1 channels rapidly remove protons from depolarized cytoplasm and maintain membrane potential, possibly playing a key role in atherosclerosis. Some studies have pointed out that Hv1 channel-mediated oxidative stress promotes excessive ROS production and foam cell formation, leading to atherosclerosis (Zheng Z, Zhang Z, Wang M. Hv1 proton channel possibly promotes atherosclerosis by regulating reactive oxygen species production. Med Hypotheses. 2020;141:109724.). In a mouse model of cerebral ischemia and a mouse model of multiple sclerosis (MS), reducing the expression of Hv1 in the brain can reduce the oxidative stress damage and inflammatory effects caused by microglia. The rat brain plays a certain protective role (Wu LJ. Microglial voltage-gated proton channel Hv1 in ischemic stroke. Transl Stroke Res. 2014; 5:99-108.).

Parkinson's disease (PD) is the second most common multifactorial neurodegenerative disease affecting 2%-3% of the elderly worldwide (Kaur R, Mehan S, Singh S. Understanding multifactorial architecture of Parkinson's disease: pathophysiology to management. J Neurol Sci. 2019, 40: 13-23). The specific pathogenesis of PD is not clear at present. The general view is that oxidative stress and inflammatory effects in the brain are the main causes of PD. Therefore, reducing oxidative stress and inflammatory effects in the brain provides a new idea for the treatment of Parkinson's disease. . Microglia play an immune function in the brain and regulate inflammatory effects (Perry VH, Nicoll JA, Holmes C. Microglia in neurodegenerative disease. Nat Rev Neurol. 2010, 6(4): 193–201.), while in microglia The highly expressed Hv1 channel in glial cells can promote oxidative stress, so inhibition of Hv1 channel may be a strategy for the treatment of PD disease.

Inflammation and inflammatory pain are highly correlated with ROS. Although Hv1 channels can control ROS overload, which can lead to inflammatory pain and neuropathic pain, the relationship between Hv1 channels and inflammatory pain and neuropathic pain has not been studied much, and there is no evidence that Hv1 channels are therapeutic New targets for inflammatory pain and neuropathic pain. Therefore, developing selective Hv1 channel inhibitors as pharmacological tools to elucidate the physiological and pathological roles of Hv1 channels in vivo and in vitro can not only provide new strategies for the treatment of stroke, atherosclerosis, Parkinson's disease and other diseases, but also reveal Whether Hv1 channel can be used as a new target for the prevention and treatment of inflammatory pain and neuropathic pain.

SUMMARY OF THE INVENTION

In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide a new target that can treat pain and inflammation, that is, the target is a voltage-gated proton channel (Hv1); through research Provide new drug binding sites for Hv1 channels and discover new compounds (inhibitors) that can be used to inhibit Hv1 channels. The present invention uses the obtained inhibitor as a probe to evaluate the application of inhibiting Hv1 in preventing/treating pain, inflammation, blocking opioid analgesic tolerance and improving pain hypersensitivity caused by opioid drugs, and found that the invention The Hv1 channel inhibitors can treat and/or alleviate these diseases, and are also used to treat diseases such as stroke, atherosclerosis and Parkinson's disease.

The present invention provides a compound represented by formula (I) (YHV98-1) or a pharmaceutically acceptable salt thereof or a compound represented by formula (II) (YHV98-4) or a pharmaceutically acceptable salt thereof Use of a salt in the preparation of a pharmaceutical composition or preparation for preventing and/or treating pain, the compound or a pharmaceutically acceptable salt thereof has Hv1 channel inhibitory activity, and the structure of the compound is as follows:

The present invention also provides a compound represented by formula (I) (YHV98-1) or a pharmaceutically acceptable salt thereof or a compound represented by formula (II) (YHV98-4) or a pharmaceutically acceptable salt thereof The use of the salt of the compound in the preparation of a pharmaceutical composition or preparation for preventing and/or treating inflammation, the compound or a pharmaceutically acceptable salt thereof has the inhibitory activity of Hv1 channel, and the structure of the compound is as follows:

The present invention also provides a compound represented by formula (I) (YHV98-1) or a pharmaceutically acceptable salt thereof or a compound represented by formula (II) (YHV98-4) or a pharmaceutically acceptable salt thereof Use of the salt of the compound in the preparation of a pharmaceutical composition or preparation for the prevention and/or treatment of opioid analgesic tolerance and hypersensitivity, the compound or a pharmaceutically acceptable salt thereof has Hv1 channel inhibitory activity, the compound The structure is as follows:

The present invention also provides a compound represented by formula (I) (YHV98-1) or a pharmaceutically acceptable salt thereof or a compound represented by formula (II) (YHV98-4) or a pharmaceutically acceptable salt thereof Use of the salt of the compound in the preparation of a pharmaceutical composition or preparation for the prevention and/or treatment of stroke, Parkinson's disease, and atherosclerosis, the compound or a pharmaceutically acceptable salt thereof has Hv1 channel inhibitory activity, and the The structure of the compound is shown below:

In another preferred embodiment, the Hv1 channel is a human or mouse Hv1 channel.

In another preferred embodiment, the Hv1 channel refers to a protein expressed by the Hvcn1 gene.

In another preferred embodiment, the Hv1 channel is used as a molecule or drug target for preventing and/or treating pain and preventing and/or treating inflammation.

In another preferred example, the drug comprises plant extracts, compounds with clear chemical structures, peptides, nucleic acids, polysaccharides, and viral vectors developed with Hv1 channel as a molecular target or drug action target. , liposome carrier or nanoparticle carrier.

In another preferred example, the compound of formula (I) is compound YHV98-1, and the two are the same compound.

In another preferred embodiment, the compound of formula (II) is compound YHV98-4, both of which are the same compound.

In another preferred embodiment, the compound of formula (I) and/or the compound of formula (II) is a compound that inhibits Hv1 channel activity.

In another preferred embodiment, the pain includes chronic pain, acute pain, and cancer pain; wherein, the chronic pain includes muscle and soft tissue pain, bone and joint pain, headache, visceral pain, and pathological neuralgia.

In another preferred embodiment, the muscle and soft tissue pain includes one or more of myofasciitis, tenosynovitis, frozen shoulder, muscle strain pain, fibromyalgia, cold pain, burn pain, and toothache.

In another preferred embodiment, the bone and joint pain includes knee joint pain, ankle joint pain, wrist joint pain, elbow joint pain, shoulder joint pain, patella joint pain, hip joint pain, femoral joint pain, and compulsive spine joint pain. Inflammation, sacroiliitis, rheumatoid arthritis, rheumatoid arthritis, gouty arthritis, intervertebral disc herniation, cervical pain, lumbar pain one or more.

In another preferred embodiment, the headache includes primary headache, secondary headache, cranial neuralgia, central and primary facial pain and other headaches.

In another preferred embodiment, the primary headache includes migraine without aura, migraine with aura, hemiplegic migraine, chronic migraine, migraine complications, migraine-related episodic syndrome, tension One or more of the types of headache, trigeminal autonomic headache and other primary headaches.

In another preferred embodiment, the secondary headache includes headache due to head and neck trauma, headache due to non-vascular intracranial disease, headache due to substance or substance withdrawal, and headache due to head and neck injury Headache of vascular disease, headache due to disturbance of internal environment, headache due to mental illness, one of head, neck, eyes, ears, nose, sinuses, teeth, mouth or other head and face structure lesions or several.

In another preferred embodiment, the cranial neuralgia, central and primary facial pain and other headaches include one or more of trigeminal neuralgia, glossopharyngeal neuralgia, intermediary neuralgia, occipital neuralgia, and optic neuritis kind.

In another preferred embodiment, the visceral pain includes pain from internal organs, and the internal organs include respiratory tract, gastrointestinal tract, pancreas, urethra, kidney, gallbladder, bladder and genitalia.

In another preferred embodiment, the pathological neuralgia includes post-herpetic neuralgia, diabetic neuralgia, painful HIV-related sensory neuropathy, burning syndrome, post-amputation pain, phantom pain, painful neuroma, Traumatic neuroma, nerve crush injury, spinal stenosis, carpal tunnel syndrome, radicular pain, sciatica, nerve avulsion, brachial plexus avulsion, complex regional pain syndrome, neuralgia due to drug therapy , One or more of neuralgia caused by cancer chemotherapy, pain after spinal cord injury, primary small fiber neuropathy, primary sensory neuropathy, and trigeminal autonomic headache.

In another preferred embodiment, the acute pain includes one or more of acute traumatic pain, postoperative pain, labor pain, visceral pain, pruritus, and postoperative pain.

In another preferred embodiment, the cancer in the cancer pain includes adenocarcinoma in glandular tissue, blastoma in organ embryo tissue, carcinoma in epithelial tissue, leukemia in blood cell-forming tissue, and lymphoid tissue Lymphoma, myeloma in bone marrow, sarcoma in connective or supporting tissue, adrenal cancer, AIDS-related lymphoma, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid tumor, cervical cancer, colon cancer, intrauterine cancer Membrane cancer, esophagus cancer, stomach cancer, head cancer, neck cancer, hepatobiliary cancer, leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's disease, non-Hodgkin's disease, nervous system tumor, oral cancer, ovarian cancer, pancreas Cancer, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, urethral cancer, bone marrow cancer, multiple myeloma, tumors that metastasize to bone, tumors infiltrating nerves and hollow organs, tumors near neural structures one or more of the tumors.

In another preferred embodiment, the inflammation refers to infectious inflammation and non-infectious inflammation, including inflammation caused by bacteria, viruses, parasitic biological pathogens, inflammation caused by high temperature, low temperature, radioactive substances, ultraviolet rays and mechanical damage, strong acid Inflammation caused by strong alkali, turpentine, mustard gas, endogenous toxic substances and metabolites in the body, inflammation caused by metals, wood chips, dust particles, and surgical sutures, inflammation caused by tissue necrosis caused by necrotic tissue, allergies Rhinitis, urticaria, glomerulonephritis, lymphocytic thyroiditis, inflammation caused by ulcerative colitis, ankylosing spondylitis, sacroiliitis, rheumatoid arthritis, rheumatoid arthritis, gouty arthritis , myofasciitis, tenosynovitis, frozen shoulder, etc.

In another preferred embodiment, the Hv1 channel is used as a molecular or drug target for preventing and/or treating opioid analgesic tolerance and hypersensitivity.

In another preferred embodiment, the opioids include morphine, heroin and the like.

In another preferred example, the administered dose of the compound of formula (I) and/or the compound of formula (II) is 0.1-50 mg/kg; preferably, it is 1-5 mg/kg.

In another preferred example, when the administered dose of the compound of formula (I) and/or compound of formula (II) is ≤50 mg/kg, there is no adverse reaction.

The present invention also provides a pharmaceutical composition comprising a compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a compound represented by formula (II) or a pharmaceutically acceptable salt thereof The salt of , where the structure of the compound is shown below:

In another preferred embodiment, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

In another preferred example, the total content of the compound of formula (I) and/or compound of formula (II) or its pharmaceutically acceptable salt is 0.001-99 wt % of the composition; preferably, it is 0.1-90 wt % %; further preferably, it is 1-80 wt%.

In another preferred embodiment, the pharmaceutical composition is used to prevent and/or treat pain.

In another preferred embodiment, the pain includes chronic pain, acute pain, and cancer pain; wherein, the chronic pain includes muscle and soft tissue pain, bone and joint pain, headache, visceral pain, and pathological neuralgia.

In another preferred embodiment, the muscle and soft tissue pain includes one or more of myofasciitis, tenosynovitis, frozen shoulder, muscle strain pain, fibromyalgia, cold pain, burn pain, and toothache.

In another preferred embodiment, the bone and joint pain includes knee joint pain, ankle joint pain, wrist joint pain, elbow joint pain, shoulder joint pain, patella joint pain, hip joint pain, femoral joint pain, and compulsive spine joint pain. Inflammation, sacroiliitis, rheumatoid arthritis, rheumatoid arthritis, gouty arthritis, intervertebral disc herniation, cervical pain, lumbar pain one or more.

In another preferred embodiment, the headache includes primary headache, secondary headache, cranial neuralgia, central and primary facial pain and other headaches.

In another preferred embodiment, the primary headache includes migraine without aura, migraine with aura, hemiplegic migraine, chronic migraine, migraine complications, migraine-related episodic syndrome, tension One or more of the types of headache, trigeminal autonomic headache and other primary headaches.

In another preferred embodiment, the secondary headache includes headache due to head and neck trauma, headache due to non-vascular intracranial disease, headache due to substance or substance withdrawal, and headache due to head and neck injury Headache of vascular disease, headache due to disturbance of internal environment, headache due to mental illness, one of head, neck, eyes, ears, nose, sinuses, teeth, mouth or other head and face structure lesions or several.

In another preferred embodiment, the cranial neuralgia, central and primary facial pain and other headaches include one or more of trigeminal neuralgia, glossopharyngeal neuralgia, intermediary neuralgia, occipital neuralgia, and optic neuritis kind.

In another preferred embodiment, the visceral pain includes pain from internal organs, and the internal organs include respiratory tract, gastrointestinal tract, pancreas, urethra, kidney, gallbladder, bladder and genitalia.

In another preferred embodiment, the pathological neuralgia includes post-herpetic neuralgia, diabetic neuralgia, painful HIV-related sensory neuropathy, burning syndrome, post-amputation pain, phantom pain, painful neuroma, Traumatic neuroma, nerve crush injury, spinal stenosis, carpal tunnel syndrome, radicular pain, sciatica, nerve avulsion, brachial plexus avulsion, complex regional pain syndrome, neuralgia due to drug therapy , One or more of neuralgia caused by cancer chemotherapy, pain after spinal cord injury, primary small fiber neuropathy, primary sensory neuropathy, and trigeminal autonomic headache.

In another preferred embodiment, the acute pain includes one or more of acute traumatic pain, postoperative pain, labor pain, visceral pain, pruritus, and postoperative pain.

In another preferred embodiment, the cancer in the cancer pain includes adenocarcinoma in glandular tissue, blastoma in organ embryo tissue, carcinoma in epithelial tissue, leukemia in blood cell-forming tissue, and lymphoid tissue Lymphoma, myeloma in bone marrow, sarcoma in connective or supporting tissue, adrenal cancer, AIDS-related lymphoma, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid tumor, cervical cancer, colon cancer, intrauterine cancer Membrane cancer, esophageal cancer, head cancer, neck cancer, hepatobiliary cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's disease, non-Hodgkin's disease, nervous system tumors, oral cancer, ovarian cancer, Pancreatic cancer, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, urethral cancer, bone marrow cancer, multiple myeloma, tumors that metastasize to bone, tumors infiltrating nerves and hollow organs, near neural structures one or more of the tumors.

In another preferred embodiment, the pharmaceutical composition is used to prevent and/or treat inflammation.

In another preferred embodiment, the inflammation refers to infectious inflammation and non-infectious inflammation, including inflammation caused by bacteria, viruses, parasitic biological pathogens, inflammation caused by high temperature, low temperature, radioactive substances, ultraviolet rays and mechanical damage, strong acid Inflammation caused by strong alkali, turpentine, mustard gas, endogenous toxic substances and metabolites in the body, inflammation caused by metals, wood chips, dust particles, and surgical sutures, inflammation caused by tissue necrosis caused by necrotic tissue, allergies Rhinitis, urticaria, glomerulonephritis, lymphocytic thyroiditis, inflammation caused by ulcerative colitis, ankylosing spondylitis, sacroiliitis, rheumatoid arthritis, rheumatoid arthritis, gouty arthritis , myofasciitis, tenosynovitis, frozen shoulder, etc.

In another preferred embodiment, the pharmaceutical composition is used for the prevention and/or treatment of opioid analgesic tolerance and hypersensitivity.

In another preferred embodiment, the pharmaceutical composition is used for preventing and/or treating stroke, Parkinson's disease, and atherosclerosis.

In another preferred embodiment, the pharmaceutical composition can also be used in combination with other drugs.

In another preferred example, the other drugs include, but are not limited to, opioid analgesics, such as morphine, heroin, etc.; non-opioid analgesics include, but are not limited to, paracetamol, acetylsalicylic acid, ibuprofen, etoric Rilene, fenbufen, fenoprofen, ketorolac, flurbiprofen, indomethacin, ketoprofen, naproxen, and their pharmaceutically acceptable salts, optically active racemates and mixtures .

In another preferred embodiment, the pharmaceutical composition can be made into an oral dosage form or an injection dosage form.

In another preferred embodiment, the oral dosage forms include tablets, capsules, films, granules and the like.

In another preferred embodiment, the pharmaceutical composition can be administered to the desired subjects (eg, human and non-human mammals) by oral administration, injection, atomization inhalation, and the like.

The present invention also provides a method for preventing and/or treating related diseases, the method comprising: administering the compound of formula (I) or compound of formula (II) as described above or a pharmaceutically acceptable salt thereof to a subject in need thereof or pharmaceutical compositions.

In another preferred embodiment, the subject includes human and non-human mammals (such as rodents and primates).

In another preferred example, the subject is a human, and the administered dose of the compound of formula (I) and/or the compound of formula (II) is 1-20 mg/kg; preferably, it is 5 mg/kg.

In another preferred embodiment, the frequency of administration is 1-4 times/day; preferably, it is 1 time/day.

In another preferred embodiment, the administration time is from 2 weeks to 5 years; preferably, from 2 months to 1 year.

In another preferred example, the administration method is oral administration, injection, atomization inhalation, etc.; the administration method is selected according to actual needs.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

The beneficial effects of the present invention include:

(a) It provides a new target for the treatment of pain diseases and has a good application prospect.

(b) A new small molecule or drug molecule binding pocket in the Hv1 channel was discovered, which improved the discovery efficiency of Hv1 channel active molecules.

(c) Compound YNV98-1 and/or compound YNV98-4 were found to have a significant inhibitory effect on Hv1 channel.

(d) Compound YNV98-1 and/or compound YNV98-4 were found to have significant anti-inflammatory effects.

(e) Compound YNV98-1 and/or compound YNV98-4 were found to have significant anti-pathological pain effects.

(f) Compound YNV98-1 and/or Compound YNV98-4 were found to significantly reduce morphine-induced pain tolerance and hypersensitivity.

Description of drawings

Figure 1 shows the new drug molecule binding pocket in Hv1 channel, and the binding model of YHV98-1 and YHV98-4 in this pocket to Hv1 channel respectively. Figure 1A shows that there is a druggable pocket in the Hv1 channel; Figure 1B shows the binding model of YHV98-1 molecule in the Hv1 channel in virtual screening; Figure 1C shows the binding model of YHV98-4 molecule in the Hv1 channel in virtual screening .

Figure 2 shows the inhibitory ability of YHV98-1 on Hv1 channel. Figure 2A is a representative current curve of 10 μM YHV98-1 inhibiting HEK-293T cells expressing Hv1 channel, and Figure 2B is a concentration-dependent curve of YHV98-1 inhibiting Hv1 channel.

Figure 3 shows the inhibitory ability of YHV98-4 on Hv1 channel. Figure 3A is a representative current curve of 10 μM YHV98-4 inhibiting HEK-293T cells expressing Hv1 channel, and Figure 3B is a concentration-dependent curve of YHV98-4 inhibiting Hv1 channel.

FIG. 4 is a graph comparing the responsiveness of Hv1 channel knockout mice and control mice to pain. WT represents the wild-type mouse group; Hv1KO represents the Hv1 channel knockout mouse group. ** means P<0.01; *** means P<0.001, which means there is a significant difference between the two groups.

Figure 5 is a line graph of the pain-relieving effect of YHV98-4 in mice von Frey silk assay after 7 days of preserved nerve injury (SNI). The intraperitoneal representative mode of administration is intraperitoneal administration. * means P<0.05; ** means P<0.01, which means there is a significant difference between the two groups.

Figure 6 is a broken line graph showing that YHV98-4 has the effect of relieving pain in the Von Frey silk test of the inflammatory pain model after 48 hours of CFA injection in mice. The intraperitoneal representative mode of administration is intraperitoneal administration. * means P<0.05; ** means P<0.01, which means there is a significant difference between the two groups.

Figure 7 is a line graph of the pain-relieving effect of YHV98-4 in mice thermal radiation assay after 7 days of preserved nerve injury (SNI). The intraperitoneal representative mode of administration is intraperitoneal administration. * means P<0.05; ** means P<0.01, which means there is a significant difference between the two groups.

Figure 8 shows that YHV98-4 has the effect of relieving pain in the thermal radiation measurement experiment of inflammatory pain model after 48 hours of CFA injection in mice. The intraperitoneal representative mode of administration is intraperitoneal administration. ** means P<0.01, which means there is a significant difference between the two groups.

组代表注射生理盐水组；Vehicle组代表注射溶剂组，YHV98-4代表注射药物组。*表示P<0.05；**表示P<0.01；***表示P<0.001，代表两组之间比较有显著差异。 Figure 9 is a bar graph showing that YHV98-4 has the effect of reducing the level of inflammatory factors in vivo in the determination of inflammatory factors in an inflammatory pain model 48 hours after CFA injection in mice.

Group represents the injection of physiological saline group; Vehicle group represents the injection of the solvent group, YHV98-4 represents the injection of the drug group. * means P<0.05; ** means P<0.01; *** means P<0.001, which means there is a significant difference between the two groups.

Figure 10 is a graph of the results of YHV98-4 compound improving morphine pain hypersensitivity and tolerance. (A) shows that YHV98-4 can significantly reduce allodynia induced by morphine in the von Frey silk experiment in which YHV98-4 is administered concurrently with morphine; (B) shows the results of morphine tolerance test, YHV98- 4 improves the dose relationship of morphine tolerance in mice. The intraperitoneal representative mode of administration is intraperitoneal administration. * means P<0.05; ** means P<0.01; *** means P<0.001, which means there is a significant difference between the two groups, and n.s. means there is no significant difference between the two groups of data.

Detailed ways

The present invention will be further described in detail with reference to the following specific embodiments and accompanying drawings. Except for the content specifically mentioned below, the process, conditions, experimental methods, etc. for implementing the present invention are all common knowledge and common knowledge in the field, and the present invention is not particularly limited.

After extensive and in-depth research, the inventors found that the pain threshold of the corresponding mice was significantly improved by knocking out the Hv1 channel technology, suggesting that the Hv1 channel can be used as a target for pain treatment. Through the combination of computer-aided drug design and electrophysiology, a small molecule or drug molecule binding site was found in the Hv1 channel for the first time, and a Hv1 inhibitory active compound was found at this site. Experiments show that the compounds of the present invention, as inhibitors of Hv1 channel, have significant analgesic effects and reduce the content of corresponding inflammatory factors. Further animal behavior experiments also show that the compounds can significantly reduce the pain tolerance and hyperactivity caused by morphine. It can be used to prevent and/or treat pain, prevent and/or treat inflammation, block morphine-induced pain tolerance and hypersensitivity. On this basis, the present invention has been completed.

Specifically, the present invention establishes an Hv1 channel gene knockout mouse model, and through pain behavior experiments, it is found that the gene knockout mouse has a significantly increased pain threshold.

The present invention uses the drug-producing pocket prediction and finds that the bottom end of S0 and S1-S4 in the Hv1 channel forms a pocket that can be used to bind small molecules or drug molecules. The activity test was carried out, and it was found that the compounds YHV98-1 and YHV98-4 have significant inhibitory effect on Hv1 channel, so they can be used as Hv1 channel inhibitors.

In the present invention, C57 mice were selected, and an anti-pain behavioral drug effect experiment was carried out, and YHV98-1 and/or YHV98-4 were injected intraperitoneally, and the result found that the compound of formula (I) YHV98-1 and/or the compound of formula (II) YHV98-4 has obvious analgesic effect and the effect of reducing inflammatory factors, so it can be used for the preparation of pain treatment and anti-inflammatory drugs.

In the present invention, different groups of C57 mice were intraperitoneally injected with different doses of morphine and YHV98-1 and YHV98-4 every day to conduct anti-pain behavioral pharmacodynamic experiments. The results found that the compounds of formula (I) YHV98-1 and formula ( II) The compound YHV98-4 has obvious effects of blocking morphine analgesic tolerance and improving pain hypersensitivity caused by morphine, so it can be used as a drug for the preparation of morphine analgesic tolerance and hypersensitivity.

As used in the present invention, the terms "compound of the present invention", "compound of formula (I)", "compound YHV98-1", or "YHV98-1" are used interchangeably to refer to the compound represented by formula (I), or its pharmacy acceptable salt.

As used in the present invention, the terms "compound of the present invention", "compound of formula (II)", "compound YHV98-4", or "YHV98-4" are used interchangeably to refer to the compound represented by formula (II), or its pharmaceutical acceptable salt.

In the present invention, the compound of formula (I) and/or the compound of formula (II) and the pharmaceutically acceptable salts of the above two compounds are also included. The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or base suitable for use as a medicament. Pharmaceutically acceptable salts include inorganic and organic salts.

A preferred class of pharmaceutically acceptable salts are the salts of the compounds of the present invention with acids. Acids suitable for salt formation include, but are not limited to, inorganic acids such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric, phosphoric; formic, acetic, propionic, oxalic, malonic, succinic, fumaric, Maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenemethanesulfonic acid, benzenesulfonic acid and other organic acids; and acidic amino acids such as aspartic acid and glutamic acid.

The pharmaceutical composition of the present invention contains a safe and effective amount of the compound of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): one or more of saline, buffer, dextrose, water, glycerol, ethanol, powder, and the like. The drug formulation should match the mode of administration.

The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The pharmaceutical combination of the present invention may also be formulated as a powder for nebulization.

The active ingredient is administered in a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 50 mg/kg body weight per day; preferably, about 5 microgram/kg body weight to about 10 mg/kg body weight; further preferably, it is About 10 micrograms/kg body weight - about 5 mg/kg body weight. In addition, the compounds of the present invention may also be used with other therapeutic agents.

The pharmaceutical compositions of the present invention can be administered to a desired subject (eg, humans and non-human mammals) by conventional means. Representative modes of administration include, but are not limited to: oral, injection, aerosol inhalation, and the like.

When a pharmaceutical composition is used, a safe and effective amount of the drug is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight; preferably, The dose is about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.

The present invention will be further described below in conjunction with specific embodiments.

Example 1 Discovery of new binding pockets of Hv1 channel can be used for active molecule or drug screening

The present invention starts from the nuclear magnetic structure of the Hv1 channel (PDB number: 5OQK), and uses Fpocket 2.0 to predict the druggable pocket in the structure of the Hv1 channel. The Druggablility score parameter is used to measure the druggability of a potential pocket, and a score greater than 0.5 means that the pocket may be druggable. After calculation and prediction, the present invention found a pocket surrounded by S0, S1-S4 bottom fragments in the intermediate state of Hv1 channel, and the Druggablility score was 0.99, indicating that the pocket may be a good drug binding pocket.

软件的Glide模块中进行。Hv1通道中间态结构用Protein Preparation模块进行结构优化和能量最小化，氢原子使用PROPKA进行调整，pH设定为7.0。优化好的蛋白做为对接结构。然后本发明对SPECS小分子化合物库进行了虚拟筛选。小分子用Maestro软件的Ligprep模块进行构象准备。准备好的小分子依次通过高通量筛选(High Throughput Virtual Screening，HTVS)和标准的精确筛选(Standard Precision，SP)，得到G-score得分前10,000的分子。本发明对这些筛选出的分子从结构的多样性、小分子结合模式以及类药性，如里宾斯基五规则、物理化学性质等方面进行了精挑细选，用于电生理检测活性。如图1显示了虚拟筛选出的YHV98-1和YHV98-4分子分别与Hv1通道中间态构象结合模式。 virtual screening at

performed in the Glide module of the software. The Hv1 channel intermediate state structure was structurally optimized and energy minimized with the Protein Preparation module, the hydrogen atoms were adjusted using PROPKA, and the pH was set at 7.0. The optimized protein is used as the docking structure. Then the present invention performs virtual screening on the SPECS small molecule compound library. Small molecules were conformationally prepared with the Ligprep module of Maestro software. The prepared small molecules were sequentially passed through High Throughput Virtual Screening (HTVS) and Standard Precision (SP) to obtain the molecules with the top 10,000 G-score scores. The present invention carefully selects these screened molecules from the aspects of structure diversity, small molecule binding mode and drug-like properties, such as Lipinski's five rules, physical and chemical properties, etc., and is used for electrophysiological detection activity. Figure 1 shows the binding modes of the YHV98-1 and YHV98-4 molecules screened out by the virtual screen to the Hv1 channel intermediate conformation, respectively.

Example 2 Detection of Hv1 inhibitory activity

Human Hv1 plasmids with green fluorescent tags were purchased from the company Vector Builder. The Hv1 plasmid was transiently transfected on HEK293T cells using Lipo3000 transfection reagent to overexpress the Hv1 channel on HEK293T cells. After 18-30 hours of transfection, cells with green fluorescence, a sign of successful transfection, were selected for electrophysiological experiments.

In electrophysiological detection of small molecule activity, the electrode inner fluid contains: 110mM MES, 65mM NMDG, 3mM MgCl ₂ , 1mM EGTA, adjusted to pH 6.0 with HCl, and the extracellular fluid contains: 180mM HEPES, 75mM NMDG, 1mM MgCl ₂ , 1mM CaCl ₂ , adjust the pH to 7.2 with HCl. During the test, the cells were clamped at -60mV for 200ms, then the voltage was returned to the test voltage of -80mV for 2s, and then returned to the clamped voltage of -60mV for 2s. During the whole 4.2s process, only the test voltage in the middle 2s is changed, and the cycle is ended after increasing to +80mV at every 10mV interval. The interval of each cycle is 10s. Whole-cell patch-clamp recording mode was used to record currents with an EPC10 (HEKA) amplifier, and the acquired data were analyzed with Clampfit 10.4 (Molecular Devices). The lower the value, the better the inhibitory activity. As shown in Figure 2, the compound YHV98-1 has a significant inhibitory ability on Hv1 channel and is an inhibitor of Hv1 channel. As shown in Figure 3, the compound YHV98-4 has significant inhibitory ability on THv1 channel and is an inhibitor of Hv1 channel.

Example 3 Effect of knockout of Hvcn1 gene on pain sensitivity in mice

Experimental mice: Hvcn1 knockout mice with C57B6 as the background were prepared and provided by Saiye Bio (China) Company using CRISPR-Cas9 technology. Wild-type (WT) mice with the same genetic background as Hvcn1 knockout mice were used as controls. All animal experiments were approved by the Ethics Committee of East China Normal University.

Data analysis: All data analysis was done using spss 22 (for mac) data processing software. Statistical comparisons were performed using one-way ANOVA or two-way ANOVA, and LSD method was used for multiple comparisons to verify the results. Data are presented as Mean±sem. One asterisk is marked when p<0.05; two asterisks are marked when p<0.01.

Von Frey silk test in mice: The Von Frey silk test is used to evaluate the tolerance response of mice to triggered mechanical pain. Use Von Frey wire kit (for 0.008g-300g stimulation), select nylon wire with appropriate thickness according to the actual situation, adjust the appropriate extension length, and stimulate the skin vertically. It can be adjusted by adjusting the elongation and replacing the nylon wire. Stimulate the force until the nylon wire bends. When the mice were mechanically stimulated, they would retract the reflex, record the intensity of the stimulation force used when raising the paw, and take the average of 3 consecutive measurements. This value was used to evaluate the sensitivity of the mice to trigger mechanical pain. As shown in Figure 4, the sensitivity of Hv1 channel knockout mice to triggered mechanical pain was significantly reduced, suggesting that Hv1 channel is a new target for the treatment of pain diseases.

Example 4 Animal pharmacology and efficacy experiment

The tested drugs used in the embodiments of the present invention include:

YHV98-4 was synthesized by conventional methods, and morphine hydrochloride injection (10mg/ml) was produced by Northeast Pharmaceutical Group Shenyang Pharmaceutical Co., Ltd., SFDA approval number: H21022436 (Liaoning, China).

A dose of YHV98-4 in the embodiment of the present invention is 10 mg/kg. The mode of administration is intraperitoneal injection, 10ml/kg.

Dissolution of Compounds:

YHV98-4 was dissolved according to the following steps: (1) 10% DMSO by injection volume was completely dissolved; (2) 10% injection volume of polysorbate 80 was added, and mixed well; (3) 80% injection volume of sulfobutyl ether was used Beta-cyclodextrin (20%) sodium chloride salt solution was diluted to final volume. According to this sequence of operations, mediate each step for 30s, followed by ultrasound for 1min, and configure and use on the same day.

Solvent control group: DMSO: polysorbate 80: sulfobutyl ether betacyclodextrin (20%) sodium chloride salt solution = 1:1:4.

Data analysis: All data analysis was done using spss 22 (for mac) data processing software. Statistical comparisons were performed using one-way ANOVA or two-way ANOVA, and LSD method was used for multiple comparisons to verify the results. Data are presented as Mean±sem. One asterisk is marked when p<0.05; two asterisks are marked when p<0.01; three asterisks are marked when p<0.001.

Experimental animals: 8-10-week-old Balb/c or C57BL/6J male mice, inbred (Inbred Mice), which are internationally used experimental mice, purchased from Shanghai Lingchang Laboratory Animal Co., Ltd.

Example 4.1 Mouse Von Frey silk experiment

The Von Frey silk test was used to evaluate the tolerance response of mice to triggered mechanical pain. Use Von Frey wire kit (for 0.008g-300g stimulation), select nylon wire with appropriate thickness according to the actual situation, adjust the appropriate extension length, and stimulate the skin vertically. It can be adjusted by adjusting the elongation and replacing the nylon wire. Stimulate the force until the nylon wire bends. When the mice were mechanically stimulated, they would retract the reflex, record the intensity of the stimulation force used when raising the paw, and take the average of 3 consecutive measurements. This value was used to evaluate the sensitivity of the mice to trigger mechanical pain. As shown in Figure 5, the mouse neuropathic pain model (Spared nerve injury (SNI) model) was established for 7-14 days and then underwent Von Frey silk experiment. After intraperitoneal injection of YHV98-4 (10 mg/kg), the The sensitivity of the mice to triggered mechanical pain was significantly reduced, suggesting that YHV98-4 could effectively relieve the triggered mechanical pain of mice. As shown in Figure 6, Balb/c mice were injected with CFA (20 μl) on the left plantar plantar for 48-72 hours to form an inflammatory pain model. Intraperitoneal injection of YHV98-4 (10 mg/kg) could significantly reduce the triggered mechanical pain in mice The degree of sensitivity of YHV98-4 can effectively alleviate the triggered mechanical hyperalgesia in mice under inflammatory pain.

Example 4.2 Mouse heat radiation experiment

Heat radiation test (Hargreaves test) was used to evaluate the tolerance response of mice to malicious heat stimuli. (1) Put the mice in the test room and adapt to the environment for 1h×3d; (2) Adapt to the plexiglass box for 30min; (3) Adjust the energy of the radiant heat source to stabilize the basic value of the stimulation intensity at 8-11s; ( 4) In order to avoid injury, the cut-off value was set to 20s; (5) The latency of raising the paw was recorded, measured three times continuously, and the average value was taken, and the time interval between two consecutive tests was 5min. As shown in Figure 7, the Hargreaves test was performed 7-14 days after the SNI model was established in mice. After intraperitoneal injection of YHV98-4 (10 mg/kg), the paw withdrawal response time of mice stimulated by thermal radiation was significantly prolonged, suggesting that YHV98-4 can Effectively relieve thermal pain in mice. As shown in Figure 8, Balb/c mice were injected with CFA (20 μl) on the left plantar plantar for 48-72 hours to form an inflammatory pain model, and intraperitoneal injection of YHV98-4 (10 mg/kg) could significantly prolong the paw withdrawal response to thermal radiation stimulation time, suggesting that YHV98-4 can significantly relieve thermal pain under inflammatory pain.

Example 4.3 Detection experiment of anti-inflammatory factors in mice

The left hind paw of the mice was subcutaneously injected with 20 μl of CFA, and the initial control group was not injected. By intraperitoneal injection, 2 injections of YHV98-4 (10 mg/kg) or control solvent were administered every 24 hours. 24-48 h after CFA injection, the skin of the hind foot of the mice was taken for protein extraction. Protein extraction was performed using ProcartaPlex Cell Lysis Extract (ThermoFisher Scientific). Protein concentration was determined using BCA protein assay kit (Beyotime). The content of mouse cytokines and chemokines was detected by mouse-derived cytokine/chemokine 26-plex kit (ThermoFisher Scientific), and the content of CXCL1 in cell culture supernatant was detected by mouse CXCL1 ELISA kit (EK10019). The kit assay was performed according to the manufacturer's instructions. As shown in Figure 9, compared with the unmodeled control group, the contents of inflammatory factors CXCL1, IL-1β, IL-9 and MIP-1β in the CFA model mice were significantly increased, while the injection of YHV98-4 significantly reduced these The content of inflammatory factors, suggesting that YHV98-4 can significantly reduce the content of inflammatory factors under inflammatory pain.

Example 4.4 Experiment on the effect of pain hypersensitivity and tolerance on morphine in mice

C57BL/6J mice were randomly divided into blank control group, model group and model administration group, with 8 mice in each group. Before administration, von Frey test was performed to detect the basic pain threshold of mice, and then blank control group: normal saline, once a day, Continuous injection for 9 days; model group mice (morphine tolerance model): intraperitoneal injection of morphine (10 mg/kg) for 6 consecutive days, once a day, 7, 8 and 9 days were injected with different doses of morphine (1 mg/kg, 5mg/kg, 10mg/kg); model administration group (morphine tolerance + YHV98-4): intraperitoneal injection of YHV98-4 (10mg/kg) 15 minutes before daily injection of morphine (10mg/kg), continuous injection for 6 days , On the 7th, 8th, and 9th days, different doses of morphine (1mg/kg, 5mg/kg, 10mg/kg) were injected respectively. From day 6 (6-9 days), von Frey test was performed daily to detect pain threshold in mice. The von Frey test was performed 30 min before (baseline) and 15 min after morphine administration. Percentage of maximum analgesia (%MPE)=(post-dose pain threshold-pre-dose pain threshold)/(termination threshold-pre-dose pain threshold)×100%. As shown in Figure 10A, the results of the von Frey test showed that the mice in the model group were more sensitive to mechanical pain-induced pain, while the mice in the model administration group had a higher threshold for mechanical pain-induced pain and could form a significant difference. It is indicated that intraperitoneal injection of YHV98-4 can improve the hyperalgesia induced by morphine administration. YHV98-4 can significantly reduce pain hypersensitivity and tolerance induced by morphine administration. As shown in Figure 10B, the maximum analgesic percentage of the mice in the model group was significantly lower than that in the model administration group and the blank control group, indicating that morphine pain tolerance was tolerated. The maximum analgesia percentage of mice in the model administration group was not significantly different from that in the blank control group, and was significantly higher than that in the model group, indicating that intraperitoneal injection of YHV98-4 could improve morphine tolerance.

It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the above-mentioned examples, the experimental methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer.

In conclusion, Hv1 channel can be used as a new target for preventing and/or treating pain, preventing and/or treating inflammation, and blocking morphine-induced pain tolerance and hypersensitivity. , neuropathic pain-related symptoms, including mechanical pain and thermal pain and other models, reduce pain response, and can significantly reduce the content of inflammatory factors in chronic inflammatory pain model mice, showing that Hv1 channel inhibitor molecular compounds have significant anti-multiple Painful and anti-inflammatory effects. All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

The protection content of the present invention is not limited to the above embodiments. Variations and advantages that can occur to those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the appended claims are the scope of protection.

Claims (15)

A use of a compound represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition or preparation for preventing and/or treating pain, characterized in that the compound or The pharmaceutically acceptable salt thereof has inhibitory activity of the voltage-gated proton channel Hv1, and the structure of the compound is shown below:

A use of a compound represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition or preparation for preventing and/or treating inflammation, characterized in that the compound or The pharmaceutically acceptable salt thereof has inhibitory activity of the voltage-gated proton channel Hv1, and the structure of the compound is shown below:

Use of a compound represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition or preparation for preventing and/or treating opioid analgesic tolerance and hypersensitivity , characterized in that the compound or a pharmaceutically acceptable salt thereof has the inhibitory activity of the voltage-gated proton channel Hv1, and the structure of the compound is as follows:

Use of a compound represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition or preparation for preventing and/or treating stroke, Parkinson's disease and atherosclerosis The use is characterized in that the compound or a pharmaceutically acceptable salt thereof has the inhibitory activity of the voltage-gated proton channel Hv1, and the structure of the compound is as follows:

The use according to any one of claims 1-4, wherein the administration dose of the compound of formula (I) is 0.1-50 mg/kg; and/or the administration dose of the compound of formula (II) 0.1-50mg/kg; and/or, the pharmaceutical composition contains 0.001-99wt% of the compound of formula (I) and/or formula (II) or a pharmaceutically acceptable salt thereof, according to the total amount of the composition weight meter. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a compound represented by formula (II) or a pharmaceutically acceptable salt thereof Salt and a pharmaceutically acceptable carrier, wherein the structure of the compound is shown below:

Use of the pharmaceutical composition according to claim 6 in the preparation of a pharmaceutical composition or preparation for preventing and/or treating pain. The use according to claim 1 or 7, wherein the pain includes chronic pain, acute pain, and cancer pain; wherein the chronic pain includes muscle and soft tissue pain, bone and joint pain, headache, and visceral pain , pathological neuralgia; the acute pain includes one or more of acute traumatic pain, postoperative pain, labor pain, visceral pain, pruritus, and postoperative pain. The use according to claim 8, wherein the muscle and soft tissue pain includes myofasciitis, tenosynovitis, frozen shoulder, muscle strain pain, fibromyalgia, cold pain, burn pain, and toothache. One or more; and/or, the bone and joint pain includes knee joint pain, ankle joint pain, wrist joint pain, elbow joint pain, shoulder joint pain, patella joint pain, hip joint pain, femoral joint pain, One or more of ankylosing spondylitis, sacroiliitis, rheumatoid arthritis, rheumatoid arthritis, gouty arthritis, intervertebral disc herniation, cervical spine pain, lumbar spine pain; and/or; said Visceral pain includes pain from internal organs including respiratory tract, gastrointestinal tract, pancreas, urethra, kidney, gallbladder, bladder and genitalia; and/or, described pathological neuralgia includes post-herpetic neuralgia, diabetes Neuropathic pain, painful HIV-related sensory neuropathy, burning syndrome, post-amputation pain, phantom pain, painful neuroma, traumatic neuroma, nerve crush injury, spinal stenosis, carpal tunnel syndrome, nerve root pain, sciatica, nerve avulsion, brachial plexus avulsion, complex regional pain syndrome, neuralgia from drug therapy, neuralgia from cancer chemotherapy, pain after spinal cord injury, primary small fiber neuropathy, One or more of primary sensory neuropathy and trigeminal autonomic headache. The use according to claim 8, wherein the headache includes primary headache, secondary headache, cranial neuralgia, central and primary facial pain and other headaches; wherein, the primary headache Headache includes migraine without aura, migraine with aura, hemiplegic migraine, chronic migraine, migraine complications, migraine-related episodic syndrome, tension-type headache, trigeminal autonomic headache, and other primary One or more of sexual headaches; the secondary headaches include headaches attributed to head and neck trauma, headaches attributed to non-vascular intracranial diseases, headaches attributed to substances or substance withdrawal, and headaches attributed to head and neck trauma Headache due to vascular disease of the neck, headache due to disturbance of the internal environment, headache due to mental illness, headache due to head, neck, eye, ear, nose, sinus, tooth, mouth or other head and face structure lesions One or more; the cranial neuralgia, central and primary facial pain and other headaches include one or more of trigeminal neuralgia, glossopharyngeal neuralgia, intermediary neuralgia, occipital neuralgia, and optic neuritis. The use according to claim 8, wherein the cancer in cancer pain comprises adenocarcinoma in glandular tissue, blastoma in organ embryo tissue, cancer in epithelial tissue, and cancer in tissue forming blood cells Leukemia, lymphoma in lymphoid tissue, myeloma in bone marrow, sarcoma in connective or supporting tissue, adrenal cancer, AIDS-related lymphoma, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid tumors, cervical cancer , colon cancer, endometrial cancer, esophageal cancer, head cancer, cervical cancer, hepatobiliary cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's disease, non-Hodgkin's disease, nervous system tumors, Oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, urethral cancer, bone marrow cancer, multiple myeloma, tumors that metastasize to bone, infiltrating nerves and hollow organs One or more of tumors near nerve structures. Use of the pharmaceutical composition according to claim 6 in the preparation of a pharmaceutical composition or preparation for preventing and/or treating inflammation. The use according to claim 2 or 12, wherein the inflammation refers to infectious inflammation and non-infectious inflammation, including inflammation caused by bacteria, viruses, parasitic biological pathogens, high temperature, low temperature, radioactive substances and ultraviolet rays Inflammation caused by and mechanical damage, inflammation caused by strong acid, strong alkali and turpentine, mustard gas and endogenous toxic substances and metabolites in the body, inflammation caused by metals, wood chips, dust particles, and surgical sutures, caused by necrotic tissue Inflammation caused by tissue necrosis, allergic rhinitis, urticaria, glomerulonephritis, lymphocytic thyroiditis, inflammation caused by ulcerative colitis, ankylosing spondylitis, sacroiliitis, rheumatoid arthritis, rheumatoid Arthritis, gouty arthritis, myofasciitis, tenosynovitis, frozen shoulder. Use of the pharmaceutical composition according to claim 6 in the preparation of a pharmaceutical composition or preparation for preventing and/or treating opioid analgesic tolerance and hypersensitivity. Use of the pharmaceutical composition according to claim 6 in the preparation of a pharmaceutical composition or preparation for preventing and/or treating stroke, Parkinson's disease and atherosclerosis.

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