WO2012139529A1

WO2012139529A1 - D-amino acid oxidase inhibitor for preventing and/or reversing opioid tolerance

Info

Publication number: WO2012139529A1
Application number: PCT/CN2012/074062
Authority: WO
Inventors: 王永祥; 龚念
Original assignee: Wang Yongxiang; Gong Nian
Priority date: 2011-04-14
Filing date: 2012-04-16
Publication date: 2012-10-18
Also published as: CN102727896A

Abstract

Provided are a D-amino acid oxidase inhibitor for preventing and/or reversing opioid tolerance, and a method using the D-amino acid oxidase inhibitor for preventing and/or reversing opioid tolerance. Also provided are a method for using the D-amino acid oxidase inhibitor for preventing and/or treating pain, and a method for identifying a compound for preventing and/or reversing opioid tolerance.

Description

Acid oxidase inhibitors prevent and/or reverse opioid tolerance

Technical field

[01] The opening relates to the prevention and/or reversal of opioid tolerance. Specifically, a D-J ^ acid oxidase inhibitor for preventing and/or reversing opioid tolerance, and a method for preventing and/or reversing opioid tolerance using a D-amino acid oxidase inhibitor are provided, and A method of preventing and/or treating pain using D-acid oxidase, and a method of identifying a compound for preventing and/or reversing opioid tolerance.

Background technique

[02] Opioid agonists (also known as opioid agonists, opioids, opioids, and opioids) such as morphine are a class of narcotic analgesics that are used over long periods of time when relatively high doses are used or although doses are low. It produces a strong drug tolerance, which leads to a decrease in the analgesic effect of the analgesic. Thus, an anesthetic analgesic should be used in an increased dose to achieve the desired analgesic effect. The increased dose of this narcotic analgesic, leading to different side effects, serious toxicity to humans, and serious clinical problems such as serious clinical problems and drug abuse, is urgently needed for development, prevention, intervention and resistance. Broken opioid-tolerant agents.

[03] Pain is one of the most common symptoms in the clinic. Many diseases or conditions have pain symptoms and seriously affect the quality of life of patients. Treatment of pain has become one of the most considered factors in health care and disease treatment. Therefore, pain has received more and more attention from the medical community. 2000-2010 has been called the pain era. Pain is an unpleasant sensation and emotional experience associated with acute or potential tissue damage that prompts people to avoid the devastating condition, thereby protecting the body and avoiding further damage. Most of the pain disappears immediately after the pain stimulus is removed, but sometimes the pain persists even though the stimulus has been eliminated and the body has recovered significantly. In addition, sometimes pain occurs despite the absence of any irritation, destruction or disease.

[04] Pain can be divided into acute pain and chronic pain. Chronic pain is a hot spot in international pain research. It can be divided into immune inflammatory pain, neuropathic pain, tumor (cancer) pain, diabetes pain and low back pain depending on the cause. The difference between chronic pain and acute pain is that some non-damaging stimuli caused by stimulation of the peripheral nerves, such as light touch and warmth, can also cause strong pain in the affected area. Chronic pain often occurs after a period of injury, with this pain Hair, randomness and persistence, and the use of commonly used analgesics is ineffective and often does not heal for life. Chronic pain involves complex mechanisms of formation. Peripheral ectopic discharge and central sensitization are currently recognized as the main mechanisms of chronic pain including neurogenic pain and tumor pain formation (Basbaum et al. Cell 139: 267- 284, 2009). The incidence of chronic pain is high, accounting for about 10% of the total population, and increases with age and prolonged prolonged disease (eg, the incidence rate is over 13% over 55 years old, and 8% in the early diagnosis of diabetes. Diagnostics up to 50% after 25 years).

[05] Pain (such as various forms of chronic pain, including cancer pain, neuropathic pain, etc.) is inadequately available in wards, intensive care units (ICUs), emergency departments, and the like. Pain can be treated with drugs, psychotherapy, physiotherapy, and traditional medical therapies. The current clinical treatment of chronic pain mainly includes tricyclic antidepressants such as amitriptyline, antiepileptic drugs such as gabapentin and pregabalin, local anesthetics such as lidocaine, opioids (such as Dihydromorphone, fentanyl and buprenorphine), adrenergic receptor antagonists such as clonidine, and 5-HT reuptake inhibitors such as duloxetine and ziconotide. However, due to the low awareness of chronic pain, most of the products available on the market are not specific to chronic pain, which is ineffective or only moderately relieved for most patients, may cause many adverse reactions, or is inconvenient to administer. . For example, the current clinical treatment of pain such as cancer pain uses a three-stage analgesic ladder therapy, that is, mild cancer pain, non-steroidal anti-inflammatory analgesics represented by aspirin and indomethacin, moderate cancer Sexual pain uses weak opioid moderate analgesics such as tramadol, codeine, and oxycodone. For severe cancer pain, an anesthetic with strong opioid receptor agonists such as morphine and chilling is required. Opioid receptor agonists, while effectively controlling severe pain, bring serious clinical problems of drug tolerance and serious social problems such as drug abuse on an international scale. In addition, the high-dose anesthetic drugs used to treat severe pain have significant side effects such as inhibition of breathing, vomiting, and visceral smooth muscle spasm, and the side effects of visceral smooth muscle spasm must be improved by using atropine and other drugs, otherwise it will become Aggravating the cause of common visceral pain such as biliary colic and renal colic, which is one of the reasons why the analgesic effect lasts for a short time.

[06] Analgesics are commonly used medical drugs to suppress or relieve acute pain. Analgesics are usually classified into narcotic and non-narcotic. Clinically, narcotic analgesics are used to treat acute pain in patients with advanced cancer because they have excellent analgesic effects on visceral pain. However, when an anesthetic analgesic is administered at a relatively high dose or although it is administered at a low dose for a long period of time, it may result in a habitual administration of the analgesic and a desire for an analgesic. Further, when the user repeatedly uses a prescribed amount of an anesthetic analgesic, the tolerance to the analgesic is rapidly developed, resulting in a decrease in the analgesic effect of the analgesic. Thus, an anesthetic analgesic should be used in an increased dose to achieve the desired analgesic effect. The increase in the dose of this narcotic analgesic causes different side effects and thus has Severe toxicity.

[07] As a representative example, opioid agonists are known to be the most commonly used analgesic analgesics, of which morphine, fentanyl and the like are currently used as opioid agonist analgesics, such as morphine. 80% share. However, repeated administration of opioid agonists such as morphine may result in a gradual decrease in analgesic effects and ultimately lead to tolerance to opioid analgesia. For these reasons, despite its excellent efficacy, the use of opioid agonists such as morphine in the clinical field is limited. Therefore, in order to utilize an opioid agonist such as morphine as an analgesic, it is very important to maintain the initial analgesic effect of the opioid agonist without reducing the analgesic effect upon repeated use. This object can be achieved by inhibiting the development of tolerance to opioid agonists.

[08] Opioid tolerance refers to the prolonged or even disappearance of drug analgesic effects after prolonged use of opioids (opioid agonists), or the need to gradually increase opioids if the same degree of analgesic effect is required. The dose, in addition to showing a decrease in the analgesic effect of the drug, and an increase in the dose required for the drug, can also lead to more severe hyperalgesia. The production of opioid tolerance (opioid tolerance) is a complex biochemical process, and a large number of experimental studies have confirmed that opioid receptor desensitization and endocytosis (Gainetdinov et al., Ann Rev Neurosci 27: 107-144, 2004) Post-receptor signal transduction (Raval et al., J Neurosci 27: 107-144, 2004) and transcription factors that cause changes in neuronal plasticity (Miyabe and Miletic, Neurosci Lett 38: 80-85, 2005) are involved. Formation of opioid tolerance.

[09] Therefore, there is an urgent need for a method of preventing and/or reversing opioid tolerance, as well as a drug capable of preventing and/or reversing opioid tolerance. In addition, it would be desirable to have other classes of drugs for the prevention and/or treatment of pain in place of opioid agonists.

Summary of the invention

[10] The inventors have unexpectedly discovered that D-amino acid oxidase inhibitors, including CBIO, AS057278 and sodium benzoate, are capable of preventing and/or reversing the tolerance produced by opioid agonist treatment.

[11] The inventors have also unexpectedly found that, contrary to the prediction according to the prior art, the acid oxidase inhibitor itself is capable of significantly preventing and/or pain.

[12] Based on the above unexpected findings, the inventors completed the present invention and obtained the following series of technical solutions.

[13] In one aspect, the present disclosure provides a method of preventing and/or reversing opioid tolerance comprising administering an effective amount of a D-amino acid oxidase inhibitor to a subject in need thereof. [14] In one embodiment, the method is for preventing and/or treating a condition which is reduced in therapeutic efficacy due to opioid tolerance, the condition being capable of achieving prevention and/or opioid tolerance by inhibiting D-amino acid oxidase Get prevented and / or treated. In particular, the condition can be pain, including but not limited to acute and/or chronic pain, especially chronic pain, such as cancer pain.

[15] In a specific embodiment, the condition that is reduced in therapeutic efficacy due to opioid tolerance is selected from the group consisting of chronic pain, cancer pain, neuropathic pain, chronic low back pain, headache, migraine, trigeminal neuralgia, Cluster headache, fibromyalgia syndrome, joint pain, inflammatory pain, arthritis pain, osteoarthritis pain, rheumatoid arthritis pain, tumor pain, cancer pain, visceral pain, somatic pain, musculoskeletal pain, bone Pain, lumbosacral pain, neck pain or upper back pain, diabetic pain, pain caused by spinal cord injury, surgical pain, postoperative pain, acute pain, or with infection, sickle cell anemia, autoimmune disease, multiple sclerosis or Inflammation-related pain, pain caused by injury or surgery, or a combination thereof.

[16] In another specific embodiment, the condition that is reduced in therapeutic efficacy due to opioid tolerance is selected from the group consisting of pain associated with diabetic neuropathy, peripheral neuralgia, post-herpetic neuralgia, waist or neck Nerve root pain, fibromyalgia, glossopharyngeal neuralgia, reflex sympathetic dystrophy, burning pain, thalamic syndrome, nerve root avulsion, phantom limb pain, pain after thoracotomy, cancer, chemical damage, toxins, nutrition Lack, viral or bacterial infection, temporomandibular joint disorder syndrome, fibromyalgia syndrome, osteoporosis, bone metastasis or other unknown causes of bone pain, gout, fibrinitis, myofascial pain, thoracic outlet syndrome , back or back pain, pelvic pain, heart chest pain, non-cardiac chest pain, spinal cord injury-related pain, central stroke pain, cancer pain, AIDS pain, anti-tumor drug-induced neuropathic pain, sickle cell pain, old-age pain , or a combination of them.

[17] In another embodiment, the D-amino acid oxidase inhibitor is selected from one or more of the following: CBIO (5-chlorobenzo [d] isoxazol-3-ol, Ferraris et al" J Med Chem 51 : 3357-3359, 2008; Hashimoto et al., Biol Psychiatry 65: 1103-1106, 2009; Horio et al., Open Clin Chem J 2: 16-21, 2009; Gong et al., J Pharmacol Exp Ther 336: 282-293, 2011), AS057278 (5-methylpyrazole-3-carboxylic acid, Adage et al" Eur Neuropsychopharmacol 18: 200-214, 2008; Gong et al., J Pharmacol Exp Ther 336: 282-293, 2011), Compound 8 (4H-thieno [3,2-b]pyrrole-5-carboxylic acid, Sparey et al" Bioorg Med Chem Lett 18: 3386-3391, 2008; Smith et al., J Pharmacol Exp Ther 328: 921-930 , 2009; Gong et al., J Pharmacol Exp Ther 336: 282-293, 2011 ), Compound 2 ( 3-hydroxyquinolin-2-(lH)-one, Duplantier et al" J Med Chem 52: 3576-3585, 2009 ; Gong et al., J Pharmacol Exp Ther 336: 282-293, 2011) and sodium benzoate (Vanoni et al., Biochemistry 36: 5624-5632, 1997; Gong et al., J Pharmacol Exp Ther 336: 282-293, 2011) and the like and derivatives thereof. D-amino acid oxidase inhibitors can be used to prevent and inhibit morphine tolerance caused by repeated administration of morphine without affecting the analgesic effect of morphine when administered alone.

[18] In another embodiment, the image exhibits tolerance to one or more opioids selected from the group consisting of: alfentanil, fenprozine, afarodidine, anilidine, benzyl Morphine, benzonitrile, buprenorphine, butorphanol, chloredazin, codeine, deoxymorphine, dexamethasone, dextrozine, dienamide, diamorphone, dihydrocodeine , dihydromorphine, dimethacamine, methadol, butylamine, morphine, pellucidum, etazocine, isoxazin, ethmidine, ethylmorphine, ethoxynidazole , etorphine, dihydroetorphine, fentanyl and derivatives, hydrocodone, hydromorphone, hydroxyperidine, isomeacone, ketomines, levorphane, levofene, Rofentanil, phenidine, meptazin, metazocine, methadone, hydromorphone, morphine, meropenine, narceine, nikomamorph, hydroxymorphin, normethapone, allymorphine, Nabate, normorphine, noripeptone, opioid, oxycodone, oxymorphone, opioid, Pentazone, benzoheptanone, hydroxyphenidate, phenazocine, phenepidine, pinidinidine, cyanamide, propheptazine, trimethylridine, meperidine, c Oxygen, sufentanil, tilidine, tramadol or a derivative thereof. For example, the opioid may be morphine or a derivative thereof.

[19] In still another embodiment, when the method is for preventing and/or treating a condition in which the therapeutic efficacy is reduced by opioid tolerance, the method further comprises administering an additional therapeutic agent to the subject. For example, when used to prevent and/or treat pain, a pain treatment agent can also be administered to the subject, including but not limited to an analgesic such as an opioid analgesic, or a combination thereof.

[20] In a specific embodiment, the additional therapeutic agent is selected from the group consisting of opioids, for example selected from one or more of the following opioids: afentanil, allylidine, alfaclodine, Anilididine, benzylmorphine, benzonitrile, buprenorphine, butorphanol, chlordazin, codeine, deoxymorphine, dexamethasin, dextrozine, dipropenamine, diamorphone, Dihydrocodeine, dihydromorphine, dimethacin, methadol, butylamine, morphine, pellucidum, etatazocin, issoxazine, methotrexate, ethylmorphine , ethoxynidazole, etorphine, dihydroetorphine, fentanyl and derivatives, hydrocodone, hydromorphone, oxypeptidine, isomezin, ketomines, levorphan , leflufendane, remifentanil, meperidine, meptazin, metazocine, methadone, hydromorphone, morphine, meropenine, narceine, nikomamorph, hydroxymorphane, formazan Methadone, allymorphine, nalbuphine, normorphine, noripeperone, opioid, oxycodone, oxymorphone , opioid, pentazocine, benzoheptanone, hydroxyphenidate, phenazocine, phenepidine, Minodine, cyanidinamide, ropheptazine, trimethylrididine, meperidine, propoxyphene, sufentanil, tilidine, tramadol or a derivative thereof. For example, the opioid may be morphine or a derivative thereof. In a specific embodiment, the opioid agonist can be a morphine drug such as morphine, hydromorphone, fentanyl, buprenorphine, and buprenorphine.

[21] In another embodiment of the method of the invention, the D-amino acid oxidase inhibitor is administered prior to, concurrently with, or after administration of the opioid.

[22] In another embodiment, the D-amino acid oxidase inhibitor is administered orally or parenterally, for example, including subcutaneous injection, spinal administration, intracerebral administration, intramuscular injection, nasal administration, intravenous Internal administration and/or intraperitoneal administration.

[23] In a second aspect, the present disclosure provides a method of preventing and/or treating pain comprising administering an opioid and a D-amino acid oxidase inhibitor to a subject in need thereof.

[24] Specifically, opioids and acid oxidase inhibitors can be used separately and/or separately. Furthermore, the D-amino acid oxidase inhibitor can be administered prior to, concurrently with, and/or after administration of the opioid.

[25] In one embodiment, the opioid is selected from one or more of the following: alfentanil, allylidine, afarodidine, anilidine, benzylmorphine, benzonitrile, Buprenorphine, butorphanol, chlordazin, codeine, deoxymorphine, dexamethasin, dextrozine, dipropenamine, diamorphone, dihydrocodeine, dihydromorphine, dimethacin , methadol, butylamine, morphine, pellucidum, etazocine, isoglucan, methotrexate, ethylmorphine, ethoxynidazole, etorphine, dihydrogen Trophine, fentanyl and derivatives, hydrocodone, hydromorphone, oxypipetiline, isomeacone, cretophanone, levorphane, levonformane, remifentanil, patidine Pyridinium, meptazin, metazocine, methadone, hydromorphone, morphine, meropenine, narceine, nikomamorph, hydroxymorphin, normethapone, allymorphine, nalbuphine, normorphine , nopiperone, opioid, oxycodone, oxymorphone, opioid, pentazocine, phenylheptanone, hydroxybenzene Ethyl, phenazocine, bepeptidin, pinidinidine, cyanamide, ropheptazine, trimethylrididine, isopropylpyrazine, propoxyphene, sufentanil, tilidine, Tramadol, its derivatives and any mixtures thereof.

[26] In another embodiment, the D-amino acid oxidase inhibitor is selected from one or more of the group consisting of CBIO, Compound 8, Compound 2, AS057278, sodium benzoate and derivatives thereof.

[27] In another embodiment, the pain is acute pain and/or chronic pain, especially chronic pain, such as cancer pain. Specifically, for example, the pain may be selected from the following one or Multiple: chronic pain, neuropathic pain, chronic low back pain, headache, migraine, trigeminal neuralgia, cluster headache, fibromyalgia syndrome, joint pain, inflammatory pain, arthritis pain, osteoarthritis pain, Rheumatoid arthritis pain, tumor pain, cancer pain, visceral pain, somatic pain, musculoskeletal pain, bone pain, lumbosacral pain, neck pain or upper back pain, diabetic pain, pain caused by spinal cord injury, surgical pain, Postoperative pain, acute pain, or pain associated with infection, sickle cell anemia, autoimmune disease, multiple sclerosis or inflammation, pain caused by injury or surgery, diabetic neuropathic pain, peripheral neuralgia, postherpetic neuralgia , lumbar or cervical nerve root pain, fibromyalgia, glossopharyngeal neuralgia, reflex sympathetic dystrophy, burning pain, thalamic syndrome, nerve root avulsion, phantom limb pain, pain after thoracotomy, cancer, chemistry Injury, Toxin, Nutritional Deficiency, Viral or Bacterial Infection, Temporomandibular Joint Disorder Syndrome, Fibromyalgia Syndrome, Bone Pine, bone metastasis or other causes of bone pain, gout, fibrinitis, myofascial pain, thoracic outlet syndrome, upper back pain, lower back pain, pelvic pain, cardiac chest pain, non-cardiac chest pain, spinal cord injury Related pain, central post-stroke pain, cancer pain, AIDS pain, anti-tumor drug-induced neuropathic pain, sickle cell pain, geriatric pain, or a combination thereof.

In a third aspect, the present disclosure provides a method of treating chronic pain, particularly cancer pain, comprising administering to a subject in need thereof an effective amount of a D-amino acid oxidase inhibitor.

[29] In one embodiment, the D-amino acid oxidase inhibitor is selected from one or more of the following: CBIO, Compound 8, Compound 2, AS057278, sodium benzoate and derivatives thereof.

[30] Based on the above findings of the present disclosure, there is also provided a method of identifying a compound capable of preventing and/or reversing opioid tolerance, comprising the steps of: providing a test compound to contact the test compound with an acid oxidase And determining the activity of the acid oxidase; if the test compound is capable of inhibiting the activity of D-amino acid oxidase, the test compound can be used as a compound for preventing and/or reversing opioid tolerance. For example, a compound identified according to the above method can be used for the prevention and/or treatment of a disease whose therapeutic efficacy is lowered due to opioid tolerance. In a specific embodiment, the condition is pain, especially chronic pain.

[31] In another aspect, the invention provides a method of identifying a compound capable of preventing and/or reversing opioid tolerance, comprising the steps of: providing a test compound and an opioid prior to administration of the opioid, Simultaneously and/or subsequent administration of the test compound to a test subject, compared to opioid activity prior to administration of the test compound and/or administration of the test compound, if the opioid is administered after administration of the test compound When the level of activity is increased, the test compound can be used as a compound for preventing and/or reversing opioid tolerance. For example, according to The compounds identified by the above methods are useful for the prevention and/or treatment of conditions which result in reduced therapeutic efficacy due to opioid tolerance. In a specific embodiment, the condition is pain, especially chronic pain.

[32] In a sixth aspect, the use of an acid oxidase inhibitor for the preparation of a medicament for preventing and/or reversing opioid tolerance is provided. In various embodiments of the use, in particular, the technical solutions corresponding to the various embodiments involved in the method of the first aspect described above are included.

[33] In a seventh aspect, the invention provides the use of an acid oxidase inhibitor in combination with an opioid for the preparation of a medicament for the prevention and/or treatment of pain. Among the various embodiments of the use, in particular, the technical solutions corresponding to the various embodiments involved in the method of the second aspect described above are included.

[34] In an eighth aspect, an acid oxidase inhibitor is provided for use in preventing and/or reversing opioid tolerance, or in combination with an opioid for preventing and/or treating pain. In various embodiments of the D-amino acid oxidase inhibitor, in particular, the technical solutions corresponding to the various embodiments involved in the methods of the first and second aspects described above are included.

[35] In a ninth aspect, an acid oxidase inhibitor for the preparation of a medicament for preventing and/or reversing opioid tolerance, or for use in combination with an opioid for the preparation of prophylaxis and/or Or a drug that treats pain. In various embodiments of the D-amino acid oxidase inhibitor, in particular, the technical solutions corresponding to the various embodiments involved in the methods of the first and second aspects described above are included.

[36] The following content merely illustrates some specific embodiments claimed, wherein the technical features described in one or more technical solutions may be combined with any one or more technical solutions, which are combined. The technical solutions are also within the scope of the present application, as these technical solutions obtained by combining have been specifically described in the present disclosure.

The invention will be exemplified with reference to the drawings and further detailed description below. It should be noted that the following description is merely illustrative of the technical solutions claimed in the present invention, and is not intended to limit the technical solutions. The scope of the invention is defined by the appended claims.

DRAWINGS

Figure 1 shows the prevention and reversal of morphine tolerance by a single or continuous subcutaneous injection of the acid oxidase inhibitor CBIO. A is a mouse formalin phase I acute pain response, and B is a phase II chronic pain response. Compared with the corresponding saline group, a represents P <0.05; In the analgesic group, b represents P < 0.05.

[39] Figure 2 shows the effect of subcutaneous injection of the D-^ acid oxidase inhibitor CBIO on the dose-effect relationship of morphine tolerance for 7 consecutive days. A is the effect of a single injection of different doses of morphine on formalin pain in mice after subcutaneous injection of normal saline for 7 days; B is a 7-day subcutaneous injection of morphine (10 m _g / k _g ), a single injection is different The effect of dose of morphine on formalin pain in mice; C was a 7-day subcutaneous injection of CBIO (10 mg/kg) + morphine (10 mg/kg), a single injection of different doses of morphine on mice formalin The effect of pain; D is the dose-effect relationship of mouse formalin phase I acute pain morphine; E is the dose-effect relationship of mouse formalin phase II chronic pain morphine.

[40] Figure 3 shows the preventive effect of subcutaneous injection of D-J-acid oxidase inhibitors AS057278 (40 mg/kg) and sodium benzoate (400 mg/kg) on morphine tolerance for 7 consecutive days. A is the acute pain response of mouse Formalin phase I, and B is the chronic pain response of phase II. Compared with the corresponding saline group, a represents P < 0.05; b represents P < 0.05 compared with the corresponding morphine analgesia group.

[41] Figure 4 shows a single intrathecal injection of acid oxidase inhibitors CBIO (30 μg), AS057278 (100 μg) and sodium benzoate (300 μg) and reactive oxygen species (ROS). The reversal effect of the scavenger PBN (1 mg) on the tolerance of morphine to the formalin pain model in rats. a indicates P<0.05 compared with the morphine tolerant group.

[42] Figure 5 shows single-dose subcutaneous injection of acid oxidase inhibitors CBIO, AS057278 and sodium benzoate in mouse hot plate iris (A, B, C) and hot plate (D, E, F) pain models Immediately reverse the dose-effect relationship of morphine tolerance.

[43] Figure 6 shows the effect of single-dose subcutaneous injection of D-acid oxidase inhibitors CBIO, AS057278 and sodium benzoate on the reversal of morphine tolerance in the mouse appendix (A) and hot plate (B) pain models. Relationship and analysis of correlations with inhibition of D-amino acid oxidase activity in rat spinal cord (C and D).

[44] Figure 7 shows the effect of spinal cord injection of D-J^ acid oxidase siRNA/DAO on formalin pain in morphine-tolerant rats for 7 consecutive days (A). Effect of spinal cord injection of D-amino acid oxidase siRNA/DAO on DAO expression in spinal cord of morphine-tolerant rats (B). Acute phase I pain map after injection of formalin in rats (C). The dose-effect relationship of the drug to the phase II pain response was calculated by the area under the curve (D).

[45] Figure 8 shows the effect of spinal cord injection of D-J^ acid oxidase Virus siRNA/DAO on formalin pain in morphine-tolerant rats for 7 consecutive days (A). Effect of spinal cord injection acid oxidase Virus siRNA/DAO on DAO expression in spinal cord of morphine-tolerant rats (B). Phase I acute pain map after injection of formalin in rats (C). Calculate the pain response of the drug to phase II by the area under the curve Dose-effect relationship (D).

detailed description

[46] The terms used herein have their ordinary meanings in the art. In order to clarify its meaning, the following is a description of some of the terms used in this article.

[47] As used in the context of the disclosure, the term "treating" means that the target disease or condition and/or any of its symptoms and/or any of its possible complications and/or sequelae of the subject to be treated are radically treated by medical intervention, Eliminating, alleviating, ameliorating, stopping exacerbation, and/or prevention, or causing the subject to be treated to obtain any positive effects in an immediate and/or potential medical sense.

[48] As used in the context of a medical condition, the term "prevention" refers to preventing, avoiding and/or delaying the occurrence of medical conditions. For example, "preventing opioid tolerance" means preventing, avoiding, and/or delaying the onset of opioid tolerance.

[49] As used in the context of a medical condition, the terms "reversal" or "disarmed" when used in a medical condition mean that the medical condition that has occurred does not occur again, and/or reduces its severity. For example, "reversing/relaxing opioid tolerance" refers to the level at which opioid tolerance that has occurred no longer occurs and/or reduces opioid tolerance.

[50] As used in the content, D-amino acid oxidase (DAO) is an oxidoreductase that oxidizes D-amino acids to the corresponding imino acids to produce ammonia. And hydrogen peroxide. DAO usually contains FAD as a cofactor, which is expressed in a wide variety of species from yeast to humans, but not in bacteria and plants.

[51] As used in the context, "inhibitor" means a substance that acts to reduce, block and/or eliminate it. An acid oxidase inhibitor" (DAO inhibitor) refers to a substance capable of reducing, blocking and/or eliminating D-amino acid oxidase activity, which is capable of inhibiting D-amino acid oxidase activity to reduce, block and/or eliminate acid Oxidase produces biological effects associated therewith. Inhibition of D-amino acid oxidase activity can be achieved in a variety of ways. In one embodiment, a DAO inhibitor can bind DAO and reduce, block and/or eliminate D-amino acid oxidase. In another embodiment, a DAO inhibitor can reduce DAO expression by gene silencing of RNA interference and/or abolish DAO expression, thereby inhibiting DAO activity. In another embodiment, the DAO inhibitor can be reduced by antisense RNA mechanism And/or eliminate DAO expression to achieve an effect of inhibiting DAO activity. In another embodiment, the DAO inhibitor can achieve inhibition of DAO activity by a gene knockout mechanism.

[52] In the context of the reference, when referring to a pharmaceutical compound, its meaning also includes its pharmaceutically acceptable Salts, hydrates and solvates.

[53] As used in the content, "opioid" can be used interchangeably with "opioids" and "opioids", which means that its pharmacological activity is exerted by acting on opioid receptors. One of the most important activities of a class of drugs is narcotic analgesia, which is used to treat a wide range of acute and chronic pain. In addition, since opioids exert their pharmacological activity by stimulating opioid receptors, they are also known as opioid receptor agonists or opioid agonists. The opioid receptor is mainly in the central nervous system (brain). In the spinal cord), it is also expressed in the peripheral nervous system such as the digestive tract. Based on this, the therapeutic efficacy and adverse reactions of opioid receptors are also mainly manifested in these systems and tissues.

[54] The opioid agonists used in the opening include, but are not limited to, alfentanil, fenprodidine, afarodidine, anilidine, benzylmorphine, benzimidate, buprenorphine, buttonol , chlordazin, codeine, deoxymorphine, dexamethasin, dextrozine, dipropenamine, diamorphone, dihydrocodeine, dihydromorphine, dimethacone, methadol, butylamine , morphine, piperazine, etazocine, isoxazin, methotrexate, ethylmorphine, ethoxynidazole, etorphine, dihydroetorphine, fentanyl and derivatives , hydrocodone, hydromorphone, hydroxyperidine, isomeacone, cretophanone, levorphane, leflufendane, remifentanil, phenidine, meptazin, beauty Sasin, methadone, hydromorphone, morphine, meropenine, narceine, nikomamorph, hydroxymorphin, normethesone, allylmorphine, nalbuphine, normorphine, noripezone, opioid, Hydroxyketone, oxymorphone, opioid, pentazocine, phenylheptanone, hydroxyphenidate, Nazocine, phenylphenididine, pinidinidine, cyanamide, propheptazine, trimethylpyridinium, propisophene, propoxyphene, sufentanil, tilidine, tramadol, Pharmaceutically acceptable salts, hydrates and solvates, mixtures of any of the foregoing, and the like.

[55] "Opioid tolerance" can be used interchangeably with "opioid tolerance" and "opioid tolerance" as used in the content. "Tolerance" refers to a process of neurological adaptation in which the receptor is less sensitive to ligands and results in a lowering of the drug's action. "Opioid tolerance" refers to a neurological adaptation process in which opioid receptors have reduced agonistic effects on opioid receptors, resulting in a corresponding decrease in their drug activity. Due to the development of opioid tolerance, the efficacy (such as analgesia) is continuously reduced, and the dose of opioids (such as morphine) has to be increased in the course of treatment (such as analgesia). Occasionally, opioid therapy is often unavoidable because of the inability to produce adequate therapeutic effects or the serious side effects due to increased doses.

[56] The inventors have unexpectedly discovered that D-amino acid oxidase inhibitors, including CBIO, AS057278 and sodium benzoate, can completely prevent or immediately completely block the tolerance of opioid receptor agonists such as morphine in the treatment of pain, The site of action in the spinal cord, with spinal cord D-amino acid oxidase activity Sexual inhibition is positively correlated. D-amino acid oxidase inhibitors prevent or block opioid tolerance.

In one aspect, the present disclosure provides a method of preventing and/or reversing opioid tolerance comprising administering an effective amount of a D-amino acid oxidase inhibitor to a subject in need thereof.

[58] In one embodiment, the method is for preventing and/or treating a condition which is reduced in therapeutic efficacy due to opioid tolerance, the condition being capable of achieving prevention and/or opioid tolerance by inhibiting D-amino acid oxidase Get prevented and / or treated. In particular, the condition can be pain, including but not limited to acute and/or chronic pain, especially chronic pain, such as cancer pain.

[59] In a specific embodiment, the condition that is reduced in therapeutic efficacy due to opioid tolerance is selected from the group consisting of chronic pain, cancer pain, neuropathic pain, chronic low back pain, headache, migraine, trigeminal neuralgia, Cluster headache, fibromyalgia syndrome, joint pain, inflammatory pain, arthritis pain, osteoarthritis pain, rheumatoid arthritis pain, tumor pain, cancer pain, visceral pain, somatic pain, musculoskeletal pain, bone Pain, lumbosacral pain, neck pain or upper back pain, diabetic pain, pain caused by spinal cord injury, surgical pain, postoperative pain, acute pain, or with infection, sickle cell anemia, autoimmune disease, multiple sclerosis or Inflammation-related pain, pain caused by injury or surgery, or a combination thereof.

[60] In another specific embodiment, the condition that is reduced in therapeutic efficacy due to opioid tolerance is selected from the group consisting of pain associated with diabetic neuropathy, peripheral neuralgia, post-herpetic neuralgia, waist or neck Nerve root pain, fibromyalgia, glossopharyngeal neuralgia, reflex sympathetic dystrophy, burning pain, thalamic syndrome, nerve root avulsion, phantom limb pain, pain after thoracotomy, cancer, chemical damage, toxins, nutrition Lack, viral or bacterial infection, temporomandibular joint disorder syndrome, fibromyalgia syndrome, osteoporosis, bone metastasis or other unknown causes of bone pain, gout, fibrinitis, myofascial pain, thoracic outlet syndrome , back or back pain, pelvic pain, heart chest pain, non-cardiac chest pain, spinal cord injury-related pain, central stroke pain, cancer pain, AIDS pain, anti-tumor drug-induced neuropathic pain, sickle cell pain, old-age pain , or a combination of them.

[61] In another embodiment, the D-amino acid oxidase inhibitor is selected from one or more of the following: CBIO (5-chlorobenzo [d] isoxazol-3-ol> Ferraris et al" J Med Chem 51 : 3357-3359, 2008; Hashimoto et al., Biol Psychiatry 65: 1103-1106, 2009; Horio et al., Open Clin Chem J 2: 16-21, 2009; Gong et al., J Pharmacol Exp Ther 336: 282-293, 2011), AS057278 (5-methylpyrazole-3-carboxylic acid, Adage et al" Eur Neuropsychopharmacol 18: 200-214, 2008; Gong et al., J Pharmacol Exp Ther 336: 282-293, 2011), Compound 8 (4H-thieno [3,2-b]pyrrole-5-carboxylic acid, Sparey et al" Bioorg Med Chem Lett 18: 3386-3391, 2008; Smith et al., J Pharmacol Exp Ther 328: 921-930, 2009; Gong et al., J Pharmacol Exp Ther 336: 282-293, 2011 ), Compound 2 ( 3-hydroxyquinolin-2-(lH)-one, Duplantier et al" J Med Chem 52: 3576- 3585, 2009; Gong et al., J Pharmacol Exp Ther 336: 282-293, 2011) and sodium benzoate (Vanoni et al., Biochemistry 36: 5624-5632, 1997; Gong et al., J Pharmacol Exp Ther 336: 282-293, 2011) and the like. D-amino acid oxidase inhibitors can be used to prevent and inhibit morphine tolerance caused by repeated administration of morphine without affecting the analgesic effect of morphine when administered alone.

[62] In another embodiment, the image exhibits tolerance to one or more opioids selected from the group consisting of: afentanil, fenprozine, afarodidine, anili Ding, benzylmorphine, benzonitrile, buprenorphine, butorphanol, chloredazin, codeine, deoxymorphine, dexamethasin, dextrozine, dienamide, diamorphone, dihydrogen Codeine, dihydromorphine, dimethacone, methadol, butylamine, morphine, pelmatone, etazocine, isosamine, metformin, ethylmorphine, B Oxynitrazole, etorphine, dihydroetorphine, fentanyl and derivatives, hydrocodone, hydromorphone, hydroxypitidine, isomezicin, ketomines, levorphane, left Fentan, remifentanil, phenidine, meptazin, metazocine, methadone, hydromorphone, morphine, meropenine, narceine, nikomamorph, hydroxymorphin, normethesone, ene Amorphine, nalbuphine, normorphine, noripeptone, opioid, oxycodone, oxymorphone, opioid Pentazone, benzoheptanone, hydroxyphenidate, phenazocine, phenepidine, pinidinidine, cyanamide, ropheptazine, trimethylidine, meperidine, c Oxygen, sufentanil, tilidine, tramadol or a derivative thereof. For example, the opioid may be morphine or a derivative thereof.

[63] In still another embodiment, when the method is for preventing and/or treating a condition in which the therapeutic efficacy is reduced by opioid tolerance, the method further comprises administering an additional therapeutic agent to the subject. For example, when used to prevent and/or treat pain, a pain treatment agent can also be administered to the subject, including but not limited to an analgesic such as an opioid analgesic, or a combination thereof.

[64] In a specific embodiment, the additional therapeutic agent is selected from the group consisting of opioids, such as one or more opioids selected from the group consisting of: alfentanil, allylidine, alfalogine, Anilididine, benzylmorphine, benzonitrile, buprenorphine, butorphanol, chlordazin, codeine, deoxymorphine, dexamethasin, dextrozine, dipropenamine, diamorphone, Dihydrocodeine, dihydromorphine, dimethacin, methadol, butylamine, morphine, pellucidum, etatazocin, issoxazine, methotrexate, ethylmorphine , ethoxynidazole, etorphine, dihydroetorphine, fentanyl and derivatives, hydrocodone, hydromorphone, oxypeptidine, isomezin, ketomines, levorphan , lefentanol, remifentanil, meperidine, meptazin, metazocine, methadone, Hydromorphone, morphine, meropenine, narceine, nikomamorph, hydroxymorphin, normethesone, allylmorphine, nalbuphine, normorphine, noripeperone, opioid, oxycodone, hydroxy Morphine, ophilide, pentazocine, benzoheptanone, hydroxyphenidate, phenazocine, phenepidine, pirimidin, cyanamide, propheptazine, trimethylidine, Isoperidine, propoxyphene, sufentanil, tilidine, tramadol or a derivative thereof. For example, the opioid may be morphine or a derivative thereof. In a specific embodiment, the opioid agonist can be a morphine drug such as morphine, hydromorphone, fentanyl, buprenorphine, and buprenorphine.

In another embodiment of the methods of the invention, the D-amino acid oxidase inhibitor is administered prior to, concurrently with, or subsequent to the administration of the opioid.

[66] In another embodiment, the D-amino acid oxidase inhibitor is administered orally or parenterally, for example, including subcutaneous injection, spinal, intracerebral administration, intramuscular injection, nasal administration, intravenous administration. And / or intraperitoneal administration.

In a second aspect, the disclosure provides a method of preventing and/or treating pain comprising administering to a subject in need thereof an opioid and a D-amino acid oxidase inhibitor.

[68] In particular, opioids and acid oxidase inhibitors may be administered separately and/or together. Furthermore, the D-amino acid oxidase inhibitor can be administered prior to, concurrently with, and/or after administration of the opioid.

[69] In one embodiment, the opioid is selected from one or more of the following: alfentanil, allylidine, afarodidine, anilidine, benzylmorphine, benzonitrile, Buprenorphine, butorphanol, chlordazin, codeine, deoxymorphine, dexamethasin, dextrozine, dipropenamine, diamorphone, dihydrocodeine, dihydromorphine, dimethacin , methadol, butylamine, morphine, piperazine, etazocine, isoxazin, metformin, ethylmorphine, ethoxynidazole, etorphine, dihydrogen Trophine, fentanyl and derivatives, hydrocodone, hydromorphone, oxypiperidine, isomeacone, cretophanone, levorphane, levonofan, rofentanil, tifenidine , meptazin, metazocine, methadone, hydromorphone, morphine, meropenine, narceine, nikomamorph, hydroxymorphin, normethapone, allymorphine, nalbuphine, normorphine, Norpeptone, opioid, oxycodone, oxymorphone, opioid, pentazocine, phenylheptanone, hydroxybenzene Morphine, phenazocine, bepeptidin, pinidinidine, cyanamide, propheptazine, trimethylridine, isopropanol, propoxyphene, sufentanil, tilidine, koji Mado, its derivatives and any mixtures thereof.

[70] In another embodiment, the D-amino acid oxidase inhibitor is selected from one or more of the following: CBIO, Compound 8, Compound 2, AS057278, sodium benzoate and its derivatives Creature.

[71] In another embodiment, the pain is acute pain and/or chronic pain, especially chronic pain, such as cancer pain. Specifically, for example, the pain may be selected from one or more of the following: chronic pain, neuropathic pain, chronic low back pain, headache, migraine, trigeminal neuralgia, cluster headache, fibromyalgia syndrome, joint Pain, inflammatory pain, arthritis pain, osteoarthritis pain, rheumatoid arthritis pain, tumor pain, cancer pain, visceral pain, somatic pain, musculoskeletal pain, bone pain, lumbosacral pain, neck pain or upper back Pain, diabetic pain, pain caused by spinal cord injury, surgical pain, postoperative pain, acute pain, or pain associated with infection, sickle cell anemia, autoimmune disease, multiple sclerosis or inflammation, caused by injury or surgery Pain, diabetic neuropathic pain, peripheral neuralgia, postherpetic neuralgia, lumbar or cervical radiculopathy, fibromyalgia, glossopharyngeal neuralgia, reflex sympathetic dystrophy, burning pain, thalamic syndrome, nerve root avulsion , phantom limb pain, pain after thoracotomy, cancer, chemical damage, toxins, nutritional deficiencies, viral or bacterial infections, Temporomandibular joint disorder syndrome, fibromyalgia syndrome, osteoporosis, bone pain caused by bone metastases or other unknown causes, gout, fibrositis, myofascial pain, thoracic outlet syndrome, upper back pain, lower back pain , pelvic pain, heart chest pain, non-cardiac chest pain, spinal cord injury-related pain, central post-stroke pain, cancer pain, AIDS pain, anti-tumor drug-induced neuropathic pain, sickle cell pain, geriatric pain, or a combination thereof .

In a third aspect, the disclosure provides a method of treating chronic pain, particularly cancer pain, comprising administering to a subject in need thereof an effective amount of a D-amino acid oxidase inhibitor.

[73] In one embodiment, the D-amino acid oxidase inhibitor is selected from one or more of the group consisting of CBIO, Compound 8, Compound 2, AS057278, sodium benzoate and derivatives thereof.

Based on the above findings of the present disclosure, there is also provided a method of identifying a compound capable of preventing and/or reversing opioid tolerance, comprising the steps of: providing a test compound to contact the test compound with an acid oxidase And determining the activity of the acid oxidase; if the test compound is capable of inhibiting the activity of D-amino acid oxidase, the test compound can be used as a compound for preventing and/or reversing opioid tolerance. For example, a compound identified according to the above method can be used for the prevention and/or treatment of a disease whose therapeutic efficacy is lowered due to opioid tolerance. In a specific embodiment, the condition is pain, especially chronic pain.

[75] In another aspect, the invention provides another method of identifying a compound capable of preventing and/or reversing opioid tolerance, comprising the steps of: providing a test compound and an opioid prior to administration of the opioid, Applying the test compound to the test subject simultaneously and/or afterwards, Comparing the activity of the opioid before administration of the test compound and/or prior to administration of the test compound, if the level of opioid activity is increased after administration of the test compound, the test compound can be used as a prophylactic / or reverse opioid-tolerant compounds. For example, a compound identified according to the above methods can be used to prevent and/or treat a condition that reduces its therapeutic efficacy due to opioid tolerance. In a specific embodiment, the condition is pain, especially chronic pain.

[76] We performed in vivo inhibitory activity studies on these acid oxidase inhibitors and found that spinal gangue and subcutaneous administration of acid oxidase inhibitors including CBIO, AS057278 and sodium benzoate were able to almost completely prevent or reverse morphine tolerance.

The invention is further illustrated by the following examples, which are not to be construed as limiting the scope of the invention. The entire contents of all of the cited references in the present application, which are hereby incorporated by reference in their entireties in their entireties in the entireties in In the following examples, the reagents and materials used are commercially available, at least analytically pure or equivalent grades, unless otherwise specified.

Example

Example 1

Effect of subcutaneous injection of DAO inhibitor CBIO on morphine tolerance in a single or 7 consecutive days

[78] Male Swiss mice (20 ~ 25 _g , Shanghai Slack Laboratory Animals Co., Ltd.), free drinking water, eating, divided into 10 groups, 4 in each group. Three groups of mice were injected subcutaneously with normal saline (10 ml/kg) twice daily for 7 days. On the 8th day, physiological saline (10 ml/kg) and CBIO (10 mg/kg, from Maybridge Chemicals, respectively) were administered. Cornwall, UK) or morphine (5 m _g /k _g from Northeast Pharmaceutical Group Shenyang First Pharmaceutical Co., Ltd.); two groups of mice were injected subcutaneously with CBIO (10 mg/kg) or morphine (10 mg/kg), CBIO (10 mg/kg) or morphine (5 mg/kg) were given twice daily for 7 days and 8 days. Group 2 mice were injected subcutaneously with CBIO (10 mg/kg) or morphine (10 mg/kg). ), twice daily for 7 days, on the 8th day, morphine (5 mg/kg) or CBIO (10 mg/kg); 2 groups of mice were injected subcutaneously with CBIO (10 mg/kg) or CBIO (10 mg) /kg ) + morphine (10 mg/kg), twice daily for 7 days, on the 8th day, saline (10 ml/k _g ) or morphine (5 mg/kg); group 1 mice subcutaneously injected with morphine (10 mg/kg), twice daily for 7 days (14 times in total), the 14th dose was given CBIO (10 mg/kg), and the 8th day was given morphine (5 mg/kg) „ Day 8 After 20 minutes of administration, the mice Injection of 10 μΐ 5% formalin on the right hind paw Pain was observed. 0-5 minutes after the formalin injection (I phase acute reaction) and 20-40 minutes (phase II chronic reaction). The mice continued to have sufficient lameness time as an index of pain. The results of the experiment are shown in Figures 1A and 1B, indicating that a single dose of morphine was injected into control mice that received subcutaneous injection of normal saline on day 7.

(5 mg/kg) inhibited formalin phase I acute pain and phase II chronic pain with inhibition rates of 92.7% and 100%, respectively; single dose injection of CBIO (10 mg/kg) for formalin Phase I acute pain did not affect, but it could effectively inhibit the chronic pain of formalin II in mice, the inhibition rate was 64.3%; subcutaneous injection of CBIO (10 mg/kg) for 7 consecutive days did not produce significant tolerance, ie continuous 7 Single dose of CBIO (10 mg/kg) after daily administration had no effect on formalin phase I acute pain, but it still effectively inhibited formalin phase II chronic pain with an inhibition rate of 62.8%; 7 days of subcutaneous Injection of morphine produced significant tolerance, ie single-dose injection of morphine after 7 consecutive days of dosing

(5 mg/kg) inhibition rate of formalin phase I acute pain and phase II chronic pain were 0% and 20.7%, respectively; subcutaneous injection of CBIO (10 mg/kg) for 7 consecutive days, single dose injection (5 Mg/kg) can effectively inhibit formalin phase I acute pain and phase II chronic pain, the inhibition rate is 88.4% and 100%, respectively, indicating that CBIO does not produce cross-tolerance to morphine; subcutaneous injection of morphine for 7 consecutive days (5 mg) /kg), single-dose injection of CBIO (10 mg/kg) has no effect on formalin phase I acute pain, can effectively inhibit formalin II phase chronic pain, inhibition rate is 57.1%, indicating that CBIO does not produce Cross-tolerance; no drug accumulation occurred on CBIO (10 mg/kg) for 7 consecutive days, no analgesic effect on day 8; CBIO and combined use for 7 days, completely prevented the tolerance of morphine production, single-dose injection Morphine inhibition rate of formalin phase I acute pain and phase II chronic pain was 81.5% and 100%, respectively; 7 days of subcutaneous injection of morphine, 14th morphine after a single dose of CBIO, can completely reverse morphine has been produced Tolerance, single dose injection of morphine on formalin I Acute pain and chronic pain phase II inhibition rate was 65.7% and 100%, respectively. The results showed that CBIO combined with it had a preventive effect on the tolerance of analgesic effects, and single-dose CBIO could completely reverse morphine tolerance.

Example 2

Effect of subcutaneous injection of D-amino acid oxidase inhibitor CBIO on dose-effect relationship of morphine tolerance for 7 consecutive days

[79] Male Swiss mice (20 ~ 25 _g , Shanghai Slack Laboratory Animals Co., Ltd.), free drinking water, eating, divided into 18 groups, 4 in each group. Six groups of mice were injected subcutaneously with normal saline (10 ml/kg) twice daily for 7 days. On the 8th day, saline (10 ml/kg) or morphine (0.1, 0.3, 1, 3 ^ 10 mg) were administered. /kg, from Northeast Pharmaceutical Group Shenyang First Pharmaceutical Co., Ltd.); 6 groups of mice were injected subcutaneously with morphine (10 m _g / k _g ) twice daily for 7 consecutive times On the 8th day, physiological saline 7j (10 ml/kg) or morphine (0.3, 1, 3, 10 and 30 mg/kg) were given respectively; 6 groups of mice were injected subcutaneously with CBIO (10 mg/kg) + morphine ( 10 mg/kg), twice daily for 7 days, on the 8th day, saline (10 ml/kg) or morphine (0.1, 0.3, 1, 3 and 10 mg/kg) were administered. After 20 minutes of dosing on day 8, mice were injected with 10 μl of 5% formalin in the right hind paw, and pain was observed 0-5 minutes after the formalin injection (phase I acute reaction) and 20-40 minutes. (Chronic phase II chronic reaction) Mice continued to have lameness time as an indicator of pain. The results of the experiment are shown in Fig. 2. In the control mice in which the saline was continuously injected subcutaneously on the 7th day, a single dose of morphine (0, 0.1, 0.3, 1, 3 and 10 mg/kg) inhibited the dose in a dose-dependent manner. Malin I phase acute pain and phase II chronic pain (Fig. 2A), inhibition of half effective dose (ED50) of 1.5 mg / kg and 1.0 mg / kg (Figure 2D and Figure 2E), the maximum inhibition rate can reach 100% Compared with the control group, the mice in the continuous subcutaneous injection of morphine (10 mg/kg) on the 7th day showed significant tolerance, and the single dose of morphine was injected on the 8th day (0.3, 1, 3, 10 and 30 m _g / k _{g ).} In a dose-dependent manner, the formalin phase I acute pain and phase II chronic pain (Fig. 2B) were inhibited, and the dose-effect relationship was significantly shifted to the right. The effective half-effectiveness (ED50) was 5.4 mg/kg and 6.0 mg/ Kg (Fig. 2D and Fig. 2E), maximal inhibition rate can also reach 100%; subcutaneous injection of CBIO (10 mg/kg) + morphine for 7 consecutive days

(10 m _g /k _g ) did not produce significant tolerance, ie, single-dose injection of morphine (0, 0.1, 0.3, 1, 3 and 10 mg/kg) on day 8 dose-dependently inhibited formalin I Acute pain and phase II chronic pain (Fig. 2C), inhibition of half effective dose (ED50) of 2.0 mg/kg and 0.5 mg/kg, respectively

(Fig. 2D and Fig. 2E), the maximum inhibition rate can reach 100%. The results showed that CBIO combined with morphine had a preventive effect on the tolerance of analgesic effects.

Example 3

Prevention of morphine resistance by subcutaneous injection of D-amino acid oxidase inhibitor AS057278 and sodium benzoate for 7 consecutive days

[80] Male Swiss mice (20 ~ 25 _g , Shanghai Slack Laboratory Animals Co., Ltd.), free drinking water, eating, divided into 5 groups, 4 in each group. Two groups of mice were injected with normal saline (10 ml/kg) twice a day for 7 days, and on the 8th day, saline (10 ml/kg) or morphine (5 mg/kg) were given respectively. Subcutaneous injection of morphine (10 mg/kg), AS057278 (40 mg/kg, from Sigma-Aldrich, St Louis, MO) + morphine (10 mg/kg) or sodium benzoate (400 mg/kg, from Sinopharm Group) , Shanghai, China) + (10 mg/kg), twice daily for 7 days, on the 8th day, morphine (5 mg/kg). After 20 minutes of dosing on day 8, mice were injected with 10 μl 5% formalin for pain in the right hind paw, and 0-5 minutes (phase I acute reaction) and 20-40 minutes after formalin injection. (phase II chronic reaction) mice continue to lick Full time as an indicator of pain. As shown in Figures 3A and 3B, the experimental results showed that in the control mice subcutaneously injected with physiological saline for 7 consecutive days, a single dose of morphine (5 mg/kg) was given to formalin phase I acute pain and phase II chronic pain. Inhibition, inhibition rate was 84.8% and 100%, respectively; 7 days of subcutaneous injection of morphine produced significant tolerance, single-dose injection of formalin phase I acute pain and phase II chronic pain inhibition rate of 12.2, respectively. % and 12.4%; AS057278 and tyrosine can prevent morphine tolerance after 7 days, and the inhibition rates of formalin phase I acute pain and phase II chronic pain by single dose morphine are 76.1% and 100%, respectively; Sodium benzoate combined with morphine for 7 days also prevented the tolerance of morphine production. The inhibition rates of formalin phase I acute pain and phase II chronic pain were 69.8% and 89.1%, respectively. The results showed that AS057278 combined with sodium benzoate and morphine can effectively prevent the tolerance of analgesia.

Example 4

Intrathecal injection of D-J-acid oxidase inhibitor CBIO (30 μg), AS057278 (100 μg) and sodium benzoate (300 μg) and Reactive oxygen species (ROS) scavenger PBN ( Effect of l mg ) on morphine tolerance

[81] Male Wistar rats (body weight 150-170 g, Shanghai Slack Laboratory Animals Co., Ltd.) were divided into 5 groups of 4 animals each. Intrathecal injection of morphine (50 μ ^ Ι μ ΐ / rat) once daily for 5 consecutive days, the fifth injection was given with normal saline (10 μl / rat), CBIO (30 g), AS057278 (100) g), sodium benzoate (300 μg) and reactive oxygen species (ROS) scavenger PBN (1 mg), given morphine (5 μg/10 μΐ/rat) on day 6, 30 minutes later Rats were injected with 50 μΐ 5% formalin for pain in the right hind paw. The rats were lifted or shaken for 1 minute every 10 minutes after 0-90 minutes after formalin injection. index. The results of the experiment are shown in Figure 4. Rats received intrathecal morphine (50 μg/10 μl/rat) for 5 days and developed significant tolerance. Single-dose administration of CBIO (30 g), AS057278 (100 g), sodium benzoate (300 μg) and PBN (1 mg) in the spinal cord can almost completely reverse the tolerance that morphine has produced, ie, a single dose of morphine. The acute pain inhibition rates of these rats in formalin phase I were 70.8%, 69.3%, 79.5% and 54.3%, respectively. The inhibition rates of phase II chronic pain were 92.6%, 95.6%, 94.9% and 92.3%, respectively. Conclusions: A single intrathecal administration of CBIO, AS057278, sodium benzoate and PBN completely reversed morphine tolerance, suggesting that D-amino acid oxidase inhibitors block morphine-tolerant sites in the spinal cord. Example 5

Single-dose subcutaneous injection of acid oxidase inhibitors CBIO, AS057278 and sodium benzoate immediately reversed the dose-effect relationship of morphine tolerance and its association with inhibition of spinal acid oxidase activity in rats

[82] According to D'Aniello detection of D-amino acid oxidase pure enzyme activity method to detect different concentrations of CBIO (0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 μ Μ), AS057278 (0, 0.1, 0.3, 1 , 3, 10, 100, 300 μ Μ ) and sodium benzoate (0, 3, 10, 30, 100, 300, 1000, 3000, 10000 μ Μ ) inhibited D-amino acid oxidase activity in rat spinal cord, dose-effect Relationship studies, calculating the half-inhibition (IC50) and the maximum inhibition rate (Ema _X ). The results showed that D-acid oxidase inhibitors CBIO, AS057278 and sodium benzoate inhibited D-amino acid oxidase activity in rat spinal cord 100%, IC50 was 1.3, 83.5 and 290.2 μ M, respectively ( Gong et al., JPET 336:282-293, 2011 )„

[83] Male Swiss mice (20 ~ 25 _g , Shanghai Slack Laboratory Animals Co., Ltd.), free drinking water, eating, divided into 15 groups, 4 in each group. Two groups of mice were injected subcutaneously with normal saline (10 ml/kg) twice a day for 7 consecutive days. The appendix and hot plate pain thresholds were measured before the 8th day of administration, and then saline (10 ml/k _{g was} administered _). 13 groups of mice were injected subcutaneously with morphine (10 m _g / k _g ) twice daily for 7 consecutive days. The appendix and hot plate pain thresholds were measured before dosing on day 8 and then given 1, 3 and 10 mg/kg CBIO, 3, 10 and 100 mg/kg AS057278 and 10, 30, 100 and 300 m _g / k _g sodium benzoate. After 15 minutes of administration of saline or D-amino acid oxidase inhibitor, all mice were examined for appendix and hot plate pain thresholds, followed by morphine (5 mg/kg), and mice were tested at 0.5, 1 and 2 hours, respectively. Tail and hot plate pain threshold. The effects of different doses of each drug on the appendix and hot plate pain in mice were compared, and the threshold net value and the area under the curve (AUC) within 2 hours were calculated. The results of the experiment are shown in Figure 5. The subcutaneous injection of morphine for 7 consecutive days produced significant tolerance. The single-dose injection did not cause analgesia on mouse tail and hot plate pain; single dose subcutaneous injection of CBIO, AS057278 and sodium benzoate It can reverse morphine tolerance in a dose-dependent manner. The dose-response analysis showed that the maximum blocking rate of CBIO, AS057278 and sodium benzoate blocking morphine tolerance was 100%, and the ED50 was 1.0, 2.5 and 40.5 mg/kg in the mouse appendicitis model, respectively. The mouse hot plate pain model was 1.1, 7.4, and 50.4 mg/kg, respectively (Fig. 6A and Fig. 6B). Further analysis showed that the inhibition of morphine production tolerance by CBIO, AS057278 and sodium benzoate in mouse appendix and hot plate pain models was positively correlated with inhibition of D-amino acid oxidase activity in rat spinal cord (Fig. 6C and Fig. 6D). . The results showed that single doses of D-amino acid oxidase inhibitors (CBIO, AS057278 and sodium benzoate) were able to immediately reverse morphine analgesia tolerance and positively correlated with D-amino acid oxidase activity inhibition. Example 6

Effects of continuous η-day spinal injection D-amino acid oxidase siRNA/DAO and Virus siRNA/DAO on formalin pain in morphine-tolerant rats

[84] Male Wistar rats (body weight 200 ~ 250 g), free to drink water, eat, divided into 4 groups. Physiological saline (10 ml/kg) or morphine (10 mg/kg) was administered subcutaneously for 7 consecutive days, twice daily; spinal cord was given PEI (7.5 Invitrogen, Shanghai, China), PEI (7.5)+Nonsense siRNA (5) And PEI ( 7.5 ) + siRNA/DAO ( 5 ), once a day. On the 8th day, the formalin pain test was performed. In the 20 minutes before the experiment, the rats in the saline (10 ml/kg) + PEI (7.5) group were injected subcutaneously with 10 ml/kg saline, and normal saline (10 ml/kg). + PEI (7.5 μ _δ ), morphine (10 mg/kg) + PEI (7.5), morphine (10 mg/kg) + PEI (7.5) + Nonsense siRNA (5) and morphine (10 mg/kg) + PEl ( 7.5) + siRNA/GLP-lr

(5) Rats were injected subcutaneously with 10 mg/kg morphine (n=4), and the experimental indicators were observed according to the formalin-induced pain model. The analgesic relationship was analyzed. The siRNA/DAO sequence is 5'-GGAGUGAAGUUCAUCCAUCUU-375'-GAUGGAUGAACUUCACU CCUU-3', the Non-sense siRNA sequence is 5,-UUC UCC GAA CGU GUC ACG UUU-375'-ACG UGA CAC GUU CGG AGA AUU-3O Experimental Results As shown in Fig. 7, after intravenous injection of saline (10 ml/kg) + PEI (7.5 μm) for 7 consecutive days, a single subcutaneous injection of morphine (10 m) was compared with the subcutaneous injection of saline in the 8th day. _g / k _g ) was effective in inhibiting formalin acute pain (I phase response, Figure 7C) and chronic pain (II phase response, Figure 7D) with inhibition rates of 90.4 and 98.9%, respectively. Rats were injected with morphine (10 mg/kg) + PEl (7.5) and morphine (10 mg/kg) + PEI (7.5 g) + Nonsense siRNA (5 g) for 7 consecutive days. Subcutaneous injection on day 8 (10 m) _g / k _g ) Rats developed significant morphine analgesic tolerance, and the inhibition rates of formalin acute pain (I phase response) and chronic pain (II phase response) in rats were reduced to 3.4% and 23.9, respectively. %, 0 and 8.5%. Injection of morphine (10 mg/kg) + PEI for 7 consecutive days

( 7.5 ) + siRNA/GLP-lr( 5 ) completely blocked the analgesic effect of subcutaneous morphine (10 mg/kg) on the 8th day, acute pain in the formalin phase I and chronic phase II The analgesic inhibition rates of pain were 68.5% and 75.6%, respectively.

[85] After completion of the formalin test, a real-time quantitative PCR experiment was performed on the spine of the rat. DAO gene expression was prepared by adding 1 ml of TRIzol solution (Invitrogen, Grand Island, NY, USA) to 100 mg tissue. 1000 rpm, 15 s), extract total tissue mRNA according to the instructions, and reverse-transcribe into total cDNA according to 1 total RNA sample using ReverTra Ace qPCR RT-Kit (Toyobo Co., Ltd., Osaka, Japan), in Mastercycler® The ep realplex (Eppendorf AG, Hamburg, Germany) machine was subjected to real-time quantitative PCR experiments using the Realmaster Mix (SYBR Green I) (Tiangen Biotech Co., Ltd., Beijing, China) method. The DAO primer sequence is: 5'-CCCTTTCTGGAAAAGCACAG-375'-CTCCTCTCACCACCTCTTC G-3', the GAPDH primer sequence is:

5,-CGGCAAGTTCAACGGCACAG-3,/5,-AGACGCCAGTAGACTCCA CGAC-3'„ Relative gene expression was calculated using the 2- ^ΔΔα formula, AACt=experimental group (Ct, Target-Ct, Gapdh)-control group (Ct, Target-Ct , Gapdh). The experimental results are shown in Fig. 7B. After 7 days of intravenous saline (10 ml/kg) + PEI (7.5), a single subcutaneous injection of morphine was compared with the subcutaneous injection of saline in the 8th day. 10 mg/kg) significantly increased DAO gene expression by approximately 38.9%; continued injection of morphine (10 mg/kg) + PEI (7.5) and morphine (10 mg/kg) + PEI (7.5) + Nonsense siRNA for 7 consecutive days ( 5) In the rats, the expression of DAO gene decreased by 17.3% and 16.4% after subcutaneous injection of morphine (10 mg/kg) on the 8th day. The injection was continued for 7 days (10 mg/kg) + PEI (7.5) + The DAO gene in the siRNA/GLP-lr (5) group was reduced by about 37%.

[86] Male Wistar rats (body weight 200 ~ 250 g), free to drink water, eat, divided into 4 groups. Respectively a single spinal administration of Virus-nonsense siRNA (30 μΐ viral titer δχΙΟ) and Virus-siRNA / DAO (30 μΐ viral titer 6.3xl0 ^1G), for 7 days then injected with normal saline (10 ml / kg) Or morphine (10 mg/kg) twice daily. On the 8th day, the formalin-induced pain test, the rats in the physiological salt 10 ml/kg 20 minutes before the experiment, were injected subcutaneously with 10 ml/kg normal saline, normal saline (10 ml/kg), and morphine (10 mg/ Kg Virus-nonsense siRNA and morphine (10 mg/kg) +Virus-siRNA/DAO rats were injected subcutaneously with 10 mg/kg morphine, and the rats were observed with formalin-induced pain model to analyze the analgesic relationship. The shRNA/DAO sequence of adenovirus was 5,-GATCCGGAGTGAAGTTCATCCATCTTCAAGAGAGAGGGATGA ACTTCACTCCTTTTTTA-3', and the shRNA/Non-sense siRNA sequence was 5'-GATCCGCCAGCTGATACTAACTCCTTCAAGAGAGGAGTTAGTA TCAGCTGGCTTTTTTA-3. The results are shown in Figure 8. After 7 days of intravenous saline injection, Subcutaneous morphine (10 mg/kg) was effective in inhibiting formalin acute pain (phase I response, Figure 8C) and chronic pain (phase II response, Figure 8D) compared with the 8th day of subcutaneous injection of saline. The inhibition rates were 90.4 and 98.9%, respectively. Rats were injected with morphine (10 mg/kg) + Virus-Nonsense siRNA for 7 consecutive days. After 8 days of subcutaneous injection of morphine (10 mg/kg), the rats developed obvious analgesia. Make Tolerance, acute pain in rats formalin (I-phase reaction) and chronic pain (II Reaction) Inhibition rate decreased to 20.5% and 5.2%, respectively, for 7 consecutive Daily injection of morphine (10 mg/kg) +Virus-siRNA/DAO completely blocked the analgesic effect of subcutaneous morphine (10 m _g /k _g ) on day 8 and acute to formalin I in rats The analgesic inhibition rates of pain and phase II chronic pain were 69.2% and 84.9%, respectively.

[87] After completion of the formalin test, real-time quantitative PCR experiments were performed on most of the spine in rats. DAO gene expression was prepared by adding 1 ml of TRIzol solution (Invitrogen, Grand Island, NY, USA) to 100 mg tissue. 1000 rpm, 15 s), extract total tissue mRNA according to the instructions, and reverse-transcribe into total cDNA according to 1 total RNA sample using ReverTra Ace qPCR RT-Kit (Toyobo Co., Ltd., Osaka, Japan), in Mastercycler® ep Realplex (Eppendorf AG, Hamburg, Germany) machine, real-time quantitative PCR experiments were performed using the Realmaster Mix (SYBR Green I) (Tiangen Biotech Co., Ltd., Beijing, China) method. The DAO primer sequence is: 5'-CCCTTTCTGGAAAAGCACAG-375'-CTCCTCTCACCACCTCTTC G-3', and the GAPDH primer sequence is:

5,-CGGCAAGTTCAACGGCACAG-3,/5,-AGACGCCAGTAGACTCCA CGAC-3' Relative gene expression was calculated using the ² - ^ΔΔα formula, ΔΔα = experimental group (Ct, Target-Ct, Gapdh) - control group (Ct, Target-Ct, Gapdh). The results of the experiment are shown in Fig. 8B. After 7 days of intravenous saline injection (10 ml/kg), a single subcutaneous injection of morphine (10 m _g /k _g ) was significant compared with the 8th day of subcutaneous injection of saline. Increased DAO gene expression by approximately 38.9%; continuous 7-day injection (10 mg/kg) + Virus-Nonsense siRNA group, subcutaneous injection of morphine (10 mg/kg) on day 8 decreased DAO gene expression in rats 13.5%; morphine morphine (10 mg/kg) for 7 consecutive days + DAO gene expression was significantly reduced by about 30.2% in the Virus-siRNA/DAO group.

The invention has been illustrated by way of specific embodiments. However, those skilled in the art can understand that the invention is not limited to the specific embodiments, and various modifications and changes may be made without departing from the spirit and scope of the invention. Such modifications and variations are within the scope of the invention.

Claims

A method of preventing and/or reversing opioid tolerance, which comprises administering an effective amount of a D-amino acid oxidase inhibitor to a subject in need thereof.

2. The method of claim 1, wherein the method is for preventing and/or treating a condition which is reduced in therapeutic efficacy due to opioid tolerance.

3. The method of claim 2, wherein the condition is pain, including but not limited to acute and/or chronic pain, especially chronic pain, such as cancer pain.

4. The method of any of the preceding claims, wherein the D-J^ acid oxidase inhibitor is selected from one or more of the following: CBIO, Compound 8, Compound 2, AS057278, sodium benzoate and derivatives thereof.

5. The method of any of the preceding claims, wherein the subject exhibits tolerance to one or more opioids selected from the group consisting of: alfentanil, fenprozine, afarodidine, anili Ding, benzylmorphine, benzonitrile, buprenorphine, butorphanol, chloredazin, codeine, deoxymorphine, dexamethasin, dextrozine, dienamide, diamorphone, dihydrogen Codeine, dihydromorphine, dimethacone, methadol, butylamine, morphine, pellucidum, etazocine, isoxazin, methotrexate, ethylmorphine, B Oxynitrazole, etorphine, dihydroetorphine, fentanyl and derivatives, hydrocodone, hydromorphone, oxypiperidine, isomezigin, ketomines, levorphane, left Fenfantadine, remifentanil, meperidine, meptazin, metazocine, methadone, hydromorphone, morphine, meropenine, narceine, nikomamorph, hydroxymorphin, normethapone, Allylmorphine, nalbuphine, normorphine, noripeptone, opioid, oxycodone, oxymorphone, Epirubicin, pentazocine, benzoheptanone, hydroxyphenidate, phenazocine, phenepidine, pirimidin, cyanide, piperazine, trimethoprim, isopropylidene Aziridine, propoxyphene, sufentanil, tilidine and tramadol.

6. The method of any of the preceding claims, wherein the D-amino acid oxidase inhibitor is administered prior to, concurrently with, or after administration of the opioid.

7. A method of preventing and/or treating pain comprising administering an opioid and an acid oxidase inhibitor separately and/or together to a subject in need thereof.

8. The method of claim 7, wherein the D-amino acid oxidase inhibitor is administered prior to, concurrently with, and/or after administration of the opioid.

9. The method of claim 7 or 8, wherein the opioid is selected from one or more of the following: alfentanil, allylidine, afarodidine, anilidine, benzylmorphine, benzonitrile Special Proporphine, butorphanol, chlordazin, codeine, deoxymorphine, dexamethasin, dextrozine, dipropenamine, diamorphone, dihydrocodeine, dihydromorphine, dimethacin , methadol, butylamine, morphine, piperazine, etazocine, isoxazin, metformin, ethylmorphine, ethoxynidazole, etorphine, dihydrogen Trophine, fentanyl and derivatives, hydrocodone, hydromorphone, hydroxyperidine, isomeacone, cretotenone, levorphane, leftanol, remifentanil, meperidine , meptazin, metazocine, methadone, hydromorphone, morphine, meropenine, narceine, nikomamorph, hydroxymorphane, normethapone, allymorphine, nalbuphine, normorphine, Noripeptazone, opioid, oxycodone, oxymorphone, opioid, pentazocine, phenylheptanone, hydroxyphenidate, phenazocine, phenidine, piridine, Cyanide bis-benzamide, propheptazine, trimethylridine, meperidine, propoxyphene, sufentanil, tilidine, trama And any mixtures thereof.

The method of any one of claims 7-9, wherein the D-glycolate oxidase inhibitor is selected from one or more of the following: CBIO, Compound 8, Compound 2, AS057278, sodium benzoate and derivatives thereof Things.

11. The method of any of claims 7-10, wherein the pain is acute pain and/or chronic pain, especially chronic pain, such as cancer pain.

The method according to any one of claims 7 to 11, wherein the pain is selected from the group consisting of: chronic pain, neuropathic pain, chronic low back pain, headache, migraine, trigeminal neuralgia, cluster headache, fibromyalgia synthesis Symptoms, joint pain, inflammatory pain, arthritis pain, osteoarthritis pain, rheumatoid arthritis pain, tumor pain, cancer pain, visceral pain, physical pain, musculoskeletal pain, bone pain, lumbosacral pain, neck pain Or upper back pain, diabetic pain, pain caused by spinal cord injury, surgical pain, postoperative pain, acute pain, or pain associated with infection, sickle cell anemia, autoimmune disease, multiple sclerosis or inflammation, by injury or Pain caused by surgery, diabetic neuropathic pain, peripheral neuralgia, postherpetic neuralgia, radiculopathy of the waist or neck, fibromyalgia, glossopharyngeal neuralgia, reflex sympathetic dystrophy, burning pain, thalamic syndrome, nerve Root avulsion, phantom limb pain, pain after thoracotomy, cancer, chemical damage, toxins, nutritional deficiencies, viral or bacterial infections, Mandibular joint disorder syndrome, fibromyalgia syndrome, osteoporosis, bone pain caused by bone metastases or other unknown causes, gout, fibrositis, myofascial pain, thoracic outlet syndrome, upper back pain, lower back pain, Pelvic pain, cardiac chest pain, non-cardiac chest pain, spinal cord injury-related pain, central post-stroke pain, cancer pain, AIDS pain, anti-tumor drug-induced neuropathic pain, sickle cell pain, geriatric pain, or a combination thereof.

13. A method of identifying a compound capable of preventing and/or reversing opioid tolerance, comprising: providing a test compound, Contacting the test compound with an acid oxidase, and

Measuring the activity of the acid oxidase,

If the test compound is capable of inhibiting the activity of D-amino acid oxidase, the test compound can be used as a compound for preventing and/or reversing opioid tolerance.

14. A method of identifying a compound capable of preventing and/or reversing opioid tolerance, comprising: providing a test compound and an opioid,

Applying the test compound to the test subject before, concurrently with, and/or after administration of the opioid, and

Determining the activity of the opioid in a test subject after administration of the test compound, as compared to opioid activity prior to administration of the test compound and/or administration of the test compound, if the test compound is administered Thereafter, the level of activity of the opioid is enhanced, and the test compound can be used as a compound for preventing and/or reversing opioid tolerance.