WO2022052996A1 - 原人参二醇类化合物在治疗疼痛和成瘾物质躯体依赖、精神依赖和成瘾的用途 - Google Patents

原人参二醇类化合物在治疗疼痛和成瘾物质躯体依赖、精神依赖和成瘾的用途 Download PDF

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WO2022052996A1
WO2022052996A1 PCT/CN2021/117518 CN2021117518W WO2022052996A1 WO 2022052996 A1 WO2022052996 A1 WO 2022052996A1 CN 2021117518 W CN2021117518 W CN 2021117518W WO 2022052996 A1 WO2022052996 A1 WO 2022052996A1
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pain
group
protopanaxadiol
active ingredient
combination
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PCT/CN2021/117518
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English (en)
French (fr)
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王永祥
阮邵穆
赵梦静
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上海交通大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones

Definitions

  • the invention relates to the technical field of medicine, in particular to the use of protopanaxadiol compounds in the treatment of pain and physical, mental dependence and addiction of addictive substances.
  • Pain is divided into acute pain and chronic pain according to its origin, nature, location and time course.
  • Acute pain refers to the pain caused by the direct activation of nociceptors in the corresponding parts by noxious stimuli under physiological conditions.
  • Acute pain is short-lived ( ⁇ 1 month) and resolves spontaneously after the injury is repaired.
  • Acute pain includes postoperative pain, post-traumatic pain, acute headache and facial pain, acute arthritic pain, etc.
  • Chronic pain is pain that persists after the lesion is repaired, which can last for several months (>1 month) or even life, or it may recur frequently.
  • Chronic pain includes lower back pain, cancer pain, pain from antineoplastic drugs and opioids, diabetic pain, neuropathic pain including post-herpetic neuralgia, trigeminal neuralgia and sciatica, inflammatory pain, phantom limbs pain, arthritis pain, fibromyalgia, musculoskeletal pain, chronic regional pain syndrome, post-traumatic neuralgia, and peripheral neuropathy.
  • NSAIDs include flurbiprofen axetil, ibuprofen, diclofenac sodium, meloxicam, naproxen, celecoxib, and precoxib;
  • Anti-epileptic drugs including carbamazepine, phenytoin and gabapentins such as gabapentin, pregabalin and mirogabalin;
  • Monoamine neurotransmitter reuptake inhibitor antidepressants including amitriptyline and duloxetine
  • Local anesthetics include lidocaine, ropivacaine, and prilocaine;
  • Opioid analgesics include codeine, dihydrocodeine, morphine, fentanyl, sufentanil, remifentanil, meperidine, oxycodone;
  • Norepinephrine ⁇ 2 receptor agonists such as clonidine, dexmedetomidine
  • MOR-NRI dual-target analgesics such as dezocine, tapentadol, pentazocine and tramadol;
  • Chinese herbal medicines include Duyiwei, Aconitum/Aconite and their active ingredients such as clathrin and gauran, and Corydalis and their active ingredients such as rotundine.
  • gabapentin and repgabalin are not specific analgesics, and their efficacy in the treatment of neuropathic pain (lowering the pain threshold by 30%) is less than 50% of the population.
  • Non-steroidal analgesics have a certain effect on headache, toothache, muscle and joint pain, etc., but are almost ineffective for traumatic pain and visceral smooth muscle colic.
  • Local anesthetics are only indicated for peripheral neuropathic pain.
  • opioids have a variety of adverse reactions such as drowsiness, respiratory depression, constipation, etc.
  • long-term use will produce analgesic tolerance, hyperalgesia, physical dependence and addiction and abuse.
  • Gabapentin and repgabalin also have serious adverse effects such as drowsiness.
  • opioids including morphine and fentanyl
  • the body develops analgesic tolerance, and the dose must be increased to obtain the same analgesic effect.
  • Long-term or repeated use of opioids can also lead to addiction, including both physical dependence (physical dependence, physical dependence) and mental dependence (psychological dependence).
  • Physical dependence is the repeated use of medication to avoid withdrawal symptoms, and due to the gradual increase in the tolerable dose, it manifests as an aversive effect in the process of addiction, which plays a negative reinforcement effect.
  • Mental dependence refers to the psychological craving of the dependent and the euphoria achieved by repeated drug use, which manifests as a rewarding effect, plays a positive reinforcement effect, and prompts the patient to relapse repeatedly.
  • Methadone, buprenorphine, clonidine, lofexidine, etc. can only improve withdrawal symptoms to a certain extent, and the efficacy is very limited, especially for mental dependence. No effect.
  • the purpose of the present invention is to provide a novel analgesic drug that can be used for a long time, has no analgesic tolerance and addiction, and can effectively treat pain and opioid-induced physical and mental dependence.
  • the present invention provides the use of protopanaxadiol compounds (such as 20(S)-protopanaxadiol) in the preparation of novel analgesic drugs for treating pain and opioid-induced physical and mental dependence.
  • protopanaxadiol compounds such as 20(S)-protopanaxadiol
  • the experiments of the present invention show that 20(S)-protopanaxadiol can also be used in combination with other active ingredients to achieve a synergistic analgesic effect.
  • 20(S)-protopanaxadiol produces analgesic effect by stimulating the expression and release of dynorphin A in spinal microglia glucocorticoid receptors (cell membrane glucocorticoid receptors).
  • an active ingredient or a preparation containing the active ingredient is selected from the group consisting of protopanaxadiol or a pharmaceutically acceptable salt or ester thereof, a protopanaxadiol or a pharmaceutically acceptable salt or ester thereof, a Panaxatriol or a pharmaceutically acceptable salt or ester thereof;
  • the active ingredient or a formulation containing the active ingredient is used to prepare:
  • the protopanaxadiol includes 20(S)-protopanaxadiol, 20(R)-protopanaxadiol, or a combination thereof.
  • the protopanaxatriol includes 20(S)-protopanaxatriol, 20(R)-protopanaxatriol, or a combination thereof.
  • the addictive substance is selected from opioids, heroin, or a combination thereof.
  • the addictive substance further includes one or more selected from the group consisting of methamphetamine, alcohol, cigarettes (nicotine), cocaine, marijuana, or a combination thereof.
  • the pain is selected from the group consisting of: neuropathic pain, inflammatory pain, arthritis pain, diabetic pain, lower back pain, spinal cord injury pain, visceral pain, fibromyalgia, chronic regional pain syndrome , musculoskeletal pain, cancer pain, pain due to antineoplastic drugs and opioids, post-surgical pain, post-traumatic pain, post-traumatic neuralgia and peripheral neuropathy, phantom limb pain, or a combination thereof.
  • the neuropathic pain includes (but is not limited to) post-herpetic neuralgia, trigeminal neuralgia and sciatica.
  • the analgesic drug is an analgesic drug other than the active ingredient, preferably, the analgesic drug is an opioid analgesic selected from the group consisting of codeine, Dihydrocodeine, morphine, fentanyl, sufentanil, remifentanil, meperidine, oxycodone, or a combination thereof.
  • the preparation further includes a second active ingredient; wherein the second active ingredient is selected from the following group:
  • (Z1) An opioid analgesic selected from the group consisting of codeine, dihydrocodeine, morphine, fentanyl, sufentanil, remifentanil, meperidine, oxycodone, or combinations thereof ;
  • (Z2) an antiepileptic drug selected from the group consisting of carbamazepine, phenytoin, gabapentinoids, or a combination thereof;
  • Z3 a non-steroidal anti-inflammatory analgesic drug selected from the group consisting of flurbiprofen axetil, ibuprofen, diclofenac sodium, meloxicam, naproxen, celecoxib, preecoxib or its combination;
  • (Z4) a monoamine neurotransmitter reuptake inhibitor antidepressant selected from the group consisting of amitriptyline, duloxetine, or a combination thereof;
  • (Z5) a local anesthetic selected from the group consisting of lidocaine, ropivacaine, prilocaine, or a combination thereof;
  • (Z6) a norepinephrine alpha2 receptor agonist selected from the group consisting of clonidine, dexmedetomidine, or a combination thereof;
  • (Z7) MOR-NRI dual-target analgesics selected from the group consisting of dezocine, tapentadol, pentazocine, tramadol, or a combination thereof;
  • the gabapentin compounds include bapentin, pregabalin and mirogabalin.
  • the unique flavor extract and its active ingredients include methyl sanjinoside and methyl 8-O-acetyl sanjinoside.
  • the aconite/aconite and its active ingredients include: clathrate, clathrate and artemisinin.
  • the Corydalis and its active ingredients include tetrahydropalmatine, serratine, Corydalmine and dehydrocorsybulbine.
  • the weight ratio of the active ingredient to the second active ingredient is 1:0.001-100, preferably 1:0.05-10, more preferably 1:0.1-5.
  • the preparation is an oral preparation or an injection.
  • the preparation includes: powder, granule, capsule, injection, tincture, oral liquid, tablet, lozenge, or drop pill.
  • the active ingredient or the formulation containing the active ingredient does not have (does not produce): (1) analgesic tolerance; (2) physical dependence; (3) mental dependence (inducing addiction).
  • composition comprising:
  • a first active ingredient selected from the group consisting of protopanaxadiol and protopanaxatriol;
  • (Z1) An opioid analgesic selected from the group consisting of codeine, dihydrocodeine, morphine, fentanyl, sufentanil, remifentanil, meperidine, oxycodone, or combinations thereof ;
  • (Z2) an antiepileptic drug selected from the group consisting of carbamazepine, phenytoin, gabapentin compounds, or a combination thereof;
  • Z3 a non-steroidal anti-inflammatory analgesic drug selected from the group consisting of flurbiprofen axetil, ibuprofen, diclofenac sodium, meloxicam, naproxen, celecoxib, preecoxib or its combination;
  • (Z4) a monoamine neurotransmitter reuptake inhibitor antidepressant selected from the group consisting of amitriptyline, duloxetine, or a combination thereof;
  • (Z5) a local anesthetic selected from the group consisting of lidocaine, ropivacaine, prilocaine, or a combination thereof;
  • (Z6) a norepinephrine alpha2 receptor agonist selected from the group consisting of clonidine, dexmedetomidine, or a combination thereof;
  • (Z7) MOR-NRI dual-target analgesics selected from the group consisting of dezocine, tapentadol, pentazocine, tramadol, or a combination thereof;
  • the weight ratio of the first active ingredient to the second active ingredient is 1:0.001-100, preferably 1:0.05-10, more preferably 1:0.1-5.
  • the first active ingredient is protopanaxadiol
  • the second active ingredient is an opioid analgesic selected from the group consisting of codeine, dihydrocodeine, morphine, fentanyl , sufentanil, remifentanil, meperidine, oxycodone, or a combination thereof.
  • the first active ingredient is protopanaxadiol
  • the second active ingredient is morphine
  • the pharmaceutical composition is administered orally or non-orally.
  • the non-oral administration mode is selected from the group consisting of: nasal feeding, anal embolization, subcutaneous injection, intramuscular injection, intravenous injection, subarachnoid injection, epidural injection, lateral ventricle injection, skin injection Topical application (patch), or a combination thereof.
  • the second active ingredient is an addictive analgesic drug
  • the pharmaceutical composition is used for preparing: for treating and/or relieving pain, and reducing the induction of the second active ingredient Medications for physical and/or mental dependence.
  • the second active ingredient is an addictive analgesic drug
  • the pharmaceutical composition is prepared for treating and/or relieving pain and reducing the analgesia induced by the second active ingredient Pain tolerance.
  • the addictive analgesic drug is an opioid analgesic selected from the group consisting of codeine, dihydrocodeine, morphine, fentanyl, sufentanil, and remifen Tanyl, pethidine, oxycodone, or a combination thereof.
  • a fourth aspect of the present invention there is provided a method for treating and/or relieving pain in vitro, comprising the steps of:
  • the cells are immune cells of the central nervous system, preferably spinal cord immune cells.
  • the cells are selected from: microglia, macrophages, monocytes, or a combination thereof.
  • the cells are spinal cord microglia.
  • the glucocorticoid is a glucocorticoid receptor agonist.
  • the glucocorticoid is a cell membrane glucocorticoid receptor agonist.
  • the method is non-diagnostic and non-therapeutic.
  • a method for treating and/or relieving pain comprising the steps of:
  • a method for inducing the expression and release of dynorphin A comprising: administering an active ingredient to a subject in need, the active ingredient being selected from the group consisting of protopanaxadiol or its pharmacy an acceptable salt or ester thereof, protopanaxatriol, or a pharmaceutically acceptable salt or ester thereof, thereby inducing dynorphin A production in the subject.
  • the method stimulates the spinal cord to increase the expression and release of dynorphin A.
  • the method is used to agonize the glucocorticoid receptor of the cells of the subject.
  • the glucocorticoid receptor is a cell membrane glucocorticoid receptor.
  • the subject is a mammal.
  • the subject includes but is not limited to mice and humans.
  • the subject is a pain patient.
  • the cells are immune cells of the central nervous system, preferably spinal cord immune cells.
  • the cells are selected from: microglia, macrophages, monocytes, or a combination thereof.
  • the cells are spinal cord microglia.
  • the method is non-diagnostic and non-therapeutic.
  • a seventh aspect of the present invention provides a method for treating and/or alleviating physical and/or mental dependence and/or addiction induced by an addictive substance, comprising the step of: administering to a subject in need an effective amount of a first active ingredient or a pharmaceutical composition containing said first active ingredient, thereby treating and/or alleviating addictive substance-induced physical and/or mental dependence and/or addiction;
  • the first active ingredient is selected from the group consisting of protopanaxadiol or a pharmaceutically acceptable salt or ester thereof, protopanaxatriol or a pharmaceutically acceptable salt or ester thereof.
  • the addictive substance is selected from one or more of the following group: opioids, heroin, methamphetamine, alcohol, cigarettes (nicotine), cocaine, marijuana, or a combination thereof.
  • the subject is a mammal, such as a human, a rat or a mouse.
  • a method for reducing the addiction and/or analgesic tolerance of analgesic drugs comprising the steps of: combining an effective amount of the addictive analgesic drugs with the first active ingredient or the containing The pharmaceutical composition of the first active ingredient is jointly administered to a subject in need, thereby reducing the addictiveness and/or analgesic tolerance of the analgesic drug;
  • the first active ingredient is selected from the group consisting of protopanaxadiol or a pharmaceutically acceptable salt or ester thereof, protopanaxatriol or a pharmaceutically acceptable salt or ester thereof.
  • the addiction refers to physical and/or mental dependence induced by the analgesic drug.
  • the addictive analgesic drug is an opioid analgesic selected from the group consisting of codeine, dihydrocodeine, morphine, fentanyl, sufentanil, remifen Tanyl, pethidine, oxycodone, or a combination thereof.
  • the weight ratio of the first active ingredient to the addictive analgesic drug is 1:0.001-100, preferably 1:0.05-10, more preferably 1:0.1-5.
  • the first active ingredient or the pharmaceutical composition containing the first active ingredient is before (eg, within 24 hours), simultaneously or after (eg, within 24 hours) of the addictive analgesic drug. Dosing.
  • the subject is a mammal, such as a human, a rat or a mouse.
  • the method is also used to reduce the analgesic tolerance of the addictive drug.
  • Figure 1 shows that oral administration of 20(S)-protopanaxadiol dose-dependently inhibits mechanical and thermal hyperalgesia in neuropathic pain.
  • Figure 2 shows the analgesic effect of oral administration of 20(S)-protopanaxadiol in models of bone cancer pain, complete Freund's adjuvant (CFA) inflammatory pain and formalin pain.
  • CFA complete Freund's adjuvant
  • Figure 3 shows the synergistic analgesic effect of oral 20(S)-protopanaxadiol in combination with gabapentin or morphine in a neuropathic pain model.
  • Figure 4 shows that oral administration of 20(S)-protopanaxadiol specifically stimulates the expression of dynorphin A gene and protein in rat spinal cord.
  • Double immunofluorescence staining shows that oral administration of 20(S)-protopanaxadiol specifically stimulates the expression of dynorphin A in rat spinal cord microglia.
  • Figure 6 shows that ex vivo administration of 20(S)-protopanaxadiol specifically stimulates the expression of dynorphin A gene and protein in primary spinal cord microglia.
  • Figure 7 shows that the microglia activation inhibitor minocycline blocks 20(S)-protopanaxadiol against neuropathic pain.
  • Figure 8 shows that dynorphin A antiserum and specific kappa-opioid receptor antagonist block 20(S)-protopanaxadiol against neuropathic pain.
  • Figure 9 shows that oral administration of 20(S)-protopanaxadiol does not produce auto-analgesic tolerance or morphine-analgesic cross-tolerance, but can prevent or inhibit morphine analgesic tolerance.
  • Figure 10 shows that oral administration of 20(S)-protopanaxadiol does not produce somatic dependence, but inhibits the somatic dependence of morphine.
  • Figure 11 shows that oral administration of 20(S)-protopanaxadiol reverses the analgesic tolerance effect of morphine.
  • Figure 12 shows that oral administration of 20(S)-protopanaxadiol does not produce psychotropic dependence, but inhibits the psychotropic effect of morphine.
  • Figure 13 shows that oral administration of 20(S)-protopanaxadiol inhibits morphine behavioral sensitization.
  • Figure 14 shows that pre-administration of a glucocorticoid receptor antagonist in the subarachnoid space inhibits the analgesic effect of oral 20(S)-protopanaxadiol.
  • Figure 15 shows that intrathecal intrathecal administration of a glucocorticoid receptor antagonist inhibits the expression of dynorphin A in the spinal cord produced by oral administration of 20(S)-protopanaxadiol.
  • Figure 16 shows that glucocorticoid receptor antagonists inhibit the expression of dynorphin A produced by 20(S)-protopanaxadiol in primary spinal cord microglia.
  • Figure 17 shows that administration of dynorphin A antiserum, kappa-opioid receptor antagonists, microglia inhibitors and glucocorticoid receptor antagonists to the nucleus accumbens inhibits the anti-morphine-dependent effects of 20(S)-protopanaxadiol.
  • Figure 18 shows that administration of dynorphin A antiserum, kappa-opioid receptor antagonist, microglia inhibitor and glucocorticoid receptor antagonist to nucleus accumbens inhibits 20(S)-protopanaxadiol against morphine-induced CPP expression effect.
  • protopanaxadiol compounds especially 20(S)-protopanaxadiol
  • Adverse reactions such as analgesic tolerance, physical dependence, and mental dependence occur. It is therefore useful in the treatment of pain and anti-addiction (eg, physical and mental dependence induced by addictive substances).
  • the present invention has been completed on this basis.
  • the examples show that the analgesic effect of the active ingredient 20(S)-protopanaxadiol does not produce self-analgesic tolerance, and can help suppress physical dependence and mental dependence induced by addictive substances while analgesic .
  • the protopanaxadiol compound can also be used in combination with other analgesic drugs to achieve synergistic analgesic effect.
  • Protopanaxadiol compounds can achieve analgesic effect by specifically stimulating the expression of dynorphin A.
  • the term “about” means that the value may vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the terms "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “consisting essentially of,” or “consisting of.”
  • the terms "physical dependence”, “physical dependence” and “physiological dependence” are used interchangeably and refer to dependence which, once discontinued from the use of the addictive drug, can trigger a withdrawal syndrome.
  • the term "withdrawal syndrome” refers to a series of symptoms, such as sweating and tearing, caused by the severe physiological response of the body due to the continuous use of addictive substances, so that once the patient with dependence has discontinued the use , yawning, chills, goosebumps, mydriasis, vomiting, diarrhea, abdominal pain, increased heart rate, blood pressure, insomnia, tremors and other complex symptoms.
  • the terms "psychological dependence” and “psychological dependence” are used interchangeably, and refer to a patient's craving for a drug in order to obtain a special pleasure after taking an addictive drug.
  • treating means at least inhibiting or ameliorating the associated symptoms affecting the individual, wherein inhibiting And amelioration is used in its broadest sense and refers to at least a reduction in the magnitude of a parameter, such as a symptom associated with the condition being treated, such as pain. Accordingly, the methods of the present invention encompass the prevention and management of a variety of pains.
  • the present invention provides the use of an active ingredient of the present invention or a formulation thereof for the treatment of pain.
  • pain is not particularly limited, and representative examples include (but are not limited to) migraine, back pain, neck pain, gynecological pain, pre-labor or labor pain, orthopaedic pain, post-stroke pain, post-surgery or Procedural pain, post-herpetic neuralgia, sickle cell crisis, interstitial cystitis, urinary pain (eg, urethritis), dental pain, headache, wounds, or surgery (eg, bursectomy or hip, knee or Pain, sutures, fracture reduction, biopsies, etc. from medical procedures for other joint replacements). Pain can also occur in patients with cancer and can be caused by a variety of factors, such as inflammation, nerve compression, and mechanical forces from tissue swelling due to tumor invasion and metastasis to bone or other tissues.
  • the pain includes (but is not limited to): peripheral neuropathic pain, central neuropathic pain, allodynia, causalgia, hyperalgesia, hyperesthesia, hyperalgesia, neuralgia, Neuritis and neuropathy.
  • Drug addiction and drug dependence is a chronic relapsing brain disease, mainly manifested as compulsive drug use behavior and uncontrollable dose of addictive drugs. If you stop taking the drug suddenly after a substance dependence condition, you may experience drug withdrawal symptoms. Many drugs originally used for medical purposes can cause substance dependence; addictive substances are called drugs if they are regulated by law and considered illegal. These addictive substances include opioids and heroin, methamphetamine, cocaine, marijuana, alcohol and nicotine, among others.
  • Methamphetamine commonly known as methamphetamine
  • methamphetamine is a highly addictive stimulant and the second most commonly used illicit drug globally.
  • the abuse of methamphetamine or other amphetamine-type stimulants has become a major public health problem.
  • traditional drugs such as heroin and cocaine
  • the synthetic process of methamphetamine is simple, the precursor is cheap and easy to obtain, the excitatory effect on the central nervous system is stronger, and the number of drugs or cumulative dose required to form an addiction is less, and the drug use is less effective. The damage caused to the body is more serious.
  • Alcohol is a psychoactive substance with highly addictive properties. There are 140 million people with alcohol dependence in the world, and its abuse and dependence have brought serious adverse effects and economic burdens to individuals and society. Worldwide, approximately 3.3 million people die each year from overuse of alcohol. Harmful use of alcohol can also lead to diseases such as alcoholic liver and cirrhosis. Alcohol abuse and alcohol addiction have become a serious public health disaster and a worldwide problem that endangers human health. It is the third largest global public health problem after cardiovascular diseases and tumors.
  • Nicotine also known as nicotine, is a potent parasympathomimetic alkaloid and the main active ingredient in cigarettes. Nicotine dependence is the main characteristic of smokers, it refers to the physical and psychological changes in individuals after repeated nicotine use, including increased craving and uncontrollable use, persistent and preferential use regardless of harmful consequences, increased tolerance and withdrawal symptoms . Tobacco dependence is one of the most serious public health problems at present. WHO pointed out that tobacco kills more than 7 million people every year, of which more than 6 million are from direct tobacco use, and about 890,000 are non-smokers exposed to second-hand smoke.
  • opioids including morphine and fentanyl
  • the body develops analgesic tolerance, and the dose must be increased to obtain the same analgesic effect.
  • Long-term or repeated use of opioids can also lead to addiction, including both physical (physical dependence) and mental (psychological) dependence.
  • Physical dependence is the repeated use of medication to avoid withdrawal symptoms, and due to the gradual increase in the tolerable dose, it manifests as an aversive effect in the process of addiction, which plays a negative reinforcement effect.
  • Mental dependence refers to the psychological craving of the dependent and the euphoria achieved by repeated drug use, which manifests as a rewarding effect, plays a positive reinforcement effect, and prompts the patient to relapse repeatedly.
  • protopanaxadiol includes 20(S)-protopanaxadiol, 20(R)-protopanaxadiol, or a combination thereof (eg, a racemate).
  • the protopanaxatriol includes 20(S)-protopanaxatriol, 20(R)-protopanaxatriol, or a combination thereof (eg, a racemate).
  • the term includes natural products or artificially synthesized or modified products.
  • the active ingredient of the present invention includes the active compound of the present invention (protopanaxadiol, protopanaxatriol, or a combination thereof), or a pharmaceutically acceptable salt or ester, enantiomer, diastereomer thereof Isomers or racemates, or prodrugs thereof. It should be understood that the active ingredients of the present invention also include crystalline forms, amorphous compounds, solvates, hydrates and the like forms of the active compounds of the present invention.
  • the “pharmaceutically acceptable salts (or esters)” are conventional non-toxic salts (or esters) formed by the reaction of the active compounds of the present invention with inorganic or organic acids.
  • conventional non-toxic salts can be prepared by reacting the active compounds of the present invention with inorganic or organic acids, including hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid, and the like.
  • Acids include citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid , Fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid acid, 2-acetoxybenzoic acid, isethionic acid, etc.; or the active compound of the present invention is combined with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid
  • Opioid analgesics include codeine, dihydrocodeine, morphine, fentanyl, sufentanil, remifentanil, meperidine, oxycodone;
  • Anti-epileptic drugs including carbamazepine, phenytoin and gabapentins such as gabapentin, pregabalin and mirogabalin;
  • NSAIDs include flurbiprofen axetil, ibuprofen, diclofenac sodium, meloxicam, naproxen, celecoxib and praecoxib;
  • Monoamine neurotransmitter reuptake inhibitor antidepressants including amitriptyline and duloxetine
  • Local anesthetics include lidocaine, ropivacaine, and prilocaine;
  • Norepinephrine ⁇ 2 receptor agonists such as clonidine and dexmedetomidine
  • MOR-NRI dual-target analgesics such as dezocine, tapentadol, pentazocine and tramadol;
  • Anti-migraine drugs such as CGRP antibodies and their receptor antagonists
  • Chinese herbal medicines include the unique extract and its active ingredients such as sanji glycoside methyl ester and 8-O-acetyl sanji glycoside methyl ester, aconite/aconite and its effective ingredients such as clathrin, clathrin and Artemisinin on Snow, as well as Corydalis and its active ingredients such as tetrahydropalmatine, seraphine, Corydalin and dehydrocoralbulbine.
  • active ingredients such as sanji glycoside methyl ester and 8-O-acetyl sanji glycoside methyl ester, aconite/aconite and its effective ingredients such as clathrin, clathrin and Artemisinin on Snow, as well as Corydalis and its active ingredients such as tetrahydropalmatine, seraphine, Corydalin and dehydrocoralbulbine.
  • compositions and methods of administration are provided.
  • the present invention also provides a composition or formulation or product containing the active ingredient of the present invention, which composition or formulation or product can be used for anti-aging.
  • Representative compositions or formulations or products include anti-aging drugs, nutraceuticals, and cosmetics.
  • a preferred composition is a pharmaceutical composition comprising an effective amount of verapamil or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • the term "effective amount” or “effective dose” refers to an amount that produces a function or activity (eg, analgesic function) in humans and/or animals and is acceptable to humans and/or animals.
  • the term "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation, and allergy), ie, a substance with a reasonable benefit/risk ratio .
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier.
  • Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration, and the dosage form of the pharmaceutical composition of the present invention is an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, and a sustained release agent.
  • it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical compositions are preferably manufactured under sterile conditions.
  • the effective amount of the active ingredient of the present invention may vary with the mode of administration, the severity of the disease to be treated, and the like. Selection of the preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the administration way etc. Generally, satisfactory results are obtained when the active ingredient of the present invention is administered at a daily dose of about 0.001-100 mg/kg animal body weight (preferably 0.01-50 mg/kg, more preferably 0.05-20 mg/kg animal body weight). . For example, several divided doses may be administered daily, or the dose may be proportionally reduced, as dictated by the exigencies of the therapeutic situation.
  • the oral dose may be 0.05-50 mg/kg, preferably 0.10-20 mg/kg.
  • the first active ingredient and the second active ingredient may be prepared in a single formulation, or may be prepared as separate formulations.
  • the first active ingredient and the second active ingredient can be administered simultaneously or sequentially.
  • the active ingredient of the present invention does not produce analgesic tolerance, physical dependence, mental dependence and addiction while being effective in analgesia.
  • Rats were rapidly anesthetized with 5% isoflurane under a respiratory anesthesia machine (anesthesia machine airflow rate of 0.3 L/min), followed by maintenance of anesthesia with 2% isoflurane.
  • An 18-cm polyethylene catheter (PE-10: outer diameter: 0.55 mm, inner diameter: 0.3 mm) was inserted from the lumbar level of the rat along the spine.
  • 10 ⁇ L of 4% lidocaine was used to detect the intubation situation. Intrathecal injection in the subarachnoid space. If the rat has bilateral hind foot paralysis and no movement disorder after recovery, it means that the intubation is successful and can be followed up. experiment.
  • Rats were acclimated in transparent observation cages measuring 23 ⁇ 35 ⁇ 19 cm for 30 minutes before the experiment.
  • the left foot of the rat was taken out and injected subcutaneously with 5% formalin solution (50 ⁇ l). After the injection, the rat was placed in the observation cage immediately, and the number of foot lifts within 60 seconds was measured every 10 minutes after the injection until 90 minutes. deadline.
  • Rats were rapidly anesthetized with 5% isoflurane under a respiratory anesthesia machine (anesthesia machine airflow rate of 0.3 L/min), followed by maintenance of anesthesia with 2% isoflurane.
  • the left muscle was bluntly dissected in the lumbar spinal cord, the L6 transverse process was exposed and removed, and the L5 nerve was exposed and tied with 6-0 silk; After the operation, the rats were kept in a single cage and recovered for one week.
  • the mechanical pain threshold of the posterior plantar was measured with a Von Frey electronic pain meter, and the model was considered successful if it was less than 8 g and no movement disorder was found, which was used for subsequent experiments.
  • Rats were placed on mechanical pain and thermal pain test racks, respectively.
  • an electromechanical pain threshold detector was used to vertically stimulate the middle of the hind limbs of the rats.
  • the detector was equipped with a 15-gauge fiber.
  • the stimulation intensity was slowly increased until the fibers were bent into an S shape, lasting 6-8 For seconds, observe whether the rat has a foot withdrawal or foot lift response.
  • the minimum threshold of foot withdrawal or foot lift was recorded as the paw withdrawal threshold (PWT). It was detected every 3 minutes, repeated three times, and the average value of the three times was taken as the mechanical pain threshold of the rat's foot.
  • the mechanical pain threshold reflects the degree of injury/pain of mechanical stimulation in rats.
  • the 10-day CPP model consists of three phases: a pre-test period, an acquisition period, and a post-test period.
  • Pre-test period (1-4 days): Male Swiss mice were allowed to travel freely between the three compartments, 2 times a day for 15 minutes each for 3 days. On day 4, the dwell time in each compartment of the mouse was recorded for 15 minutes as the mice freely shuttled through the three compartments.
  • Acquisition period (5-9 days): Mice were given alternating subcutaneous injections of morphine (10 mg/kg) or saline (10 mL/kg) every 6 hours (9:00 am and 3:00 pm) for 5 days, followed by immediate Enter the compartment and train for 45 minutes.
  • morphine (10 mg/kg) was injected subcutaneously at 9:00 am and normal saline (10 mL/kg) was injected subcutaneously at 3:00 pm, respectively, and placed in the morphine concomitant medicine box and physiological 45 minutes of training in the saline paired box.
  • days 6 and 8 the injection times of morphine and saline were exchanged.
  • Conditioned place preference scores were calculated by subtracting the time spent in the saline paired box from the time the mice spent in the companion box.
  • the shuttle activity of mice in each compartment was captured by a 3CCD camera, and the time that mice stayed in each compartment was recorded using EthoVision XT 8.0 software.
  • Behavioral sensitization refers to the enhancement of certain behavioral effects caused by repeated opioid use and is associated with compulsive drug-seeking and relapse.
  • the method of establishing mouse behavior sensitization is briefly described as follows: The mice were placed in a motion detection chamber (40 ⁇ 40 ⁇ 35cm) under the video tracking system, and the data were automatically analyzed by ANY-maze software. Morphine-induced behavioral sensitization includes an adaptive phase (1-3 days) and a formative phase (4-8 days). During the adaptation period (1-3 days), all mice were injected subcutaneously with normal saline, and immediately placed in the detection room to move freely, 1 hour a day, and the movement distance of the mice was recorded for 3 consecutive days.
  • microglia When preparing microglia, put the culture flask into a shaker and shake it at 37°C (260rpm) for 1.5-2 hours, collect the cell suspension, centrifuge, resuspend the cells, and then inoculate it in a new cell culture plate. Unadherent cells were washed away with warm PBS. The obtained microglia were more than 95% pure by immunofluorescence assay of the microglia marker protein Iba-1.
  • the cultured cells were discarded from the medium, washed twice with PBS, and then added with 0.05% EDTA-containing trypsin. Oligodendrocytes were removed by digestion at 37°C for 3 minutes, the digestion was terminated and the cell suspension was removed, and the remaining adherent monolayers of astrocytes were further passaged by trypsinization for subsequent use.
  • the obtained astrocytes were more than 90% pure by immunofluorescence assay of the astrocyte marker protein GFAP.
  • neuronal cells To prepare neuronal cells, the cell suspension was filtered through a 40- ⁇ m mesh, seeded into a 10-cm cell culture dish, and cultured in a cell incubator for 30 minutes. The non-adherent upper cell suspension was then aspirated and plated on poly-lysine plates. After culturing for 1.5-2 hours, DMEM was replaced with Neurobasal medium containing 1 ⁇ B27 neurotrophic factor and 0.5 mM glutamine, and the culture was continued for 3-4 days. By immunofluorescence assay of neuronal cell marker protein NeuN, the obtained neuronal cells were more than 85% pure.
  • RNA precipitation RNA precipitation
  • a reverse transcription kit was used to run the corresponding program on a common PCR machine, and the extracted total RNA was reverse transcribed into cDNA and stored at -20°C for later use.
  • Subsequent real-time quantitative PCR operations used SYBR qPCR mix to detect precursor dynorphin (PDYN), precursor endorphin (POMC), precursor enkephalin (PNOC), and Nociceptin/OrphaninFQ precursor gene ( PENK) Ct value, with GAPDH as the internal reference gene, the 2- ⁇ Ct method was used to calculate the relative expression of the target gene.
  • PYN precursor dynorphin
  • POMC precursor endorphin
  • PNOC precursor enkephalin
  • PENK Nociceptin/OrphaninFQ precursor gene
  • the rat spinal cord lumbar enlargement (L3-L5) tissue was removed, homogenized (4,000rpm, 15 seconds) with 10mM Tris-HCl (5mL/1g tissue), and centrifuged (5000rpm) at 4°C for 15 minutes. of the supernatant.
  • primary cells derived from neonatal rat spinal cord were administered and cultured for 2 hours, and the cell culture supernatant was collected. According to the instructions of the ELISA kit, the content of dynorphin A and ⁇ -endorphin in the supernatant of cell culture and spinal cord tissue was determined.
  • Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg), and the thoracic cavity was opened along the lower border of the sternal xiphoid process to expose and free the heart. Quickly insert the needle into the aorta through the left ventricle, fix the needle with No. 4-0 surgical suture, and cut the right atrial appendage. Slowly perfuse 100ml of normal saline to flush the blood, and then continue to perfuse 60ml of 4% formaldehyde solution.
  • the lumbar spinal cord (L3-L5) was taken out and placed in 4% formaldehyde fixative solution at 4°C overnight, followed by dehydration in a gradient of sucrose solution, embedding, frozen section (30 ⁇ m thickness) and -20°C for future use.
  • the cryopreserved tissue sections were rewarmed and blocked with blocking solution at room temperature for 1 hour, followed by the preparation of primary antibodies (dynorphin A antibody, microglia marker Iba-1, astrocyte marker GFAP and neuronal cells) using the blocking solution. Labeled NeuN) was incubated at 4°C for 18-24 hours.
  • the blocking solution prepared with the secondary antibody was added and incubated at 37 °C for 1 hour, then mounted with anti-fluorescence quenching mounting medium, and stored at -20 °C in the dark for future use. Images were taken with a Leica TCS SP8 confocal microscope, and fluorescence quantification and colocalization analysis of fluorescent staining were performed with Image J image processing software.
  • Example 1 Analgesic effect of oral administration of 20(S)-protopanaxadiol in a rat model of neuropathic pain
  • Neuropathic pain rats with L5/L6 spinal nerve ligation were selected and randomly divided into six groups (6 rats in each group).
  • the paw withdrawal response threshold or paw withdrawal latency of rats to mechanical stimulation and thermal radiation stimulation were measured (thermal radiation stimulation was performed 10 minutes after mechanical stimulation). ).
  • the mechanical pain threshold and thermal radiation pain threshold of each metering group were measured at 1 hour to calculate the %maximal possible effect (%MPE), and then a dose-response analysis was performed.
  • oral 20(S)-protopanaxadiol can inhibit neuropathic pain, and the degree of this inhibitory effect is positively dependent on the dose of 20(S)-protopanaxadiol taken.
  • the analgesic effect of oral 20(S)-protopanaxadiol is also very obvious.
  • Example 2 Oral analgesic effect of 20(S)-protopanaxadiol in rat models of pain caused by different etiologies.
  • Two groups of rats with bone cancer pain (6 rats in each group) and two groups of rats with CFA-induced inflammatory pain (6 rats in each group) were orally administered with solvent (6.5 mL/kg) or 20(S)-protopanaxadiol ( 100 mg/kg), before administration and 0.5, 1, 2, and 4 hours after administration, the withdrawal response threshold or the withdrawal latency of rats to mechanical stimulation and thermal radiation stimulation were determined (thermal radiation stimulation in mechanical stimulation). 10 minutes after stimulation).
  • Formalin can induce both phase I and phase II foot licking responses in rats.
  • Oral administration of 20(S)-protopanaxadiol inhibited formalin-induced phase II foot licking in rats, but had no effect on phase I pain (Fig. 2E).
  • the paw withdrawal response threshold or paw withdrawal latency of each rat to mechanical stimulation and thermal radiation stimulation was measured (thermal radiation stimulation was performed 10 minutes after mechanical stimulation). conduct).
  • Example 4 The specific stimulating effect of oral administration of 20(S)-protopanaxadiol on the expression of dynorphin A in rat spinal cord microglia
  • neuropathic pain rats with L5/L6 spinal nerve ligation (6 rats in each group) were orally administered with solvent (6.5 mL/kg) or 20(S)-protopanaxadiol (100 mg/kg) for one hour, respectively.
  • the head was decapitated and the spinal cord tissue of the operative side of the enlarged lumbar spine (L3-L5) was collected.
  • the gene expression levels of PDYN, POMC, PENK and PNOC were determined by real-time quantitative PCR, and the results are shown in Fig. 4A.
  • the levels of dynorphin A and ⁇ -endorphin in the supernatant of spinal cord homogenate were determined by enzyme-linked immunofluorescence assay.
  • primary spinal cord microglia were treated with different concentrations of 20(S)-protopanaxadiol (1, 3, 10, 30 and 100 ⁇ M), and after culturing for 2 hours, the microglia dynorphin A gene was detected and protein expression, as shown in Figures 6A and 6B; primary spinal cord astrocytes and neuronal cells were treated with 20(S)-protopanaxadiol (100 ⁇ M), and astrocytes were detected after 2 hours of culture and neuronal cell dynorphin A gene and protein expression, as shown in Figures 6C and 6D.
  • dynorphin A is also co-expressed with astrocyte marker protein GFAP or neuron cell marker protein NeuN in rat spinal cord.
  • the co-stained area of dynorphin A and GFAP in the oral administration of 20(S)-protopanaxadiol did not change significantly compared with the administration of physiological saline (control group).
  • 20(S)-protopanaxadiol treatment increased the expression of dynorphin A gene and protein in spinal microglia in a dose-dependent manner, with ED 50 values of 13 and 19.8 ⁇ M, respectively.
  • 20(S)-protopanaxadiol did not significantly change the expression of dynorphin A gene or protein in spinal cord astrocytes or neurons.
  • Example 5 Pre-administration of the microglia activation inhibitor minocycline, dynorphin A antiserum and specific ⁇ -opioid receptor antagonists in the subarachnoid space for analgesia of 20(S)-protopanaxadiol inhibitory effect
  • neuropathic pain rats (6 rats in each group) were used to pre-inject normal saline (10 ⁇ L) or microglia activation inhibitor minocycline (100 ⁇ g) into the subarachnoid space. After 4 hours, both groups were orally administered 20(S)-protopanaxadiol (100 mg/kg). Before the first administration, before the second administration and 0.5, 1, 2 and 4 hours after administration, the paw withdrawal response threshold of the hind paw to mechanical stimulation and the paw withdrawal response latency to thermal radiation were measured. The results are shown in Fig. 7A and 7B.
  • neuropathic pain rats (6 rats in each group) were pre-injected with blank rabbit serum (10 ⁇ L), dynorphin A antiserum (1:10, 10 ⁇ L) or ⁇ -endorphin in the subarachnoid space, respectively. Antiserum (1:10, 10 ⁇ L). After 0.5 hours, all three groups were orally administered 20(S)-protopanaxadiol (100 mg/kg). The paw withdrawal response threshold of the rat hind paw to mechanical stimulation and the paw withdrawal response latency to thermal radiation were measured, and the results are shown in Figures 8A and 8B.
  • Dynorphin A is an endogenous ⁇ -opioid receptor agonist.
  • the following verifications were made: four groups of neuropathic pain were used. Rats (6 rats in each group) were injected intrathecally with normal saline (10 ⁇ L), ⁇ -opioid receptor antagonist CTAP (10 ⁇ g), ⁇ -opioid receptor antagonist GNTI (50 ⁇ g) or ⁇ -opioid receptor antagonist. The body antagonist naltrindole (5 ⁇ g). After 0.5 hours, the four groups of rats were all orally administered with 20(S)-protopanaxadiol (100 mg/kg). The results are shown in Figures 8C and 8D.
  • Example 6 Inhibitory effect of oral 20(S)-protopanaxadiol on morphine analgesic tolerance and physical dependence
  • the rats in the four groups were orally administered 20(S)-protopanaxadiol (30 mg/kg).
  • all four groups of rats were administered morphine (3 mg/kg), and the hindpaw pain threshold of the rats was measured for the next 4 hours.
  • the results are shown in Figures 9C and 9D.
  • single-dose subcutaneous injection of morphine could not produce analgesic effect in rats that were morphine-tolerant by continuous morphine administration for one week; And 20(S)-protopanaxadiol + morphine co-administration group, and then single-dose subcutaneous injection of morphine can produce significant analgesic effect.
  • Figures 10A-10E show that, compared with the normal saline control group, oral administration of 20(S)-protopanaxadiol did not produce physical dependence, while morphine produced significant physical dependence, and the combined application of 20(S)-protopanaxadiol ( 30 mg/kg) significantly reduced morphine-related withdrawal symptoms, including tremors (Fig. 10A), jumping (Fig. 10B), teeth chattering (Fig. 10C), diarrhea (Fig. 10D) and wet dog shaking (Fig. 10E).
  • FIG. 11A and 11B show that the acute analgesic effect of morphine in the paw-licking and tail-flicking models gradually decreased and completely disappeared 7 days after administration.
  • the two groups of morphine-tolerant rats were given the solvent (6.5 mL/kg) and 20(S)-protopanaxadiol (30 mg/kg) by gavage, respectively.
  • a single dose of morphine (3 mg/kg) was injected, and the pain threshold of the rats was measured simultaneously.
  • Figures 11C and 11D show that within 1 hour of gavage with vehicle or 20(S)-protopanaxadiol (the time point is the peak of 20(S)-protopanaxadiol inhibiting neuropathic pain in rats) on hot plate and No analgesic effect was shown in the tail-flick response.
  • Example 7 Inhibitory effect of oral 20(S)-protopanaxadiol on morphine conditioned place preference (CPP) acquisition
  • mice Two groups of mice (10 mice in each group) were given oral administration of solvent (10 mL/kg) or 20(S)-protopanaxadiol (100 mg/kg) alternately every day for 5 consecutive days, followed by conditioned place preference test, the results are shown in Figure 12A shown.
  • mice Another four groups of mice (10 mice in each group) were injected subcutaneously with saline (10 mL/kg) or morphine (10 mg/kg) daily for 5 days, and 50 minutes before the last injection, the mice received a single oral dose of the solvent (10 mL/kg) or 20(S)-protopanaxadiol (100 mg/kg), followed immediately by a 15-minute place preference test, as shown in Figure 12B.
  • Example 8 Inhibitory effect of oral 20(S)-protopanaxadiol on behavioral sensitization of morphine
  • mice During the 5-day conditioning period, 4 groups of 10 mice were administered with normal saline (10 mL/kg) or 20 (S )-protopanaxadiol (100 mg/kg), and then immediately put the mice into the box to record the free movement distance of the mice in the box within 1 hour. As shown in Figure 13, the free movement distance of the mice in the saline group was maintained at a low level, while the free movement distance of the mice in the morphine injection group for 5 consecutive days increased significantly, and the daily movement distance was gradually increased compared with the previous day. Administration of 20(S)-protopanaxadiol for 5 consecutive days attenuated the morphine-induced behavioral sensitization effect of mice, and the daily movement distance of mice gradually decreased.
  • Example 9 Inhibitory effect of pre-administration of glucocorticoid receptor antagonist in subarachnoid space on 20(S)-protopanaxadiol analgesia
  • Two groups of rats with neuropathic pain (6 rats in each group) were pre-injected with solvent (10 ⁇ L) or non-specific glucocorticoid receptor antagonist RU1486 (10 nmol) in the subarachnoid space. After 0.5 hours, both groups of rats were orally administered 20(S)-protopanaxadiol (100 mg/kg). Before the first administration, before the second administration and at 0.5, 1, 2 and 4 hours after administration, the paw withdrawal response thresholds of the hind paws of rats to mechanical stimulation were determined.
  • the other two groups of neuropathic pain rats (6 rats in each group) were pre-injected with solvent (10 ⁇ L) or the specific glucocorticoid receptor antagonist dexamethasone 21-mesylate (Dex-21-mesylate, 10 nmol). After 0.5 hours, both groups of rats were orally administered 20(S)-protopanaxadiol (100 mg/kg). Before the first administration, before the second administration and at 0.5, 1, 2 and 4 hours after administration, the paw withdrawal response thresholds of the hind paws of rats to mechanical stimulation were determined. Oral administration of 20(S)-protopanaxadiol produces time-dependent analgesia.
  • Dex-21-mesylate does not affect the basal threshold of pain, but completely inhibits the analgesia produced by 20(S)-protopanaxadiol ( Figure 14B).
  • two groups of neuropathic pain rats (6 rats in each group) were pre-injected with solvent (10 ⁇ L) or Dex-21-mesylate (10 nmol) in the subarachnoid space. After 0.5 hours, both groups of rats were subcutaneously injected with bulleaconitine A (BAA, 300 ⁇ g/kg). Before the first administration, before the second administration and at 0.5, 1, 2 and 4 hours after administration, the paw withdrawal response thresholds of the hind paws of rats to mechanical stimulation were determined. Oral subcutaneous injection of fenugreek produced time-dependent analgesia, but Dex-21-mesylate did not affect the analgesia produced by fenugreek ( Figure 14C).
  • neuropathic pain rats (6 rats in each group) were pre-injected with solvent (10 ⁇ L) or estrogen receptor antagonist G15 (10 nmol or 1 ⁇ mol) in the subarachnoid space, respectively. After 0.5 hours, the three groups of rats were orally administered 20(S)-protopanaxadiol (100 mg/kg). Determination of the paw withdrawal response threshold of rat hind paws to mechanical stimuli. Oral administration of 20(S)-protopanaxadiol produced time-dependent analgesia, and G15 neither affected the basal threshold of pain nor the analgesia produced by 20(S)-protopanaxadiol ( Figure 14D). .
  • the other two groups of neuropathic pain rats (6 rats in each group) were pre-injected with solvent (10 ⁇ L) or aldosterone receptor antagonist eperenone (10 nmol) in the subarachnoid space. After 0.5 hours, the three groups of rats were orally administered 20(S)-protopanaxadiol (100 mg/kg). Determination of the paw withdrawal response threshold of rat hind paws to mechanical stimuli. Oral administration of 20(S)-protopanaxadiol produced time-dependent analgesia, and eperenone neither affected the basal threshold of pain nor the analgesia produced by 20(S)-protopanaxadiol ( Figure 14E). .
  • Example 10 Inhibitory effect of glucocorticoid receptor antagonists on the expression of dynorphin A stimulated by 20(S)-protopanaxadiol
  • primary cultured spinal cord microglia were administered with solvent or glucocorticoid receptor antagonist Dex-21-mesylate (100 nM) for 0.5 hours, then administered with 20(S)-protopanaxadiol (100 ⁇ M), The specific glucocorticoid receptor agonist Dex (100 nM) or the membrane-impermeable Dex and bovine serum albumin (BSA) conjugate Dex-BSA (10 nM) was incubated for 2 hours, and then the microglia were detected for Orphan A gene and protein expression.
  • Dex-21-mesylate 100 nM
  • 20(S)-protopanaxadiol 100 ⁇ M
  • the specific glucocorticoid receptor agonist Dex (100 nM) or the membrane-impermeable Dex and bovine serum albumin (BSA) conjugate Dex-BSA (10 nM) was incubated for 2 hours, and then the microglia were detected for Orphan A gene and
  • 20(S)-protopanaxadiol, Dex and Dex-BSA significantly enhanced microglia dynorphin A gene expression; while Dex 21-melylate did not affect microglia dynorphin gene basal expression, but completely inhibited the stimulatory effects of 20(S)-protopanaxadiol, Dex and Dex-BSA on dynorphin A gene expression.
  • Dex-21-melylate did not affect the basal expression of dynorphin A protein in microglia, but completely inhibited the effect of 20(S)-protopanaxadiol, Dex and Dex-BSA on dynorphin A protein. expression stimulation.
  • Example 11 Nucleus accumbens administration of microglia inhibitor, dynorphin A antiserum, ⁇ -opioid receptor antagonist and glucocorticoid receptor antagonist inhibits the anti-morphine somatic dependence of 20(S)-protopanaxadiol
  • mice in each group were injected into the nucleus accumbens and administered by gavage according to the following groups: normal saline (1 ⁇ L) + normal saline (10 mL/kg), dynorphin A antiserum (1:30 dilution) , 1 ⁇ L) + normal saline (10 mL/kg), normal saline (1 ⁇ L) + 20(S)-protopanaxadiol (100 mg/kg) and dynorphin A antiserum (1:30 dilution, 1 ⁇ L) + 20(S) - protopanaxadiol (100 mg/kg).
  • Normal saline (10 mL/kg) or 20(S)-protopanaxadiol (100 mg/kg) was administered by gavage 30 minutes after the nucleus accumbens injection, and naloxone (5 mg/kg) was intraperitoneally injected 40 minutes after the intragastric administration. ) and immediately observe for withdrawal symptoms within 30 minutes.
  • 20(S)-protopanaxadiol 100 mg/kg gavage in morphine-dependent mice alleviated morphine withdrawal symptoms, including reduced forepaw tremors, decreased wet dog shaking frequency, Decreased cunnilingus frequency, decreased jumping frequency, increased diarrhea, and relieved weight loss, and a single injection of dynorphin A antiserum (1:30 dilution, 1 ⁇ L) into the nucleus accumbens had no significant effect on morphine withdrawal, but could induce 20(S) -Reappearance of protopanaxadiol antimorphine withdrawal symptoms.
  • mice in each group were also given the first nucleus accumbens injection and the second intragastric administration: normal saline (1 ⁇ L) + normal saline (10 mL/kg), GNTI (5 ⁇ g, 1 ⁇ L) ) + normal saline (10mL/kg), normal saline (6 ⁇ L)+20(S)-protopanaxadiol (100mg/kg) and GNTI (5 ⁇ g, 1 ⁇ L)+20(S)-protopanaxadiol (100mg/ kg).
  • the second intragastric administration was performed 30 minutes after the nucleus accumbens injection, and naloxone (5 mg/kg) was intraperitoneally injected 40 minutes after the intragastric administration, and the withdrawal phenomenon was observed immediately.
  • naloxone 5 mg/kg
  • GNTI injection into the nucleus accumbens did not affect morphine withdrawal but restores 20(S)-protopanaxadiol to anti-morphine withdrawal.
  • mice in each group were also injected into the nucleus accumbens and administered by gavage: normal saline (1 ⁇ L) + normal saline (10 mL/kg), minocycline (10 ⁇ g) + normal saline (10 mL/kg), normal saline (1 ⁇ L) + 20(S)-protopanaxadiol (100 mg/kg) and minocycline (10 ⁇ g) + 20(S)-protopanaxadiol (100 mg/kg).
  • a second gavage of normal saline (10 mL/kg) or 20(S)-protopanaxadiol (100 mg/kg) was performed 4 hours after the first nucleus accumbens injection. After 40 minutes, intraperitoneal injection of naloxone (5 mg/kg) was used to observe the withdrawal phenomenon. Oral administration of 20(S)-protopanaxadiol in morphine-dependent mice could significantly reduce morphine withdrawal symptoms. Minocycline had no effect on morphine withdrawal symptoms, but could make 20(S)-protopanaxadiol inhibit the Withdrawal symptoms reappeared in mice (Figure 17M-R).
  • mice in each group were also injected into the nucleus accumbens and administered by gavage: normal saline (1 ⁇ L) + normal saline (10 mL/kg), Dex 21-mesylate (10 nmol, 1 ⁇ L) + Normal Saline (10mL/kg), Normal Saline (1 ⁇ L)+20(S)-protopanaxadiol (100mg/kg) and Dex 21-mesylate (10nmol, 1 ⁇ L)+20(S)-protopanaxadiol ( 100mg/kg).
  • Example 12 Nucleus accumbens administration of dynorphin A antiserum, kappa-opioid receptor antagonists, microglia inhibitors and glucocorticoid receptor antagonists inhibits 20(S)-protopanaxadiol against morphine-induced CPP expression effect
  • mice in each group were given the first nucleus accumbens injection before the test, and the second intragastric administration 4 hours later: normal saline (1 ⁇ L) + normal saline ( 10mL/kg), dynorphin A antiserum (1:30 dilution, 1 ⁇ L) + normal saline (10mL/kg), normal saline (1 ⁇ L) + 20(S)-protopanaxadiol (100mg/kg) and dynorphin A antiserum Serum (1:30 dilution, 1 ⁇ L) + 20(S)-protopanaxadiol (100 mg/kg).
  • CPP test was performed 50 minutes after gavage of normal saline (10 mL/kg) or 20(S)-protopanaxadiol (100 mg/kg). As shown in Figure 18A, gavage of 20(S)-protopanaxadiol completely attenuated morphine-induced CPP expression. Injection of dynorphin A antiserum into the nucleus accumbens had no effect on CPP expression, but could completely restore the inhibitory effect of 20(S)-protopanaxadiol on morphine-induced CPP expression, and the CPP score increased again.
  • mice in each group were given the first nucleus accumbens injection before the test, and the second intragastric administration 30 minutes later: normal saline (1 ⁇ L) + normal saline (10mL/kg), GNTI (5 ⁇ g, 1 ⁇ L) + saline (10mL/kg), saline (6 ⁇ L) + 20(S)-protopanaxadiol (100mg/kg) and GNTI (5 ⁇ g, 1 ⁇ L) + 20 (S)-protopanaxadiol (100 mg/kg).
  • the CPP test was also performed 50 minutes after gavage.
  • mice in each group were also injected into the nucleus accumbens and administered by gavage: normal saline (1 ⁇ L) + normal saline (10 mL/kg), minocycline (10 ⁇ g) + normal saline (10 mL/kg), normal saline (1 ⁇ L) + 20(S)-protopanaxadiol (100 mg/kg) and minocycline (10 ⁇ g) + 20(S)-protopanaxadiol (100 mg/kg).
  • the second gavage with normal saline (10 mL/kg) or 20(S)-protopanaxadiol (100 mg/kg) was performed 50 minutes after the first nucleus accumbens injection.
  • Oral administration of 20(S)-protopanaxadiol in morphine-dependent mice could significantly inhibit the expression of CPP.
  • Minocycline injection into the nucleus accumbens had no effect on CPP expression, but was able to re-emerge mouse CPP expression suppressed by 20(S)-protopanaxadiol ( Figure 18C).
  • mice in each group were also given the first injection into the nucleus accumbens, respectively, and the second intragastric administration 30 minutes later: normal saline (1 ⁇ L) + normal saline (10 mL/kg), Dex 21-mesylate (10nmol, 1 ⁇ L) + saline (10mL/kg), saline (1 ⁇ L) + 20(S)-protopanaxadiol (100mg/kg) and Dex21-mesylate (10nmol, 1 ⁇ L) + 20( S)-protopanaxadiol (100 mg/kg), CPP score test after 50 minutes.
  • Dex 21-mesylate injection into the nucleus accumbens had no effect on CPP expression, but completely reversed 20(S)-protopanaxadiol against morphine-induced CPP expression (Figure 18D).
  • the present invention provides that 20(S)-protopanaxadiol has significant analgesic effect in rat/mouse models of neuropathic pain, cancer pain, inflammatory pain and formalin pain; 20(S)-protopanaxadiol has a significant analgesic effect Long-term administration of panaxadiol does not produce analgesic tolerance, physical dependence and mental dependence (expressed as conditioned place preference and behavioral sensitization); 20(S)-protopanaxadiol effectively inhibits morphine-induced analgesia Pain tolerance, physical dependence, and mental dependence.
  • the inventor's research shows that the main site of analgesia of 20(S)-protopanaxadiol is in the spinal cord, and the main site of anti-addiction effect is in the nucleus accumbens.
  • 20(S)-protopanaxadiol is extremely potent in promoting dynorphin A expression and release by activating spinal cord or nucleus accumbens microglia glucocorticoid receptors (probably cell membrane glucocorticoid receptors)
  • Unexpectedly produce analgesic effects and withdrawal effects such as physical and mental dependence of opioids (or other addictive substances).

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Abstract

原人参二醇类化合物(尤其是20(S)-原人参二醇)及其药学上可接受的盐或酯，在制备a)治疗和/或缓解疼痛的药物；b)治疗和/或缓解成瘾物质尤其是阿片类药物诱导的躯体和/或精神依赖的药物中的用途。实验表明，20(S)-原人参二醇可通过激动脊髓或伏隔核小胶质细胞糖皮质激素受体刺激强啡肽A表达和释放，从而达到治疗和/或缓解疼痛以及抗成瘾物质诱导的躯体和/或精神依赖作用的效果，并可以与其他镇痛药物尤其是加巴喷丁类和阿片类药物联用达到协同镇痛的作用。

Description

原人参二醇类化合物在治疗疼痛和成瘾物质躯体依赖、精神依赖和成瘾的用途 技术领域
本发明涉及医药技术领域，具体涉及原人参二醇类化合物在治疗疼痛和成瘾物质躯体、精神依赖和成瘾的用途。
背景技术
疼痛，是一种与伤害及痛苦相关联的令人厌恶的复合感受。在正常生理条件下，疼痛能够在躯体收到威胁时提供报警信号，是一种不可或缺的生命保护功能。但在病理条件下，疼痛是大多数疾病具有的共同症状，与自主神经活动、运动反射、心理和情绪反应等交织在一起，给患者带来痛苦。
根据起因、性质、部位和时程的不同，疼痛分为急性疼痛和慢性疼痛。急性疼痛是指生理状态下，伤害性刺激直接激活相应部位的伤害性感受器而引起的疼痛。急性疼痛持续时间不长(<1月)，在损伤修复后，疼痛则自行消失。急性疼痛包括术后疼痛、创伤后疼痛、急性头疼和面部疼痛、急性关节炎疼痛等。
慢性疼痛则是在病灶修复后，疼痛依然持续存在，可长达数月(>1月)甚至终生，或可经常复发。慢性疼痛包括下背部疼痛、癌症疼痛、抗肿瘤药物和阿片类药物引起的疼痛、糖尿病性疼痛、神经病理性疼痛包括带状疱疹后遗神经痛、三叉神经痛和坐骨神经痛、炎性疼痛、幻肢痛、关节炎疼痛、纤维肌痛、肌肉骨骼疼痛、慢性区域性疼痛综合征、创伤后神经痛和周围神经病等。
根据国际疼痛研究学会(International Association for the Study of Pain,IASP)数据，全世界慢性疼痛的患病率为10％(Scholz et al.,Pain,160:53-59,2019)。仅在西欧，据报道就有8.0％的人口患有慢性疼痛。在中国，约有20％的糖尿病患者被诊断为糖尿病周围神经病，三分之一的带状疱疹病毒患者发展为带状疱疹后遗神经痛，超过一百万的人患有癌性疼痛。疼痛给患者带来痛苦，严重影响了家庭生活或工作，也给公共卫生系统带来巨大负担。
常用治疗疼痛药物有：1)非甾体抗炎镇痛药物包括氟比洛芬酯、布洛芬、双氯芬酸钠、美洛昔康、萘普生、塞来昔布和普瑞昔布；
2)抗癫痫药物包括卡马西平、苯妥英钠和加巴喷丁类药物如加巴喷丁、普瑞巴林和米罗巴林；
3)单胺类神经递质重摄取抑制剂抗抑郁药包括阿米替林和度洛西丁；
4)局部麻醉药包括利多卡因、罗哌卡因、丙胺卡因；
5)阿片类镇痛药包括可待因、双氢可待因、吗啡、芬太尼、舒芬太尼、瑞芬太尼、哌替啶、羟考酮；
6)去甲肾上腺素α2受体激动剂如可乐定、右美托咪定；
7)MOR-NRI双靶点镇痛药如地佐辛、他喷他多、喷他佐辛和曲马多；
8)中草药包括独一味、乌头/附子及其有效成分如草乌甲素和高乌甲素，以及延胡索及其有效成分如罗通定。
但这些药物治疗疼痛或疗效有限或产生较多较重不良反应。如加巴喷丁和瑞普巴林不是特异性镇痛药，其治疗神经病理性疼痛的有效(降低疼痛阈值30％)人群低于50％。非甾体类镇痛药对头痛、牙痛、肌肉和关节痛等有一定疗效，而对外伤剧痛和内脏平滑肌绞痛几乎无效。局部麻醉药仅适用于外周神经病理性疼痛。
再如阿片类药物有多种不良反应如嗜睡、呼吸抑制、便秘等，长期服用会产生镇痛耐受、痛觉过敏、躯体依赖性和成瘾及滥用。加巴喷丁和瑞普巴林也有嗜睡等严重不良反应。反复使用阿片类药物包括吗啡和芬太尼，机体会产生镇痛耐受，必须增加剂量才能获得相同的镇痛效果。长期或反复使用阿片类药物也可导致成瘾，包括躯体依赖(身体依赖、生理依赖)和精神依赖(心理依赖)两个方面。
躯体依赖性是为避免戒断症状而反复用药，且由于耐受性剂量逐渐加大，在成瘾过程中表现为厌恶效应，起到负强化效应。精神依赖性是指依赖者的心理觅药的渴求和重复用药达到的欣快感，表现为奖赏作用，起到正强化效应，促使患者屡次复吸。目前解决阿片类药物成瘾方法和途径仍有较大局限，美沙酮、丁丙诺啡、可乐定、洛非西定等仅在一定程度上改善戒断症状，疗效十分有限，尤其对精神依赖几无疗效。
因此，疼痛治疗目前仍然是临床一大难题，急需研发可长期使用且自身无镇痛耐受和成瘾性，能有效治疗疼痛和阿片类药物诱导的躯体和精神依赖的新型镇痛药物。
发明内容
本发明的目的是提供一种可长期使用且自身无镇痛耐受和成瘾性，能有效治疗疼痛和阿片类药物诱导的躯体和精神依赖的新型镇痛药物。
具体地，本发明提供了原人参二醇类化合物(如20(S)-原人参二醇)在制备治疗疼痛和阿片类药物诱导的躯体和精神依赖的新型镇痛药物中的应用。本发明实验表明，20(S)-原人参二醇还可以和其他活性成分联用，达到协同镇痛的作用。并且，20(S)-原人参二醇通过激动脊髓小胶质细胞糖皮质激素受体(细胞膜糖皮质激素受体)表达和释放强啡肽A，从而产生镇痛作用。
在本发明的第一方面，提供了一种活性成分或含所述活性成分的制剂的用途，所述活性成分选自下组：原人参二醇或其药学上可接受的盐或酯、原人参三醇或其药学上可接受的盐或酯；
并且，所述活性成分或含所述活性成分的制剂用于制备：
(a)治疗和/或缓解疼痛的药物；和/或
(b)治疗和/或缓解成瘾物质诱导的躯体和/或精神依赖和/或成瘾的药物；和/或
(c)预防、抑制和/或逆转镇痛药物诱导的镇痛耐受作用的药物。
在另一优选例中，所述的原人参二醇包括20(S)-原人参二醇、20(R)-原人参二醇、或其组合。
在另一优选例中，所述的原人参三醇包括20(S)-原人参三醇、20(R)-原人参三醇、或其组合。
在另一优选例中，所述成瘾物质选自：阿片类药物、海洛因、或其组合。
在另一优选例中，所述成瘾物质还包括选自下组的一种或多种：冰毒、酒精、香烟(尼古丁)、可卡因、大麻、或其组合。
在另一优选例中，所述疼痛选自：神经病理性疼痛、炎症性疼痛、关节炎疼痛、糖尿病性疼痛、下背部疼痛、脊髓损伤性疼痛、内脏疼痛、纤维肌痛、慢性局部疼痛综合征、肌肉骨骼疼痛、癌症疼痛、抗肿瘤药物和阿片类药物引起的疼痛、手术后疼痛、创伤后疼痛、创伤后神经痛和周围神经病、幻肢痛、或其组合。
在另一优选例中，所述神经病理性疼痛包括(但不限于)带状疱疹后遗神经痛、三叉神经痛和坐骨神经痛。
在另一优选例中，所述镇痛药物为所述活性成分之外的镇痛药物，较佳地，所述镇痛药为：选自下组的阿片类镇痛药：可待因、双氢可待因、吗啡、芬太尼、舒芬太尼、瑞芬太尼、哌替啶、羟考酮或其组合。
在另一优选例中，所述制剂还包括第二活性成分；其中第二活性成分选自下组：
(Z1)选自下组的阿片类镇痛药：可待因、双氢可待因、吗啡、芬太尼、舒芬太尼、瑞芬太尼、哌替啶、羟考酮或其组合；
(Z2)选自下组的抗癫痫药物：卡马西平、苯妥英钠、加巴喷丁类(gabapentinoids)化合物、或其组合；
(Z3)选自下组的非甾体抗炎镇痛药物：氟比洛芬酯、布洛芬、双氯芬酸钠、美洛昔康、萘普生、塞来昔布、普瑞昔布或其组合；
(Z4)选自下组的单胺类神经递质重摄取抑制剂抗抑郁药：阿米替林、度洛西丁、或其组合；
(Z5)选自下组的局部麻醉药：利多卡因、罗哌卡因、丙胺卡因、或其组合；
(Z6)选自下组的去甲肾上腺素α2受体激动剂：可乐定、右美托咪定、或其组合；
(Z7)选自下组的MOR-NRI双靶点镇痛药：地佐辛、他喷他多、喷他佐辛、曲马多、或其组合；
(Z8)选自下组的抗偏头痛药：CGRP抗体及其受体拮抗剂；
(Z9)选自下组的中草药：独一味提取物及其有效成分、乌头/附子及其有效成分、以及延胡索及其有效成分、或其组合；
(Z10)上述Z1～Z9的任意组合。
在另一优选例中，所述加巴喷丁类化合物包括巴喷丁、普瑞巴林和米罗巴林(mirogabalin)。
在另一优选例中，所述独一味提取物及其有效成分包括山栀子苷甲酯、8-O-乙酰山栀子苷甲酯。
在另一优选例中，所述乌头/附子及其有效成分包括：草乌甲素、高乌甲素和雪上一支蒿甲素。
在另一优选例中，所述延胡索及其有效成分包括四氢巴马汀、千金藤啶碱、紫堇达明和脱氢紫堇鳞茎碱。
在另一优选例中，所述活性成分与第二活性成分的重量比为1：0.001-100，较佳地，1:0.05-10，更佳地1:0.1-5。
在另一优选例中，所述制剂为口服制剂、或注射剂。
在另一优选例中，所述制剂包括：粉剂、颗粒剂、胶囊剂、针剂、酊剂、口服液、片剂、含片、或滴丸。
在另一优选例中，所述活性成分或含所述活性成分的制剂不具有(不产生)：(1)镇痛耐受性；(2)躯体依赖性；(3)精神依赖性(成瘾性)。
在本发明的第二方面，提供了一种药物组合物，所述药物组合物包含：
(i)第一活性成分，所述第一活性成分选自下组：原人参二醇、原人参三醇；
(ii)第二活性成分，所述第二活性成分选自：
(Z1)选自下组的阿片类镇痛药：可待因、双氢可待因、吗啡、芬太尼、舒芬太尼、瑞芬太尼、哌替啶、羟考酮或其组合；
(Z2)选自下组的抗癫痫药物：卡马西平、苯妥英钠、加巴喷丁类化合物、或其组合；
(Z3)选自下组的非甾体抗炎镇痛药物：氟比洛芬酯、布洛芬、双氯芬酸钠、美洛昔康、萘普生、塞来昔布、普瑞昔布或其组合；
(Z4)选自下组的单胺类神经递质重摄取抑制剂抗抑郁药：阿米替林、度洛西丁、或其组合；
(Z5)选自下组的局部麻醉药：利多卡因、罗哌卡因、丙胺卡因、或其组合；
(Z6)选自下组的去甲肾上腺素α2受体激动剂：可乐定、右美托咪定、或其组合；
(Z7)选自下组的MOR-NRI双靶点镇痛药：地佐辛、他喷他多、喷他佐辛、曲马多、或其组合；
(Z8)选自下组的抗偏头痛药：CGRP抗体及其受体拮抗剂；
(Z9)选自下组的中草药：独一味提取物及其有效成分、乌头/附子及其有效成分、以及延胡索及其有效成分、或其组合；
(Z10)上述Z1～Z9的任意组合；
(iii)药学上可接受的载体和/或赋形剂。
在另一优选例中，所述第一活性成分与第二活性成分的重量比为1：0.001-100，较佳地，1:0.05-10，更佳地1:0.1-5。
在另一优选例中，所述第一活性成分为原人参二醇，第二活性成分为选自下组的阿片类镇痛药：可待因、双氢可待因、吗啡、芬太尼、舒芬太尼、瑞芬太尼、哌替啶、羟考酮或其组合。
在另一优选例中，所述第一活性成分为原人参二醇，第二活性成分为吗啡。
在另一优选例中，所述药物组合物给药方式是口服的或非口服的。
在另一优选例中，所述非口服的给药方式选自下组：鼻饲、肛门栓塞、皮下注射、肌肉注射、静脉注射、蛛网膜下腔注射、硬膜外注射，侧脑室注射、皮肤外敷(贴剂)、或其组合。
在本发明的第三方面，提供了一种第二方面所述的药物组合物的用途，所述药物组合物用于制备：
(a)治疗和/或缓解疼痛的药物；
(b)治疗和/或缓解成瘾物质诱导的躯体和/或精神依赖的药物。
在另一优选例中，所述第二活性成分为具有成瘾性的镇痛药物，所述药物组合物用于制备：用于治疗和/或缓解疼痛，且降低所述第二活性成分诱导的躯体和/或精神依赖的药物。
在另一优选例中，所述第二活性成分为具有成瘾性的镇痛药物，所述药物组合物制备：用于治疗和/或缓解疼痛，且降低所述第二活性成分诱导的镇痛耐受作用。
在另一优选例中，具有成瘾性的镇痛药物为选自下组的阿片类镇痛药：可待因、双氢可待因、吗啡、芬太尼、舒芬太尼、瑞芬太尼、哌替啶、羟考酮或其组合。
在本发明的第四方面，提供了一种体外的治疗和/或缓解疼痛的方法，包括步骤：
(1)在含有有效量的第一活性成分或含所述活性成分的制剂或如第二方面所述的药物组合物的体系中培养细胞，其中所述第一活性成分选自下组：原人参二醇或其药学上可接受的盐或酯、原人参三醇或其药学上可接受的盐或酯；
(2)激动所述细胞糖皮质激素受体表达强啡肽A。
在另一优选例中，所述细胞为中枢神经系统的免疫细胞，优选地脊髓免疫细胞。
在另一优选例中，所述细胞选自：小胶质细胞、巨噬细胞、单核细胞、或其组合。
在另一优选例中，所述细胞为脊髓小胶质细胞。
在另一优选例中，所述糖皮质激素为糖皮质激素受体激动剂。
在另一优选例中，所述糖皮质激素为细胞膜糖皮质激素受体激动剂。
在另一优选例中，所述方法为非诊断性非治疗性的。
在本发明的第五方面，提供了一种治疗和/或缓解疼痛的方法，包括步骤：
给有需要的对象施用医学有效量的第一活性成分或含所述活性成分的制剂或第 二方面所述的药物组合物，其中所述第一活性成分选自下组：原人参二醇或其药学上可接受的盐或酯、原人参三醇或其药学上可接受的盐或酯；从而治疗和/或缓解疼痛。
在本发明的第六方面，提供了一种诱导产生强啡肽A表达和释放的方法，包括：给需要的对象施用活性成分，所述活性成分选自下组：原人参二醇或其药学上可接受的盐或酯、原人参三醇或其药学上可接受的盐或酯，从而诱导所述对象产生强啡肽A。
在另一优选例中，所述的方法刺激脊髓增加强啡肽A的表达和释放。
在另一优选例中，所述方法用于激动所述对象细胞的糖皮质激素受体。
在另一优选例中，所述糖皮质激素受体为细胞膜糖皮质激素受体。
在另一优选例中，所述对象为哺乳动物。
在另一优选例中，所述对象包括但不限于老鼠、人。
在另一优选例中，所述对象为疼痛患者。
在另一优选例中，所述细胞为中枢神经系统的免疫细胞，优选地脊髓免疫细胞。
在另一优选例中，所述细胞选自：小胶质细胞、巨噬细胞、单核细胞、或其组合。
在另一优选例中，所述细胞为脊髓小胶质细胞。
在另一优选例中，所述方法为非诊断性和非治疗性的。
本发明第七方面，提供了一种治疗和/或缓解成瘾物质诱导的躯体和/或精神依赖和/或成瘾的方法，包括步骤：给予给有需要的对象有效量的第一活性成分或含所述第一活性成分的的药物组合物，从而治疗和/或缓解成瘾物质诱导的躯体和/或精神依赖和/或成瘾；
其中所述第一活性成分选自下组：原人参二醇或其药学上可接受的盐或酯、原人参三醇或其药学上可接受的盐或酯。
在另一优选例中，所述成瘾物质选自下组的一种或多种：阿片类药物、海洛因、冰毒、酒精、香烟(尼古丁)、可卡因、大麻、或其组合。
在另一优选例中，所述对象是哺乳动物，如人，大鼠或小鼠。
本发明第八方面，提供了一种降低镇痛药物的成瘾性和/或镇痛耐受作用的方法，包括步骤：将有效量的成瘾性镇痛药物和第一活性成分或含所述第一活性成分的的药物组合物联合给予有需要的对象，从而降低所述镇痛药物的成瘾性和/或镇痛耐受作用；
其中所述第一活性成分选自下组：原人参二醇或其药学上可接受的盐或酯、原人参三醇或其药学上可接受的盐或酯。
在另一优选例中，所述成瘾性指所述镇痛药物诱导的躯体和/或精神依赖。
在另一优选例中，所述成瘾性镇痛药物为选自下组的阿片类镇痛药：可待因、双氢可待因、吗啡、芬太尼、舒芬太尼、瑞芬太尼、哌替啶、羟考酮或其组合。
在另一优选例中，所述第一活性成分与成瘾性镇痛药物的重量比为1：0.001-100， 较佳地，1:0.05-10，更佳地1:0.1-5。
在另一优选例中，所述第一活性成分或含所述第一活性成分的的药物组合物在所述成瘾性镇痛药物之前(如24h内)、同时或之后(如24h内)给药。
在另一优选例中，所述对象是哺乳动物，如人，大鼠或小鼠。
在另一优选例中，所述方法还用于降低所述成瘾性药物的镇痛耐受作用。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1表明口服20(S)-原人参二醇呈剂量依赖式地抑制神经病理性疼痛的机械痛敏和热痛敏。
图2显示口服20(S)-原人参二醇在骨癌疼痛、完全弗氏佐剂(CFA)炎症性疼痛和福尔马林疼痛模型中镇痛作用。
图3显示口服20(S)-原人参二醇联合加巴喷丁或吗啡在神经病理性疼痛模型中协同镇痛作用。
图4显示口服20(S)-原人参二醇特异性地刺激大鼠脊髓表达强啡肽A基因和蛋白质。
图5免疫荧光双染色显示口服20(S)-原人参二醇特异性地刺激大鼠脊髓小胶质细胞表达强啡肽A。
图6显示离体给予20(S)-原人参二醇特异性地刺激原代脊髓小胶质细胞表达强啡肽A基因和蛋白。
图7显示小胶质细胞活化抑制剂米诺环素阻断20(S)-原人参二醇抗神经病理性疼痛。
图8显示强啡肽A抗血清和特异性κ-阿片受体拮抗剂阻断20(S)-原人参二醇抗神经病理性疼痛。
图9显示口服20(S)-原人参二醇不产生自身镇痛耐受性或吗啡镇痛交叉耐受性，但可预防或抑制吗啡镇痛耐受作用。
图10显示口服20(S)-原人参二醇不产生躯体依赖性，但抑制吗啡躯体依赖作用。
图11显示口服20(S)-原人参二醇逆转吗啡镇痛耐受作用。
图12显示口服20(S)-原人参二醇不产生精神依赖性，但抑制吗啡精神依赖作用。图13显示口服20(S)-原人参二醇抑制吗啡行为敏化作用。
图14显示蛛网膜下腔鞘内预先给予糖皮质激素受体拮抗剂抑制口服20(S)-原人参二醇的镇痛作用。
图15显示蛛网膜下腔鞘内预先给予糖皮质激素受体拮抗剂抑制口服20(S)-原人参二醇在脊髓所产生的强啡肽A表达作用。
图16显示糖皮质激素受体拮抗剂抑制20(S)-原人参二醇在原代脊髓小胶质细胞所产生的强啡肽A表达作用。
图17显示伏隔核给予dynorphin A抗血清、κ-阿片受体拮抗剂、小胶质细胞抑制剂和糖皮质激素受体拮抗剂抑制20(S)-原人参二醇抗吗啡躯体依赖作用。
图18显示伏隔核给予dynorphin A抗血清、κ-阿片受体拮抗剂、小胶质细胞抑制剂和糖皮质激素受体拮抗剂抑制20(S)-原人参二醇抗吗啡诱导的CPP表达作用。
具体实施方式
本发明人经过广泛而深入地研究，首次意外发现，原人参二醇类化合物(尤其是20(S)-原人参二醇)可显著地抑制疼痛，并具有明显的抗成瘾作用，并且不产生镇痛耐受性、躯体依赖性、精神依赖性等不良反应。因此可用于治疗疼痛和抗成瘾(如成瘾物质诱导的躯体和精神依赖)。在此基础上完成本发明。
具体地，实施例表明，活性成分20(S)-原人参二醇的镇痛作用不产生自身镇痛耐受性，并且能在镇痛同时帮助抑制成瘾物质诱导的躯体依赖、精神依赖作用。原人参二醇类化合物还可以合其他镇痛药物联用达到协同镇痛的作用。原人参二醇类化合物通过特异性刺激强啡肽A的表达，从而达到镇痛的作用。
术语
除非另有定义，否则本文中所用的全部技术术语和科学术语均具有如本发明所属领域普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
在本发明中，术语“躯体依赖(physical dependence)”、“身体依赖”和“生理依赖”可互换使用，指一旦中断使用成瘾药物，可引发戒断综合征的依赖性。
在本发明中，术语“戒断综合征”指由于连续使用成瘾物质，从而使产生依赖性的患者一旦中断使用，身体所产生的剧烈生理反应而出现的一系列症状，如出汗、流泪、打哈欠、寒战、起鸡皮疙瘩、瞳孔散大、呕吐、腹泻、腹痛、心律增加、血压上升、失眠、震颤等综合症状。
在本发明中，术语“精神依赖(psychological dependence)”与“心理依赖”可互换使用，是指患者对药物的渴求，以期获得服用成瘾药物后的特殊快感。
通过“治疗(treating)”或“治疗(treatment)”和/或“预防(preventing)”或“预防(prevention)”，作为一个整体，是指至少抑制或改善影响个体的相关症状，其中，抑 制和改善使用其广义，指至少降低参数的量级，所述参数如所治疗的病症相关的症状，如疼痛。因此，本发明的方法包括预防和处理各种不同的疼痛。
疼痛
在本发明提供将本发明活性成分或其制剂用于治疗疼痛的应用。
在本发明中，疼痛没有特别限制，代表性的例子包括(但并不限于)偏头痛、背痛、颈痛、妇科疼痛、分娩前或分娩疼痛、矫形疼痛、中风后疼痛、外科手术后或操作性疼痛、疱疹后神经痛、镰状细胞危象、间质性膀胱炎、泌尿疼痛(如尿道炎)、牙齿疼痛、头痛、伤口或如外科手术(如囊炎切除术或臀部、膝盖或其他关节置换术)的医疗操作产生的疼痛、缝合、骨折复位、活检等。疼痛还可出现在患有癌症的患者中，这可能由多方面原因造成，如炎症、神经压迫和因肿瘤入侵和肿瘤转移到骨骼或其他组织而产生的组织肿胀所造成的机械力。
在另一优选例中，所述的疼痛包括(但并不限于)：周围神经性疼痛、中枢神经性疼痛、异常性疼痛、灼性神经痛、痛觉过敏、感觉过敏、痛觉过度、神经痛、神经炎和神经病变。
成瘾
药物成瘾和药物依赖是一种慢性复发性脑部疾病，主要表现为对成瘾药物的强迫用药行为和药量的不可控性。出现物质依赖状况后，若突然停止服用药物，可能出现药物戒断症状。许多原本用于医学用途的药品，可能造成物质依赖现象；成瘾物质若是被法律管制，视为非法者，则被称为毒品。这些成瘾物质包括阿片类药物和海洛因、冰毒(甲基苯丙胺)、可卡因、大麻、酒精和尼古丁等。
甲基苯丙胺，俗称冰毒，是一种高度成瘾的兴奋剂，是全球最常用的第二大非法药物。滥用甲基苯丙胺或其他安非他明类兴奋剂已成为公共卫生的重要问题。相对于海洛因、可卡因等传统毒品而言，甲基苯丙胺合成工艺简单、前体廉价易得，对中枢神经系统的兴奋作用更强，形成成瘾所需用药次数或累计用药量更少，对吸毒者身体造成的损害更为严重。
酒精是一种具有高度成瘾特性的精神活性物质。全球酒精依赖患者达到1.4亿，其滥用和依赖给个人和社会带来了严重的不良影响和经济负担。全世界每年约330万人因过度使用酒精导致死亡。酒精的有害使用还会导致酒精肝，肝硬化等疾病。酒精滥用，酒精成瘾已经成为严峻的公共卫生灾难和危害人类健康的世界性难题，是仅次于心血管疾病和肿瘤之后，位居第三位的全球公共卫生问题。
尼古丁，又名烟碱，是一种强效拟副交感神经生物碱，是香烟内的主要有效成分。尼古丁依赖是吸烟者的主要特征，它指个体在反复使用尼古丁后导致生理和心理变化，包括使用渴求增强且难以控制，不计危害后果而持续且优先地使用，耐受性增 加且出现戒断症状。烟草依赖是目前非常严重的公共卫生问题之一。WHO指出，烟草每年使700多万人失去生命，其中有600多万人源于直接使用烟草，约89万人是接触二手烟的非吸烟者。
反复使用阿片类药物包括吗啡和芬太尼，机体会产生镇痛耐受，必须增加剂量才能获得相同的镇痛效果。长期或反复使用阿片类药物也可导致成瘾，包括躯体依赖(生理依赖)和精神依赖(心理依赖)两个方面。躯体依赖性是为避免戒断症状而反复用药，且由于耐受性剂量逐渐加大，在成瘾过程中表现为厌恶效应，起到负强化效应。精神依赖性是指依赖者的心理觅药的渴求和重复用药达到的欣快感，表现为奖赏作用，起到正强化效应，促使患者屡次复吸。
活性成分
如本文多用，“本发明的活性成分”、“本发明的活性化合物”可互换使用，均指原人参二醇类化合物，包括原人参二醇、原人参三醇。其中，原人参二醇包括20(S)-原人参二醇、20(R)-原人参二醇、或其组合(如外消旋物)。所述的原人参三醇包括20(S)-原人参三醇、20(R)-原人参三醇、或其组合(如外消旋物)。此外，所述术语包括天然产物或人工合成或修饰的产物。
应理解，本发明的活性成分包括本发明的活性化合物(原人参二醇、原人参三醇、或其组合)、或其药学上可接受的盐或酯、对映异构体、非对映异构体或外消旋体、或其前药。应理解，本发明的活性成分还包括本发明的活性化合物的晶型、无定形化合物、溶剂化物、水合物等形式。
所述“药学上可接受的盐(或酯)”为本发明的活性化合物与无机酸或有机酸反应形成常规的无毒盐(或酯)。例如，常规的无毒盐可通过本发明的活性化合物与无机酸或有机酸反应制得，所述无机酸包括盐酸、氢溴酸、硫酸、硝酸、胺基磺酸和磷酸等，所述有机酸包括柠檬酸、酒石酸、乳酸、丙酮酸、乙酸、苯磺酸、对甲苯磺酸、甲磺酸、萘磺酸、乙磺酸、萘二磺酸、马来酸、苹果酸、丙二酸、富马酸、琥珀酸、丙酸、草酸、三氟乙酸、硬酯酸、扑酸、羟基马来酸、苯乙酸、苯甲酸、水杨酸、谷氨酸、抗坏血酸、对胺基苯磺酸、2-乙酰氧基苯甲酸和羟乙磺酸等；或者本发明的活性化合物与丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、天冬氨酸或谷氨酸形成酯后再与无机碱形成的钠盐、钾盐、钙盐、铝盐或铵盐；或者本发明的活性化合物与赖氨酸、精氨酸、鸟氨酸形成酯后再与盐酸、氢溴酸、氢氟酸、硫酸、硝酸或磷酸形成的对应的无机酸盐或与甲酸、乙酸、苦味酸、甲磺酸或乙磺酸形成的对应的有机酸盐。
其他镇痛类药物
1)阿片类镇痛药包括可待因、双氢可待因、吗啡、芬太尼、舒芬太尼、瑞芬太尼、 哌替啶、羟考酮；
2)抗癫痫药物包括卡马西平、苯妥英钠和加巴喷丁类药物如加巴喷丁、普瑞巴林和米罗巴林；
3)非甾体抗炎镇痛药物包括氟比洛芬酯、布洛芬、双氯芬酸钠、美洛昔康、萘普生、塞来昔布和普瑞昔布；
4)单胺类神经递质重摄取抑制剂抗抑郁药包括阿米替林和度洛西丁；
5)局部麻醉药包括利多卡因、罗哌卡因、丙胺卡因；
6)去甲肾上腺素α2受体激动剂如可乐定和右美托咪定；
7)MOR-NRI双靶点镇痛药如地佐辛、他喷他多、喷他佐辛和曲马多；
8)抗偏头痛药如CGRP抗体及其受体拮抗剂；
9)中草药包括独一味提取物及其有效成分如山栀子苷甲酯和8-O-乙酰山栀子苷甲酯、乌头/附子及其有效成分如草乌甲素、高乌甲素和雪上一支蒿甲素，以及延胡索及其有效成分如四氢巴马汀、千金藤啶碱、紫堇达明和脱氢紫堇鳞茎碱。
药物组合物及施用方法
本发明还提供了含有本发明活性成分的组合物或制剂或产品，所述组合物或制剂或产品可用于抗衰老。代表性的组合物或制剂或产品包括抗衰老的药物、保健品、和化妆品。
一种优选的组合物是药物组合物，它含有有效量的维拉帕米或其药学上可接受的盐和药学上可接受的载体。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性(如镇痛功能)的且可被人和/或动物所接受的量。
如本文所用，术语“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
本发明的药物组合物含有安全有效量的本发明的活性成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物的剂型为注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、缓释剂。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。
本发明所述的活性成分的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本发明的活性成分每天以约0.001-100mg/kg动 物体重(较佳的0.01-50mg/kg，更佳地0.05-20mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
典型地，当以口服方式给予本发明的活性成分，优选地，施用对象为人时，口服剂量可以为0.05-50mg/kg，较佳地0.10-20mg/kg。
本发明中，第一活性成分和第二活性成分可以以单一制剂制备，或者分别制成制剂。第一活性成分和第二活性成分可以同时给药或顺序给药。
本发明的主要优点包括：
a)本发明的活性成分在有效镇痛的同时，不产生镇痛耐受性、躯体依赖性、精神依赖性和成瘾。
b)本发明的活性成分有效镇痛的同时，可以治疗或缓解阿片类药物诱导的成瘾包括躯体以及精神依赖性。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook等，分子克隆：实验室手册，New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实验通用方法
1大鼠鞘内置管：
大鼠在呼吸麻醉机下用5％异氟烷快速麻醉(麻醉机气流速度为0.3L/min)，随后由2％异氟烷维持麻醉。将18-cm的聚乙烯导管(PE-10：外径：0.55mm,内径：0.3mm)从大鼠腰部水平处沿脊柱处插入。在进行药物治疗前一周，用10μL 4％利多卡因检测插管情况，蛛网膜下腔鞘内注射若大鼠出现双侧后足瘫软，且恢复后无运动障碍，说明插管成功可进行后续实验。
2大鼠福尔马林疼痛模型：
实验前将大鼠在尺寸为23×35×19cm的透明观察笼中适应30分钟。取出大鼠左侧足部皮下注射5％的福尔马林溶液(50μl)，注射后大鼠立刻放入观察笼，在注射后每隔10分钟测定60秒内的抬脚次数，直到90分钟截止。
3大鼠脊神经结扎神经病理性疼痛模型：
大鼠在呼吸麻醉机下用5％异氟烷快速麻醉(麻醉机气流速度为0.3L/min)，随后由2％异氟烷维持麻醉。在腰脊髓部钝性分离左侧肌肉，暴露和去除L6横突，暴露L5神经并用6-0丝线扎紧；去除骶骨夹角下方附近筋膜，挑出L6神经并用6-0丝线扎紧。术后大 鼠单笼饲养恢复一周。用Von Frey电子测痛仪测定后足底机械痛阈值，＜8g且无运动障碍视为造模成功，用于后续实验。
4大鼠骨癌疼痛模型：
雌性大鼠按50mg/kg腹腔注射戊巴比妥钠进行麻醉，在大鼠胫骨处开一0.5-cm切口，钝性分离肌肉后暴露胫骨，使用5号针头在胫骨中间轻轻钻孔。随后微量注射器注射10μL Walker 256乳腺癌细胞(4×10 5个/mL)，注射完毕后停针30秒，拔出针头立刻用无菌骨蜡封住伤口，随后消毒、缝合和放入笼中。两周后测定足机械痛阈值，＜8g大鼠视为造模成功，用于后续实验。
5大鼠炎性疼痛模型：
异氟烷麻醉动物，在左后爪的胫跗骨关节中缓慢注入100μL CFA。CFA注射两天后测定机械痛和热痛阈值。
6大鼠机械痛和热痛阈值测定：
将大鼠分别置于机械痛和热痛检测架上。对于大鼠机械痛测定，用电子机械痛阈检测仪垂直刺激大鼠后肢足底中部，检测仪安装了15号纤维，测量时，缓慢增加刺激力度，直至纤维弯曲成S形，持续6-8秒，观察大鼠是否出现缩足或抬脚反应。记录大鼠缩足或抬脚的最小阈值作为足收缩阈值(paw withdrawal threshold,PWT)。每隔3分钟检测一次，重复三次，三次的平均值作为大鼠该足的机械疼痛阈值。机械疼痛阈值反映大鼠机械刺激伤害/疼痛程度。对于大鼠热痛测定，打开垂直放置于大鼠后肢脚掌处的玻璃板下方的辐射热源进行检测，观察大鼠足底开始接受辐射热源到突然舔足，抬足或缩足的总时长为其受辐射热刺激后痛觉阈值，用缩足反应潜伏期(paw withdrawal latency,PWL)表示。以30秒为最大测量阈值，每隔5分钟检测一次，重复三次，三次的平均值作为大鼠该足辐射热痛阈值。辐射热疼痛阈值反映大鼠热刺激伤害/疼痛程度。
7小鼠条件位置偏好(conditioned place preference,CPP)模型建立：
为期10天的CPP模型包括三个阶段:前测试期，获得期和后测试期。前测试期(1-4天)：允许雄性Swiss小鼠在三个隔室自由穿梭，每天2次，每次15分钟，共3天。在第4天，记录小鼠自由穿梭于三个隔室15分钟内每个隔室的停留时间。获得期(5-9天)：小鼠每隔6小时(上午9:00和下午3:00)进行皮下交替注射5天吗啡(10mg/kg)或生理盐水(10mL/kg)，接着立刻放入隔室内，训练45分钟。在获得期的第5、7和9天，分别上午9:00皮下注射吗啡(10mg/kg)和下午3:00皮下注射生理盐水(10mL/kg)，并放入吗啡伴药箱中和生理盐水配对箱中训练45分钟。在第6和8天，交换吗啡和生理盐水的注射时间。后测试期(第10天)：允许小鼠自由进出三个隔室15分钟进行测试。条件位置偏好得分由小鼠在伴药箱中停留的时间减去在生理盐水配对箱中所花费的时间。本实验由3CCD相机拍摄小鼠在每个隔室的穿梭活动，并使用EthoVision XT 8.0软件记录小鼠在每个隔室停留的时间。
8小鼠吗啡行为敏化模型
行为敏化是指重复服用阿片类药物所导致的某些行为效应的增强，与强迫性觅药和复吸有关。建立小鼠行为敏化方法简述如下：将小鼠置于视频跟踪系统下的运动检测室中(40×40×35cm)，使用ANY-maze软件自动分析数据。吗啡诱发的行为敏化过程包括适应性阶段(1-3天)和形成期(4-8天)。在适应期(1-3天)，所有小鼠皮下注射生理盐水，立即放置于检测室中自由活动，每天1小时，连续3天记录小鼠的运动距离。
9原代细胞培养：
出生24小时内任一性别的Wistar大鼠分离获得脊髓，剥膜剪碎后加入0.05％胰酶在5％二氧化碳培养箱中消化7-9分钟。离心去上清，将离心管内底部消化后的组织用10％FBS、1％双抗的DMEM培养基吹散重悬，依次70-和40-μm筛网过滤，接种至多聚赖氨酸(0.1mg/ml)包被的75cm 2培养瓶中，放入5％二氧化碳培养箱培养10天。
制备小胶质细胞时，培养瓶放入摇床在37℃摇荡(260rpm)1.5-2小时，收集细胞悬浮液、离心、重悬细胞，后接种于新的细胞培养板中，次日用预温的PBS洗去未黏附细胞。通过小胶质细胞标记蛋白Iba-1的免疫荧光测定，获得的小胶质细胞纯度大于95％。
制备星形胶质细胞时，培养的细胞弃去培养基，经PBS洗两遍后加入0.05％含EDTA的胰酶。在37℃条件下消化3分钟除去少突胶质细胞，终止消化并去除细胞悬浮液，剩下的贴壁单层星形胶质细胞继续用胰酶消化传代以供后续使用。通过星形胶质细胞标记蛋白GFAP的免疫荧光测定，获得的星形胶质细胞纯度大于90％。
制备神经元细胞时，细胞悬浮液经40-μm筛网过滤后，接种于10-cm的细胞培养皿中，放入细胞培养箱培养30分钟。然后吸取未贴壁的上层细胞悬液，将其接种于多聚赖氨酸培养板中。培养1.5-2小时后，将DMEM更换成含1×B27神经营养因子及0.5mM谷氨酰胺的Neurobasal培养基，继续培养3-4天。通过神经元细胞标记蛋白NeuN的免疫荧光测定，获得的神经元细胞纯度大于85％。
10细胞及组织总RNA提取及实时定量PCR测定：
戊巴比妥钠(50mg/kg，i.p.)麻醉大鼠用，迅速断头放血，取出脊髓腰膨大(L3-L5)组织，按比例加入Trizol试剂(50mg/ml)匀浆以萃取、沉淀RNA，最后根据RNA沉淀量加入适量DEPC水使其溶解。采用微量酶标仪测定提取RNA的浓度及纯度。
采用逆转录试剂盒在普通PCR仪上运行相应的程序，将提取的总RNA逆转录成cDNA后-20℃保存备用。后续的实时定量PCR操作采用SYBR qPCR mix，检测强啡肽前体基因(PDYN),内啡肽前体基因(POMC),脑啡肽前体基因(PNOC),和Nociceptin/OrphaninFQ前体基因(PENK)Ct值，以GAPDH作为内参基因，采用2 -ΔΔCt方法计算目标基因相对表达量。
11强啡肽A和β-内啡肽蛋白质含量测定：
大鼠取出脊髓腰膨大处(L3-L5)组织，用10mM Tris-HCl(5mL/1g组织)均浆化(4,000rpm，15秒)，并在4℃条件下离心(5000rpm)15分钟后获得的上清液。此外，新生大鼠脊髓来源的原代细胞给药处理培养2小时后收集细胞培养上清液。根据酶联免疫试剂盒说 明书，测定细胞培养和脊髓组织上清液中强啡肽A和β-内啡肽含量。
12组织免疫荧光染色：
大鼠腹腔注射戊巴比妥钠(50mg/kg)麻醉，沿胸骨剑突下缘打开胸腔，暴露和游离心脏。迅速经左心室将针头插入主动脉，用4-0号手术缝合丝线固定针头，剪开右心耳。缓慢灌注100ml生理盐水将血液冲洗后，继续灌注60ml 4％甲醛溶液。然后取出脊髓腰膨大部分(L3-L5)置于4％甲醛固定液4℃过夜，然后依次经蔗糖溶液梯度脱水、包埋、冰冻切片(厚度为30μm)和-20℃保存备用。冻存的组织切片复温后用封闭液室温封闭1小时，随后使用封闭液配制一抗(强啡肽A抗体、小胶质细胞标记Iba-1、星形胶质细胞标记GFAP和神经元细胞标记NeuN)在4℃孵育18-24小时。一抗孵育结束后，加入配制二抗的封闭液在37℃条件下培养1小时，随后用抗荧光淬灭封片剂封片，-20℃避光保存备用。使用Leica TCS SP8激光共聚焦显微镜拍摄成像，并用Image J图像处理软件进行荧光定量和荧光染色共定位分析。
实施例1 口服20(S)-原人参二醇在神经病理性疼痛模型大鼠模型中镇痛作用
方法
选取L5/L6脊神经结扎的神经病理性疼痛大鼠，随机分为六组(每组6只)。其中一组口服溶剂(乙醇：丙烯乙二醇：蒸馏水＝2:7:1比例,6.5mL/kg)，另外5组分别口服不同剂量的20(S)-原人参二醇(五组口服剂量分别为1、3、10、30或100mg/kg)。在给药前及给药后0.5、1、2、4小时不同时间点测定大鼠对机械刺激和热辐射刺激的缩足反应阈值或缩足潜伏时间(热辐射刺激在机械刺激10分钟后进行)。在1小时测得各计量组机械痛阈值和热辐射痛阈值以计算％最大可能效应(％maximal possible effect,％MPE)，然后进行剂量-反应分析。
结果
在4小时的检测时间内，生理盐水组大鼠健侧和手术患侧的机械痛阈值及热辐射痛阈值基本不变，而口服20(S)-原人参二醇可剂量依赖性地抑制大鼠手术患侧的机械痛敏(图1A和1B)和热辐射痛敏(图1C和1D)，但不影响健侧疼痛阈值。镇痛作用可持续约3小时，镇痛效果最强时间点为1小时。
由图1B和1D的剂量-反应分析结果显示，口服20(S)-原人参二醇在神经结扎引起的机械痛敏和热辐射痛敏中的最大镇痛效应分别为E max为61％和68％MPE，ED 50分别为5.8和4.5mg/kg。
综上，口服20(S)-原人参二醇可以抑制神经病理性疼痛，且这种抑制作用的程度和服用的20(S)-原人参二醇剂量呈正向依赖性。并且，口服20(S)-原人参二醇镇痛效果也非常明显。
实施例2 口服20(S)-原人参二醇在不同病因引起的疼痛大鼠模型中的镇痛作用。
方法
为验证20(S)-原人参二醇对其他不同病因疼痛模型的镇痛作用，我们采用了骨癌疼痛模型、CFA引发的炎性疼痛模型和福尔马林诱发疼痛模型。
两组骨癌疼痛大鼠(每组6只)和两组CFA引发的炎性疼痛大鼠(每组6只)分别口服溶剂(6.5mL/kg)或20(S)-原人参二醇(100mg/kg)，在给药前及给药后0.5、1、2、4小时不同时间点测定大鼠对机械刺激和热辐射刺激的缩足反应阈值或缩足潜伏时间(热辐射刺激在机械刺激10分钟后进行)。
此外，两组大鼠(每组6只)在足底注射5％福尔马林前30分钟口服溶剂(6.5mL/kg)或20(S)-原人参二醇(100mg/kg)。
结果
口服20(S)-原人参二醇可以显著缓解大鼠骨癌疼痛(图2A和2B)和CFA引发的炎性疼痛(图2C和2D)。
福尔马林可以引起大鼠I相和II相舔足反应。口服20(S)-原人参二醇可以抑制大鼠福尔马林引起的II相舔足次数，但对I相疼痛并无影响(图2E)。
实施例3 口服20(S)-原人参二醇联合加巴喷丁或吗啡的协同镇痛效应
方法
为验证20(S)-原人参二醇与加巴喷丁或吗啡之间的相互作用，给予最小有效剂量的20(S)-原人参二醇(3mg/kg)，加巴喷丁(10mg/kg)和吗啡(0.3mg/kg)。第一个实验四组大鼠(每组6只)，每组分别服用溶剂(6.5mL/kg)+生理盐水(3mL/kg)，20(S)-原人参二醇(3mg/kg)+生理盐水(3mL/kg)，溶剂(6.5mL/kg)+加巴喷丁(10mg/kg)和20(S)-原人参二醇(3mg/kg)+加巴喷丁(10mg/kg)。
第二个实验四组大鼠(每组6只)，每组分别服用溶剂(6.5mL/kg)+生理盐水(3mL/kg)，20(S)-原人参二醇(3mg/kg)+生理盐水(3mL/kg)，溶剂(6.5mL/kg)+吗啡(0.3mg/kg)和20(S)-原人参二醇(3mg/kg)+吗啡(0.3mg/kg)。
在给药前及给药后0.5、1、2、4小时不同时间点测定各大鼠对机械刺激和热辐射刺激的缩足反应阈值或缩足潜伏时间(热辐射刺激在机械刺激10分钟后进行)。
结果
单独给予小剂量20(S)-原人参二醇或加巴喷丁在给药后1小时抗机械痛敏作用分别为20％MPE和22％MPE，抗热痛敏作用分别为20％MPE和30％MPE，在该剂量下镇痛效果较弱。但巴喷丁和20(S)-原人参二醇联合给药，相比任一单独给药，均显著增强抗机械痛敏达到55％MPE，热痛敏作用增强达到62.2％MPE。采用金氏公式(q＝(E A+B)/(E A+E B-E A*E B)计算(金正均，中国药理学报，1：70-76，1980；金正均、张效文，上海第二医学院学报，1：15-18，1981)，q值分别为1.46和1.40，均大于1.15。这表明加巴喷丁和20(S)-原人参二醇联合给药，显著增强缓解大鼠机械痛敏和热辐射 痛敏作用，表现为协同镇痛效应(图3A和3B)。
同样，小剂量吗啡或20(S)-原人参二醇单独给药，产生较弱的镇痛作用，在给药1小时后其抗机械痛敏作用分别为20％MPE和25％MPE，抗热痛敏作用分别为20％MPE和27％MPE。二者联合使用，抗机械痛敏作用增加到54％MPE，热辐射痛敏作用增加到74％MPE。采用金氏公式计算，它们的q值分别为1.35和1.78，均大于1.15。这表明二药联合使用产生显著的协同镇痛作用(图3C和3D)。
实施例4 口服20(S)-原人参二醇对大鼠脊髓小胶质细胞表达强啡肽A的特异性刺激作用
方法
采用两组L5/L6脊神经结扎的神经病理性疼痛大鼠(每组6只)，分别口服溶剂(6.5mL/kg)或20(S)-原人参二醇(100mg/kg)，给药一小时后断头并取腰椎膨大处(L3-L5)手术侧脊髓组织。通过实时定量PCR测定PDYN，POMC，PENK和PNOC基因表达水平，结果如图4A所示。同时通过酶联免疫荧光法测定脊髓匀浆上清液强啡肽A和β-内啡肽水平。结果如图4B所示。另外采用两组假手术大鼠口服生理盐水(6.5mL/kg)或20(S)-原人参二醇(100mg/kg)，给药一小时后断头并取腰椎膨大处(L3-L5)手术侧脊髓组织，以测定阿片样肽基因和蛋白表达，结果如图4C和4D所示。
选用两组神经病理性疼痛大鼠，采用免疫荧光染色法对脊髓切片进行共染实验：强啡肽A与小胶质细胞标记蛋白Iba-1、星形胶质细胞标记蛋白GFAP或神经元细胞标记蛋白NeuN的共染图像如图5A-5N所示。
进一步，用不同浓度的20(S)-原人参二醇(1、3、10、30和100μM)处理原代脊髓小胶质细胞，培养2小时后，检测小胶质细胞强啡肽A基因和蛋白表达，如图6A和6B所示；用20(S)-原人参二醇(100μM)处理原代脊髓星形胶质细胞和神经元细胞，培养2小时后，检测星形胶质细胞和神经元细胞强啡肽A基因和蛋白表达，如图6C和6D所示。
结果
由图4A可知，在脊神经结扎的神经病理性疼痛的大鼠中，服用20(S)-原人参二醇使得脊髓PDYN表特异性升高达，而不影响POMC，PENK和PNOC表达。且明服用20(S)-原人参二醇可以特异性刺激脊髓释放强啡肽A，而不影响β-内啡肽释放(图4B)。
由图4C可知，在假手术大鼠模型中，口服20(S)-原人参二醇也显著增加脊髓PDYN基因表达，而不增加POMC，PENK或PNOC基因表达水平。与此同时，口服20(S)-原人参二醇显著增加脊髓强啡肽A蛋白表达，而不影响β-内啡肽蛋白表达(图4D)
由图5A-5D可知，强啡肽A与小胶质细胞Iba-1在脊髓有免疫荧光共表达，口服20(S)-原人参二醇明显增加脊髓强啡肽A和Iba-1共表达。通过ImageJ定量分析，给 药组的共染区域面积为对照组的2.1倍(图5E)。
由图5F-5I和5K-5N可知，同样在大鼠脊髓，强啡肽A与星形胶质细胞标记蛋白GFAP或神经元细胞标记蛋白NeuN均有共表达。
由图5J可知，与服用生理盐水(对照组)相比，口服20(S)-原人参二醇(给药组)的强啡肽A与GFAP的共染面积没有明显改变。
同样地，由图5O可知，口服20(S)-原人参二醇不明显改变强啡肽A与NeuN的共染面积。
如图6A和6B可知，20(S)-原人参二醇处理呈剂量依赖性地增加脊髓小胶质细胞表达强啡肽A基因和蛋白质，ED 50值分别为13和19.8μM。而由图6C和6D可知，20(S)-原人参二醇不明显改变脊髓星形胶质细胞或神经元表达强啡肽A基因或蛋白。
实施例5 蛛网膜下腔鞘内预先给予小胶质活化抑制剂米诺环素、强啡肽A抗血清和特异性κ-阿片受体拮抗剂对20(S)-原人参二醇镇痛的抑制作用
方法
采用两组神经病理性疼痛大鼠(每组6只)，分别蛛网膜下腔鞘内预先注射生理盐水(10μL)或小胶质细胞活化抑制剂米诺环素(minocycline,100μg)。4小时后，两组均口服20(S)-原人参二醇(100mg/kg)。第一次给药前，第二次给药前及给药后0.5、1、2和4小时测定大鼠后足对机械刺激的缩足反应阈值和热辐射的缩足反应潜伏期，结果如图7A、7B所示。
采用三组神经病理性疼痛大鼠(每组6只)，分别蛛网膜下腔鞘内预先注射空白兔血清(10μL)、强啡肽A抗血清(1:10，10μL)或β-内啡肽抗血清(1:10，10μL)。0.5小时后，三组均口服20(S)-原人参二醇(100mg/kg)。测定大鼠后足对机械刺激的缩足反应阈值和热辐射的缩足反应潜伏期，结果如图8A、8B所示。
强啡肽A是内源性κ-阿片受体激动剂，为验证20(S)-原人参二醇镇痛作用是否通过激动κ-阿片受体达到，作以下验证：采用四组神经病理性疼痛大鼠(每组6只)分别蛛网膜下腔鞘内注射生理盐水(10μL)、μ-阿片受体拮抗剂CTAP(10μg)、κ-阿片受体拮抗剂GNTI(50μg)或δ-阿片受体拮抗剂naltrindole(5μg)。0.5小时后，四组大鼠均口服20(S)-原人参二醇(100mg/kg)，结果如图8C和8D所示。
结果
由图7A、7B可知，口服20(S)-原人参二醇产生时间依赖式的镇痛作用，米诺环素不影响疼痛的基础阈值，但完全抑制20(S)-原人参二醇所产生的镇痛作用。
由图8A、8B可知，口服20(S)-原人参二醇产生时间依赖式的镇痛作用，强啡肽A抗血清不影响疼痛的基础阈值，但完全抑制20(S)-原人参二醇所产生的镇痛作用，而β-内啡肽抗血清不能阻断20(S)-原人参二醇产生的镇痛作用。
由图8C、8D可知，口服20(S)-原人参二醇(100mg/kg)产生时间依赖式的镇痛作 用，GNTI不影响疼痛的基础阈值，但完全抑制20(S)-原人参二醇产生的镇痛作用，而CTAP或naltrindole未能阻断20(S)-原人参二醇所产生的镇痛作用。
实施例6 口服20(S)-原人参二醇对吗啡镇痛耐受和躯体依赖的抑制作用
采用四组神经病理性疼痛大鼠(每组6只)，每一组分别连续每日两次给予溶剂(6.5mL/kg)+生理盐水(1mL/kg)，20(S)-原人参二醇(30mg/kg)+生理盐水(1mL/kg)，溶剂(6.5mL/kg)+吗啡(3mg/kg)或20(S)-原人参二醇(30mg/kg)+吗啡(3mg/kg)，连续7天。每次早晨给药1小时后，测定大鼠后足对机械刺激的缩足反应阈值和热辐射的缩足反应潜伏期，结果如图9A和9B所示。
在第8天早晨，四组大鼠均口服20(S)-原人参二醇(30mg/kg)，给药前及给药后0.5、1、2和4小时测定大鼠后足对机械刺激的缩足反应阈值和热辐射的缩足反应潜伏期。在给予20(S)-原人参二醇6小时后，四组大鼠均给予吗啡(3mg/kg)，测定接下来4小时大鼠后足疼痛阈值。结果如图9C和9D所示。
对上述大鼠再持续口服给药3日，在最后一日早晨给药后4小时，大鼠腹腔注射纳洛酮(5mg/kg)，随后立即观察30分钟内大鼠戒断症状，结果如图10A-10E所示。
由图9A和9B可知，20(S)-原人参二醇在7天内的镇痛作用保持不变，而吗啡镇痛作用在7天内逐渐产生耐受并最终完全消失。与单独使用20(S)-原人参二醇或吗啡相比，同时给予20(S)-原人参二醇和吗啡不仅产生明显的镇痛协同作用，而且还能够完全抑制吗啡镇痛耐受反应。
由图9C和9D可知，对于分别连续一周给予生理盐水、20(S)-原人参二醇、吗啡、20(S)-原人参二醇+吗啡联合的四组大鼠，再单剂量口服20(S)-原人参二醇后，大鼠均产生显著的时间依赖式镇痛作用。此外，单剂量皮下注射吗啡对于连续一周吗啡给药从而产生吗啡耐受的大鼠，不能产生镇痛作用；但对于连续一周生理盐水给药组、20(S)-原人参二醇给药组和20(S)-原人参二醇+吗啡联合给药组，再进行单剂量皮下注射吗啡能产生显著的镇痛作用。
图10A-10E表明，与生理盐水对照组比较，口服20(S)-原人参二醇不产生躯体依赖性，而吗啡产生明显的躯体依赖性，联合应用20(S)-原人参二醇(30mg/kg)显著减少吗啡相关的戒断症状，包括颤抖(图10A)，跳跃(图10B)，牙齿颤动(图10C)，腹泻(图10D)和湿狗样抖动(图10E)。
采用两组正常大鼠，每日皮下两次注射吗啡(3mg/kg,bid)，连续7日，同时采用热板法和甩尾法测定急性热疼痛阈值。图11A和11B表明，吗啡在舔爪和甩尾模型中急性镇痛作用逐渐降低，并在给药后7日完全消失。在给药第8日，两组吗啡耐受大鼠分别灌胃给予溶剂(6.5mL/kg)和20(S)-原人参二醇(30mg/kg)，1小时后两组大鼠均皮下注射单剂量吗啡(3mg/kg)，并同时测定大鼠疼痛阈值。图11C和11D表明，在灌胃溶剂或20(S)-原人参二醇1小时内(该时间点是20(S)-原人参二醇抑制大鼠神经病理性疼 痛作用高峰)在热板和甩尾反应中不显示镇痛作用。同时在提前给予溶剂的吗啡耐受大鼠组，皮下注射吗啡不产生镇痛作用，但在提前给予20(S)-原人参二醇的吗啡耐受大鼠组，皮下注射吗啡产生明显的镇痛作用，20(S)-原人参二醇完全逆转吗啡镇痛耐受作用。
实施例7 口服20(S)-原人参二醇对吗啡条件性位置偏好(CPP)获得的抑制作用
两组小鼠(每组10只)每天交替口服溶剂(10mL/kg)或20(S)-原人参二醇(100mg/kg)，连续5日，随后进行条件位置偏好测试，结果如图12A所示。
另外四组小鼠(每组10只)每天皮下注射生理盐水(10mL/kg)或吗啡(10mg/kg)5天，在最后一次注射前50分钟，小鼠单剂口服溶剂(10mL/kg)或20(S)-原人参二醇(100mg/kg)，随后立即进行15分钟位置偏好测试，如图12B所示。
由图12A可知，两组小鼠在前测试期和后测试期均未获得条件位置偏好，表明长期口服20(S)-原人参二醇不产生条件位置偏好。
由图12B可知，在前测试期，四组小鼠均未显示条件位置偏好。在后测试期，生理盐水组未出现条件位置偏好，但皮下注射吗啡组显示出明显的条件位置偏好。单剂口服20(S)-原人参二醇(100mg/kg)不影响生理盐水组条件位置偏好反应，但完全阻断吗啡诱导的条件位置偏好的获得。
实施例8 口服20(S)-原人参二醇对吗啡行为敏化作用的抑制作用
4组小鼠，每组10只，在5日的条件化期间，每天注射生理盐水(10mL/kg)或吗啡(10mg/kg)前20分钟灌胃生理盐水(10mL/kg)或20(S)-原人参二醇(100mg/kg)，然后立刻将小鼠放入箱体记录小鼠1小时内在箱体中自由活动距离。如图13所示，生理盐水组小鼠自由活动距离维持在一个较低的水平，而连续5天注射吗啡组小鼠自由活动距离显著增加，并且每天活动距离相比前一天都是逐步递增。连续5天给予20(S)-原人参二醇则减弱了吗啡诱导的小鼠行为敏化效应，小鼠每天活动距离逐步降低。
实施例9 蛛网膜下腔鞘内预先给予糖皮质激素受体拮抗剂对20(S)-原人参二醇镇痛的抑制作用
两组神经病理性疼痛大鼠(每组6只)，分别蛛网膜下腔鞘内预先注射溶剂(10μL)或非特异性糖皮质激素受体拮抗剂RU1486(10nmol)。0.5小时后，两组大鼠均口服20(S)-原人参二醇(100mg/kg)。第一次给药前、第二次给药前及给药后0.5、1、2和4小时测定大鼠后足对机械刺激的缩足反应阈值。口服20(S)-原人参二醇产生时间依赖式的镇痛作用，RU1486不影响疼痛的基础阈值，但完全抑制20(S)-原人参二醇所产生的镇痛作用(图14A)。
另外两组神经病理性疼痛大鼠(每组6只)，分别蛛网膜下腔鞘内预先注射溶剂(10 μL)或特异性糖皮质激素受体拮抗剂dexamethasone 21-mesylate(Dex-21-mesylate,10nmol)。0.5小时后，两组大鼠均口服20(S)-原人参二醇(100mg/kg)。第一次给药前、第二次给药前及给药后0.5、1、2和4小时测定大鼠后足对机械刺激的缩足反应阈值。口服20(S)-原人参二醇产生时间依赖式的镇痛作用，Dex-21-mesylate不影响疼痛的基础阈值，但完全抑制20(S)-原人参二醇所产生的镇痛作用(图14B)。此外两组神经病理性疼痛大鼠(每组6只)，分别蛛网膜下腔鞘内预先注射溶剂(10μL)或Dex-21-mesylate(10nmol)。0.5小时后，两组大鼠均皮下注射草乌甲素(bulleyaconitine A,BAA,300μg/kg)。第一次给药前、第二次给药前及给药后0.5、1、2和4小时测定大鼠后足对机械刺激的缩足反应阈值。口服皮下注射草乌甲素产生时间依赖式的镇痛作用，但Dex-21-mesylate不影响草乌甲素所产生的镇痛作用(图14C)。
三组神经病理性疼痛大鼠(每组6只)，分别蛛网膜下腔鞘内预先注射溶剂(10μL)或雌激素受体拮抗剂G15(10nmol或1μmol)。0.5小时后，三组大鼠均口服20(S)-原人参二醇(100mg/kg)。测定大鼠后足对机械刺激的缩足反应阈值。口服20(S)-原人参二醇产生时间依赖式的镇痛作用，G15既不影响疼痛的基础阈值，也不影响20(S)-原人参二醇所产生的镇痛作用(图14D)。另外二组神经病理性疼痛大鼠(每组6只)，分别蛛网膜下腔鞘内预先注射溶剂(10μL)或醛固酮受体拮抗剂eperenone(10nmol)。0.5小时后，三组大鼠均口服20(S)-原人参二醇(100mg/kg)。测定大鼠后足对机械刺激的缩足反应阈值。口服20(S)-原人参二醇产生时间依赖式的镇痛作用，eperenone既不影响疼痛的基础阈值，也不影响20(S)-原人参二醇所产生的镇痛作用(图14E)。
结果说明，20(S)-原人参二醇与草乌甲素相反，通过特异性激动脊髓糖皮质激素受体产生镇痛作用。
实施例10 糖皮质激素受体拮抗剂对20(S)-原人参二醇刺激强啡肽A表达的抑制作用
四组L5/L6脊神经结扎的神经病理性疼痛大鼠(每组6只)分别蛛网膜下腔鞘内预先注射溶剂(10μL)或特异性糖皮质激素受体拮抗剂Dex-21-mesylate(10nmol)。0.5小时后，四组大鼠分别口服溶剂(6.5mL/kg)或20(S)-原人参二醇(100mg/kg)。给药一小时后断头并取腰椎膨大处(L3-L5)手术侧脊髓组织。通过实时定量PCR测定PDYN基因表达水平。结果表明，20(S)-原人参二醇特异性升高脊髓强啡肽A基因PDYN表达，Dex-21-mesylate不影响脊髓基础PDYN表达，但完全阻断20(S)-原人参二醇诱导的PDYN表达(图15A)。同时通过酶联免疫荧光法测定脊髓匀浆上清液强啡肽A水平。结果表明，口服20(S)-原人参二醇显著增加脊髓强啡肽A蛋白表达，Dex-21-mesylate不影响脊髓基础强啡肽A表达，但完全阻断20(S)-原人参二醇诱导的强啡肽A表达(图15B)。
进一步，原代培养的脊髓小胶质细胞给与溶剂或糖皮质激素受体拮抗剂Dex-21-mesylate(100nM)0.5小时后，分别给与20(S)-原人参二醇(100μM)、特异性糖 皮质激素受体激动剂Dex(100nM)或不透过细胞膜的Dex与牛血清白蛋白(BSA)的缀合物Dex-BSA(10nM)并培养2小时，随后检测小胶质细胞强啡肽A基因和蛋白表达。如图16A和16B可知，20(S)-原人参二醇、Dex和Dex-BSA显著增强小胶质细胞强啡肽A基因表达；而Dex 21-melylate不影响小胶质细胞强啡肽基因的基础表达，但完全抑制20(S)-原人参二醇、Dex和Dex-BSA对强啡肽A基因表达的刺激作用。而由图16B可知，Dex-21-melylate不影响小胶质细胞的强啡肽A蛋白基础表达，但完全抑制20(S)-原人参二醇、Dex和Dex-BSA对强啡肽A蛋白表达的刺激作用。
上述结果说明，20(S)-原人参二醇通过激动脊髓小胶质细胞糖皮质激素受体(可能是细胞膜糖皮质激素受体)，刺激小胶质细胞表达和释放强啡肽A。
实施例11 伏隔核给予小胶质细胞抑制剂、dynorphin A抗血清、κ-阿片受体拮抗剂和糖皮质激素受体拮抗剂抑制20(S)-原人参二醇抗吗啡躯体依赖作用
四组吗啡依赖小鼠(每组10只)按照以下分组分别进行伏隔核注射和灌胃给药：生理盐水(1μL)+生理盐水(10mL/kg)，dynorphin A抗血清(1:30稀释，1μL)+生理盐水(10mL/kg)，生理盐水(1μL)+20(S)-原人参二醇(100mg/kg)和dynorphin A抗血清(1:30稀释，1μL)+20(S)-原人参二醇(100mg/kg)。灌胃生理盐水(10mL/kg)或20(S)-原人参二醇(100mg/kg)在伏隔核注射后30分钟进行，灌胃给药后40分钟腹腔注射纳洛酮(5mg/kg)，立刻观察30分钟内的戒断症状。如图17A-F所示，吗啡依赖组小鼠灌胃20(S)-原人参二醇(100mg/kg)可以缓解吗啡戒断症状，包括前爪震颤次数减少、湿狗样抖动频率降低、舔阴次数降低、跳跃次数减少、腹泻增加和体重减轻缓解，伏隔核单次注射dynorphin A抗血清(1:30稀释，1μL)对吗啡戒断现象无明显影响，但能够使20(S)-原人参二醇抗吗啡戒断症状重新出现。
四组吗啡依赖小鼠(每组10只)也分别进行第一次伏隔核注射，第二次灌胃给药：生理盐水(1μL)+生理盐水(10mL/kg)，GNTI(5μg，1μL)+生理盐水(10mL/kg)，生理盐水(6μL)+20(S)-原人参二醇(100mg/kg)和GNTI(5μg，1μL)+20(S)-原人参二醇(100mg/kg)。第二次灌胃给药在伏隔核注射后30分钟进行，灌胃给药后40分钟腹腔注射纳洛酮(5mg/kg)，立刻观察戒断现象。图17G-L所示，伏隔核注射GNTI不会影吗啡戒断现象，但却恢复20(S)-原人参二醇抗吗啡戒断症状。
另外四组吗啡依赖小鼠(每组10只)同样分别进行伏隔核注射和灌胃给药：生理盐水(1μL)+生理盐水(10mL/kg)，米诺环素(10μg)+生理盐水(10mL/kg)，生理盐水(1μL)+20(S)-原人参二醇(100mg/kg)和米诺环素(10μg)+20(S)-原人参二醇(100mg/kg)。第二次灌胃生理盐水(10mL/kg)或20(S)-原人参二醇(100mg/kg)在第一次伏隔核注射后4小时进行。40分钟后腹腔注射纳洛酮(5mg/kg)，观察戒断现象。吗啡依赖组小鼠灌胃20(S)-原人参二醇可以显著减轻吗啡戒断症状,米诺环素对吗啡戒断症状无影响，但能够使20(S)-原人参二醇抑制的小鼠戒断症状再次出现(图17M-R)。
最后，四组吗啡依赖小鼠(每组10只)同样分别进行伏隔核注射和灌胃给药：生理盐水(1μL)+生理盐水(10mL/kg)，Dex 21-mesylate(10nmol，1μL)+生理盐水(10mL/kg)，生理盐水(1μL)+20(S)-原人参二醇(100mg/kg)和Dex 21-mesylate(10nmol，1μL)+20(S)-原人参二醇(100mg/kg)。第一次伏隔核注射生理盐水(1μL)或Dex 21-mesylate(10nmol，1μL)后30分钟后进行第二次灌胃生理盐水(10mL/kg)或20(S)-原人参二醇(100mg/kg)。40分钟后腹腔注射纳洛酮(5mg/kg)，观察戒断现象。吗啡依赖组小鼠灌胃给予20(S)-原人参二醇可以显著减轻吗啡戒断症状,Dex 21-mesylate逆转了20(S)-原人参二醇抗小鼠吗啡戒断症状(图17S-X)。
实施例12伏隔核给予dynorphin A抗血清、κ-阿片受体拮抗剂、小胶质细胞抑制剂和糖皮质激素受体拮抗剂抑制20(S)‐原人参二醇抗吗啡诱导的CPP表达作用
四组吗啡诱导的CPP小鼠，每组10只，在测试前按照以下分组分别进行第一次伏隔核注射，4小时后第二次灌胃给药：生理盐水(1μL)+生理盐水(10mL/kg)，dynorphin A抗血清(1:30稀释，1μL)+生理盐水(10mL/kg)，生理盐水(1μL)+20(S)-原人参二醇(100mg/kg)和dynorphin A抗血清(1:30稀释，1μL)+20(S)-原人参二醇(100mg/kg)。灌胃生理盐水(10mL/kg)或20(S)-原人参二醇(100mg/kg)50分钟后进行CPP测试。如图18A所示，灌胃20(S)-原人参二醇可以完全减弱吗啡诱导的CPP表达。伏隔核注射dynorphin A抗血清对CPP表达无影响，但可以完全恢复20(S)-原人参二醇对吗啡诱导的CPP表达的抑制作用，CPP得分重新升高。
另外四组吗啡诱导的CPP小鼠，每组10只，在测试前按照以下分组分别进行第一次伏隔核注射，30分钟后第二次灌胃给药：生理盐水(1μL)+生理盐水(10mL/kg)，GNTI(5μg，1μL)+生理盐水(10mL/kg)，生理盐水(6μL)+20(S)-原人参二醇(100mg/kg)和GNTI(5μg，1μL)+20(S)-原人参二醇(100mg/kg)。同样灌胃50分钟后进行CPP测试。灌胃20(S)-原人参二醇可以完全抑制吗啡诱导的CPP表达。伏隔核注射GNTI对CPP表达无显著影响，但可完全减弱20(S)-原人参二醇对吗啡诱导的CPP表达的抑制作用(图18B)。
接着四组吗啡依赖小鼠，每组10只，同样分别进行伏隔核注射和灌胃给药：生理盐水(1μL)+生理盐水(10mL/kg)，米诺环素(10μg)+生理盐水(10mL/kg)，生理盐水(1μL)+20(S)-原人参二醇(100mg/kg)和米诺环素(10μg)+20(S)-原人参二醇(100mg/kg)。第二次灌胃生理盐水(10mL/kg)或20(S)-原人参二醇(100mg/kg)在第一次伏隔核注射后50分钟进行。吗啡依赖组小鼠灌胃20(S)-原人参二醇可以显著抑制CPP表达,。伏隔核注射米诺环素对CPP表达无影响，但能够使20(S)-原人参二醇抑制的小鼠CPP表达再次出现(图18C)。
最后，四组吗啡依赖小鼠，每组10只，也分别进行第一次伏隔核注射，30分钟 后第二次灌胃给药：生理盐水(1μL)+生理盐水(10mL/kg)，Dex 21-mesylate(10nmol，1μL)+生理盐水(10mL/kg)，生理盐水(1μL)+20(S)-原人参二醇(100mg/kg)和Dex21-mesylate(10nmol，1μL)+20(S)-原人参二醇(100mg/kg)，50分钟后进行CPP得分测试。伏隔核注射Dex 21-mesylate对CPP表达无影响，但完全逆转20(S)-原人参二醇抗吗啡诱导的CPP表达(图18D)。
讨论
20(S)-原人参二醇的生物活性已有一些报道，但本发明之前目前未报道过20(S)-原人参二醇具有治疗疼痛的用途，也未公开过可用于治疗阿片类药物(或成瘾物质)的躯体依赖和精神依赖。本发明提供，20(S)-原人参二醇在大鼠/小鼠神经病理性疼痛、癌症疼痛、炎性疼痛和福尔马林疼痛等模型有显著的镇痛作用；20(S)-原人参二醇长期给药不产生镇痛耐受作用、躯体依赖性和精神依赖性(表现为条件性位置偏好和行为敏化作用)；20(S)-原人参二醇有效抑制吗啡诱导的镇痛耐受性、躯体依赖性和精神依耐性。
本发明人研究表明，20(S)-原人参二醇的镇痛主要部位在脊髓，抗成瘾作用主要部位在伏隔核。20(S)-原人参二醇通过激动脊髓或伏隔核小胶质细胞糖皮质激素受体(可能是细胞膜糖皮质激素受体)极其有效地促进强啡肽A表达和释放，从而出乎意料地产生镇痛作用和戒除治疗阿片类药物(或其他成瘾物质)的躯体依赖和精神依赖等作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (13)

  1. 一种活性成分或含所述活性成分的制剂的用途，其特征在于，所述活性成分选自下组：原人参二醇或其药学上可接受的盐或酯、原人参三醇或其药学上可接受的盐或酯；
    并且，所述活性成分或含所述活性成分的制剂用于制备：
    (a)治疗和/或缓解疼痛的药物；和/或
    (b)治疗和/或缓解成瘾物质诱导的躯体和/或精神依赖和/或成瘾的药物；和/或
    (c)预防、抑制和/或逆转镇痛药物诱导的镇痛耐受作用的药物。
  2. 如权利要求1所述的用途，所述成瘾物质选自：阿片类药物、海洛因、或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述疼痛选自：神经病理性疼痛、炎症性疼痛、关节炎疼痛、糖尿病性疼痛、下背部疼痛、脊髓损伤性疼痛、内脏疼痛、纤维肌痛、慢性局部疼痛综合征、肌肉骨骼疼痛、癌症疼痛、抗肿瘤药物和阿片类药物引起的疼痛、手术后疼痛、创伤后疼痛、创伤后神经痛和周围神经病、幻肢痛、或其组合。
  4. 如权利要求1所述的用途，其特征在于，所述制剂还包括第二活性成分；其中第二活性成分选自下组：
    (Z1)选自下组的阿片类镇痛药：可待因、双氢可待因、吗啡、芬太尼、舒芬太尼、瑞芬太尼、哌替啶、羟考酮或其组合；
    (Z2)选自下组的抗癫痫药物：卡马西平、苯妥英钠、加巴喷丁类(gabapentinoids)化合物、或其组合；
    (Z3)选自下组的非甾体抗炎镇痛药物：氟比洛芬酯、布洛芬、双氯芬酸钠、美洛昔康、萘普生、塞来昔布、普瑞昔布或其组合；
    (Z4)选自下组的单胺类神经递质重摄取抑制剂抗抑郁药：阿米替林、度洛西丁、或其组合；
    (Z5)选自下组的局部麻醉药：利多卡因、罗哌卡因、丙胺卡因、或其组合；
    (Z6)选自下组的去甲肾上腺素α2受体激动剂：可乐定、右美托咪定、或其组合；
    (Z7)选自下组的MOR-NRI双靶点镇痛药：地佐辛、他喷他多、喷他佐辛、曲马多、或其组合；
    (Z8)选自下组的抗偏头痛药：CGRP抗体及其受体拮抗剂；
    (Z9)选自下组的中草药：独一味提取物及其有效成分、乌头/附子及其有效成分、以及延胡索及其有效成分、或其组合；
    (Z10)上述Z1～Z9的任意组合。
  5. 如权利要求1所述的用途，其特征在于，所述制剂为口服制剂、或注射剂。
  6. 一种药物组合物，其特征在于，所述药物组合物包含：
    (i)第一活性成分，所述第一活性成分选自下组：原人参二醇、原人参三醇；
    (ii)第二活性成分，所述第二活性成分选自：
    (Z1)选自下组的阿片类镇痛药：可待因、双氢可待因、吗啡、芬太尼、舒芬太尼、瑞芬太尼、哌替啶、羟考酮或其组合；
    (Z2)选自下组的抗癫痫药物：卡马西平、苯妥英钠、加巴喷丁类化合物、或其组合；
    (Z3)选自下组的非甾体抗炎镇痛药物：氟比洛芬酯、布洛芬、双氯芬酸钠、美洛昔康、萘普生、塞来昔布、普瑞昔布或其组合；
    (Z4)选自下组的单胺类神经递质重摄取抑制剂抗抑郁药：阿米替林、度洛西丁、或其组合；
    (Z5)选自下组的局部麻醉药：利多卡因、罗哌卡因、丙胺卡因、或其组合；
    (Z6)选自下组的去甲肾上腺素α2受体激动剂：可乐定、右美托咪定、或其组合；
    (Z7)选自下组的MOR-NRI双靶点镇痛药：地佐辛、他喷他多、喷他佐辛、曲马多、或其组合；
    (Z8)选自下组的抗偏头痛药：CGRP抗体及其受体拮抗剂；
    (Z9)选自下组的中草药：独一味提取物及其有效成分、乌头/附子及其有效成分、以及延胡索及其有效成分、或其组合；
    (Z10)上述Z1～Z9的任意组合；
    (iii)药学上可接受的载体和/或赋形剂。
  7. 如权利要求6所述的药物组合物，其特征在于，所述第一活性成分为原人参二醇，第二活性成分为选自下组的阿片类镇痛药：可待因、双氢可待因、吗啡、芬太尼、舒芬太尼、瑞芬太尼、哌替啶、羟考酮或其组合。
  8. 一种如权利要求7所述的药物组合物的用途，其特征在于，所述药物组合物用于制备：
    (a)治疗和/或缓解疼痛的药物；
    (b)治疗和/或缓解成瘾物质诱导的躯体和/或精神依赖的药物。
  9. 一种治疗和/或缓解疼痛的方法，其特征在于，包括步骤：
    给有需要的对象施用医学有效量的第一活性成分或含所述活性成分的制剂或权利要求6所述的药物组合物，其中所述第一活性成分选自下组：原人参二醇或其药学上可接受的盐或酯、原人参三醇或其药学上可接受的盐或酯；从而治疗和/或缓解疼痛。
  10. 一种诱导产生强啡肽A表达和释放的方法，其特征在于，包括：给需要的对象施用活性成分，所述活性成分选自下组：原人参二醇或其药学上可接受的盐或酯、原人参三醇或其药学上可接受的盐或酯，从而诱导所述对象产生强啡肽A。
  11. 一种治疗和/或缓解成瘾物质诱导的躯体和/或精神依赖和/或成瘾的方法，包括步骤：给予给有需要的对象有效量的第一活性成分或含所述第一活性成分的的药物 组合物，从而治疗和/或缓解成瘾物质诱导的躯体和/或精神依赖和/或成瘾；
    其中所述第一活性成分选自下组：原人参二醇或其药学上可接受的盐或酯、原人参三醇或其药学上可接受的盐或酯。
  12. 一种降低镇痛药物的成瘾性和/或镇痛耐受作用的方法，包括步骤：将有效量的成瘾性镇痛药物和第一活性成分或含所述第一活性成分的的药物组合物联合给予有需要的对象，从而降低所述镇痛药物的成瘾性和/或镇痛耐受作用；
    其中所述第一活性成分选自下组：原人参二醇或其药学上可接受的盐或酯、原人参三醇或其药学上可接受的盐或酯。
  13. 如权利要求12所述的方法，其特征在于，所述成瘾性镇痛药物为选自下组的阿片类镇痛药：可待因、双氢可待因、吗啡、芬太尼、舒芬太尼、瑞芬太尼、哌替啶、羟考酮或其组合。
PCT/CN2021/117518 2020-09-10 2021-09-09 原人参二醇类化合物在治疗疼痛和成瘾物质躯体依赖、精神依赖和成瘾的用途 WO2022052996A1 (zh)

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