CN114159450A

CN114159450A - Use of protopanoxadiol compounds in treating pain and addiction to substances physical dependence, mental dependence and addiction

Info

Publication number: CN114159450A
Application number: CN202010949695.3A
Authority: CN
Inventors: 王永祥; 阮邵穆; 赵梦静
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2020-09-10
Filing date: 2020-09-10
Publication date: 2022-03-11
Anticipated expiration: 2040-09-10
Also published as: CN114159450B; WO2022052996A1

Abstract

The present invention provides the use of protopanoxadiol compounds in the treatment of pain and addictive substance physical and mental dependence. Specifically, the present invention provides protopanaxadiol compounds (especially 20(S) -protopanaxadiol) and pharmaceutically acceptable salts or esters thereof, for use in the preparation of a) a medicament for treating and/or relieving pain; b) use in a medicament for the treatment and/or alleviation of addictive substances, in particular opioid-induced physical and/or mental dependence. Experiments show that 20(S) -protopanaxadiol can stimulate the expression and release of dynorphin A (dynorphin A) by exciting a glucocorticoid receptor of a spinal microglia cell so as to achieve the effect of treating and/or relieving pain, and can be combined with other analgesic drugs, particularly gabapentin and opioid drugs, to achieve the synergistic analgesic effect.

Description

Use of protopanoxadiol compounds in treating pain and addiction to substances physical dependence, mental dependence and addiction

Technical Field

The invention relates to the technical field of medicines, in particular to application of protopanoxadiol compounds in treating pain and addiction to physical, mental and addictive substances.

Background

Pain, a compound sensation of aversion associated with injury and suffering. Under normal physiological conditions, pain can provide an alarm signal when the body is threatened, which is an indispensable life protection function. However, under pathological conditions, pain is a common symptom of most diseases, and is mixed with autonomic nervous activity, motor reflex, psychological and emotional reactions and the like, so that pain is brought to patients.

Pain is classified into acute pain and chronic pain according to cause, nature, location and time course. Acute pain refers to pain caused by directly activating nociceptors at the corresponding site by nociceptive stimulation under physiological conditions. Acute pain does not last long (<1 month), and pain self-disappears after injury repair. Acute pain includes postoperative pain, post-traumatic pain, acute headache and facial pain, acute arthritis pain, etc.

Chronic pain is when following focal repair, pain persists for months (>1 month) or even for life, or may recur frequently. Chronic pain includes low back pain, cancer pain, pain caused by antineoplastic drugs and opioids, diabetic pain, neuropathic pain including postherpetic neuralgia, trigeminal neuralgia and sciatica, inflammatory pain, phantom limb pain, arthritic pain, fibromyalgia, musculoskeletal pain, chronic regional pain syndrome, posttraumatic neuralgia, peripheral neuropathy, and the like.

According to the International Association for the Study of Pain (IASP) data, the prevalence of chronic Pain worldwide is 10% (Scholz et al, Pain,160:53-59,2019). In western europe alone, 8.0% of the population is reported to suffer from chronic pain. In china, about 20% of diabetic patients are diagnosed with diabetic peripheral neuropathy, and one third of patients with herpes zoster virus develop postherpetic neuralgia, with over one million people suffering from cancer pain. Pain brings pain to patients, seriously affects family life or work, and brings huge burden to public health systems.

The commonly used pain-treating drugs are: 1) the nonsteroidal anti-inflammatory analgesic drugs comprise flurbiprofen axetil, ibuprofen, diclofenac sodium, meloxicam, naproxen, celecoxib and prorexib;

2) antiepileptic drugs include carbamazepine, phenytoin sodium, and gabapentin drugs such as gabapentin, pregabalin, and milopalin;

3) monoamine neurotransmitter reuptake inhibitor antidepressants including amitriptyline and doxoxetine;

4) local anesthetics include lidocaine, ropivacaine, prilocaine;

5) opioid analgesics including codeine, dihydrocodeine, morphine, fentanyl, sufentanil, remifentanil, pethidine, oxycodone;

6) norepinephrine alpha 2 receptor agonists such as clonidine, dexmedetomidine;

7) MOR-NRI dual-target analgesics such as dezocine, tapentadol, pentazocine and tramadol;

8) the Chinese herbal medicine comprises radix Lamiophlomidis Rotatae, radix Aconiti Kusnezoffii/radix Aconiti lateralis Preparata and its effective components such as bulleyaconitine A and lappaconitine A, and rhizoma corydalis and its effective components such as rotundine.

However, the medicines have limited curative effect or generate more serious adverse reactions when used for treating pain. Such as gabapentin and rappalene, are not specific analgesics, and their effectiveness in treating neuropathic pain (lowering the pain threshold by 30%) is less than 50% in people. The non-steroidal analgesic has certain curative effect on headache, toothache, muscle and joint pain and the like, but almost has no effect on traumatic severe pain and visceral smooth muscle colic. Local anesthetics are only indicated for peripheral neuropathic pain.

For another example, opioid drugs have various adverse reactions such as lethargy, respiratory depression, constipation, etc., and can produce analgesic tolerance, hyperalgesia, physical dependence, addiction and abuse after long-term administration. Gabapentin and rapibalin also have serious adverse reactions such as lethargy. Repeated use of opioids, including morphine and fentanyl, leads to analgesic tolerance and requires increased doses to achieve the same analgesic effect. Chronic or repeated use of opioids can also lead to addiction, including both physical (physical, physiological) and mental (psychological) dependence.

Physical dependence is repeated for the avoidance of withdrawal symptoms, and as the tolerance dose is gradually increased, it manifests as an aversive effect in the course of addiction, playing a role in negative reinforcement. The mental dependence refers to the craving of psychological drug-seeking of the depended and the euphoria achieved by repeated drug-seeking, and is expressed as a reward effect, plays a positive reinforcement effect and promotes the repeated drug-seeking of the depended. At present, the method and the approach for solving the addiction of the opioid are still greatly limited, methadone, buprenorphine, clonidine, lofexidine and the like only improve the withdrawal symptoms to a certain extent, the curative effect is very limited, and particularly, the method has no curative effect on the psychic dependence.

Therefore, pain treatment is still a great clinical problem, and there is an urgent need to develop novel analgesic drugs which can be used for a long time, have no analgesic tolerance and addiction and can effectively treat pain and physical and mental dependence induced by opioid drugs.

Disclosure of Invention

The invention aims to provide a novel analgesic drug which can be used for a long time, has no analgesic tolerance and addiction and can effectively treat pain and physical and mental dependence induced by opioid drugs.

Specifically, the invention provides the application of protopanoxadiol compounds (such as 20(S) -protopanaxadiol) in preparing novel analgesic drugs for treating pain and opioid-induced physical and mental dependence. The experiment of the invention shows that 20(S) -protopanoxadiol can be used together with other active ingredients to achieve the effect of synergistic analgesia. Also, 20(S) -protopanaxadiol produces an analgesic effect by agonizing the expression and release of dynorphin a at the glucocorticoid receptor (cell membrane glucocorticoid receptor) of spinal microglia.

In a first aspect of the invention there is provided the use of an active ingredient or a formulation containing said active ingredient, said active ingredient being selected from the group consisting of: protopanaxadiol or a pharmaceutically acceptable salt or ester thereof, protopanaxatriol or a pharmaceutically acceptable salt or ester thereof;

and, the active ingredient or a formulation containing the active ingredient is used for the preparation of:

(a) drugs for the treatment and/or alleviation of pain; and/or

(b) A medicament for the treatment and/or alleviation of physical and/or mental dependence and/or addiction induced by an addictive substance.

In another preferred embodiment, the protopanaxadiol comprises 20(S) -protopanaxadiol, 20(R) -protopanaxadiol, or a combination thereof.

In another preferred embodiment, the protopanaxatriol comprises 20(S) -protopanaxatriol, 20(R) -protopanaxatriol, or a combination thereof.

In another preferred embodiment, the addictive substance is selected from the group consisting of: an opioid, heroin, or a combination thereof.

In another preferred embodiment, the addictive substance further comprises one or more selected from the group consisting of: methamphetamine, alcohol, cigarette (nicotine), cocaine, cannabis, or combinations thereof.

In another preferred embodiment, the pain is selected from: neuropathic pain, inflammatory pain, arthritic pain, diabetic pain, low back pain, spinal cord injury pain, visceral pain, fibromyalgia, chronic regional pain syndrome, musculoskeletal pain, cancer pain, pain caused by antineoplastic and opioid drugs, post-operative pain, post-traumatic neuralgia and peripheral neuropathy, phantom limb pain, or a combination thereof.

In another preferred embodiment, the neuropathic pain includes, but is not limited to, postherpetic neuralgia, trigeminal neuralgia, and sciatica.

In another preferred embodiment, the formulation further comprises a second active ingredient; wherein the second active ingredient is selected from the group consisting of:

(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 sodium, gabapentin (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, pricelecoxib, or combinations thereof;

(Z4) monoamine neurotransmitter reuptake inhibitor antidepressants selected from the group consisting of: amitriptyline, doxoxetine, or a combination thereof;

(Z5) a local anesthetic selected from the group consisting of: lidocaine, ropivacaine, prilocaine, or a combination thereof;

(Z6) a norepinephrine alpha 2 receptor agonist selected from the group consisting of: clonidine, dexmedetomidine, or combinations thereof;

(Z7) a MOR-NRI dual-target analgesic selected from the group consisting of: dezocine, tapentadol, pentazocine, tramadol, or combinations thereof;

(Z8) an anti-migraine agent selected from the group consisting of: CGRP antibodies and receptor antagonists thereof;

(Z9) a herbal medicine selected from the group consisting of: radix Lamiophlomidis Rotatae extract and its effective component, radix Aconiti Kusnezoffii/radix Aconiti lateralis and its effective component, and rhizoma corydalis and its effective component, or their combination;

(Z10) any combination of the above Z1 to Z9.

In another preferred embodiment, the gabapentin-like compound includes gabapentin, pregabalin, and milobalin (miroarabalin).

In another preferred embodiment, the radix Lamiophlomidis Rotatae extract and its effective components comprise shanzhiside methyl ester, and 8-O-acetyl shanzhiside methyl ester.

In another preferred embodiment, the aconite/aconite and its effective components include: bulleyaconitine A, lappaconitine A and radix Aconiti Brachypodi Aconitifoliae A.

In another preferred embodiment, the corydalis tuber and its effective components include tetrahydropalmatine, stephanine, corydalmine and dehydrocorydaline.

In another preferred embodiment, the formulation is an oral formulation, or an injection.

In another preferred embodiment, the formulation comprises: powder, granule, capsule, injection, tincture, oral liquid, tablet, buccal tablet, or dripping pill.

In another preferred embodiment, the active ingredient or the formulation containing the active ingredient does not have: (1) (ii) analgesic tolerance; (2) (ii) a somatic dependence; (3) mental dependence (addiction).

In a second aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) a first active ingredient selected from the group consisting of: protopanaxadiol, protopanaxatriol;

(ii) a second active ingredient selected from the group consisting of:

(Z2) an antiepileptic drug selected from the group consisting of: carbamazepine, phenytoin sodium, gabapentin, or a combination thereof;

(Z10) any combination of Z1 to Z9;

(iii) a pharmaceutically acceptable carrier and/or excipient.

In another preferred embodiment, the pharmaceutical composition is administered orally or non-orally.

In another preferred embodiment, the non-oral administration is selected from the group consisting of: nasal feeding, anal embolization, subcutaneous injection, intramuscular injection, intravenous injection, subarachnoid injection, epidural injection, lateral ventricular injection, external application of skin (patch), or a combination thereof.

In a third aspect of the invention, there is provided a use of the pharmaceutical composition of the second aspect for the preparation of:

(a) drugs for the treatment and/or alleviation of pain;

(b) a medicament for the treatment and/or alleviation of addiction material induced physical and/or mental dependence.

In a fourth aspect of the invention, there is provided an in vitro method of treating and/or alleviating pain, comprising the steps of:

(1) culturing the cells in a system comprising an effective amount of a first active ingredient or a formulation comprising said active ingredient or a pharmaceutical composition according to the second aspect, wherein said 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;

(2) agonizing the cellular glucocorticoid receptor to express dynorphin a.

In another preferred embodiment, the cell is an immune cell of the central nervous system, preferably a spinal cord immune cell.

In another preferred embodiment, the cell is selected from the group consisting of: microglia, macrophages, monocytes, or a combination thereof.

In another preferred embodiment, the cells are spinal microglia.

In another preferred embodiment, the glucocorticoid is a glucocorticoid receptor agonist.

In another preferred embodiment, the glucocorticoid is a cell membrane glucocorticoid receptor agonist.

In another preferred embodiment, the method is non-diagnostic, non-therapeutic.

In a fifth aspect of the invention, there is provided a method of treating and/or alleviating pain comprising the steps of:

administering to a subject in need thereof a medically effective amount of a first active ingredient or a formulation comprising said active ingredient or a pharmaceutical composition according to the second aspect, wherein said 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; thereby treating and/or relieving pain.

In a sixth aspect of the invention, there is provided a method of inducing expression and release of dynorphin a, comprising: administering to a subject in need thereof an active ingredient selected from the group consisting of: protopanaxadiol or a pharmaceutically acceptable salt or ester thereof, protopanaxatriol or a pharmaceutically acceptable salt or ester thereof, thereby inducing the subject to produce dynorphin A.

In another preferred embodiment, the method stimulates the expression and release of myeloproliferative dynorphin a.

In another preferred embodiment, the method is for agonizing a glucocorticoid receptor of cells of the subject.

In another preferred embodiment, the glucocorticoid receptor is a cell membrane glucocorticoid receptor.

In another preferred embodiment, the subject is a mammal.

In another preferred example, the subject includes, but is not limited to, a mouse, a human.

In another preferred embodiment, the subject is a pain patient.

In another preferred embodiment, the cells are spinal microglia.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows that oral administration of 20(S) -protopanaxadiol inhibits the mechanical and thermal nociceptive effects of neuropathic pain in a dose-dependent manner.

Figure 2 shows the analgesic effect of oral 20(S) -protopanaxadiol in bone cancer pain, complete freund' S adjuvant (CFA) inflammatory pain and formalin pain models.

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 spinal cord expression of dynorphin a gene and protein in rats.

Figure 5 immunofluorescence double staining shows that oral 20(S) -protopanaxadiol specifically stimulates rat spinal microglia to express dynorphin a.

Figure 6 shows that ex vivo administration of 20(S) -protopanaxadiol specifically stimulates expression of dynorphin a genes and proteins by primary spinal microglia.

Figure 7 shows that the microglial activation inhibitor minocycline blocks 20(S) -protopanaxadiol against neuropathic pain.

Figure 8 shows that dynorphin a antiserum and specific kappa-opioid receptor antagonists block 20(S) -protopanaxadiol against neuropathic pain.

Figure 9 shows that oral administration of 20(S) -protopanaxadiol did not produce self-analgesic tolerance, but inhibited morphine analgesic tolerance.

Figure 10 shows that oral administration of 20(S) -protopanaxadiol does not produce physical dependence, but inhibits morphine physical dependence.

Figure 11 shows that oral administration of 20(S) -protopanaxadiol does not produce psychodependence, but inhibits the psychotropic effects of morphine.

FIG. 12 shows that intrathecal pre-administration of a glucocorticoid receptor antagonist into the subarachnoid space inhibits the analgesic effect of oral 20(S) -protopanaxadiol.

Figure 13 shows that intrathecal pre-administration of glucocorticoid receptor antagonist into the subarachnoid space inhibits the effect of oral administration of 20(S) -protopanaxadiol on dynorphin a expression in spinal cord.

Figure 14 shows that glucocorticoid receptor antagonists inhibit dynorphin a expression produced by 20(S) -protopanaxadiol on primary spinal microglia.

Detailed Description

The inventor of the present invention has conducted extensive and intensive studies and has surprisingly found for the first time that protopanoxadiol compounds (especially 20(S) -protopanaxadiol) can significantly inhibit pain, have a significant anti-addiction effect, and do not produce adverse reactions such as analgesic tolerance, physical dependence, mental dependence, and the like. Thus, can be used for treating pain and resisting addiction (such as physical and mental dependence induced by addictive substances). The present invention has been completed based on this finding.

Specifically, the examples show that the analgesic effect of the active ingredient 20(S) -protopanaxadiol does not produce self-analgesic tolerance, and can help suppress the physical dependence and mental dependence induced by addictive substances while relieving pain. The protopanaxadiol compound can be combined with other analgesic drugs to achieve synergistic analgesic effect. The protopanoxadiol compound specifically stimulates the expression of dynorphin A, so that the analgesic effect is achieved.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

In the present invention, the terms "physical dependence", "physical dependence" and "physiological dependence" are used interchangeably to refer to the dependence that can trigger withdrawal syndrome once the use of an addictive drug is discontinued.

In the present invention, the term "withdrawal syndrome" refers to a series of symptoms caused by the severe physiological reactions of the body, such as sweating, lacrimation, yawning, chills, keloids, mydriasis, vomiting, diarrhea, abdominal pain, increased heart rate, increased blood pressure, insomnia, tremor, etc., once the use of a patient who has developed a dependency is discontinued due to the continuous use of addictive substances.

In the present invention, the terms "psychological dependence" and "psychological dependence" are used interchangeably to refer to a patient's craving for a drug in order to obtain a particular pleasure after taking an addictive drug.

By "treating" or "treatment" and/or "preventing" or "prevention", as a whole, is meant at least inhibiting or ameliorating the relevant symptoms affecting the individual, wherein inhibiting and ameliorating is used in a broad sense to mean at least reducing the magnitude of a parameter, such as a symptom associated with the condition being treated, e.g., pain. Thus, the methods of the invention include the prevention and management of a variety of different pain conditions.

Pain (due to cold or dampness)

The invention provides the use of an active ingredient of the invention or a formulation thereof for the treatment of pain.

In the present invention, pain is not particularly limited, and representative examples include (but are not limited to) migraine, back pain, neck pain, gynecological pain, pre-or labor-delivery pain, orthopedic pain, post-stroke pain, post-surgical or operative pain, post-herpetic neuralgia, sickle cell crisis, interstitial cystitis, urinary pain (e.g., urethritis), dental pain, headache, wound or pain resulting from medical procedures such as surgery (e.g., capsuliectomy or hip, knee or other joint replacement), suturing, fracture reduction, biopsy, and the like. Pain may also occur in patients with cancer, which may be caused by a variety of causes, such as inflammation, nerve compression, and mechanical forces due to tissue swelling resulting from tumor invasion and metastasis to bone or other tissues.

In another preferred embodiment, the pain includes (but is not limited to): peripheral neuropathic pain, central neuropathic pain, allodynia, causalgia, hyperalgesia, hyperesthesia, hyperpathia, neuralgia, neuritis and neuropathy.

Addiction (addiction)

Drug addiction and drug dependence are chronic recurrent brain diseases, which are mainly characterized by the uncontrollable behavior and dosage of the compulsive medication of the addictive drugs. After the substance dependence occurs, if the administration of the drug is stopped suddenly, the drug withdrawal symptoms may occur. Many drugs, originally intended for medical use, may cause substance-dependent phenomena; an addictive substance is called drug if it is judged to be illegal by law. These include opioids and heroin, methamphetamine, cocaine, cannabis, alcohol, nicotine, and the like.

Methamphetamine, commonly known as methamphetamine, is a highly addictive stimulant and the second most commonly used illegal drug worldwide. Abuse of methamphetamine or other amphetamine-like stimulants has become a significant public health problem. Compared with traditional drugs such as heroin, cocaine and the like, methamphetamine has the advantages of simple synthesis process, cheap and easily obtained precursor, stronger excitation effect on the central nervous system, less drug administration times or accumulated drug administration amount required by addiction formation and more serious damage to the body of a drug user.

Alcohol is a psychoactive substance with high addictive properties. Global alcohol dependence reaches 1.4 billion, and its abuse and dependence bring serious adverse effects and economic burden to the individual and society. About 330 million people die each year worldwide due to excessive use of alcohol. The harmful use of alcohol can also cause diseases such as alcoholic liver, liver cirrhosis and the like. Alcohol abuse, alcohol addiction have become a serious public health disaster and a worldwide problem endangering human health, and are the third global public health problems after cardiovascular diseases and tumors.

Nicotine, also known as nicotine, is a potent parasympathomimetic alkaloid, which is the main active ingredient in cigarettes. Nicotine dependence is a major characteristic of smokers and refers to the physiological and psychological changes that an individual causes after repeated nicotine use, including increased craving and difficulty in controlling use, sustained and preferential use without compromising outcome, increased tolerance and withdrawal symptoms. Tobacco dependence is one of the very serious public health problems at present. WHO states that tobacco is life-deprived every year for over 700 million people, of which over 600 are derived from direct tobacco use, and about 89 million are non-smokers exposed to second-hand smoke.

Repeated use of opioids, including morphine and fentanyl, leads to analgesic tolerance and requires increased doses to achieve the same analgesic effect. Chronic or repeated use of opioids can also lead to addiction, both in physical dependence (physical dependence) and in mental dependence (psychological dependence). Physical dependence is repeated for the avoidance of withdrawal symptoms, and as the tolerance dose is gradually increased, it manifests as an aversive effect in the course of addiction, playing a role in negative reinforcement. The mental dependence refers to the craving of psychological drug-seeking of the depended and the euphoria achieved by repeated drug-seeking, and is expressed as a reward effect, plays a positive reinforcement effect and promotes the repeated drug-seeking of the depended.

Active ingredient

As used herein, "active ingredient of the present invention" and "active compound of the present invention" are used interchangeably and refer to protopanaxadiol compounds, including protopanaxadiol and protopanaxatriol. Wherein the protopanoxadiol comprises 20(S) -protopanaxadiol, 20(R) -protopanaxadiol, or a combination thereof (e.g., a racemate). The protopanaxatriol comprises 20(S) -protopanaxatriol, 20(R) -protopanaxatriol, or a combination thereof (e.g., a racemate). In addition, the term includes natural products or artificially synthesized or modified products.

It is to be understood that the active ingredients of the present invention include the active compounds of the present invention (protopanaxadiol, protopanaxatriol, or a combination thereof), or pharmaceutically acceptable salts or esters, enantiomers, diastereomers, or racemates thereof, or prodrugs thereof. It is to be understood that the active ingredients of the present invention also include crystalline, amorphous, solvate, hydrate, etc. forms of the active compounds of the present invention.

The "pharmaceutically acceptable salts (or esters)" are the conventional non-toxic salts (or esters) formed by reacting the active compounds of the present invention with inorganic or organic acids. For example, conventional non-toxic salts can be prepared by reacting the active compounds of the present invention with inorganic acids including hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid and the like, or organic acids including 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, 2-acetoxybenzoic acid, isethionic acid and the like; or sodium, potassium, calcium, aluminum or ammonium salts of the active compounds of the invention which are esterified with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid and then with an inorganic base; or the corresponding inorganic acid salt formed by the active compound of the invention and lysine, arginine and ornithine after forming ester and then hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid, or the corresponding organic acid salt formed by the active compound of the invention and formic acid, acetic acid, picric acid, methanesulfonic acid or ethanesulfonic acid.

Other analgesic drugs

1) Opioid analgesics including codeine, dihydrocodeine, morphine, fentanyl, sufentanil, remifentanil, pethidine, oxycodone;

3) the nonsteroidal anti-inflammatory analgesic drugs comprise flurbiprofen axetil, ibuprofen, diclofenac sodium, meloxicam, naproxen, celecoxib and prorexib;

4) monoamine neurotransmitter reuptake inhibitor antidepressants including amitriptyline and doxoxetine;

5) local anesthetics include lidocaine, ropivacaine, prilocaine;

6) norepinephrine alpha 2 receptor agonists such as clonidine and dexmedetomidine;

8) anti-migraine agents such as CGRP antibodies and receptor antagonists thereof;

9) the Chinese herbal medicine comprises radix Lamiophlomidis Rotatae extract and its effective components such as shanzhiside methyl ester and 8-O-acetyl shanzhiside methyl ester, radix Aconiti Kusnezoffii/radix Aconiti lateralis and its effective components such as bulleyaconitine A, lappaconitine A and radix Aconiti Brachypodi Aconitum, and rhizoma corydalis and its effective components such as tetrahydropalmatine, stephanine, corydaline and dehydrocorydaline.

Pharmaceutical compositions and methods of administration

The invention also provides a composition or a preparation or a product containing the active ingredients of the invention, and the composition or the preparation or the product can be used for resisting aging. Representative compositions or formulations or products include anti-aging drugs, nutraceuticals, and cosmetics.

One preferred composition is a pharmaceutical composition comprising an effective amount of verapamil or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity (i.e., anti-aging function) in a human and/or animal and is acceptable to the human and/or animal.

As used herein, an ingredient of the term "pharmaceutically acceptable" is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "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 vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical composition of the invention can be prepared into injections, oral preparations (tablets, capsules, oral liquids), transdermal agents and sustained-release agents. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: 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 route of administration, and the like. In general, satisfactory results are obtained when the active ingredients of the invention are administered at a daily dosage of about 0.001 to 100mg/kg of animal body weight (preferably 0.01 to 50mg/kg, more preferably 0.05 to 20 mg/kg). For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

Typically, when the active ingredient of the invention is administered orally, preferably in humans, the oral dose may be 0.05-50mg/kg, preferably 0.10-20 mg/kg.

The main advantages of the invention include:

a) the active ingredients of the invention have the advantages of effective analgesia and no generation of analgesia tolerance, physical dependence, mental dependence and addiction.

b) The active ingredients of the invention can treat or relieve addiction induced by opioid drugs, including physical and mental dependence, while effectively relieving pain.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for which specific conditions are not indicated in the following examples are generally carried out according to conventional conditions, for example as described in (Sambrook et al, molecular cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press,1989), or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

General method for experiment

1 rat intrathecal catheter:

rats were rapidly anesthetized with 5% isoflurane under a respiratory anesthesia machine (anesthesia machine airflow rate 0.3L/min), followed by maintaining anesthesia with 2% isoflurane. An 18-cm polyethylene catheter (PE-10: outer diameter: 0.55mm, inner diameter: 0.3mm) was inserted along the spinal column from the level of the lumbar region of the rat. One week before drug treatment, intubation condition was detected with 10 μ L of 4% lidocaine, and if rats were paralyzed on both sides and were not dyskinesia after recovery, the success of intubation was demonstrated in the subsequent experiments by intrathecal injection under arachnoid.

2 rat formalin pain model:

rats were acclimated for 30 minutes in transparent observation cages of 23X 35X 19cm size prior to the experiment. The left foot of the rat was removed and injected subcutaneously with 5% formalin solution (50 μ l), and immediately after injection the rat was placed in an observation cage and the number of foot lifts within 60 seconds was measured every 10 minutes after injection until the 90 minute cut-off.

3 rat spinal nerve ligation neuropathic pain model:

rats were rapidly anesthetized with 5% isoflurane under a respiratory anesthesia machine (anesthesia machine airflow rate 0.3L/min), followed by maintaining anesthesia with 2% isoflurane. Blunt dissection of the left muscle at the lumbar spinal cord, exposure and removal of the L6 transverse process, exposure of the L5 nerve and tightening with 6-0 silk; the fascia was removed near the angle below the sacrum, and the L6 nerve was teased out and secured with 6-0 silk thread. The rats were fed in a single cage for one week after surgery. The mechanical pain threshold of the hind sole is measured by a Von Frey electronic pain measuring instrument, and the model is successfully made if the threshold is less than 8g and no dyskinesia exists, and the model is used for subsequent experiments.

4 rat bone cancer pain model:

female rats were anesthetized with 50mg/kg intraperitoneal injection of pentobarbital sodium, a 0.5-cm incision was made in the tibia, the tibia was exposed after blunt muscle dissection, and holes were gently drilled in the middle of the tibia using a 5-gauge needle. Followed by microinjector injection of 10. mu.L Walker 256 Breast cancer cells (4X 10)⁵one/mL), the needle was stopped for 30 seconds after the injection was completed, the needle was withdrawn, and the wound was immediately sealed with sterile bone wax, and then sterilized, sutured, and placed in a cage. After two weeks, the mechanical pain threshold of the feet was determined, and rats < 8g were considered as successful in modeling and used for subsequent experiments.

5 rat inflammatory pain model:

isoflurane anesthetized animals, 100 μ L CFA was slowly injected in the tibiotarsal joint of the left hind paw. The mechanical and thermal pain thresholds were determined two days after CFA injection.

6 rat mechanical and thermal pain threshold determination:

rats were placed on mechanical and thermal pain testing racks, respectively. For rat mechanical pain determination, an electronic mechanical pain threshold detector is used for vertically stimulating the middle part of the sole of the hind limb of a rat, No. 15 fibers are installed on the detector, during measurement, the stimulation force is slowly increased until the fibers are bent into S-shaped, the duration is 6-8 seconds, and whether the rat has a foot contraction or foot lifting reaction is observed. The minimum threshold for the contraction or lifting of the rat foot was recorded as the foot contraction threshold (PWT). The test was performed every 3 minutes in triplicate, and the average of the triplicates was used as the mechanical pain threshold for that foot in the rat. The mechanical pain threshold reflects the degree of mechanical stimulation injury/pain in the rat. For rat thermal pain measurement, a radiant heat source vertically placed below a glass plate at the palm of the hind limb of a rat is turned on for detection, the total time from the beginning of receiving the radiant heat source to the sudden foot licking of the rat foot is observed as a pain threshold value after the foot is stimulated by the radiant heat, and the pain threshold value is expressed by a paw with paw side latency. The measurement was repeated three times every 5 minutes with 30 seconds as the maximum measurement threshold, and the average of the three times was used as the radiant heat pain threshold of the foot of the rat. The radiant heat pain threshold reflects the degree of thermal stimulation injury/pain in the rat.

7 mouse Conditional Place Preference (CPP) model establishment:

the 10-day CPP model includes three phases, a pre-test phase, an acquisition phase and a post-test phase. Pre-test period (1-4 days): male Swiss mice were allowed to freely shuttle in three compartments, 2 times daily for 15 minutes each, for 3 days. On day 4, the residence time of the mice freely shuttled to each of the three compartments within 15 minutes was recorded. Acquisition period (5-9 days): mice were injected subcutaneously with morphine (10mg/kg) or saline (10mL/kg) for 5 days alternately every 6 hours (9: 00 am and 3:00 pm), immediately placed in the compartment, and trained for 45 minutes. On

days

5, 7 and 9 of the acquisition period, morphine (10mg/kg) and saline (10mL/kg) were subcutaneously injected at 9:00 am and 3:00 pm, respectively, and put in a morphine companion box and a saline counterpart box for training for 45 minutes. On

days

6 and 8, the injection time of morphine and saline was exchanged. Post-test period (day 10): mice were allowed free access to the three compartments for 15 minutes for testing. The conditional site preference score is the time the mouse spends in the partner box minus the time spent in the saline counterpart box. The shuttling activity of the mice in each compartment was photographed by a 3CCD camera and the residence time of the mice in each compartment was recorded using EthoVision XT 8.0 software.

8, primary cell culture:

wistar rats of either sex were isolated within 24 hours of birth to obtain spinal cords, which were then denuded, minced, and digested with 0.05% trypsin in a 5% carbon dioxide incubator for 7-9 minutes. Centrifuging to remove supernatant, blowing off and resuspending the tissue digested at bottom in the centrifuge tube with 10% FBS and 1% double-resistant DMEM medium, sequentially filtering with 70-and 40- μm sieves, and inoculating to polylysine (0.1mg/ml) coated 75cm²The mixture was placed in a 5% carbon dioxide incubator and cultured for 10 days.

When preparing microglia, the culture bottle is put into a shaker to shake (260rpm) at 37 ℃ for 1.5-2 hours, cell suspension is collected, centrifuged, cells are resuspended, then the cell suspension is inoculated into a new cell culture plate, and non-adhered cells are washed away by pre-warmed PBS the next day. The purity of the obtained microglia is more than 95 percent through immunofluorescence assay of the microglia labeled protein Iba-1.

In the preparation of astrocytes, the cultured cells were discarded from the medium, washed twice with PBS, and then 0.05% of pancreatin containing EDTA was added. Digestion at 37 ℃ for 3 minutes removes oligodendrocytes, terminates digestion and removes the cell suspension, leaving adherent monolayers of astrocytes that continue to be passaged with pancreatin for subsequent use. The purity of the obtained astrocytes is more than 90% by immunofluorescence assay of the astrocyte marker protein GFAP.

When neuronal cells were prepared, the cell suspension was filtered through a 40- μm mesh screen, inoculated into a 10-cm cell culture dish, and placed in a cell incubator for 30 minutes. The nonadherent supernatant cell suspension was then aspirated and seeded into polylysine plates. After 1.5-2 hours of culture, DMEM was replaced with Neurobasal medium containing 1 XB 27 neurotrophic factor and 0.5mM glutamine, and culture was continued for 3-4 days. The purity of the obtained neuron cells is more than 85 percent through immunofluorescence assay of a neuron cell marker protein NeuN.

9 total RNA extraction and real-time quantitative PCR determination of cells and tissues:

pentobarbital sodium (50mg/kg, i.p.) was anesthetized, the head was rapidly broken and bleeding was released, the tissue of enlarged spinal cord and lumbar (L3-L5) was removed, Trizol reagent (50mg/ml) was added proportionally to the homogenate to extract and precipitate RNA, and finally, an appropriate amount of DEPC water was added to dissolve it according to the amount of RNA precipitated. And (3) determining the concentration and purity of the extracted RNA by adopting a micro enzyme-labeling instrument.

The reverse transcription kit is adopted to run corresponding programs on a common PCR instrument, and the extracted total RNA is reversely transcribed into cDNA and then stored at the temperature of minus 20 ℃ for later use. The subsequent real-time quantitative PCR was performed by using SYBR qPCR mix to detect Ct values of dynorphin precursor gene (PDYN), endorphin precursor gene (POMC), enkephalin precursor gene (PNOC), and Nociceptin/orphaninFQ precursor gene (PENK), using GAPDH as a reference gene and 2^-ΔΔCtThe method calculates the relative expression level of the target gene.

10-dynorphin a and beta-endorphin protein content determination:

the rat removed the tissue of the spinal cord lumbar enlargement (L3-L5), homogenized (4,000rpm, 15 seconds) with 10mM Tris-HCl (5mL/1g tissue), and the supernatant obtained after centrifugation (5000rpm) at 4 ℃ for 15 minutes. Furthermore, primary cell administration treatment of neonatal rat spinal cord origin cell culture supernatants were collected after 2 hours of culture. According to the specification of the enzyme linked immunosorbent assay kit, the content of dynorphin A and beta-endorphin in cell culture and spinal cord tissue supernatant is determined.

11 tissue immunofluorescence staining:

rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50mg/kg) and the chest was opened along the inferior margin of the sternal xiphoid process, exposing and freeing the heart. The needle was quickly inserted into the aorta via the left ventricle, the needle was secured with a number 4-0 surgical suture, and the right atrial appendage was cut. After the blood was washed by slowly pouring 100ml of physiological saline, 60ml of 4% formaldehyde solution was continuously poured. Then taking out the spinal cord lumbar enlargement part (L3-L5), placing in 4% formaldehyde stationary liquid at 4 ℃ overnight, and then sequentially carrying out gradient dehydration, embedding and frozen section (the thickness is 30 mu m) through sucrose solution and preserving at-20 ℃ for later use. The cryopreserved tissue sections were rewarming and then blocked with blocking solution for 1 hour at room temperature, followed by incubation of primary antibodies (dynorphin A antibody, microglia marker Iba-1, astrocyte marker GFAP and neuronal cell marker NeuN) with blocking solution for 18-24 hours at 4 ℃. After the primary antibody incubation is finished, adding a blocking solution for preparing a secondary antibody, culturing for 1 hour at 37 ℃, then sealing by using an anti-fluorescence quenching sealing agent, and storing at-20 ℃ in a dark place for later use. Images were taken using a Leica TCS SP8 confocal laser microscope and analyzed for fluorescence quantification and fluorescent staining co-localization using Image J Image processing software.

Example 1 analgesic Effect of oral 20(S) -Protopanaxadiol in a model rat model of neuropathic pain

Method

The neuropathic pain rats with L5/L6 spinal nerve ligation were selected and randomly divided into six groups (6 per group). One group of oral solvents (2: 7:1 ratio of ethanol to propylene glycol: distilled water, 6.5mL/kg) and 5 groups of 20(S) -protopanaxadiol were orally administered at different doses (five groups of oral PPD doses were 1, 3, 10, 30 or 100mg/kg, respectively). The paw withdrawal response threshold or paw withdrawal latency time of rats to mechanical and thermal stimuli was determined at various time points before and at 0.5, 1, 2, 4 hours after dosing (thermal stimuli were performed 10 minutes after mechanical stimuli). The mechanical pain threshold and thermal radiation pain threshold were measured for each dosing group at 1 hour to calculate the% maximum possible effect (% maximum possible effect,% MPE), and then dose-response analysis was performed.

Results

The mechanical pain threshold and thermal pain threshold of rats in the normal saline group were substantially unchanged from the healthy side and the surgically affected side within the test period of 4 hours, while oral administration of 20(S) -protopanaxadiol inhibited the mechanical pain sensitivity (FIGS. 1A and 1B) and thermal pain sensitivity (FIGS. 1C and 1D) of rats in the surgically affected side dose-dependently, but did not affect the healthy side pain threshold. The analgesic effect can last for about 3 hours, and the time point with the strongest analgesic effect is 1 hour.

The maximum analgesic effect of oral 20(S) -protopanaxadiol in mechanical and thermal nociceptive pain caused by nerve ligation was E, respectively, as shown by the results of dose-response analysis of FIGS. 1B and 1D _max61% and 68% MPE, ED₅₀5.8 and 4.5mg/kg, respectively.

In conclusion, the oral administration of 20(S) -protopanaxadiol can inhibit neuropathic pain, and the extent of this inhibition is positively dependent on the dose of 20(S) -protopanaxadiol administered. In addition, the analgesic effect of the oral 20(S) -protopanoxadiol is very obvious.

Example 2 analgesic effect of oral 20(S) -protopanaxadiol in rat model of pain caused by different etiologies.

Method

To verify the analgesic effect of 20(S) -protopanaxadiol on other pain models of different etiologies, we used a bone cancer pain model, a CFA-induced inflammatory pain model, and a formalin-induced pain model.

Two groups of bone cancer pain rats (6 per group) and two groups of CFA-induced inflammatory pain rats (6 per group) were orally administered either vehicle (6.5mL/kg) or 20(S) -protopanaxadiol (100mg/kg), respectively, and the withdrawal thresholds or withdrawal latencies of the rats to mechanical and thermal radiation stimuli were determined at different time points before and after administration, 0.5, 1, 2, 4 hours (thermal radiation stimulation was performed 10 minutes after mechanical stimulation).

In addition, two groups of rats (6 per group) were orally administered either solvent (6.5mL/kg) or 20(S) -protopanaxadiol (100mg/kg) 30 minutes prior to plantar injection of 5% formalin.

Results

Oral administration of 20(S) -protopanaxadiol significantly alleviated rat bone cancer pain (FIGS. 2A and 2B) and CFA-induced inflammatory pain (FIGS. 2C and 2D).

Formalin can elicit phase I and phase II licking responses in rats. Oral administration of 20(S) -protopanaxadiol inhibited phase II licking times in rat formalin but had no effect on phase I pain (FIG. 2E).

Example 3 synergistic analgesic Effect of oral administration of 20(S) -Protopanaxadiol in combination with gabapentin or morphine

Method

To verify the interaction between 20(S) -protopanaxadiol and gabapentin or morphine, the minimum effective dose of 20(S) -protopanaxadiol (3mg/kg), gabapentin (10mg/kg) and morphine (0.3mg/kg) was administered. The first experiment four groups of rats (6 per group) were dosed with solvent (6.5mL/kg) + physiological saline (3mL/kg), 20(S) -protopanaxadiol (3mg/kg) + physiological saline (3mL/kg), solvent (6.5mL/kg) + gabapentin (10mg/kg) and 20(S) -protopanaxadiol (3mg/kg) + gabapentin (10mg/kg), respectively.

A second experiment four groups of rats (6 per group) were administered solvent (6.5mL/kg) + physiological saline (3mL/kg), 20(S) -protopanaxadiol (3mg/kg) + physiological saline (3mL/kg), solvent (6.5mL/kg) + morphine (0.3mg/kg) and 20(S) -protopanaxadiol (3mg/kg) + morphine (0.3mg/kg), respectively.

The paw withdrawal response threshold or paw withdrawal latency to mechanical and thermal stimuli (thermal stimuli were performed 10 minutes after mechanical stimuli) was determined for each rat at various time points, pre-dose and 0.5, 1, 2, 4 hours after dose administration.

Results

Small doses of 20(S) -protopanaxadiol or gabapentin administered alone had 20% MPE and 22% MPE, respectively, for anti-mechanociceptive effects 1 hour after administration, and 20% MPE and 22% MPE, respectively, for anti-thermnociceptive effects30% MPE, the analgesic effect is weaker at this dose. However, when the combination of the gabapentin and the 20(S) -protopanaxadiol is administrated, compared with any single administration, the combination can obviously enhance the mechanical pain sensitivity resistance to 55 percent of MPE and enhance the thermal pain sensitivity to 62.2 percent of MPE. Using the formula of King (q ═ E)_A+B)/(E_A+E_B-E_A*E_B) Calculation (jinzhengzhang, chinese pharmacology, 1: 70-76, 1980; jinzhengyun, zhangxianwu, written by shanghai second college of medicine, 1: 15-18, 1981), q values of 1.46 and 1.40, respectively, both greater than 1.15. This indicates that gabapentin and 20(S) -protopanaxadiol administered in combination significantly enhanced the relief of mechanical and thermal hyperalgesia in rats, showing a synergistic analgesic effect (fig. 3A and 3B).

Similarly, small doses of morphine or 20(S) -protopanaxadiol administered alone, produced a weaker analgesic effect with 20% MPE and 25% MPE respectively for the anti-mechanociceptive effect and 20% MPE and 27% MPE respectively for the anti-thermal nociceptive effect 1 hour after administration. The combination of the two increases the anti-mechanical hyperalgesia effect to 54% MPE and the thermal hyperalgesia effect to 74% MPE. Calculated by using the gold formula, the q values of the two are respectively 1.35 and 1.78 which are both larger than 1.15. This indicates that the combination of the two drugs produced significant synergistic analgesic effect (fig. 3C and 3D).

Example 4 specific stimulation of rat spinal microglia cell expression of dynorphin A by oral administration of 20(S) -protopanoxadiol

Method

Two groups of L5/L6 neuropathic pain rats (6 rats in each group) with spinal nerve ligation were orally administered with solvent (6.5mL/kg) or 20(S) -protopanaxadiol (100mg/kg), respectively, one hour after the administration, the head was broken and the side spinal cord tissue was operated at the lumbar vertebra bulge (L3-L5). The PDYN, POMC, PENK and PNOC gene expression levels were determined by real-time quantitative PCR and are shown in FIG. 4A. And simultaneously, the levels of dynorphin A and beta-endorphin in spinal cord homogenate supernatant are determined by an enzyme-linked immunosorbent assay. The results are shown in FIG. 4B. Two groups of sham-operated rats were additionally orally administered with physiological saline (6.5mL/kg) or 20(S) -protopanaxadiol (100mg/kg), decapitated one hour after administration and lateral spinal cord tissue was surgically removed from the lumbar enlargement (L3-L5) to determine opioid peptide gene and protein expression, the results of which are shown in FIGS. 4C and 4D.

Two groups of neuropathic pain rats are selected, and a co-staining experiment is carried out on spinal cord sections by adopting an immunofluorescence staining method: images of the co-staining of dynorphin A with microglia marker protein Iba-1, astrocyte marker protein GFAP or neuronal marker protein NeuN are shown in FIGS. 5A-5N.

Further, primary spinal microglia were treated with different concentrations of 20(S) -protopanaxadiol (1, 3, 10, 30 and 100 μ M), and after 2 hours of culture, microglial dynorphin a gene and protein expression were detected, as shown in fig. 6A and 6B; primary spinal astrocytes and neuronal cells were treated with 20(S) -protopanaxadiol (100 μ M) and after 2 hours of culture, astrocytes and neuronal cells were examined for dynorphin a gene and protein expression, as shown in fig. 6C and 6D.

Results

As can be seen from FIG. 4A, in the rats with neuropathic pain with spinal nerve ligation, administration of 20(S) -protopanoxadiol resulted in a specific elevation of spinal cord PDYN expression without affecting POMC, PENK and PNOC expression. And the administration of 20(S) -protopanaxadiol specifically stimulated the release of dynorphin A from the spinal cord without affecting the release of beta-endorphin (FIG. 4B).

As can be seen from fig. 4C, oral administration of 20(S) -protopanaxadiol also significantly increased spinal cord PDYN gene expression without increasing POMC, PENK or PNOC gene expression levels in the sham-operated rat model. At the same time, oral administration of 20(S) -protopanaxadiol significantly increased myelodyrphin A protein expression without affecting beta-endorphin protein expression (FIG. 4D)

As can be seen from FIGS. 5A-5D, both dynorphin A and microglia Iba-1 were immunofluorescent co-expressed in spinal cord, and oral administration of 20(S) -protopanaxadiol significantly increased the co-expression of both dynorphin A and Iba-1 in spinal cord. The co-stained area of the administered group was 2.1 times as large as that of the control group by ImageJ quantitative analysis (fig. 5E).

As can be seen from FIGS. 5F-5I and 5K-5N, dynorphin A was co-expressed with either the astrocytic marker protein GFAP or the neuronal marker protein NeuN, also in rat spinal cord.

As seen in FIG. 5J, the co-staining area of dynorphin A and GFAP was not significantly changed in the case of oral administration of 20(S) -protopanaxadiol (administered group) as compared with the case of administration of physiological saline (control group).

Similarly, as can be seen from FIG. 5O, oral administration of 20(S) -protopanoxadiol did not significantly change the co-staining area of dynorphin A and NeuN.

As can be seen in FIGS. 6A and 6B, treatment with 20(S) -protopanaxadiol dose-dependently increased spinal microglial expression of dynorphin A gene and protein, ED₅₀The values were 13 and 19.8. mu.M, respectively. As can be seen in fig. 6C and 6D, 20(S) -protopanaxadiol did not significantly alter spinal astrocytes or neurons expression of dynorphin a gene or protein.

EXAMPLE 5 intrathecal Pre-administration of the Microglia activation inhibitor minocycline, dynorphin A antiserum and specific kappa-opioid receptor antagonists in the subarachnoid space for the inhibition of analgesia by 20(S) -protopanaxadiol

Method

Two groups of neuropathic pain rats (6 per group) were pre-injected intrathecally with physiological saline (10 μ L) or minocycline (100 μ g) the microglial activation inhibitor, respectively. After 4 hours, 20(S) -protopanaxadiol (100mg/kg) was administered orally to both groups. The results of measuring the paw withdrawal response threshold to mechanical stimulation and the paw withdrawal response latency to thermal radiation in rats before the first dose, before the second dose and at 0.5, 1, 2 and 4 hours after the dose are shown in fig. 7A and 7B.

Three groups of neuropathic pain rats (6 per group) were pre-injected intrathecally with either blank rabbit serum (10 μ L), dynorphin a antiserum (1:10, 10 μ L) or β -endorphin antiserum (1:10, 10 μ L) in the subarachnoid space. After 0.5 hours, 20(S) -protopanaxadiol (100mg/kg) was orally administered to three groups. The results of measuring the withdrawal response threshold of rat hind paw to mechanical stimulation and withdrawal response latency of thermal radiation are shown in FIGS. 8A and 8B.

Dynorphin a is an endogenous kappa-opioid receptor agonist, and in order to verify whether the analgesic effect of 20(S) -protopanoxadiol is achieved by agonizing kappa-opioid receptors, the following verification was made: four groups of neuropathic pain rats (6 per group) were injected intrathecally with saline (10 μ L), the μ -opioid receptor antagonist CTAP (10 μ g), the κ -opioid receptor antagonist GNTI (50 μ g) or the δ -opioid receptor antagonist naltrindole (5 μ g) separately in the subarachnoid space. After 0.5 hour, 20(S) -protopanaxadiol (100mg/kg) was orally administered to four groups of rats, and the results are shown in FIGS. 8C and 8D.

Results

As can be seen from fig. 7A and 7B, oral administration of 20(S) -protopanaxadiol produced time-dependent analgesia, while minocycline did not affect the basal threshold of pain, but completely inhibited the analgesia produced by 20(S) -protopanaxadiol.

As can be seen from fig. 8A and 8B, oral administration of 20(S) -protopanaxadiol produced time-dependent analgesia, and dynorphin a antiserum did not affect the basal threshold of pain, but completely inhibited the analgesia produced by 20(S) -protopanaxadiol, whereas β -endorphin antiserum failed to block the analgesia produced by 20(S) -protopanaxadiol.

As can be seen from fig. 8C and 8D, oral administration of 20(S) -protopanaxadiol (100mg/kg) produced time-dependent analgesic effect, GNTI did not affect the basal threshold of pain, but completely inhibited the analgesic effect produced by 20(S) -protopanaxadiol, whereas CTAP or naltrindole failed to block the analgesic effect produced by 20(S) -protopanaxadiol.

Example 6 inhibition of analgesic tolerance and physical dependence of morphine by oral administration of 20(S) -protopanaxadiol

Four groups of neuropathic pain rats (6 per group) were used, and each group was administered with solvent (6.5mL/kg) + physiological saline (1mL/kg), 20(S) -protopanaxadiol (30mg/kg) + physiological saline (1mL/kg), solvent (6.5mL/kg) + morphine (3mg/kg) or 20(S) -protopanaxadiol (30mg/kg) + morphine (3mg/kg) twice daily for 7 consecutive days. The paw withdrawal response threshold to mechanical stimulation and the paw withdrawal response latency to thermal radiation were measured 1 hour after each morning dose and the results are shown in fig. 9A and 9B.

On the morning of day 8, four groups of rats were orally administered 20(S) -protopanaxadiol (30mg/kg), and the hind paw withdrawal thresholds for mechanical stimulation and the withdrawal latencies for thermal radiation were measured before and at 0.5, 1, 2 and 4 hours after administration. Four groups of rats were given morphine (3mg/kg) 6 hours after 20(S) -protopanaxadiol administration, and the hindfoot pain threshold was determined for the next 4 hours. The results are shown in FIGS. 9C and 9D.

The oral administration was continued for another 3 days to the above rats, and at 4 hours after the last morning administration, the rats were intraperitoneally injected with naloxone (5mg/kg), and then immediately observed for withdrawal symptoms of the rats within 30 minutes, with the results shown in FIGS. 10A to 10E.

As can be seen from fig. 9A and 9B, the analgesic effect of 20(S) -protopanaxadiol remained unchanged for 7 days, whereas the analgesic effect of morphine gradually became tolerated and finally completely disappeared within 7 days. Compared with the single use of 20(S) -protopanoxadiol or morphine, the simultaneous administration of 20(S) -protopanaxadiol and morphine can not only produce obvious analgesic synergistic effect, but also completely inhibit morphine analgesic tolerance reaction.

As can be seen from fig. 9C and 9D, four groups of rats, each administered with physiological saline, 20(S) -protopanoxadiol, morphine, and a combination of 20(S) -protopanaxadiol + morphine continuously for one week, all produced significant time-dependent analgesia in the rats after a single oral dose of 20(S) -protopanaxadiol. Furthermore, a single dose of subcutaneous morphine failed to produce analgesia in rats that were morphine-resistant given a week of continuous morphine administration; however, a single dose of subcutaneous morphine injection was given to the group administered with physiological saline, the group administered with 20(S) -protopanaxadiol, and the group administered with 20(S) -protopanaxadiol + morphine for one week continuously, which resulted in significant analgesic effects.

Figures 10A-10E show that oral administration of 20(S) -protopanaxadiol produced no physical dependence, while morphine produced significant physical dependence, and that the combined use of 20(S) -protopanaxadiol (30mg/kg) significantly reduced morphine-related withdrawal symptoms, including tremor (figure 10A), jumping (figure 10B), tooth tremor (figure 10C), diarrhea (figure 10D) and wet dog-like tremor (figure 10E), compared to saline control.

Example 7 inhibition of conditional positional preference for morphine (CPP) by oral administration of 20(S) -protopanaxadiol

Two groups of mice (10 per group) were orally administered either solvent (10mL/kg) or 20(S) -protopanaxadiol (100mg/kg) alternately daily for 5 consecutive days, followed by the conditional location preference test, and the results are shown in FIG. 11A.

Four additional groups of mice (10 per group) were injected subcutaneously with either normal saline (10mL/kg) or morphine (10mg/kg) daily for 5 days, 50 minutes prior to the last injection, with a single oral dose of either solvent (10mL/kg) or 20(S) -protopanaxadiol (100mg/kg), followed immediately by 15 minutes of site preference testing, as shown in figure 11B.

As can be seen from fig. 11A, both groups of mice did not obtain conditional positional preference in both the pre-test period and the post-test period, indicating that long-term oral administration of 20(S) -protopanoxadiol did not produce conditional positional preference.

As can be seen in fig. 11B, none of the four groups of mice showed a conditional positional preference during the previous test period. In the post-test period, the saline group showed no conditional site preference, but the group injected subcutaneously with morphine showed a clear conditional site preference. Oral administration of 20(S) -protopanaxadiol (100mg/kg) in a single dose did not affect the conditioned place preference response in the saline group, but completely blocked the acquisition of morphine-induced conditioned place preference.

EXAMPLE 8 INHIBITING EFFECT OF INTENCALLY PRE-ADMINISTERING OF GLUCOCORTICOID RECEPTOR ANTAGONISTS TO ANALGESIC 20(S) -PROPANEDIOL

Two groups of neuropathic pain rats (6 per group) were pre-injected intrathecally with solvent (10 μ L) or the non-specific glucocorticoid receptor antagonist RU1486(10nmol), respectively, in the subarachnoid space. After 0.5 hours, 20(S) -protopanaxadiol (100mg/kg) was orally administered to both groups of rats. The paw withdrawal response thresholds of the rat hind paw to mechanical stimulation were determined before the first dose, before the second dose and at 0.5, 1, 2 and 4 hours after the dose. Oral administration of 20(S) -protopanaxadiol produced time-dependent analgesia, and RU1486 did not affect the basal threshold of pain, but completely inhibited the analgesia produced by 20(S) -protopanaxadiol (fig. 12A).

Two additional groups of neuropathic pain rats (6 per group) were pre-injected intrathecally with solvent (10 μ L) or the specific glucocorticoid receptor antagonist dexamethasone 21-mesylate (Dex-21-mesylate,10nmol), respectively. After 0.5 hours, 20(S) -protopanaxadiol (100mg/kg) was orally administered to both groups of rats. The paw withdrawal response thresholds of the rat hind paw to mechanical stimulation were determined before the first dose, before the second dose and at 0.5, 1, 2 and 4 hours after the dose. Oral administration of 20(S) -protopanaxadiol produced time-dependent analgesia, and Dex-21-mesylate did not affect the basal threshold of pain, but completely inhibited the analgesia produced by 20(S) -protopanaxadiol (FIG. 12B). In addition, two groups of neuropathic pain rats (6 per group) were pre-injected intrathecally with solvent (10. mu.L) or Dex-21-mesylate (10nmol), respectively, in the subarachnoid space. After 0.5 hours, both groups of rats were injected subcutaneously with bulleyaconine A (BAA, 300. mu.g/kg). The paw withdrawal response thresholds of the rat hind paw to mechanical stimulation were determined before the first dose, before the second dose and at 0.5, 1, 2 and 4 hours after the dose. Oral subcutaneous injection of bulleyaconitine A produced time-dependent analgesia, but Dex-21-mesylate did not affect the analgesia produced by bulleyaconitine A (FIG. 12C).

Three groups of neuropathic pain rats (6 per group) were pre-injected intrathecally with solvent (10 μ L) or estrogen receptor antagonist G15(10nmol or 1 μmol), respectively, in the subarachnoid space. After 0.5 hours, three groups of rats were orally administered 20(S) -protopanaxadiol (100 mg/kg). The withdrawal response threshold of the rat hind paw to mechanical stimulation was determined. Oral administration of 20(S) -protopanaxadiol produced time-dependent analgesia, and G15 affected neither the basal threshold of pain nor the analgesia produced by 20(S) -protopanaxadiol (fig. 12D). Another group of neuropathic pain rats (6 per group) were pre-injected intrathecally with solvent (10. mu.L) or the aldosterone receptor antagonist eperenone (10nmol), respectively. After 0.5 hours, three groups of rats were orally administered 20(S) -protopanaxadiol (100 mg/kg). The withdrawal response threshold of the rat hind paw to mechanical stimulation was determined. Oral administration of 20(S) -protopanaxadiol produced a time-dependent analgesic effect, with eporenone affecting neither the basal threshold of pain nor the analgesic effect produced by 20(S) -protopanaxadiol (fig. 12E).

The results show that 20(S) -protopanaxadiol produces analgesia by specifically exciting the glucocorticoid receptor of the spinal cord, in contrast to bulleyaconitine A.

Example 9 inhibition of 20(S) -Protopanaxadiol-stimulated dynorphin A expression by glucocorticoid receptor antagonists

Four groups of L5/L6 spinal nerve-ligated neuropathic pain rats (6 per group) were pre-injected intrathecally with solvent (10 μ L) or the specific glucocorticoid receptor antagonist Dex-21-mesylate (10nmol), respectively, subarachnoid space. After 0.5 hour, four groups of rats were orally administered either solvent (6.5mL/kg) or 20(S) -protopanoxadiol (100mg/kg), respectively. After one hour of administration, the head was broken and the spinal cord tissue was surgically harvested from the lumbar spine bulge (L3-L5). The expression level of PDYN gene was determined by real-time quantitative PCR. The results showed that 20(S) -protopanoxadiol specifically elevated PDYN expression in myelodynorphin A gene, and Dex-21-mesylate did not affect spinal cord-based PDYN expression, but completely blocked PDYN expression induced by 20(S) -protopanaxadiol (FIG. 13A). And simultaneously, the level of dynorphin A in spinal cord homogenate supernatant is determined by an enzyme-linked immunosorbent assay. The results show that oral administration of 20(S) -protopanaxadiol significantly increased spinal dynorphin a protein expression, and that Dex-21-mesylate did not affect spinal cord-based dynorphin a expression, but completely blocked 20(S) -protopanaxadiol-induced dynorphin a expression (fig. 13B).

Further, after 0.5 hour of the administration of the solvent or glucocorticoid receptor antagonist Dex-21-mesylate (100nM) to the primary cultured spinal microglia cells, 20(S) -protopanaxadiol (100 μ M), specific glucocorticoid receptor agonist Dex (100nM), or cell membrane-impermeable conjugate Dex-BSA (10nM) of Bovine Serum Albumin (BSA) was administered and cultured for 2 hours, respectively, followed by the detection of the gene and protein expression of dynorphin a in the microglia cells. As can be seen in fig. 14A and 14B, 20(S) -protopanaxadiol, Dex, and Dex-BSA significantly enhanced the expression of the dynorphin a gene in microglia; while Dex 21-melylate did not affect the basal expression of dynorphin gene in microglia, but completely inhibited the stimulatory effect of 20(S) -protopanoxadiol, Dex and Dex-BSA on dynorphin A gene expression. FIG. 14B shows that Dex-21-melylate did not affect the basal expression of dynorphin A protein in microglia, but completely inhibited the stimulatory effect of 20(S) -protopanaxadiol, Dex and Dex-BSA on dynorphin A protein expression.

The above results demonstrate that 20(S) -protopanaxadiol produces analgesia by stimulating the glucocorticoid receptor (probably the cell membrane glucocorticoid receptor) of spinal microglia, stimulating the expression and release of dynorphin a by microglia.

Discussion of the related Art

The biological activity of 20(S) -protopanaxadiol has been reported, but the use of 20(S) -protopanaxadiol for treating pain has not been reported before in the present invention, and physical dependence and mental dependence useful for treating opioid (or addictive substance) have not been disclosed. The invention provides that 20(S) -protopanaxadiol has obvious analgesic effect on models such as neuropathic pain, cancer pain, inflammatory pain, formalin pain and the like of rats/mice; long-term administration of 20(S) -protopanaxadiol does not produce analgesic tolerance, physical dependence and conditioned site preference (mental dependence); 20(S) -protopanaxadiol is effective in inhibiting morphine-induced analgesic tolerance, physical dependence and psychotropic tolerance.

The research of the inventor shows that the main analgesic part of 20(S) -protopanaxadiol is in spinal cord, and 20(S) -protopanaxadiol can extremely effectively promote the expression and release of dynorphin A by exciting a glucocorticoid receptor (possibly a cell membrane glucocorticoid receptor) of spinal microglia cells, thereby unexpectedly producing the analgesic effect and the effect of quitting the physical dependence and the mental dependence of treating opioid (or other addictive substances).

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of an active ingredient or a formulation containing said active ingredient, wherein said 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;

(a) drugs for the treatment and/or alleviation of pain; and/or

2. The use of claim 1, wherein the addictive substance is selected from the group consisting of: an opioid, heroin, or a combination thereof.

3. The use according to claim 1, wherein the pain is selected from the group consisting of: neuropathic pain, inflammatory pain, arthritic pain, diabetic pain, low back pain, spinal cord injury pain, visceral pain, fibromyalgia, chronic regional pain syndrome, musculoskeletal pain, cancer pain, pain caused by antineoplastic and opioid drugs, post-operative pain, post-traumatic neuralgia and peripheral neuropathy, phantom limb pain, or a combination thereof.

4. The use of claim 1, wherein the formulation further comprises a second active ingredient; wherein the second active ingredient is selected from the group consisting of:

(Z10) any combination of the above Z1 to Z9.

5. The use of claim 1, wherein the formulation is an oral formulation, or an injection.

6. A pharmaceutical composition, comprising:

(ii) a second active ingredient selected from the group consisting of:

(Z10) any combination of Z1 to Z9;

(iii) a pharmaceutically acceptable carrier and/or excipient.

7. Use of a pharmaceutical composition according to claim 6 for the preparation of:

(a) drugs for the treatment and/or alleviation of pain;

8. An in vitro method of treating and/or alleviating pain, comprising the steps of:

(1) culturing cells in a system comprising an effective amount of a first active ingredient or a formulation comprising said active ingredient or a pharmaceutical composition according to claim 6, wherein said 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;

(2) agonizing the cellular glucocorticoid receptor to express dynorphin a.

9. A method of treating and/or alleviating pain, comprising the steps of:

administering to a subject in need thereof a medically effective amount of a first active ingredient or a formulation comprising said active ingredient or a pharmaceutical composition according to claim 6, wherein said 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; thereby treating and/or relieving pain.

10. A method for inducing expression and release of dynorphin a, comprising: administering to a subject in need thereof an active ingredient selected from the group consisting of: protopanaxadiol or a pharmaceutically acceptable salt or ester thereof, protopanaxatriol or a pharmaceutically acceptable salt or ester thereof, thereby inducing the subject to produce dynorphin A.