CN114159450B

CN114159450B - Use of protopanaxadiol compounds for treating physical dependence, mental dependence and addiction of pain and addiction substances

Info

Publication number: CN114159450B
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: 2023-10-27
Anticipated expiration: 2040-09-10
Also published as: WO2022052996A1; CN114159450A

Abstract

The present invention provides the use of protopanaxadiol compounds in the treatment of pain and addictive substances somatic and mental dependencies. In particular, the invention provides protopanaxadiol compounds (especially 20 (S) -protopanaxadiol) and pharmaceutically acceptable salts or esters thereof, for preparing a) a medicament for treating and/or alleviating pain; b) Use of a medicament for the treatment and/or alleviation of addictive substances, in particular opioid-induced physical and/or mental dependencies. Experiments show that the 20 (S) -protopanaxadiol can stimulate the expression and release of dynorphin A (dynorphin A) by exciting the spinal cord microglial glucocorticoid receptor, so that the effect of treating and/or relieving pain is achieved, and the 20 (S) -protopanaxadiol can be used together with other analgesic drugs, especially gabapentin and opioid drugs to achieve the synergistic effect of relieving pain.

Description

Use of protopanaxadiol compounds for treating physical dependence, mental dependence and addiction of pain and addiction substances

Technical Field

The invention relates to the technical field of medicines, in particular to application of protopanaxadiol compounds in treating pain and addiction substances such as somatic body, mental dependence and addiction.

Background

Pain is a compound, unpleasant sensation associated with injury and pain. Pain, under normal physiological conditions, can provide an alarm signal when the body is threatened, and is an indispensable life protection function. However, in pathological conditions, pain is a common symptom of most diseases, and is interwoven with autonomic nervous activities, motor reflex, psychological and emotional reactions, etc., which brings pain to patients.

Pain is classified into acute pain and chronic pain according to the cause, nature, location and time course. Acute pain refers to pain caused by direct activation of nociceptors in the corresponding sites by nociceptive stimuli in physiological conditions. Acute pain is not long in duration (< 1 month) and self-disappears after injury repair. Acute pain includes postoperative pain, post-traumatic pain, acute headache and facial pain, acute arthritic pain, and the like.

Chronic pain is one in which pain persists after focal repair, which can last for months (> 1 month) or even for life, or can 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, post-traumatic neuralgia and peripheral neuropathy, etc.

According to the International Pain institute (International Association for the Study of Pain, IASP) data, the prevalence of chronic Pain is 10% worldwide (Scholz et al, paint, 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 diabetics are diagnosed with diabetic peripheral neuropathy, and one third of herpes zoster virus patients develop postherpetic neuralgia, with more than one million people suffering from cancer pain. Pain brings pain to patients, seriously affects family life or work, and also brings great burden to public health systems.

The common pain treatment drugs are: 1) Non-steroidal anti-inflammatory analgesics include flurbiprofen axetil, ibuprofen, diclofenac sodium, meloxicam, naproxen, celecoxib, and primixib;

2) Antiepileptic drugs include carbamazepine, phenytoin sodium and gabapentin drugs such as gabapentin, pregabalin and milabalin;

3) Monoamine neurotransmitter re-uptake inhibitor antidepressants include amitriptyline and duloxetine;

4) Local anesthetics include lidocaine, ropivacaine, prilocaine;

5) Opioid analgesics include codeine, dihydrocodeine, morphine, fentanyl, sufentanil, remifentanil, pethidine, oxycodone;

6) Norepinephrine α2 receptor agonists such as clonidine, dexmedetomidine;

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

8) The Chinese herbal medicines comprise radix Lamiophlomidis Rotatae, aconite/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, these drugs have limited pain or efficacy or produce more serious adverse effects. As gabapentin Ding Herui pregabalin is not a specific analgesic, its effectiveness in treating neuropathic pain (lowering pain threshold by 30%) is less than 50% of the population. The nonsteroidal analgesic has certain curative effects on headache, toothache, muscle and joint pain, and the like, but has almost no effect on traumatic severe pain and visceral smooth muscle angina. Local anesthetics are only suitable for peripheral neuropathic pain.

For example, opioid drugs have various adverse reactions such as sleepiness, respiratory depression, constipation, etc., and can produce analgesic tolerance, hyperalgesia, physical dependence and addiction and abuse after long-term administration. Gabapentin Ding Herui pregabalin also has severe adverse effects such as somnolence. The repeated use of opioids, including morphine and fentanyl, produces analgesic tolerance in the body, and the dosage must be increased to achieve the same analgesic effect. Prolonged or repeated use of opioids can also lead to addiction, including both physical dependence (physical dependence, physiological dependence) and mental dependence (psychological dependence).

Physical dependence is repeated administration to avoid withdrawal symptoms, and as tolerance dosage increases gradually, aversion effect is exhibited in the addiction process, and negative strengthening effect is achieved. Mental dependency refers to the craving of psychological foraging of a relying person and euphoria achieved by repeated administration of drugs, and the psychological dependence is expressed as a rewarding effect, plays a positive strengthening effect and promotes frequent re-inhalation of patients. The existing method and approach for solving the addiction of opium still have great limitation, and methadone, buprenorphine, coladine, lofexidine and the like only improve withdrawal symptoms to a certain extent, have very limited curative effect, and especially have few curative effects on mental dependence.

Therefore, pain treatment is still a clinical problem at present, and development of novel analgesic drugs which can be used for a long time and have no analgesic tolerance and addiction per se and can effectively treat pain and opioid-induced physical and mental dependence is urgently needed.

Disclosure of Invention

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

In particular, the invention provides the application of protopanaxadiol compounds (such as 20 (S) -protopanaxadiol) in preparing novel analgesic drugs for treating pains and opioid-induced physical and mental dependencies. Experiments show that the 20 (S) -protopanoxadiol can be combined with other active ingredients to achieve the synergistic analgesic effect. And, 20 (S) -protopanaxadiol expresses and releases dynorphin a by agonizing spinal cord microglial glucocorticoid receptor (cell membrane glucocorticoid receptor), thereby producing an analgesic effect.

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 pharmaceutically acceptable salt or ester thereof, protopanaxatriol or pharmaceutically acceptable salt or ester thereof;

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

(a) A medicament for treating and/or alleviating pain; and/or

(b) Drugs for treating and/or alleviating physical and/or mental dependencies and/or addiction induced by addictive substances.

In another preferred embodiment, the protopanoxadiol comprises 20 (S) -protopanoxadiol, 20 (R) -protopanoxadiol, 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: opioids, heroin, or combinations thereof.

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

In another preferred embodiment, the pain is selected from the group consisting of: neuropathic pain, inflammatory pain, arthritic pain, diabetic pain, lower back pain, spinal cord injury pain, visceral pain, fibromyalgia, chronic regional pain syndrome, musculoskeletal pain, cancer pain, pain caused by antineoplastic drugs and opioids, postoperative pain, post-traumatic neuralgia and peripheral neuropathy, phantom limb pain, or combinations thereof.

In another preferred embodiment, the neuropathic pain includes, but is not limited to, post-herpetic 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, pethidine, oxycodone, or combinations thereof;

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

(Z3) a non-steroidal anti-inflammatory analgesic drug selected from the group consisting of: flurbiprofen axetil, ibuprofen, sodium diclofenac, meloxicam, naproxen, celecoxib, apraxib, or a combination thereof;

(Z4) a monoamine neurotransmitter re-uptake 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 noradrenergic 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 a combination thereof;

(Z8) an antimigraine 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 components, radix Aconiti lateralis Preparata and its effective components, rhizoma corydalis and its effective components, or their combination;

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

In another preferred embodiment, the gabapentin compound includes gabapentin, pregabalin and milobalin (mirogalin).

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

In another preferred embodiment, the aconite/aconite and its active ingredients comprise: bulleyaconitine A, lappaconitine A and radix Aconiti Brachypodi A.

In another preferred example, the corydalis tuber and its active ingredients include tetrahydropalmatine, stephanine, corydaline, 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) analgesic tolerance; (2) body dependence; (3) mental dependency (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: protopanoxadiol and 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 combinations thereof;

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

(iii) Pharmaceutically acceptable carriers and/or excipients.

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

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

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

(a) A medicament for treating and/or alleviating pain;

(b) Drugs for treating and/or alleviating physical and/or mental dependencies induced by addictive substances.

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 cells in a system comprising an effective amount of a first active ingredient or a formulation comprising the active ingredient or a pharmaceutical composition according to the second aspect, wherein the first active ingredient is selected from the group consisting of: protopanaxadiol or pharmaceutically acceptable salt or ester thereof, protopanaxatriol or 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 combinations 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 and 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 pharmaceutically effective amount of a first active ingredient or a formulation comprising the active ingredient or a pharmaceutical composition according to the second aspect, wherein the first active ingredient is selected from the group consisting of: protopanaxadiol or pharmaceutically acceptable salt or ester thereof, protopanaxatriol or pharmaceutically acceptable salt or ester thereof; thereby treating and/or alleviating pain.

In a sixth aspect of the invention there is provided a method of inducing dynorphin a expression and release 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 spinal cord to increase expression and release of enkephalin a.

In another preferred embodiment, the method is for agonizing a glucocorticoid receptor in a cell 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 embodiment, 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.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Figure 1 shows that oral administration of 20 (S) -protopanoxadiol dose-dependently inhibits mechanical and thermal pain in neuropathic pain.

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 stimulated expression of dynorphin a gene and protein in rat spinal cord.

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

Figure 6 shows that ex vivo administration of 20 (S) -protopanaxadiol specifically stimulated primary spinal cord microglia to express dynorphin a genes and proteins.

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

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

Fig. 9 shows that oral administration of 20 (S) -protopanaxadiol does not produce self-analgesic tolerance, but inhibits morphine analgesic tolerance.

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

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

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

Fig. 13 shows the effect of pre-administration of a glucocorticoid receptor antagonist in the subarachnoid space to inhibit dynorphin a expression in the spinal cord produced by oral administration of 20 (S) -protopanaxadiol.

Fig. 14 shows that glucocorticoid receptor antagonists inhibit the expression of dynorphin a produced by 20 (S) -protopanaxadiol in primary spinal cord microglia.

Detailed Description

The present inventors have studied extensively and intensively, and have found for the first time that protopanaxadiol compounds (especially 20 (S) -protopanaxadiol) can significantly inhibit pain and have an obvious anti-addiction effect without causing adverse reactions such as analgesic tolerance, physical dependence, mental dependence, etc. Thus can be used for treating pain and anti-addiction (such as physical and mental dependence induced by addictive substances). The present invention has been completed on the basis of this.

In particular, 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 and mental dependence effects induced by addictive substances while simultaneously easing pain. The protopanaxadiol compound can also be combined with other analgesic drugs to achieve the synergistic analgesic effect. The protopanaxadiol compound can achieve the effect of easing pain by specifically stimulating the expression of the dynorphin A.

Terminology

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

As used herein, the term "comprising" or "including" 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 "somatic dependence (physical dependence)", "physical dependence" and "physiological dependence" are used interchangeably to refer to the dependence that can cause withdrawal syndrome upon discontinuation of the addictive drug.

In the present invention, the term "withdrawal syndrome" refers to a series of symptoms such as sweating, tearing, yawning, chills, chicken skin, mydriasis, vomiting, diarrhea, abdominal pain, increased heart rhythm, increased blood pressure, insomnia, tremors, etc., which occur due to continuous use of addictive substances, so that a patient who develops dependency once discontinues use, generates severe physiological reactions by his body.

In the present invention, the term "mental dependency (psychological dependence)" is used interchangeably with "psychological dependency" and refers to the craving of a patient for a drug in order to obtain a particular feeling of well-being after taking an addictive drug.

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

Pain and pain

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 post-partum 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, pain resulting from wounds or medical procedures such as surgery (e.g., capsulotomy or hip, knee or other joint replacement), suturing, fracture reduction, biopsy, and the like. Pain can also occur in patients with cancer, which can be caused by a variety of causes such as inflammation, nerve compression, and mechanical forces caused by tumor invasion and tissue swelling due to tumor metastasis to bone or other tissue.

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

Addiction to drugs

Drug addiction and drug dependence are chronic recurrent brain diseases, and are mainly manifested by forced administration behavior of addictive drugs and uncontrollable drug dosage. After the substance-dependent condition, if the taking of the medicine is stopped suddenly, the withdrawal symptoms of the medicine may occur. Many drugs originally used for medical purposes may cause substance-dependent phenomena; addictive substances are known as drugs if they are regulated by law and considered as illegal. Such addictive substances include opioids and heroin, methamphetamine, cocaine, marijuana, alcohol, nicotine, and the like.

Methamphetamine, commonly known as methamphetamine, is a highly addictive stimulant, the second most commonly used illegal drug worldwide. Abuse of methamphetamine or other amphetamine-type agonists has become an important public health issue. Compared with traditional drugs such as heroin, cocaine and the like, the methamphetamine has the advantages of simple synthesis process, low cost and easy availability of precursors, stronger excitation effect on a central nervous system, fewer times of drug administration or accumulated drug administration required for forming addiction and more serious damage to the body of a drug addict.

Alcohol is a psychoactive substance with highly addictive properties. Global alcohol dependence patients reach 1.4 billion, and their abuse and dependence impose serious adverse effects and economic burden on individuals and society. About 330 tens of thousands of people worldwide die each year due to excessive alcohol use. The harmful use of alcohol can also lead to diseases such as alcoholic liver, liver cirrhosis and the like. Alcohol abuse, alcohol addiction, has become a serious public health disaster and a worldwide problem of jeopardizing human health, being a third global public health problem, next to cardiovascular diseases and tumors.

Nicotine, also called nicotine, is a potent parasympathetic alkaloid and is the main active ingredient in cigarettes. Nicotine dependency is a major characteristic of smokers and it refers to individuals who, after repeated use of nicotine, cause physiological and psychological changes, including enhanced craving and difficult control of use, sustained and preferential use regardless of the consequences of harm, increased tolerance and withdrawal symptoms. Tobacco dependence is one of the most serious public health problems at present. WHO notes that tobacco is lost to more than 700 tens of thousands of people each year, of which 600 are from direct use of tobacco and about 89 are non-smokers WHO are exposed to second-hand smoke.

The repeated use of opioids, including morphine and fentanyl, produces analgesic tolerance in the body, and the dosage must be increased to achieve the same analgesic effect. Prolonged or repeated use of opioids can also lead to addiction, including both physical dependence (physiological dependence) and mental dependence (psychological dependence). Physical dependence is repeated administration to avoid withdrawal symptoms, and as tolerance dosage increases gradually, aversion effect is exhibited in the addiction process, and negative strengthening effect is achieved. Mental dependency refers to the craving of psychological foraging of a relying person and euphoria achieved by repeated administration of drugs, and the psychological dependence is expressed as a rewarding effect, plays a positive strengthening effect and promotes frequent re-inhalation of patients.

Active ingredient

As used herein, "active ingredients of the present invention" and "active compounds of the present invention" are used interchangeably and refer to protopanaxadiol compounds, including protopanaxadiol and protopanaxatriol. Wherein the protopanaxadiol comprises 20 (S) -protopanaxadiol, 20 (R) -protopanaxadiol, or a combination (e.g., racemate) thereof. The protopanaxatriol comprises 20 (S) -protopanaxatriol, 20 (R) -protopanaxatriol, or a combination (such as racemate) thereof. Furthermore, 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 a pharmaceutically acceptable salt or ester, enantiomer, diastereomer or racemate thereof, or a prodrug thereof. It is to be understood that the active ingredients of the present invention also include crystalline, amorphous, solvated, hydrated, etc. forms of the active compounds of the present invention.

The "pharmaceutically acceptable salts (or esters)" are those which are formed by reacting the active compounds of the present invention with inorganic or organic acids to form conventional non-toxic salts (or esters). 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 with 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 the active compounds of the invention form esters 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 form sodium, potassium, calcium, aluminum or ammonium salts with inorganic bases; or the active compounds according to the invention form esters with lysine, arginine, ornithine and then with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid or with formic acid, acetic acid, picric acid, methanesulfonic acid or ethanesulfonic acid.

Other analgesic drugs

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

3) Non-steroidal anti-inflammatory analgesics include flurbiprofen axetil, ibuprofen, diclofenac sodium, meloxicam, naproxen, celecoxib, and primixib;

4) Monoamine neurotransmitter re-uptake inhibitor antidepressants include amitriptyline and duloxetine;

5) Local anesthetics include lidocaine, ropivacaine, prilocaine;

6) Norepinephrine α2 receptor agonists such as clonidine and dexmedetomidine;

8) Antimigraine 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 methyl shanzhiside and 8-O-acetyl shanzhiside, aconite root/radix Aconiti lateralis Preparata and its effective components such as bulleyaconitine A, lappaconitine and radix Aconiti Brachypodi A, and rhizoma corydalis and its effective components such as tetrahydropalmatine, stephanine, corydaline and dehydrocorydaline.

Pharmaceutical compositions and methods of administration

The invention also provides compositions or formulations or products containing the active ingredients of the invention, which compositions or formulations or products are useful for anti-aging. Representative compositions or formulations or products include anti-aging drugs, health products, 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.

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

As used herein, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., commensurate with 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 compositions of the present invention contain a safe and effective amount of the active ingredients 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. Generally, the pharmaceutical preparation is matched with the administration mode, and the dosage forms of the pharmaceutical composition are injection, oral preparation (tablet, capsule and oral liquid), transdermal agent and sustained release agent. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. The pharmaceutical compositions are preferably manufactured under sterile conditions.

The effective amount of the active ingredient described herein may vary depending upon the mode of administration, the severity of the condition being treated, and the like. The selection of the 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 in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. Generally, satisfactory results are obtained when the active ingredient of the present invention is administered at a daily dose of about 0.001-100mg/kg of animal body weight (preferably 0.01-50mg/kg, more preferably 0.05-20mg/kg of animal body weight). For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

Typically, when the active ingredient of the present invention is administered orally, preferably, the administration subject is a human, the oral dosage may be 0.05-50mg/kg, preferably 0.10-20mg/kg.

The main advantages of the invention include:

a) The active ingredients of the invention can effectively relieve pain and simultaneously do not produce analgesic tolerance, physical dependence, mental dependence and addiction.

b) The active ingredients of the invention can effectively relieve pain and simultaneously treat or relieve opioid-induced addiction including physical and mental dependencies.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, in which no specific conditions are noted in the examples below, is generally followed by conventional conditions, for example those described in (Sambrook et al, molecular cloning: A laboratory Manual, new York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations. Percentages and parts are weight percentages and parts unless otherwise indicated.

Experimental general method

1 rat sheath inner tube:

rats were rapidly anesthetized with 5% isoflurane under a respiratory anesthesia machine (anesthesia machine airflow rate 0.3L/min), followed by maintenance of anesthesia with 2% isoflurane. An 18-cm polyethylene catheter (PE-10: outer diameter: 0.55mm, inner diameter: 0.3 mm) was inserted from the lumbar level of the rat along the spinal column. One week prior to drug treatment, the cannulation was examined with 10 μl of 4% lidocaine, and if rats were given bilateral hindfoot flaccidity following intrathecal injection, and no dyskinesia was observed following recovery, indicating that the cannulation was successful and a follow-up trial was performed.

2 formalin pain model in rats:

rats were acclimatized for 30 minutes in transparent observation cages of 23X 35X 19cm in size prior to the experiment. The left foot of the rat was removed and injected subcutaneously with 5% formalin (50 μl) and immediately after injection the rat was placed in the observation cage and the number of lifts in 60 seconds was measured every 10 minutes after injection until 90 minutes had expired.

3 model of neuropathic pain of spinal nerve ligation in rats:

rats were rapidly anesthetized with 5% isoflurane under a respiratory anesthesia machine (anesthesia machine airflow rate 0.3L/min), followed by maintenance of anesthesia with 2% isoflurane. Blunt separation of left muscle at lumbar medulla, exposure and removal of L6 transverse processes, exposure of L5 nerve and tightening with 6-0 silk thread; the fascia surrounding the lower sacral angle was removed, and the L6 nerve was picked up and tied with 6-0 silk. The single cage rearing of the rats after operation is recovered for one week. The mechanical pain threshold of the sole after the measurement is carried out by using a Von Frey electronic pain measuring instrument, the mechanical pain threshold of the sole is less than 8g, and no dyskinesia is regarded as successful molding and is used for subsequent experiments.

4 model of bone cancer pain in rats:

female rats were anesthetized with 50mg/kg intraperitoneal injection of pentobarbital sodium, a 0.5-cm incision was made in the tibia of the rats, the tibia was exposed after blunt muscle separation, and a 5-gauge needle was used to gently drill a hole in the middle of the tibia. Along with it Post-microinjector 10. Mu.L Walker 256 breast cancer cells (4X 10) ⁵ and/mL), stopping the needle for 30 seconds after the injection is completed, immediately sealing the wound with sterile bone wax by pulling out the needle, and then sterilizing, suturing and placing the wound in a cage. After two weeks the mechanical pain threshold of the foot was determined and < 8g rats were considered successful in modeling for subsequent experiments.

5 model of inflammatory pain in rats:

isoflurane anesthetized animals, 100 μl CFA was slowly injected in the tibialis joint of the left hindpaw. Mechanical pain and thermal pain thresholds were determined two days after CFA injection.

6 measurement of mechanical pain and thermal pain threshold in rats:

rats were placed on mechanical pain and thermal pain test racks, respectively. For the mechanical pain measurement of rats, an electronic mechanical pain threshold detector is used for vertically stimulating the midfoot of hind limbs of the rats, the 15 # fiber is arranged on the detector, and the stimulation force is slowly increased until the fiber is bent into an S shape for 6-8 seconds during measurement, so as to observe whether the rats have foot contraction or foot lifting reaction. The minimum threshold for foot contractions or lifts of the rats was recorded as foot contractions threshold (paw withdrawal threshold, PWT). The test was repeated three times every 3 minutes, and the average of the three times was used as the mechanical pain threshold of the foot of the rat. The mechanical pain threshold reflects the degree of mechanical stimulus injury/pain in the rat. For the rat thermal pain measurement, the radiation heat source vertically arranged below the glass plate at the hind limb sole of the rat is turned on for detection, the rat sole is observed to start receiving the radiation heat source until suddenly licking the foot, and the total length of time for lifting or shrinking the foot is the pain threshold after the rat is stimulated by the radiation heat, and the pain threshold is expressed by the foot shrinking response latency (paw withdrawal latency, PWL). The measurement was repeated three times every 5 minutes with 30 seconds as the maximum measurement threshold, and the average value of the three times was used as the radiation heat pain threshold of the foot of the rat. The radiant heat pain threshold reflects the degree of thermal-stimulated injury/pain in the rat.

7 mouse conditional position preference (conditioned place preference, CPP) model establishment:

the CPP model for 10 days included 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 per day for 15 minutes for 3 days. On day 4, the residence time of the mice freely shuttled through each of the three compartments for 15 minutes was recorded. Acquisition period (5-9 days): mice were injected subcutaneously with 5-day morphine (10 mg/kg) or normal saline (10 mL/kg) alternately every 6 hours (9:00 am and 3:00 pm), then immediately placed in the compartments and trained for 45 minutes. On days 5, 7 and 9 of the acquisition phase, morphine (10 mg/kg) was subcutaneously injected at 9:00 am and physiological saline (10 mL/kg) was subcutaneously injected at 3:00 pm, respectively, and placed in morphine concomitant and physiological saline paired boxes for training for 45 minutes. On days 6 and 8, the injection times of morphine and physiological saline were exchanged. Post test period (day 10): mice were allowed to freely enter and exit the three compartments for 15 minutes for testing. The conditional location preference score is calculated by subtracting the time spent in the physiological saline paired bin from the time the mice remained in the concomitant bin. The shuttle movement of the mice in each compartment was photographed by a 3CCD camera and the time the mice remained in each compartment was recorded using EthoVision XT 8.0 software.

8 primary cell culture:

wistar rats of any sex were isolated from spinal cord within 24 hours of birth, and after membrane stripping and shearing, 0.05% pancreatin was added to digest for 7-9 minutes in a 5% carbon dioxide incubator. Centrifuging to remove supernatant, blowing and resuspending digested tissue in centrifuge tube bottom with 10% FBS and 1% double-antibody DMEM medium, sequentially filtering with 70-and 40- μm screens, inoculating to polylysine (0.1 mg/ml) coated 75cm ² The flask was placed in a 5% carbon dioxide incubator for 10 days.

When microglial cells are prepared, the culture flask is put into a shaking table to shake (260 rpm) at 37 ℃ for 1.5-2 hours, cell suspension is collected, centrifuged and resuspended, then the cells are inoculated into a new cell culture plate, and the non-adherent cells are washed off by PBS (phosphate buffered saline) with pre-temperature in the next day. The purity of the obtained microglial cells is more than 95 percent through immunofluorescence measurement of microglial cell marker protein Iba-1.

When astrocytes were prepared, the cultured cells were discarded in the medium, washed twice with PBS, and 0.05% of pancreatin containing EDTA was added. The oligodendrocytes were removed by digestion at 37 ℃ for 3 minutes, the digestion was stopped and the cell suspension removed, and the remaining adherent monolayer astrocytes were passaged with pancreatin for subsequent use. The purity of the obtained astrocytes is more than 90% by immunofluorescence measurement of the astrocyte marker protein GFAP.

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

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

sodium pentobarbital (50 mg/kg, i.p.) is used for anesthetizing rats, rapidly breaking the head and bleeding, taking out spinal cord and waist enlargement (L3-L5) tissues, adding Trizol reagent (50 mg/ml) in proportion for homogenizing to extract and precipitate RNA, and finally adding a proper amount of DEPC water according to the precipitation amount of the RNA to dissolve the RNA. And (5) measuring the concentration and purity of the extracted RNA by adopting a micro enzyme-labeled instrument.

And (3) performing reverse transcription on the extracted total RNA into cDNA by adopting a reverse transcription kit on a common PCR instrument, and preserving at-20 ℃ for later use. Subsequent real-time quantitative PCR procedures used SYBR qPCR mix to detect dynorphin precursor gene (PDYN), endorphin precursor gene (POMC), enkephalin precursor gene (PNOC), and Nociceptin/OrphaninFQ precursor gene (PENK) Ct values with GAPDH as the reference gene, using 2 ^-ΔΔCt The method calculates the relative expression quantity of the target gene.

10 dynorphin a and beta-endorphin protein content assay:

rats were removed from spinal cord lumbar expansion (L3-L5) tissue, homogenized (4,000 rpm,15 seconds) with 10mM Tris-HCl (5 mL/1g tissue), and the supernatant obtained after centrifugation (5000 rpm) at 4℃for 15 minutes. In addition, cell culture supernatants were collected after 2 hours of treatment culture of primary cells derived from spinal cord of neonatal rats. The dynorphin a and beta-endorphin content in cell culture and spinal tissue supernatants was determined according to the enzyme-linked immunosorbent assay kit instructions.

11 tissue immunofluorescent staining:

rats were anesthetized with sodium pentobarbital (50 mg/kg) and the chest was opened along the lower edge of the xiphoid process of the sternum, exposing and freeing the heart. The needle was inserted into the aorta through the left ventricle rapidly, the needle was fixed with a surgical suture thread No. 4-0, and the right atrial appendage was cut open. After the blood was washed by slowly pouring 100ml of normal saline, 60ml of 4% formaldehyde solution was continuously poured. Then taking out the spinal cord lumbar expansion part (L3-L5), placing the spinal cord lumbar expansion part in 4% formaldehyde fixing solution at 4 ℃ overnight, and then sequentially carrying out gradient dehydration, embedding, frozen section (with the thickness of 30 mu m) and preservation at-20 ℃ for later use. The frozen tissue sections were incubated with blocking solution for 1 hour at room temperature, followed by preparation of primary antibodies (dynorphin A antibody, microglial marker Iba-1, astrocyte marker GFAP and neuronal cell marker NeuN) using blocking solution and incubation at 4℃for 18-24 hours. After the incubation of the primary antibody is finished, adding a secondary antibody prepared sealing solution, culturing for 1 hour at 37 ℃, and then sealing the sealing tablet by using an anti-fluorescence quenching sealing tablet, and preserving the sealing tablet at-20 ℃ in a dark place for standby. Imaging was performed using a Leica TCS SP8 laser confocal microscope and fluorescence quantification and fluorescence staining co-localization analysis was performed with Image J Image processing software.

EXAMPLE 1 analgesic effect of oral 20 (S) -Protopanaxadiol in neuropathic pain model rat models

Method

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

Results

During the test period of 4 hours, the mechanical pain threshold and the thermal radiation pain threshold of the healthy side and the operation affected side of the rats in the physiological saline group were substantially unchanged, whereas oral administration of 20 (S) -protopanaxadiol can dose-dependently inhibit mechanical pain sensitivity (fig. 1A and 1B) and thermal radiation pain sensitivity (fig. 1C and 1D) of the operation affected side of the rats, but does 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.

From the results of the dose-response analysis of FIGS. 1B and 1D, it was shown that oral administration of 20 (S) -protopanoxadiol had E as the maximum analgesic effect in mechanical and thermal radiation pain caused by nerve ligation, respectively _max 61% and 68% MPE, ED ₅₀ 5.8 and 4.5mg/kg, respectively.

Taken orally, 20 (S) -protopanoxadiol inhibits neuropathic pain, and the extent of this inhibition is positively dependent on the 20 (S) -protopanoxadiol dose taken. In addition, the analgesic effect of the oral administration of 20 (S) -protopanaxadiol is very obvious.

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

Method

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

Two groups of bone cancer pain rats (6 each) and two groups of CFA-induced inflammatory pain rats (6 each) were orally administered solvent (6.5 mL/kg) or 20 (S) -protopanaxadiol (100 mg/kg), respectively, and the paw withdrawal response threshold or paw withdrawal latency time (heat radiation stimulation was performed 10 minutes after mechanical stimulation) of the rats was measured at different time points of 0.5, 1, 2, and 4 hours before and after administration.

In addition, two groups of rats (6 each) were orally administered solvent (6.5 mL/kg) or 20 (S) -protopanoxadiol (100 mg/kg) 30 minutes before plantar injection of 5% formalin.

Results

Oral administration of 20 (S) -protopanaxadiol significantly reduced bone cancer pain in rats (fig. 2A and 2B) and CFA-induced inflammatory pain (fig. 2C and 2D).

Formalin can cause phase I and phase II licking responses in rats. Oral administration of 20 (S) -protopanaxadiol inhibited phase II licking times induced by formalin in rats, but had no effect on phase I pain (fig. 2E).

Example 3 synergistic analgesic effects of oral 20 (S) -protopanoxadiol in combination with gabapentin or morphine

Method

To verify the interaction between 20 (S) -protopanaxadiol and gabapentin or morphine, minimum effective doses of 20 (S) -protopanaxadiol (3 mg/kg), gabapentin (10 mg/kg) and morphine (0.3 mg/kg) were administered. Four groups of rats (6 each) were treated with solvent (6.5 mL/kg) +physiological saline (3 mL/kg), 20 (S) -protopanaxadiol (3 mg/kg) +physiological saline (3 mL/kg), solvent (6.5 mL/kg) +gabapentin (10 mg/kg), and 20 (S) -protopanaxadiol (3 mg/kg) +gabapentin (10 mg/kg), respectively.

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

The footshrink response threshold or footshrink latency time of each rat to mechanical and thermal radiation stimulation (thermal radiation stimulation was performed 10 minutes after mechanical stimulation) was measured before and at different time points of 0.5, 1, 2, 4 hours after administration.

Results

The small dose of 20 (S) -protopanaxadiol or gabapentin administered alone had 20% mpe and 22% mpe for antimechanical sensitization at 1 hour after administration, 20% mpe and 30% mpe for thermal sensitization, respectively, and the analgesic effect was weak at this dose. However, the combination of the barpentadine and the 20 (S) -protopanoxadiol significantly enhances the anti-mechanociception to 55% MPE and enhances the thermal nociception to 62.2% MPE compared with any single administration. Using the gold equation (q= (E) _A+B )/(E _A +E _B -E _A *E _B ) Calculation (Jinzhengzhong, chinese pharmacology report, 1:70-76, 1980; jin Zhengjun, zhang Xiaowen, second medical school of Shanghai, 1:15-18, 1981) Q values are 1.46 and 1.40, respectively, each greater than 1.15. This suggests that the co-administration of gabapentin and 20 (S) -protopanaxadiol significantly enhances the relief of mechanical and thermal radiation pain sensitivity in rats, exhibiting a synergistic analgesic effect (FIGS. 3A and 3B).

Also, administration of small doses of morphine or 20 (S) -protopanaxadiol alone resulted in weaker analgesic effects, 20% MPE and 25% MPE for antimechanical pain sensitivity, and 20% MPE and 27% MPE for thermal pain sensitivity, respectively, 1 hour after administration. When the two are combined, the anti-mechanociception effect is increased to 54% MPE, and the thermal radiation pain sensitivity effect is increased to 74% MPE. Calculated by the golden formula, q values of the two are respectively 1.35 and 1.78, which are both larger than 1.15. This suggests that the combination of the two agents produces a significant synergistic analgesic effect (fig. 3C and 3D).

EXAMPLE 4 specific stimulation of oral 20 (S) -Protopanaxadiol on the expression of dynorphin A by rat spinal cord microglia

Method

Two groups of L5/L6 spinal nerve-ligated neuropathic pain rats (6 per group) were orally administered with solvent (6.5 mL/kg) or 20 (S) -protopanaxadiol (100 mg/kg), respectively, and one hour after the administration, the spinal cord tissue was broken and the lumbar vertebra enlargement site (L3-L5) was harvested. PDYN, POMC, PENK and PNOC gene expression levels were determined by real-time quantitative PCR and the results are shown in fig. 4A. Meanwhile, the levels of dynorphin A and beta-endorphin in the supernatant fluid of the spinal cord homogenate are measured by an enzyme-linked immunofluorescence method. The results are shown in FIG. 4B. Two additional groups of sham operated rats were orally administered normal saline (6.5 mL/kg) or 20 (S) -protopanaxadiol (100 mg/kg), and one hour after administration, the spinal cord tissue was broken and the lumbar enlargement (L3-L5) operated on side was taken to determine opioid peptide gene and protein expression, and the results are shown in FIGS. 4C and 4D.

Two groups of neuropathic pain rats are selected, and the spinal cord section is subjected to co-staining experiments by adopting an immunofluorescence staining method: co-staining images of dynorphin A with microglial marker protein Iba-1, astrocyte marker protein GFAP or neuronal cell marker protein NeuN are shown in FIGS. 5A-5N.

Further, primary spinal cord microglia were treated with different concentrations of 20 (S) -protopanaxadiol (1, 3, 10, 30 and 100 μm), and after 2 hours of culture, microglial dynorphin a gene and protein expression were detected as shown in fig. 6A and 6B; primary spinal cord astrocytes and neuronal cells were treated with 20 (S) -protopanaxadiol (100 μm) 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 rats with neuropathic pain with spinal nerve ligation, administration of 20 (S) -protopanaxadiol resulted in specific elevation of spinal PDYN surface without affecting POMC, PENK and PNOC expression. And the administration of 20 (S) -protopanaxadiol in the clear stimulated spinal cord release of dynorphin a specifically without affecting beta-endorphin release (fig. 4B).

As can be seen from fig. 4C, oral administration of 20 (S) -protopanaxadiol also significantly increased spinal 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 spinal cord dynorphin a protein expression without affecting beta-endorphin protein expression (fig. 4D)

From FIGS. 5A-5D, it can be seen that dynorphin A and microglial Iba-1 are immunofluorescent co-expressed in spinal cord, and oral administration of 20 (S) -protopanaxadiol significantly increases the co-expression of dynorphin A and Iba-1. The co-stained area of the dosing group was 2.1 times 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 astrocyte marker protein GFAP or the neuronal cell marker protein NeuN in the rat spinal cord as well.

As can be seen from fig. 5J, the co-staining area of dynorphin a and GFAP was not significantly changed when 20 (S) -protopanaxadiol (administration group) was orally taken as compared with normal saline (control group).

Similarly, as can be seen from fig. 5O, oral administration of 20 (S) -protopanaxadiol did not significantly alter the co-dye area of dynorphin a with NeuN.

As can be seen in FIGS. 6A and 6B, 20 (S) -protopanaxadiol treatment dose-dependently increased spinal cord microglial expression of dynorphin A gene and protein, ED ₅₀ The values were 13 and 19.8. Mu.M, respectively. And from FIG. 6CAnd 6D, it was found that 20 (S) -protopanaxadiol did not significantly alter spinal cord astrocytes or neurons to express dynorphin A gene or protein.

EXAMPLE 5 inhibition of 20 (S) -Protopanaxadiol analgesia by pre-administration of the microglial activation inhibitor minocycline, dynorphin A antiserum and specific kappa-opioid receptor antagonist in the subarachnoid sheath

Method

Two groups of neuropathic pain rats (6 per group) were pre-injected with saline (10 μl) or microglial activation inhibitor minocycline (100 μg) respectively, in subarachnoid sheaths. After 4 hours, both groups were orally administered 20 (S) -protopanoxadiol (100 mg/kg). The paw withdrawal response threshold of the hind paw to mechanical stimulus and the paw withdrawal response latency of the heat radiation were measured before the first dose, before the second dose and at 0.5, 1, 2 and 4 hours after the second dose, and the results are shown in fig. 7A and 7B.

Three groups of neuropathic pain rats (6 per group) were pre-injected with empty rabbit serum (10 μl), dynorphin a antiserum (1:10, 10 μl) or β -endorphin antiserum (1:10, 10 μl) respectively, in subarachnoid space sheath. After 0.5 hour, all three groups were orally administered 20 (S) -protopanoxadiol (100 mg/kg). The hindfoot response threshold to mechanical stimulation and the hindfoot response latency to thermal radiation were measured and the results are shown in fig. 8A and 8B.

Dynorphin a is an endogenous kappa-opioid receptor agonist, to verify whether 20 (S) -protopanaxadiol analgesic effect 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 physiological saline (10. Mu.L), the mu-opioid receptor antagonist CTAP (10. Mu.g), the kappa-opioid receptor antagonist GNTI (50. Mu.g) or the delta-opioid receptor antagonist naltrindole (5. Mu.g), respectively. After 0.5 hour, four groups of rats were orally administered 20 (S) -protopanaxadiol (100 mg/kg), 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 produces a time-dependent analgesic effect, and minocycline does not affect the basal threshold of pain, but completely inhibits the analgesic effect produced by 20 (S) -protopanaxadiol.

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

As can be seen from fig. 8C and 8D, oral administration of 20 (S) -protopanaxadiol (100 mg/kg) produced a 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 somatic dependence of oral 20 (S) -protopanaxadiol

Four groups of neuropathic pain rats (6 per group) were used, each group being given solvent (6.5 mL/kg) +physiological saline (1 mL/kg), 20 (S) -protopanoxadiol (30 mg/kg) +physiological saline (1 mL/kg), solvent (6.5 mL/kg) +morphine (3 mg/kg) or 20 (S) -protopanoxadiol (30 mg/kg) +morphine (3 mg/kg) twice daily, respectively, for 7 consecutive days. The paw withdrawal response threshold of the hind paw to mechanical stimulus and the paw withdrawal response latency of the heat radiation were measured 1 hour after each morning administration and the results are shown in figures 9A and 9B.

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

The rats were continuously administered orally for 3 days, and 4 hours after the last morning administration, the rats were intraperitoneally injected with naloxone (5 mg/kg) and immediately observed for withdrawal symptoms within 30 minutes, and the results are 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, while the analgesic effect of morphine gradually developed tolerance and eventually completely disappeared within 7 days. The simultaneous administration of 20 (S) -protopanaxadiol and morphine not only produces a significant analgesic synergy, but also completely inhibits morphine analgesic tolerance reactions, as compared to 20 (S) -protopanaxadiol or morphine alone.

As can be seen from fig. 9C and 9D, for four groups of rats to which physiological saline, 20 (S) -protopanaxadiol, morphine, 20 (S) -protopanaxadiol+morphine were administered for one continuous week, the rats developed significant time-dependent analgesic effects after oral administration of 20 (S) -protopanaxadiol in a single dose, respectively. Furthermore, single-dose subcutaneous injections of morphine do not produce analgesia in rats that are morphine-tolerant by continuous one week administration of morphine; however, for the physiological saline administration group, the 20 (S) -protopanaxadiol administration group and the 20 (S) -protopanaxadiol+morphine combination administration group which are continued for one week, the single subcutaneous injection of morphine can produce a remarkable analgesic effect.

Figures 10A-10E demonstrate that oral administration of 20 (S) -protopanaxadiol does not produce a physical dependence, whereas morphine produces a significant physical dependence, and that the combined use of 20 (S) -protopanaxadiol (30 mg/kg) significantly reduces morphine-related withdrawal symptoms, including shivering (figure 10A), jumping (figure 10B), tooth tremors (figure 10C), diarrhea (figure 10D) and wet dog-like tremors (figure 10E), as compared to the saline control group.

EXAMPLE 7 inhibition of morphine-Conditional Position Preference (CPP) by oral administration of 20 (S) -protopanoxadiol

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

Four additional groups of mice (10 each) were subcutaneously injected daily with normal saline (10 mL/kg) or morphine (10 mg/kg) for 5 days, and 50 minutes prior to the last injection, the mice were given a single dose of oral solvent (10 mL/kg) or 20 (S) -protopanoxadiol (100 mg/kg) immediately followed by a 15 minute position preference test, as shown in FIG. 11B.

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

As can be seen from fig. 11B, none of the four groups of mice showed conditional positional preference during the pre-test period. During the post-test period, no conditional positional preference appears in the saline group, but the subcutaneous morphine group showed a clear conditional positional preference. Single dose oral administration of 20 (S) -protopanaxadiol (100 mg/kg) did not affect the physiological saline group conditional site-specific response, but completely blocked morphine-induced acquisition of conditional site-specific responses.

EXAMPLE 8 inhibition of 20 (S) -Protopanaxadiol analgesia by Pre-administration of glucocorticoid receptor antagonist within the subarachnoid space sheath

Two groups of neuropathic pain rats (6 per group) were pre-injected with solvent (10 μl) or non-specific glucocorticoid receptor antagonist RU1486 (10 nmol) in the subarachnoid space sheath, respectively. After 0.5 hour, both groups of rats were orally administered 20 (S) -protopanoxadiol (100 mg/kg). The paw withdrawal thresholds of the rat hind paw to mechanical stimulus were measured 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 a time-dependent analgesic effect, RU1486 did not affect the basal threshold of pain, but inhibited the analgesic effect produced by 20 (S) -protopanaxadiol completely (fig. 12A).

Two additional groups of neuropathic pain rats (6 per group) were pre-injected with solvent (10 μl) or specific glucocorticoid receptor antagonist dexamethasone 21-mesylate (Dex-21-mesylate, 10 nmol) respectively, within the subarachnoid space sheath. After 0.5 hour, both groups of rats were orally administered 20 (S) -protopanoxadiol (100 mg/kg). The paw withdrawal thresholds of the rat hind paw to mechanical stimulus were measured 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 a time-dependent analgesic effect, dex-21-mesylate did not affect the basal threshold of pain, but completely inhibited the analgesic effect produced by 20 (S) -protopanaxadiol (fig. 12B). In addition, two groups of neuropathic pain rats (6 per group) were pre-injected with solvent (10. Mu.L) or Dex-21-mesylate (10 nmol), respectively, in the subarachnoid space sheath. After 0.5 hours, both groups of rats were subcutaneously injected with bulleyaconitine A (bulleyaconitine A, BAA, 300. Mu.g/kg). The paw withdrawal thresholds of the rat hind paw to mechanical stimulus were measured 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 analgesic effects, but Dex-21-mesylate did not affect the analgesic effects produced by bulleyaconitine A (FIG. 12C).

Three groups of neuropathic pain rats (6 per group) were pre-injected with solvent (10. Mu.L) or estrogen receptor antagonist G15 (10 nmol or 1. Mu. Mol) in the subarachnoid space sheath, respectively. After 0.5 hour, all three groups of rats were orally administered 20 (S) -protopanoxadiol (100 mg/kg). The paw withdrawal response threshold of the rat hind paw to mechanical stimulus was determined. Oral administration of 20 (S) -protopanaxadiol produced a time-dependent analgesic effect, and G15 did not affect either the basal threshold of pain or the analgesic effect produced by 20 (S) -protopanaxadiol (fig. 12D). Two additional groups of neuropathic pain rats (6 per group) were pre-injected with solvent (10 μl) or aldosterone receptor antagonist eperenone (10 nmol) in the subarachnoid space sheath, respectively. After 0.5 hour, all three groups of rats were orally administered 20 (S) -protopanoxadiol (100 mg/kg). The paw withdrawal response threshold of the rat hind paw to mechanical stimulus was determined. Oral administration of 20 (S) -protopanaxadiol produced a time-dependent analgesic effect, and eperenone did not affect either the basal threshold of pain or the analgesic effect produced by 20 (S) -protopanaxadiol (fig. 12E).

The results demonstrate that 20 (S) -protopanaxadiol, in contrast to bulleyaconitine a, produces analgesic effects by specific agonism of the spinal glucocorticoid receptor.

EXAMPLE 9 inhibition of 20 (S) -Protopanaxadiol by glucocorticoid receptor antagonists on the stimulation of dynorphin A expression

Four groups of L5/L6 spinal nerve-ligated neuropathic pain rats (6 per group) were pre-injected with solvent (10. Mu.L) or specific glucocorticoid receptor antagonist Dex-21-mesylate (10 nmol) in the subarachnoid space sheath, respectively. After 0.5 hours, four groups of rats were orally administered either solvent (6.5 mL/kg) or 20 (S) -protopanoxadiol (100 mg/kg), respectively. One hour after administration, the ends were broken and spinal cord tissue was harvested from the lumbar enlargement (L3-L5) at the surgical side. The PDYN gene expression level was determined by real-time quantitative PCR. The results showed that 20 (S) -protopanaxadiol specifically increased the spinal cord dynorphin a gene PDYN expression, and Dex-21-mesylate did not affect spinal cord basal PDYN expression, but completely blocked 20 (S) -protopanaxadiol-induced PDYN expression (fig. 13A). Meanwhile, the level of dynorphin A in the supernatant fluid of the spinal cord homogenate is measured by an enzyme-linked immunofluorescence method. The results showed that oral administration of 20 (S) -protopanaxadiol significantly increased spinal cord dynorphin a protein expression, dex-21-mesylate did not affect spinal cord basal dynorphin a expression, but completely blocked 20 (S) -protopanaxadiol-induced dynorphin a expression (fig. 13B).

Further, after primary cultured spinal cord microglia were administered with solvent or glucocorticoid receptor antagonist Dex-21-merylate (100 nM) for 0.5 hours, 20 (S) -protopanaxadiol (100. Mu.M), specific glucocorticoid receptor agonist Dex (100 nM) or conjugate of Dex, which does not permeate cell membrane, and Bovine Serum Albumin (BSA) Dex-BSA (10 nM) were administered, respectively, and cultured for 2 hours, followed by detection of microglial dynorphin A gene and protein expression. As can be seen in fig. 14A and 14B, 20 (S) -protopanaxadiol, dex and Dex-BSA significantly enhanced microglial dynorphin a gene expression; whereas Dex 21-melylate did not affect basal expression of microglial dynorphin gene, but completely inhibited stimulation of dynorphin A gene expression by 20 (S) -protopanaxadiol, dex and Dex-BSA. As can be seen from FIG. 14B, however, 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 stimulates microglial expression and release of dynorphin a by agonizing spinal cord microglial glucocorticoid receptor (possibly cell membrane glucocorticoid receptor), thereby producing an analgesic effect.

Discussion of the invention

20 The biological activity of (S) -protopanaxadiol has been reported, but the use of 20 (S) -protopanaxadiol for the treatment of pain has not been reported until the present invention, nor has physical and mental dependencies been disclosed as useful for the treatment of opioids (or addictives). The invention provides that 20 (S) -protopanaxadiol has remarkable analgesic effect in the models of neuropathic pain, cancer pain, inflammatory pain, formalin pain and the like of rats/mice; 20 (S) -protopanaxadiol does not produce analgesic tolerance, somatic dependence and conditional site-preference effects (mental dependence) upon chronic administration; 20 (S) -protopanaxadiol is effective in inhibiting morphine-induced analgesic tolerance, somatic dependence and mental retardation.

The study of the present inventors shows that 20 (S) -protopanaxadiol has an analgesic main part in spinal cord, and that 20 (S) -protopanaxadiol extremely effectively promotes expression and release of dynorphin A by exciting spinal cord microglial glucocorticoid receptor (possibly cell membrane glucocorticoid receptor), thereby unexpectedly producing analgesic effect and effects of abstaining from physical dependence and mental dependence of opioid (or other addictive substances) treatment.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. A pharmaceutical composition, characterized in that it consists of:

(i) A first active ingredient, the first active ingredient being 20 (S) -protopanoxadiol;

(ii) A second active ingredient selected from the group consisting of: morphine or gabapentin;

(iii) Pharmaceutically acceptable carriers and/or excipients.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is an oral formulation or an injectable formulation.

3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is selected from the group consisting of: powder, granule, capsule, injection, tincture, oral liquid, tablet, buccal tablet or dripping pill.

4. Use of a pharmaceutical composition according to claim 1 for the preparation of a medicament for the treatment and/or alleviation of pain.

5. The use according to claim 4, wherein the pain is selected from the group consisting of: neuropathic pain, inflammatory pain, diabetic pain, lower back pain, spinal cord injury pain, visceral pain, fibromyalgia, chronic localized pain syndrome, musculoskeletal pain, cancer pain, pain caused by antineoplastic agents, postoperative pain, posttraumatic neuralgia and peripheral neuropathy, phantom limb pain, or combinations thereof.

6. The use of claim 5, wherein the neuropathic pain is selected from the group consisting of: postherpetic neuralgia, trigeminal neuralgia and sciatica.