Application of MCU in treatment of addictive diseases

Abstract

The invention provides an application of MCU (Microprogrammed Control Unit) in treating addictive diseases. By means of a conditional knockout dopamine system mouse model, it is observed that motonous nerve excitability caused by heroin, morphine and methamphetamine is remarkably reduced due to deficiency of MCU. In addition, behavioral experiments further prove that the MCU knockout significantly reduces the addiction desire of mice to heroin and methamphetamine. By applying MCU inhibitors, namely mitoxantrone (MTX) and Ru265, the inhibitors are found to be capable of remarkably inhibiting motor nerve excitability caused by heroin and reducing addiction and relapse behaviors of mice on heroin. Mechanism research reveals that MCU knockout inhibits dopamine release caused by opioids and methamphetamine in a nucleus septum (NAc) area, and it is indicated that MCU may serve as an effective target for treating addiction of multiple drugs. The invention provides a brand-new treatment strategy for inhibiting MCU as a clinical treatment for opioid (morphine) or drug (heroin) and methamphetamine addiction for the first time.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of MCU in treating addictive diseases. Background Art

[0002] Drug addiction is a chronic, highly recurrent, complex brain disease, characterized by the progressive development of impulsive drug seeking caused by the first use of the drug into compulsive drug seeking, forming strong physical and mental dependence. Common drug addictions include opioid analgesics: such as morphine, pethidine, oxycodone, etc. These drugs mainly act on the central nervous system, produce analgesics and improve the unpleasant emotions caused by pain, but they are also addictive. Common drug addictions mainly include opioid drugs: including heroin (commonly known as "white powder"), morphine, opium, etc. These drugs are extremely addictive and difficult to quit. Long-term use will cause serious harm to people's physical and mental health. Methamphetamine (ice): a synthetic stimulant, long-term use can cause brain damage and induce serious mental illnesses such as schizophrenia. Cocaine: also known as cocaine, is an extremely dangerous drug. Abuse of cocaine may cause arrhythmia, shortness of breath, and even death. In addition, smoking and drinking addiction are also global public health issues. Nicotine and alcohol can both produce tolerance and dependence, bringing the risk of addiction.

[0003] Long-term abuse of drugs or narcotics can lead to physical and mental dependence, increased tolerance and withdrawal symptoms such as nausea, vomiting, anxiety, depression, etc. In severe cases, drug addiction may also lead to organ damage, mental illness and even death. Drug addicts may have psychological problems such as social disorders, anxiety, depression, and some addicts may also have other mental illnesses. Therefore, drug addiction increases social costs, such as public health costs, increased drug-related crimes and mortality, and damage to family and social structures.

[0004] Drug addiction is a chronic, relapsing brain disease that is difficult to treat and has a high relapse rate. Current drug treatments are mainly targeted at specific types of addiction, using different drugs to relieve withdrawal symptoms or reduce drug cravings. For example, opioid addicts can use opioid receptor agonists such as methadone and buprenorphine. These drugs can replace exogenous opioid compounds and help patients achieve physiological detoxification by decreasing the dose. However, opioid receptor agonists themselves may be addictive, with a narrow treatment window, requiring strict evaluation and management. Opioid receptor antagonists can also be used to treat acute poisoning in opioid addiction: such as naloxone and naltrexone, which can relieve symptoms such as respiratory depression, but use alone may cause patients to experience immediate withdrawal symptoms, poor patient compliance and high relapse rates. There is currently no effective treatment for methamphetamine and cocaine.

[0005] Existing strategies for treating drug and substance addiction directly target the reward system itself, such as GABA receptors or dopamine receptors. However, these treatments can cause significant side effects, including psychosis, nervous system toxicity, respiratory depression, immune and endocrine system deficiencies, withdrawal symptoms, drug dependence, and addiction. Therefore, finding new safe and effective targets for treating drug addiction is of great significance for global public health and human health. Summary of the Invention

[0006] To address the deficiencies of the prior art, the present invention provides the use of MCU in the treatment of addictive diseases.

[0007] To achieve the above objective, the present invention adopts the following technical solutions: The first aspect of the present invention provides the use of an inhibitor of MCU in the preparation of a pharmaceutical composition for preventing / treating drug addiction.

[0008] Furthermore, the inhibitor of MCU includes nucleic acid inhibitors, protein inhibitors, and compounds.

[0009] Furthermore, the compounds include mitoxantrone (MTX), Ru265, RU360, RuR, DS16570511, KB-R7943, NecroX-5, Minocycline, MCU-i4, MCU-i11, and Doxycycline.

[0010] Furthermore, the drugs include opioid drugs, amphetamine drugs, alcohol, and tobacco.

[0011] Furthermore, the opioid drugs include heroin, morphine, methadone, and fentanyl.

[0012] Furthermore, the amphetamine drugs include methamphetamine, ecstasy, and yaba.

[0013] The second aspect of the present invention provides the use of an inhibitor of MCU in the preparation of a pharmaceutical composition for inhibiting relapse after discontinuing addictive drugs.

[0014] Furthermore, the inhibitor of MCU includes nucleic acid inhibitors, protein inhibitors, and compounds.

[0015] Furthermore, the compounds include mitoxantrone, Ru265, RU360, RuR, DS16570511, KB-R7943, NecroX-5, Minocycline, MCU-i4, MCU-i11, and Doxycycline.

[0016] Furthermore, the addictive drugs include opioid drugs, amphetamine drugs, alcohol, and tobacco.

[0017] Furthermore, the opioid drugs include heroin, morphine, methadone, and fentanyl.

[0018] Furthermore, the amphetamine drugs include methamphetamine, ecstasy, and "maguo".

[0019] The third aspect of the present invention provides the use of an inhibitor of MCU in the preparation of a pharmaceutical composition for inhibiting the excitability caused by addictive drugs.

[0020] Furthermore, the inhibitor of MCU includes nucleic acid inhibitors, protein inhibitors, and compounds.

[0021] Furthermore, the compounds include mitoxantrone, Ru265, RU360, RuR, DS16570511, KB-R7943, NecroX-5, Minocycline, MCU-i4, MCU-i11, and Doxycycline.

[0022] Furthermore, the drugs include opioid drugs, amphetamine drugs, alcohol, and tobacco.

[0023] Furthermore, the opioid drugs include heroin, morphine, methadone, and fentanyl.

[0024] Furthermore, the amphetamine drugs include methamphetamine, ecstasy, and "maguo".

[0025] The fourth aspect of the present invention provides the use of an inhibitor of MCU in the preparation of a pharmaceutical composition for inhibiting dopamine release.

[0026] Furthermore, the dopamine is dopamine in the nucleus accumbens brain region.

[0027] Furthermore, the inhibitor of MCU includes nucleic acid inhibitors, protein inhibitors, and compounds.

[0028] Furthermore, the compounds include mitoxantrone, Ru265, RU360, RuR, DS16570511, KB-R7943, NecroX-5, Minocycline, MCU-i4, MCU-i11, and Doxycycline.

[0029] Furthermore, the dopamine release is dopamine release caused by drugs.

[0030] Furthermore, the drugs include opioid drugs, amphetamine drugs, alcohol, and tobacco.

[0031] Furthermore, the opioid drugs include heroin, morphine, methadone, and fentanyl.

[0032] Further, the amphetamine drugs include methamphetamine, ecstasy, and MDMA.

[0033] The fifth aspect of the present invention provides a pharmaceutical composition for preventing / treating drug addiction / inhibiting relapse after withdrawal of addictive drugs / inhibiting excitatory / inhibitory dopamine release caused by addictive drugs, and the pharmaceutical composition includes an inhibitor of MCU.

[0034] Further, the dopamine release is dopamine release caused by drugs.

[0035] Further, the pharmaceutical composition further includes other drugs for preventing / treating drug addiction or inhibiting relapse after withdrawal of addictive drugs / inhibiting excitatory / inhibitory dopamine release caused by addictive drugs.

[0036] Further, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0037] Further, the administration routes of the pharmaceutical composition include intravenous administration, oral administration, mucosal administration, nasal administration, and rectal administration.

[0038] Further, the dosage forms of the pharmaceutical composition include liquid dosage forms and solid dosage forms.

[0039] Further, the liquid dosage forms include tablets, suppositories, capsules, microparticles, and powders.

[0040] Further, the liquid dosage forms include injections, aerosols, emulsions, syrups, elixirs, suspensions, and solutions.

[0041] The sixth aspect of the present invention provides the use of MCU as a target in screening candidate drugs for preventing / treating drug addiction / inhibiting relapse after withdrawal of addictive drugs / inhibiting excitatory / inhibitory dopamine release caused by addictive drugs.

[0042] Further, the screening method for the candidate drugs includes: treating a culture system expressing or containing the MCU gene or its encoded protein with a substance to be screened; and detecting the expression or activity of the MCU gene or its encoded protein in the system; wherein, when the substance to be screened inhibits the expression level or activity of the MCU gene or its encoded protein, the substance to be screened is a candidate drug for preventing / treating drug addiction / inhibiting relapse after withdrawal of addictive drugs / inhibiting excitatory.

[0043] The seventh aspect of the present invention provides a method for screening candidate drugs for preventing / treating drug addiction / suppressing relapse after withdrawal of addictive drugs / suppressing excitability caused by addictive drugs / suppressing dopamine release, the method comprising: treating a culture system expressing or containing the MCU gene or the protein encoded thereby with a substance to be screened; and detecting the expression or activity of the MCU gene or the protein encoded thereby in the system; wherein, when the substance to be screened inhibits the expression level or activity of the MCU gene or the protein encoded thereby, the substance to be screened is a candidate drug for preventing / treating drug addiction / suppressing relapse after withdrawal of addictive drugs / suppressing excitability caused by addictive drugs.

[0044] The eighth aspect of the present invention provides the use of a reagent for detecting the expression level of MCU in the preparation of a product for diagnosing drug addiction.

[0045] Furthermore, the reagent is selected from a probe specifically recognizing MCU, a primer specifically amplifying MCU, or a binder specifically binding to the protein encoded by the MCU gene.

[0046] Furthermore, the drugs include opioid drugs, amphetamine drugs, alcohol, and tobacco.

[0047] Furthermore, the opioid drugs include heroin, morphine, methadone, and fentanyl.

[0048] Furthermore, the amphetamine drugs include methamphetamine, ecstasy, and yaba.

[0049] Furthermore, the product includes a chip, a test strip, a kit, or a nucleic acid membrane strip.

[0050] Advantages and beneficial effects of the present invention: In this study, we found that mitochondrial calcium uniporter (MCU), a key molecule in mitochondrial energy metabolism, could be a potential target for treating addiction to various drugs and narcotics. Using a conditional knockout mouse model of the dopamine system, we observed that the deletion of MCU significantly reduced the motor nerve excitability induced by heroin, morphine, and methamphetamine (ice). In addition, through a place preference experiment, we found that the knockout of MCU significantly reduced the craving for heroin- and ice-related addictive behaviors in mice.

[0051] Furthermore, we explored the potential role of the MCU inhibitors mitoxantrone (MTX) and Ru265 in addiction treatment. The experimental results showed that these two compounds significantly inhibited the motor nerve excitability induced by heroin and reduced the relapse behavior of mice addicted to heroin. At the molecular mechanism level, we found that the knockout of MCU inhibited the dopamine release induced by opioid drugs and ice in the nucleus accumbens (NAc) brain region, indicating that MCU may be an effective target for treating addiction to various drugs.

[0052] In summary, 1) This study for the first time reveals that inhibiting the key molecule MCU of mitochondrial energy metabolism may provide a new treatment strategy for the clinical treatment of opioid drugs or drug (such as morphine, heroin) addiction.

[0053] 2) It is found that intervening in the key molecule MCU of mitochondrial energy metabolism can be used as a new and effective target for the treatment of amphetamine-type poisons and drugs (such as methamphetamine) addiction. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 is the mating diagram of MCU conditional knockout mice with dopamine neuron specificity; Figure 2 is the diagram for evaluating the effect of MCU knockout on the excitability caused by drugs in the acute drug administration model; Figure 3 is the diagram for evaluating the effect of MCU knockout on drug reward behavior in the conditioned place preference model; Figure 4 is the diagram for evaluating the effect of the MCU inhibitor - mitoxantrone and Ru265 on the excitability caused by drugs in the acute drug administration model; Figure 5 is the diagram for evaluating the effect of the MCU inhibitor - mitoxantrone and Ru265 on drug reward behavior in the conditioned place preference model; Figure 6 is the diagram showing that MCU knockout inhibits dopamine release caused by opioid drugs and methamphetamine. DETAILED DESCRIPTION OF THE INVENTION

[0055] The following provides definitions of some terms used in this specification. Unless otherwise specified, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

[0056] The present invention provides the use of an inhibitor of MCU in the preparation of a pharmaceutical composition for preventing / treating drug addiction.

[0057] In one embodiment, MCU includes wild type, mutant or a fragment thereof. This term encompasses full-length, unprocessed MCU, as well as naturally occurring variants of MCU (such as splice variants or allelic variants). This term encompasses, for example, human MCU and MCU from any other vertebrate source, including mammals such as primates and rodents (such as mice and rats), gene ID: 90550.

[0058] In one embodiment, drug addiction is a substance-induced disorder and generally refers to the symptoms caused when an addictive drug is administered or taken. Drug addiction is considered a form of drug dependence and can be defined as a disorder of emotional state, such as a physical or mental disorder, loss of self-control, and a state of continuously seeking or taking drugs. Drug addiction can also be defined as long-term exposure to drugs.

[0059] In one embodiment, an inhibitor refers to any substance that can reduce the activity of the MCU protein, decrease the stability of the MCU gene or protein, downregulate the expression of the MCU protein, reduce the effective action time of the MCU protein, or inhibit the transcription and translation of the MCU gene. These substances can all be used in this application as substances useful for inhibiting MCU and can thus be used to treat drug addiction.

[0060] In one embodiment, inhibitors include nucleic acid inhibitors, protein inhibitors, and compounds.

[0061] In one embodiment, nucleic acid inhibitors include reagents capable of inhibiting or knocking out the expression of the MCU gene, and the reagents include gene editing systems and RNA interference agents.

[0062] In one embodiment, gene editing systems include, but are not limited to, CRISPR / Cas, TALEN, ZFN, transposon technology, PASTE technology, PE technology, base editors, and any combination thereof.

[0063] Among them, the CRISPR / Cas system includes a Cas effector protein (including, but not limited to, Cas9, Cas12a (Cpf1), Cas12b (C2c1), Cas13a (C2c2), C2c3, Cas13b) and the corresponding guide RNA (gRNA). The CRISPR / Cas system recruits the Cas enzyme protein to the target locus through the guide RNA (gRNA) to complete the modification. The gRNA contains a guide sequence complementary to the target sequence in the target locus. In certain embodiments, the gRNA can be a chimeric guide RNA or a single guide RNA (sgRNA). In certain embodiments, the gRNA contains a guide sequence and a tracr pairing sequence (or direct repeat). In certain embodiments, the gRNA contains a guide sequence, a tracr pairing sequence (or direct repeat), and a tracr sequence. In certain embodiments, the CRISPR-Cas system does not contain and / or is not dependent on the presence of the tracr sequence (e.g., if the Cas protein is Cas12a).

[0064] In one embodiment, an RNA interfering agent refers to any agent that inhibits the expression of a target gene through the RNA interference (RNAi) mechanism. RNA interference (RNAi) is an evolutionarily conserved process in which the expression or introduction of RNA with a sequence identical or highly similar to that of the target gene results in sequence-specific degradation of messenger RNA (mRNA) transcribed from the target gene or specific post-transcriptional gene silencing (PTGS), thereby inhibiting the expression of the target gene.

[0065] In one embodiment, the RNA interfering agent includes, but is not limited to, shRNA, siRNA, ribozyme, and antisense oligonucleotide.

[0066] In one embodiment, the protein inhibitor includes a substance that specifically binds to the MCU protein, such as an antibody or ligand that can inhibit the activity of the MCU protein.

[0067] In one embodiment, the compound includes any substance that can inhibit MCU, including but not limited to mitoxantrone (CAS No.: 65271-80-9), Ru265 (CAS No.: 27056-01-5), Ruthenium 360 (RU360, CAS No.: 133399-54-9), Ruthenium Red (RuR, CAS No.: 11103-72-3), DS16570511 (CAS No.: 2446154-84-1), KB-R7943 (CAS No.: 182004-64-4), NecroX-5 (CAS No.: 1383718-29-3), Minocycline (CAS No.: 10118-90-8), MCU-i4 (CAS No.: 371924-24-2), MCU-i11 (CAS No.: 902903-59-7), Doxycycline (CAS No.: 564-25-0), Ln 3+ salts, [Co(NH3)5(H2O)] 3 、[Co(NH3)6] 3+ 、[Co(en)3] 3+ 、[Cr(en)3] 3+ 、[Rh(en)3] 3+ 、[Co(NH3)5(NH2(CH2)5NH3)] 4+ , or derivatives / metabolites of the above compounds.

[0068] In one embodiment, the derivatives of the above compounds include pharmaceutically acceptable salts of the above compounds. Pharmaceutically acceptable salts are suitable for contact with patient tissues within the scope of sound medical judgment and do not produce undue toxicity, irritation, allergic reactions, etc. The main body of pharmaceutically acceptable salts is well-known in the art. If pharmaceutically acceptable salts of the above compounds are used in these compositions, those salts are preferably derived from inorganic acids or organic acids and bases. Such acid salts include, but are not limited to, the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, mesylate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, toluenesulfonate, undecanoate, hydrohalides (e.g., hydrochloride and hydrobromide), sulfate, phosphate, nitrate, sulfamate, malonates, salicylate, methylene-bis-b-hydroxynaphthoate, gentisate, hydroxyethanesulfonate, di-p-toluoyl tartrate, ethanesulfonate, cyclohexylaminosulfonate, quinate, etc. Pharmaceutically acceptable base addition salts include, but are not limited to, those derived from alkali metal or alkaline earth metal bases or conventional organic bases, such as triethylamine, pyridine, piperidine, morpholine, N-methylmorpholine, ammonium salts, alkali metal salts (such as sodium salts and potassium salts), alkaline earth metal salts (such as calcium salts and magnesium salts), salts with organic bases (such as dicyclohexylamine salts, N-methyl-D-glucamine), and salts with amino acids such as arginine and lysine.

[0069] In a specific embodiment, the compound is selected from mitoxantrone, RU265.

[0070] In one embodiment, the drugs include, but are not limited to, opioid drugs, amphetamine drugs, alcohol, tobacco.

[0071] Dopamine is a neurotransmitter produced in the brain. In drug addiction, dopamine plays an important role. Substances prone to addiction such as alcohol, tobacco, opioids, and amphetamines directly or indirectly promote dopamine release, which can damage key components of the "brain reward system" over time, leading to an individual's dependence on substances prone to addiction such as alcohol, tobacco, opioids, and amphetamines (Pharmacology of nicotine: addiction, smoking-induced disease, and therapeutics; Long-term alcohol consumption alters dorsal striatal dopamine release and regulation by D2 dopamine receptors in rhesus macaques).

[0072] In one embodiment, the opioids include, but are not limited to, heroin, morphine, methadone, fentanyl, pethidine, and codeine.

[0073] In a preferred embodiment, the opioids are selected from heroin, morphine, methadone, and fentanyl.

[0074] Among them, methadone, fentanyl, and morphine are all μ-opioid receptor agonists, and their pharmacological effects are similar.

[0075] In one embodiment, the amphetamines include, but are not limited to, methamphetamine (ice), ecstasy, and yaba.

[0076] The present invention provides a pharmaceutical composition for preventing / treating drug addiction / inhibiting relapse after withdrawal of addictive drugs / inhibiting excitability caused by addictive drugs / releasing dopamine, and the pharmaceutical composition includes an inhibitor of MCU.

[0077] In one embodiment, the pharmaceutical composition further includes other drugs for treating drug addiction or inhibiting relapse after withdrawal of addictive drugs / inhibiting excitability caused by addictive drugs. The other drugs for treating drug addiction or inhibiting relapse after withdrawal of addictive drugs / inhibiting excitability caused by addictive drugs include, but are not limited to, opium tincture, dextropropoxyphene, and buprenorphine.

[0078] The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0079] In one embodiment, a pharmaceutically acceptable carrier is used to refer to a material that is compatible with the recipient, preferably a mammal, more preferably a human, and is suitable for delivering an active agent to the target site without terminating the activity of the agent. The toxicity or side effects (if any) associated with the pharmaceutically acceptable carrier are preferably commensurate with the reasonable risk / benefit ratio for the intended use of the active agent.

[0080] Pharmaceutically acceptable carriers include, but are not limited to, diluents, binders, surfactants, humectants, adsorbent carriers, lubricants, fillers, and disintegrants. These carriers are used as needed to assist in the stability of the formulation or to enhance the activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration. The formulations that can be used in such drugs can be in the form of the original compound itself or optionally in the form of its pharmaceutically acceptable salts. The pharmaceutical compositions so formulated can be administered in any suitable manner known to those skilled in the art as needed.

[0081] Among them, diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, and water.

[0082] Binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and its salts, xanthan gum, hydroxypropylcellulose, and hydroxypropylmethylcellulose.

[0083] Surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters, sodium dodecyl sulfate, monoglyceride stearate, and cetyl alcohol.

[0084] Humectants include, but are not limited to, glycerol.

[0085] Adsorbent carriers include, but are not limited to, bentonite, silica gel, kaolin, and saponite.

[0086] Lubricants include, but are not limited to, zinc stearate, monoglyceride stearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, sucrose monolaurate, sodium lauryl sulfate, magnesium lauryl sulfate, and sodium dodecyl sulfate.

[0087] Fillers include, but are not limited to, mannitol (granular or powdered), xylitol, sorbitol, maltose, erythritol, microcrystalline cellulose, polymeric sugars, coupled sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminaran powder, agar powder, calcium carbonate, and sodium bicarbonate.

[0088] Disintegrants include, but are not limited to, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methylcellulose, cross-linked sodium carboxymethyl cellulose, and soy polysaccharide.

[0089] In one embodiment, the drug can be administered via different routes, including but not limited to intravenous administration, oral administration, mucosal administration, nasal administration, rectal administration, brain region administration, peritoneal administration, subcutaneous administration, intramuscular administration.

[0090] In a preferred embodiment, the administration route of the pharmaceutical composition is selected from intravenous administration, oral administration, and brain region administration.

[0091] In one embodiment, the dosage form of the pharmaceutical composition includes liquid dosage forms and solid dosage forms.

[0092] Among them, solid dosage forms include, but are not limited to, granules, precipitates or microparticles, powders (including lyophilized powders, rotary dried powders or spray dried powders, amorphous powders), tablets, suppositories, capsules.

[0093] Liquid dosage forms include injections, aerosols, emulsions, syrups, elixirs, suspensions or solutions.

[0094] In a preferred embodiment, the dosage form of the pharmaceutical composition is selected from liquid dosage forms.

[0095] In a specific embodiment, the liquid dosage form is selected from injections.

[0096] In one embodiment, according to known techniques, suitable dispersants or wetting agents and suspending agents can be used to formulate injectables, such as sterile injectable aqueous or oily suspensions. Sterile injectable preparations can also be sterile injectable solutions, suspensions or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile non-volatile oils are usually used as solvents or suspending media. For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used in injectable preparations. The injectable formulation can be sterilized, for example, by filtering through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that is soluble in sterile water or other sterile injectable media before use. Drugs formulated for parenteral administration can be administered by rapid injection or by timed bolus injection, or can be administered by continuous infusion.

[0097] In one embodiment, the pharmaceutical composition can be used in the treatment of a target disease in a therapeutically effective amount. As used herein, unless otherwise mentioned, treatment refers to reversing or alleviating one or more symptoms of a disease or disorder in need of treatment or inhibiting or preventing the progression of symptoms.

[0098] In one embodiment, a therapeutically effective amount refers to the amount of an active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal, or human, as determined by a researcher, veterinarian, physician, or other clinician. A therapeutically effective amount is intended to include an amount that causes a reduction in the symptoms of the disease or disorder mentioned. The effective amount of the active ingredient in the pharmaceutical composition will vary depending on the desired effect and will be apparent to those skilled in the art.

[0099] Thus, those skilled in the art can readily determine the optimal amount of the pharmaceutical composition, which can vary depending on a variety of factors, including the type and severity of the disease, the amount of other ingredients in the composition, the type of formulation, the age, weight, general health, sex, and diet of the patient, the time and route of administration, the excretion rate of the composition, the duration of treatment, and the presence of concomitant medications.

[0100] The present invention provides a method for screening candidate drugs for preventing / treating drug addiction / inhibiting relapse after withdrawal of addictive drugs / inhibiting dopamine release, the method comprising: treating a culture system expressing or containing the MCU gene or the protein encoded thereby with a substance to be screened; and detecting the expression or activity of the MCU gene or the protein encoded thereby in the system; wherein when the substance to be screened inhibits the expression level or activity of the MCU gene or the protein encoded thereby, the substance to be screened is a candidate drug for preventing / treating drug addiction / inhibiting relapse after withdrawal of addictive drugs / inhibiting the excitability caused by addictive drugs.

[0101] In one embodiment, the candidate drug refers to a substance having therapeutic activity against drug addiction and is intended to include extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and molecules of various compounds. The candidate drug can be a cell transfected with a liposome or a vector. Transfection can be carried out by microinjection, calcium phosphate co-precipitation, electroporation, or liposome fusion, but is not limited thereto.

[0102] In one embodiment, prevention refers to all actions that inhibit or delay the development of drug addiction and its symptoms. Treatment refers to all actions that relieve or beneficially modify the drug addiction symptoms and withdrawal symptoms caused by drug addiction.

[0103] The present invention provides the use of a reagent for detecting the expression level of MCU in the preparation of a product for diagnosing drug addiction.

[0104] In one embodiment, the expression level or level refers to the absolute or relative amount of MCU in the present application. The expression level of MCU in the present invention can be determined by a variety of techniques. In particular, the absolute or relative amount of MCU in the present invention can be detected by using methods well known to those skilled in the art.

[0105] The reagent is selected from a probe that specifically recognizes MCU, a primer that specifically amplifies MCU, or a binder that specifically binds to the protein encoded by the MCU gene.

[0106] In one embodiment, the probe that specifically recognizes MCU can be DNA, RNA, a DNA-RNA chimera, PNA, or other derivatives. There is no limitation on the length of the probe, and any length can be used as long as it can complete specific hybridization and specifically bind to the target nucleotide sequence. The length of the probe can be as short as 25, 20, 15, 13, or 10 base lengths. Similarly, the length of the probe can be as long as 60, 80, 100, 150, 300 bases or longer, or even the entire gene.

[0107] In one embodiment, a primer refers to a short nucleic acid molecule, such as a DNA oligonucleotide, which can anneal to a complementary target nucleic acid molecule through nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid strand. The primer can be extended along the target nucleic acid molecule by a polymerase. Therefore, a primer can be used to amplify a target nucleic acid molecule, where the sequence of the primer is specific for the target nucleic acid molecule, for example, the primer will hybridize to the target nucleic acid molecule under very high stringency hybridization conditions.

[0108] In one embodiment, the primer or probe can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. It can also be modified using many means known in the art. Non-limiting examples of these modifications include methylation, capping, substitution with one or more analogs of natural nucleotides, and modifications between nucleotides. For example, modifying an uncharged linker (such as methyl phosphate, phosphotriester, phosphoramidite, carbamate, etc.) or a charged linker (such as phosphorothioate, dithiophosphonate, etc.).

[0109] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. Without departing from the scope of the present invention, the main features of the present invention can be used in various embodiments.

[0110] Example 1 Inhibition of MCU can reduce the locomotor excitability caused by various addictive drugs or narcotics 1. Experimental materials (1) Spontaneous activity behavior box [Anlai Scientific Instruments (Ningbo) Co., Ltd.] (2) MCUflox / flox The mice and DAT-IRES-Cre mice were purchased from Cyagen Biosciences Inc. (3) Heroin (from the National Institutes for Food and Drug Control, China), methamphetamine (METH, from the National Institutes for Food and Drug Control, China), morphine (from Qinghai Pharmaceutical Factory), cocaine (from Qinghai Pharmaceutical Factory).

[0111] 2. Experimental methods Obtaining dopamine neuron-specific conditional knockout MCU mice First, DAT-IRES-Cre mice and MCU flox / flox mice were crossed to obtain DAT-Cre + / - ;MCU flox / + and DAT-Cre - / - ;MCU flox / + offspring. Subsequently, DAT-Cre + / - ;MCU flox / + and DAT-Cre - / - ;MCU flox / + mice were crossed to obtain DAT-Cre + / - ;MCU flox / flox knockout mice. DAT-Cre + / - ; MCU + / + mice were used as controls. The specific process is as Figure 1 shown.

[0112] Open field test (1) One hour before the experiment, the mice were placed in the functional laboratory to adapt. The software was set, and wood shavings were laid at the bottom of the behavior chamber.

[0113] (2) The mice were placed in the open field to adapt for 60 min (30 min for acute cocaine administration adaptation), and the movement trajectories of the mice were recorded.

[0114] (3) The mice were intraperitoneally injected with a single dose of drug (10 mg / kg heroin, 30 mg / kg morphine, 2 mg / kg METH, or 20 mg / kg cocaine), and the movement trajectories of the mice were continuously recorded for 180 min (60 min for cocaine).

[0115] (4) According to the movement distances of each group of mice, the experimental data were analyzed.

[0116] An acute drug administration model was used to evaluate the effect of inhibiting MCU on the excitability induced by drugs.

[0117] The acute drug administration model is to first allow the mice to adapt to an open field for a period of time, and then inject a shot of drug into the mouse's peritoneum. Due to the excitatory effect of the drug, the mouse will move vigorously in the open field, and the excitability caused by the drug is quantified by recording the movement distance of the mouse.

[0118] 3. Experimental results Figure 2 The horizontal axis is time, and the vertical axis represents the distance the mice move in ten minutes. The black line represents wild-type mice, and the red line represents knockout mice.

[0119] It can be seen that after the injection of heroin, the movement distance of wild-type mice increased significantly, but after knocking out MCU, the high activity caused by heroin / meth / morphine was suppressed. However, if the drug is replaced with cocaine in the same model, knocking out MCU does not affect the excitability caused by cocaine, indicating that targeting MCU is drug-specific for the high excitatory movement caused by drugs ( Figure 2 , heroin: number of mice (control) n=9, number of mice (knockout group) n=10; methamphetamine: number of mice (control) n=10, number of mice (knockout group) n=11; morphine: number of mice (control) n=10, number of mice (knockout group) n=11; cocaine: number of mice (control) n=9, number of mice (knockout group) n=11).

[0120] Example 2 Inhibition of MCU can alleviate addictive behaviors caused by various addictive drugs or narcotics 1. Experimental Materials (1) Behavior box for conditional place preference experiment [Anlai Scientific Instrument (Ningbo) Co., Ltd.] (2) DAT-Cre + / - ;MCU flox / flox Mice and DAT-Cre + / - ;MCU + / + How to obtain it, see Figure 1 (3) Heroin (China Food and Drug Inspection Institute), methamphetamine (China Food and Drug Inspection Institute), and cocaine (Qinghai Pharmaceutical Factory).

[0121] 2. Experimental methods Mouse conditioned place preference experiment (1) Pre-adaptation period: Day 1 to Day 3 of the experiment: insert the partition into the slot between the two chambers (U-shaped door facing down), put the animals in from the white box, and allow them to move freely between the two chambers. Adapt once a day for 30 minutes each time, and the position preference video analysis system will record it at the same time. The data of the second and third days of the experiment were statistically analyzed, and mice that stayed in the box on one side for more than 70% of the total time were eliminated (serious self-preference). The remaining mice were randomly grouped and subjected to subsequent experiments.

[0122] (2)Animal grouping: The remaining animals were evenly and randomly grouped according to the difference in residence time in the black and white boxes (absolute value of CPP score).

[0123] (3)Conditioning training period: From the 4th day to the 11th day of the experiment: Reinsert the partition (U-shaped door facing up) to confine the animals in the black or white box. Each animal received one task training per day. And so on, for 8 consecutive days. Training was conducted once a day for a total of 4 cycles.

[0124] During the training in the drug-paired box, each group was put into the drug-paired box for training after drug administration. During the training in the non-drug-paired box, each group was put into the non-drug-paired box for training after receiving the solvent, and the time interval between drug administration and training was the same as that in the drug-paired box training. During the training period, the body weight of the animals was measured once a day before drug administration for calculating the drug administration volume.

[0125] (4)Expression period: On the 12th day of the experiment: The day after the training ended, insert the partition (U-shaped door facing down) into the card slot, put the animals in from the white box, and record the residence time (s) of the animals in the drug-paired box and the non-drug-paired box within 30 min respectively, and calculate the CPP score of the animals. The calculation formula is: CPP score (s) = residence time of the animal in the drug-paired box (s) - residence time of the animal in the non-drug-paired box (s).

[0126] The classical addiction model - conditioned place preference model was used to evaluate the effect of MCU inhibition on drug reward behavior.

[0127] In this experiment, the mice were first placed in the middle of the behavior box, and the mice had no preference for either side. Then the mice were trained. On the first day, the mice were injected with a dose of drug and then placed in the left box. On the second day, the mice were injected with a dose of normal saline and then placed in the right box. After repeating this 4 cycles. On the ninth day, the mice were placed in the middle box, and the mice would prefer to run into the box where they were previously administered the drug. The longer it stayed in the drug-administered box, the stronger its addiction and the more eager it was for the drug.

[0128] 3. Experimental results Figure 3The results of CPP experiments of different drugs. The horizontal axis represents mice of different genotypes before and after drug administration, and the vertical axis represents the difference between the time mice stayed in the drug-administered box and the time mice stayed in the drug-free box. The first two columns represent that before drug administration, both groups of mice had no preference for the two boxes. However, after heroin training, WT group mice preferred the drug-administered box. However, after knocking out MCU, the addiction to heroin / meth was significantly reduced. However, in the CPP formed by cocaine drugs, knocking out MCU had no effect, which once again showed that the MCU target was specifically involved in the heroin / meth reward behavior (heroin: number of mice (control) n=9, number of mice (knockout group) n=9; methamphetamine: number of mice (control) n=8, number of mice (knockout group) n=8; cocaine: number of mice (control) n=9, number of mice (knockout group) n=9).

[0129] Example 3 Brain administration of MCU inhibitors - mitoxantrone (MTX) and Ru265 inhibits heroin-induced motor excitability 1. Experimental Materials (1) Spontaneous activity behavior box [Anlai Scientific Instrument (Ningbo) Co., Ltd.] (2) C57 / 6N wild-type mice were purchased from Saiye Biotechnology Co., Ltd. (3) Heroin (China Food and Drug Inspection Institute) and mitoxantrone were purchased from Medicilon Biopharmaceutical Technology Co., Ltd. Ru265 (Sigma-Aldrich, SML2991) (4) Stereotaxic apparatus, microscope, cold light source, warming pad, surgical pad, cannula holder, surgical instrument kit, alcohol cotton balls, dry cotton balls, cranial drill, isoflurane gas anesthesia system, cannula drug delivery system (catheter, injection inner tube, catheter cap, PE tube, small screw), micro-drug delivery system (micro-injection pump, micro-syringe) etc. were purchased from Reward Life Science Co., Ltd.

[0130] 2. Experimental methods Cannula implantation surgery ① Anesthetize the animal, shave the hair on the mouse's head with a shaver, and fix the head to a stereotaxic apparatus. Level the skull, and then locate the target brain area NAc (AP: +1.6, ML: ±0.8, DV: -4.5). Use a cranial drill to drill holes (at the same time, drill 2-3 small holes near the hole for screwing in the skull pins, which serve as points of force for dental cement to prevent the dental cement from falling off. At the same time, the diameter of these small holes needs to be smaller than the skull pins, so that the skull pins can be tightened to provide adhesion), then use a needle to slightly puncture the dura mater above the target brain area to expose the skull; the dura mater does not need to be punctured at the location where the skull pins are fixed. Screw the skull pin into the hole for about three turns, then slowly implant the prepared catheter into the skull through the clamp, and then wrap the screw and catheter with dental cement to fix them.

[0131] ② After waiting for the dental cement to solidify (about 15 minutes), remove the clamp (pay attention not to drive the catheter), and slowly insert the catheter cap and tighten it; judge whether it is necessary to suture the skin according to the actual situation.

[0132] ③ During the animal recovery period, it can recover in about 2 - 3 days. During this period, roxithromycin ointment can be applied around the skin to prevent the animal from getting inflamed and infected; at the same time, pay attention to the animal's condition and inject sugar and salt intraperitoneally in a timely manner to enable the animal to obtain sufficient energy. The next experiment starts 7 days after the operation.

[0133] Open - field test ① Place the mice in the functional laboratory 1 h before the experiment to adapt, set the software, and lay wood shavings on the bottom of the behavior chamber.

[0134] ② Use a microsyringe to inject mitoxantrone solution (4.5 mM), or Ru265 solution (50 μM), or the control solvent into the NAc brain region of the mice through the indwelling catheter implanted during the previous surgery. Specifically, assemble the injection inner tube, PE tube, locking nut, and syringe in advance. Use an injection pump to aspirate the MCU inhibitor solution or the control solvent, and mark the position of the liquid medicine on the PE tube (mainly used to observe whether the liquid level of the medicine drops during the drug injection process). Then remove the catheter cap, slowly insert the injection inner tube into the catheter, and lock it with the locking nut. Set the injection volume of the injection pump to 1 μL and the injection speed to 0.2 μL / min, and start the injection. After the injection is completed, slowly pull out the injection inner tube, re - insert the catheter cap, place the mice in the open - field for 60 min to adapt, and record the movement trajectory of the mice; tighten.

[0135] ③ Inject a single dose of drug (10 mg / kg heroin) into the abdominal cavity of the mice, and continue to record the movement trajectory of the mice within 180 min.

[0136] ④ Analyze the experimental data according to the movement distance of each group of mice.

[0137] 3. Experimental results As Figure 4 Statistics of the motility of mice before and after drug administration stimulation: Mitoxantrone can significantly inhibit the locomotor excitability of wild mice induced by heroin (wild mice: control n = 10, mitoxantrone n = 10). Ru265 can also significantly inhibit the locomotor excitability of wild mice induced by heroin (wild mice: control n = 9, Ru265 n = 9). The above results indicate that inhibiting MCU can reduce the locomotor excitability caused by the opioid drug heroin.

[0138] Example 4 Effects of intracerebral administration of MCU inhibitors - mitoxantrone (MTX) and Ru265 on heroin relapse 1. Experimental materials (1) The conditioned place preference experiment (CPP) system was purchased from Anlai Scientific Instruments (Ningbo) Co., Ltd. (2) C57 / 6N mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (3) Heroin was purchased from China Food and Drug Administration (4) Heroin (China Food and Drug Inspection Institute), mitoxantrone was purchased from Medicilon Biopharmaceutical Technology Co., Ltd., Ru265 (Sigma-Aldrich, SML2991) (5) Stereotaxic apparatus, microscope, cold light source, warming pad, surgical pad, cannula holder, surgical instrument kit, alcohol cotton balls, dry cotton balls, cranial drill, isoflurane gas anesthesia system, cannula drug delivery system (catheter, injection inner tube, catheter cap, PE tube, small screw), micro-drug delivery system (micro-injection pump, micro-syringe), etc. were purchased from Reward Life Science Co., Ltd.

[0139] 2. Experimental methods Cannula implantation surgery ① Anesthetize the animal, shave the hair on the mouse's head with a shaver, and fix the head to a stereotaxic apparatus. Level the skull, and then locate the target brain area NAc (AP: +1.6, ML: ±0.8, DV: -4.5). Use a cranial drill to drill holes (at the same time, drill 2-3 small holes near the hole for screwing in the skull pins, which serve as points of force for dental cement to prevent the dental cement from falling off. At the same time, the diameter of these small holes needs to be smaller than the skull pins, so that the skull pins can be tightened to provide adhesion), then use a needle to slightly puncture the dura mater above the target brain area to expose the skull; the dura mater does not need to be punctured at the location where the skull pins are fixed. Screw the skull pin into the hole for about three turns, then slowly implant the prepared catheter into the skull through the clamp, and then wrap the screw and catheter with dental cement to fix them.

[0140] ② After waiting for the dental cement to solidify (about 15 minutes), remove the clamp (be careful not to move the catheter), and slowly insert the catheter cap and tighten it; determine whether the skin needs to be sutured based on the actual situation.

[0141] ③ The animal will recover in about 2-3 days. During this period, roxithromycin ointment can be applied around the skin to prevent inflammation and infection of the animal. At the same time, pay attention to the animal's condition and inject sugar and salt into the abdominal cavity in time to make the animal get enough energy. The next experiment will start 7 days after the operation.

[0142] Heroin conditioned place preference experiment in mice ① Pre - adaptation period: From the 1st day to the 3rd day of the experiment, the partition was removed, and the animals were placed in from the white box, enabling them to move freely between the two chambers. They were adapted once a day for 30 minutes each time, and the position preference video analysis system recorded simultaneously. The data of the 2nd and 3rd days of the experiment were statistically analyzed, and mice that stayed on one side of the box for more than 70% of the total time (with severe self - preference) were excluded. The remaining mice were randomly grouped according to the random principle and subsequent experiments were carried out. Animal grouping: The remaining animals were evenly and randomly grouped according to the difference in residence time in the black and white boxes (absolute value of CPP score).

[0143] ② Conditional training period: From the 4th day to the 11th day of the experiment, the partition was re - inserted. After intraperitoneal injection of 3 mg / kg heroin on the first day, the animals were restricted in the drug - paired box (depending on the grouping, the drug - paired box for different mice was either the white box or the black box) for 30 minutes; after intraperitoneal injection of normal saline on the second day, the animals were restricted in the other box for 30 minutes. Training was carried out once a day, with a 2 - day cycle, for a total of 4 cycles.

[0144] ③ Expression period: On the 12th day of the experiment, the day after the training ended, the partition was removed, and the animals were randomly placed in from one box. The residence time (s) of the animals in the drug - paired box and the non - drug - paired box within 30 minutes was recorded respectively, and the CPP score of the animals was calculated. The calculation formula was: CPP score (s) = residence time of the animal in the drug - paired box (s) - residence time of the animal in the non - drug - paired box (s).

[0145] ④ Withdrawal period: From the 13th to the 25th day of the experiment, the mice underwent a 12 - day withdrawal. The withdrawal process was the same as the pre - adaptation period. The mice moved freely in the two behavior boxes for 30 minutes every day, and the CPP scores of the mice were statistically analyzed. When the CPP score of the mice tended to 0, they entered the next stage.

[0146] ⑤ Drug - induced relapse period: On the 26th day of the experiment, the injection inner tube, PE tube, locking nut, and syringe were assembled in advance. Mitoxantrone solution (4.5 mM), or Ru265 solution (50 μM), or the control solvent was aspirated using an injection pump, and the position of the liquid medicine was marked on the PE tube (mainly used to observe whether the liquid level of the drug decreased during the drug injection process). Subsequently, the catheter cap was removed, the injection inner tube was slowly inserted into the catheter, and tightened with the locking nut. The injection volume of the injection pump was set to 1 μL and the injection speed was set to 0.2 μL / min, and injection began. After injection, it was left stationary for about 10 minutes until the drug was fully diffused, then the injection inner tube was slowly pulled out, the catheter cap was re - inserted and tightened. The mice were placed back in the breeding cage to recover for 30 min, and then 1.5 mg / kg heroin was injected. The mice were randomly placed in from one box, and the residence time (s) of the animals in the drug - paired box and the non - drug - paired box within 30 minutes was recorded respectively, and the CPP score of the animals was calculated.

[0147] 3. Experimental results As Figure 5 The results showed that there were no significant differences between the mice in the drug administration group and the control group during the test period, expression period, and withdrawal period, and they were comparable. During the drug-induced relapse period, compared with the mice in the solvent intracerebral region administration group, the CPP scores of the mice in the mitoxantrone and Ru265 intracerebral region administration groups were significantly decreased, and the difference was statistically significant (P < 0.05, Figure 5 , n = 10 in the control group, n = 10 in the mitoxantrone group, n = 10 in the Ru265 group). Therefore, intracerebral region administration of the MCU inhibitor can inhibit heroin-induced relapse behavior in mice, indicating that MCU in the NAc brain region is an effective target for the treatment of drug addiction relapse.

[0148] Example 5 Dopamine release induced by opioid drugs and methamphetamine 1. Experimental materials (1) Spontaneous activity behavior box [Anlai Scientific Instruments (Ningbo) Co., Ltd.] (2) Mice were purchased from Cyagen Biosciences Inc. (3) Heroin (heroin, National Institutes for Food and Drug Control), methamphetamine (METH, National Institutes for Food and Drug Control), cocaine (cocaine, Qinghai Pharmaceutical Factory) (4) AAV-Syn-DA2m dopamine probe was purchased from Vigene Biosciences Inc. (5) Fiber optic probe, three-color single-channel fiber optic recording system (both purchased from Nanjing Qianao Star Technology).

[0149] 2. Experimental methods 1) Anesthetize the animals, remove the hair on the heads of the mice with a hair clipper, and fix the heads to the stereotaxic apparatus. Level the skull, and then locate the target brain region NAc (AP: +1.6, ML: +0.8, DV: -4.5). After drilling a hole in the skull, use a microinjector to inject 50 nl of AAV-Syn-DA2m (3×10 13 vg / ml) virus at a speed of 50 nl / min. After standing for 5 min, implant a fiber optic probe 0.1 mm above the above NAc site. Then fix it with dental cement.

[0150] 2) Wait for the dental cement to solidify (about 15 minutes), remove the clamp (note not to drive the catheter), and judge whether it is necessary to suture the skin according to the actual situation.

[0151] 3) During the animal recovery period, it can recover in about 2 - 3 days. During this period, roxithromycin ointment can be applied around the skin to prevent the animals from getting inflamed and infected; at the same time, pay attention to the animal's condition and inject glucose and saline intraperitoneally in a timely manner to enable the animals to obtain enough energy. The next experiment will start 14 days after the operation.

[0152] 4) Place the mice in the functional laboratory 1 h before the experiment to acclimatize, set up the software, and lay wood shavings at the bottom of the behavior chamber.

[0153] 5) Subsequently, connect the fiber optic probe on the mouse's head to the three-color single-channel fiber optic recording system through an optical fiber, and start collecting signals excited at 470 nm. Then place it in the open field to acclimatize for 60 min (30 min for acute cocaine administration acclimatization), and record the movement trajectory of the mice.

[0154] 6) Inject a single dose of drug intraperitoneally into the mice (10 mg / kg heroin, 30 mg / kg morphine, 2 mg / kg METH, or 20 mg / kg cocaine), and continue to record the changes in the dopamine fluorescence signal in the mouse brain within 30 min.

[0155] 7) Then perform data analysis and plotting using the three-color single-channel analysis software.

[0156] 3. Experimental results The results are as Figure 6 shown. Knockout of MCU inhibits dopamine release induced by opioid drugs and methamphetamine, but has no effect on the dopamine elevation induced by cocaine. This experiment suggests that inhibiting MCU has an inhibitory effect on potential addictive substances that specifically induce dopamine release (such as heroin, morphine, methamphetamine, fentanyl, methadone, nicotine, alcohol, etc.).

[0157] The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Use of MCU inhibitors in the preparation of pharmaceutical compositions for preventing / treating drug addiction.

2. The use according to claim 1, characterized in that: The inhibitors of the MCU include nucleic acid inhibitors, protein inhibitors, and compounds; Preferably, the compound includes mitoxantrone, Ru265, RU360, RuR, DS16570511, KB-R7943, NecroX-5, Minocycline, MCU-i4, MCU-i11, and Doxycycline.

3. The use according to claim 1, characterized in that: Said drugs include opioids, amphetamines, alcohol, and tobacco; Preferably, the opioids include heroin, morphine, methadone, and fentanyl; Preferably, the amphetamine drugs include methamphetamine, ecstasy, and magu.

4. Use of MCU inhibitors in the preparation of pharmaceutical compositions for inhibiting relapse after withdrawal of addictive drugs; Preferably, the inhibitor of MCU includes nucleic acid inhibitors, protein inhibitors, and compounds; Preferably, the compound includes Mitoxantrone, Ru265, RU360, RuR, DS16570511, KB-R7943, NecroX-5, Minocycline, MCU-i4, MCU-i11, Doxycycline; Preferably, the addictive drugs include opioids, amphetamines, alcohol, and tobacco; Preferably, the opioids include heroin, morphine, methadone, and fentanyl; Preferably, the amphetamine drugs include methamphetamine, ecstasy, and magu.

5. Use of MCU inhibitors in the preparation of pharmaceutical compositions for inhibiting excitability caused by addictive drugs; Preferably, the inhibitor of MCU includes nucleic acid inhibitors, protein inhibitors, and compounds; Preferably, the compound includes Mitoxantrone, Ru265, RU360, RuR, DS16570511, KB-R7943, NecroX-5, Minocycline, MCU-i4, MCU-i11, Doxycycline; Preferably, the drugs include opioids, amphetamines, alcohol, and tobacco; Preferably, the opioids include heroin, morphine, methadone, and fentanyl; Preferably, the amphetamine drugs include methamphetamine, ecstasy, and magu.

6. Use of MCU inhibitors in the preparation of pharmaceutical compositions for inhibiting dopamine release; Preferably, the dopamine is dopamine in the nucleus accumbens brain region; Preferably, the inhibitor of MCU includes nucleic acid inhibitors, protein inhibitors, and compounds; Preferably, the compound includes Mitoxantrone, Ru265, RU360, RuR, DS16570511, KB-R7943, NecroX-5, Minocycline, MCU-i4, MCU-i11, Doxycycline; Preferably, the dopamine release is drug-induced dopamine release; Preferably, the drugs include opioids, amphetamines, alcohol, and tobacco; Preferably, the opioids include heroin, morphine, methadone, and fentanyl; Preferably, the amphetamine drugs include methamphetamine, ecstasy, and magu.

7. A pharmaceutical composition for preventing / treating drug addiction / inhibiting relapse after drug withdrawal / inhibiting excitability caused by addictive drugs / inhibiting dopamine release, characterized in that: The pharmaceutical composition includes an inhibitor of MCU; Preferably, the dopamine release is drug-induced dopamine release; Preferably, the pharmaceutical composition further comprises other drugs for preventing / treating drug addiction or inhibiting relapse after withdrawal of addictive drugs / inhibiting excitability caused by addictive drugs / inhibiting dopamine release; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; Preferably, the administration routes of the pharmaceutical composition include intravenous administration, oral administration, mucosal administration, nasal administration, and rectal administration; Preferably, the dosage form of the pharmaceutical composition includes a liquid dosage form and a solid dosage form; Preferably, the liquid dosage form includes tablets, suppositories, capsules, microgranules, powders; Preferably, the liquid dosage form includes injection, aerosol, emulsion, syrup, elixir, suspension, solution.

8. Application of MCU as a target in screening candidate drugs for preventing / treating drug addiction / inhibiting relapse after withdrawal of addictive drugs / inhibiting excitability caused by addictive drugs / inhibiting dopamine release; Preferably, the method for screening candidate drugs comprises: Treating a culture system expressing or containing the MCU gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the MCU gene or the protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the MCU gene or the protein encoded by it, the substance to be screened is a candidate drug for preventing / treating drug addiction / inhibiting relapse after withdrawal of addictive drugs / inhibiting excitability caused by addictive drugs.

9. A method for screening candidate drugs for preventing / treating drug addiction / inhibiting relapse after drug withdrawal / inhibiting excitability caused by addictive drugs / inhibiting dopamine release, characterized in that: The method comprises: treating a culture system expressing or containing an MCU gene or a protein encoded by the MCU gene with a substance to be screened; and detecting the expression or activity of the MCU gene or the protein encoded by the MCU gene in the system; wherein, when the substance to be screened inhibits the expression level or activity of the MCU gene or the protein encoded by the MCU gene, the substance to be screened is a candidate drug for preventing / treating drug addiction / inhibiting relapse after withdrawal of addictive drugs / inhibiting excitability caused by addictive drugs.

10. Application of reagents for detecting MCU expression levels in the preparation of products for diagnosing drug addiction; Preferably, the reagent is selected from a probe that specifically recognizes MCU, a primer that specifically amplifies MCU, or a binding agent that specifically binds to a protein encoded by the MCU gene; Preferably, the drugs include opioids, amphetamines, alcohol, and tobacco; Preferably, the opioids include heroin, morphine, methadone, and fentanyl; Preferably, the amphetamine drugs include methamphetamine, ecstasy, and ecstasy; Preferably, the product includes a chip, a test paper, a test kit or a nucleic acid membrane strip.

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