WO2020024071A1

WO2020024071A1 - Pharmaceutical composition for the treatment of addiction to psychoactive substances and/or the prevention of relapse to said addiction

Info

Publication number: WO2020024071A1
Application number: PCT/CL2018/050063
Authority: WO
Inventors: Fernando EZQUER; Marcelo EZQUER; Yedy ISRAEL JACARD; María Elena QUINTANILLA GONZÁLEZ; Paola MORALES RETAMALES; Mario Herrera Marschitz
Original assignee: Universidad Del Desarrollo; Universidad De Chile
Priority date: 2018-08-03
Filing date: 2018-08-03
Publication date: 2020-02-06
Also published as: UY38319A; AR115883A1

Abstract

The present invention related to a pharmaceutical composition for the treatment of chronic addiction to psychoactive substances and/or the prevention of relapse to an addiction to psychoactive substances, said composition comprising secretome and/or exosomes derived from preconditioned mesenchymal stem cells; and the use of said composition for the treatment of addiction to psychoactive substances and prevention of relapse to said addiction.

Description

PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF ADDICTION TO PSYCHOACTIVE SUBSTANCES AND / OR FOR PREVENTION

OF THE PURCHASES TO SUCH ADDICTION

Technical Field

The present invention relates to the technical field of pharmaceutical compositions for use in medical treatments, particularly it relates to a composition for the treatment of addiction to psychoactive substances and prevention of relapse to said addiction, which comprises secretoma and / or exosomes. derivatives of preconditioned mesenchymal stem cells; and the use of said composition for the treatment of addiction to psychoactive substances and prevention of relapses to said addiction.

Background

Addiction to psychoactive substances or drugs is a chronic disease that consists of physical and / or psychological dependence caused by compulsive consumption of these substances, which affects the person in their physical, mental and social health. The constant consumption of the substance generates physiological changes and activates the reward pathways in the brain (such as the release of dopamine) that translates into an uncontrollable need to continue using this drug despite the damage it produces. (Drugs, Brains and Behavior: The Science of Addiction. 2018. U. S. Department of Health and Human Services. NIH Publication No. 18-DA-5605).

The most common drugs of abuse, both legal and illegal, include, among others, alcohol, nicotine, cocaine, amphetamines, cannabinoids and opioids. Chronic consumption of these substances also generates serious health consequences such as heart disease, lung disease, cancer in different organs, liver cirrhosis, decreased fertility, and mental conditions such as depression, anxiety and insomnia, among others.

The treatment of drug addiction consists of approaching the person in an integral way, with drugs and cognitive-behavioral therapies to eliminate consumption and prevent the person from subsequently reoccurring addiction. There are different stages of treatment, which initially consists in detoxifying the subject, then treating withdrawal symptoms, that is, treating all those symptoms produced by abrupt stopping of drug use, achieving adherence to treatment to treat addiction. chronic consumption, and finally prevent the subject from falling into addiction.

As for the use of drugs during this therapy, these will depend on the drug to which the person is addicted and the stage of therapy in which they are. For example, methadone, buprenorphine, naltrexone and lofexidine are currently used for opioid addiction; replacement therapies (patches, inhalers or chewing gum), bupropion and varenicline are used for nicotine addiction, and naltrexone and acamprosate are used for alcohol addiction, both to prevent relapse to addiction, disulfiram for the stage of chronic addiction (as it causes exacerbations of unpleasant symptoms associated with alcohol consumption), and topiramate that is useful for treating symptoms associated with withdrawal symptoms. However, all these medications have a large number of adverse effects, very low adhesion, since they must be administered continuously several times a day, and very reduced therapeutic effect. For example, acamprosate, which is one of the best available drugs, prevents approximately only 18% of relapses of subjects (Hartung DM, et al. (2014). J Subst Abuse Treat 47: 113-121; Donoghue K, et al . (2015). Addiction 110: 920-930; Kranzler HR et al. (2004). Alcohol Clin Exp Res 28: 1051-1059).

Because of this, scientists have recently focused on the development of new drug alternatives for the treatment of addiction and other diseases, based on the use of mesenchymal stem cells, which have the potential to treat diseases as varied as heart failure. , neurodegenerative diseases, lung diseases, inflammatory diseases, among others.

Particularly, for the treatment of addiction, patent document CL 201700237 discloses an injectable composition that allows reducing the alcohol consumption of a person suffering from addiction or blocking relapse induced by deprivation or associated with the withdrawal process, which comprises Mesenchymal stem cells (MSC) without stimulation or preconditioned MSCs, which are mentioned to be used interchangeably. Specifically, this document teaches that the specific use of 5 x 10 ⁵ MSCs injected intracranially decreases alcohol consumption in a model of alcoholic rats.

On the other hand, US patent document 2017/0258843 A1 describes means for inducing neuroregeneration and / or neuroprotection in subjects with damage to the nervous system. In particular, it indicates the use of positive CD34 MSCs obtained from placenta, or exosomes derived from them. He mentions only once in the specification that the neurodegenerative condition may be drug addiction, but does not teach or suggest any pharmaceutical formulation or specific treatment method to treat subjects with addiction to psychoactive substances.

WO 2018/083700 A1, discloses pharmaceutical compositions comprising populations of MSCs, extracellular vesicles secreted by said population or a combination thereof, and methods of using treatments or diseases. In particular, he mentions that these pharmaceutical compositions are potentially useful for the treatment of neurological, muscular, autoimmune, inflammatory, digestive, energy homeostasis, fibrosis, aging, radiation-induced wounds, cell transplant rejection and proliferative diseases. Among the many diseases of such diverse origin, he casually mentions that such composition could be useful for the treatment of drug addiction. However, it does not set forth any example of embodiment in which it demonstrates that the MSCs or extracellular vesicles secreted by said population or a combination of these are effective for the treatment of drug addiction, nor does it suggest the method of administration, dosage regimen , or a specific pharmaceutical formulation for the treatment of this disease.

It is important to mention that there are strong controversies over the use of stem cell therapies, due to the potential risks that are involved, which includes the possible formation of tumors, development of immune responses and / or unwanted biological effects, in addition to the low level of control of the effects they produce, and the possibility of accidentally transmitting harmful agents by administering these cells to the subject. This type of therapy also has technical difficulties associated with the management, culture and storage of cells, which makes its application very unfeasible at the industrial level. For this reason, the use of secrecy and exosomes appears as an advantageous therapeutic alternative to replace the use of stem cells.

Consequently, there are no effective medicines to date for the treatment of addiction to psychoactive substances that serve as a complementary pharmacological treatment during the entire therapy process to overcome the addiction, both in the period of chronic consumption of the drug and in the period of critical time to avoid relapse to addiction.

Summary of the invention A first object of the present invention relates to a pharmaceutical composition for the treatment of addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances, which comprises between 0.05 to 50 mg. of total exosome proteins derived from preconditioned mesenchymal stem cells during their culture; and a pharmaceutically acceptable excipient.

Preferably, the pharmaceutical composition is a composition for intranasal or intravenous administration, more preferably, the composition for intranasal administration comprises between 0.5 and 5 mg of total proteins and the composition for intravenous administration comprises between 0, 5 and 5 mg of total proteins.

In a preferred embodiment, the psychoactive substance is selected from the group consisting of alcohol, nicotine, opioids and cocaine.

In another preferred embodiment, mesenchymal stem cells are obtained from mesenchymal stem cells of adipose tissue. A second object of the present invention relates to a pharmaceutical composition for the treatment of addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances, characterized in that it comprises between 0.08 to 80 mg of total protein of secretion of preconditioned mesenchymal stem cells during culture; and a pharmaceutically acceptable excipient.

Preferably, said pharmaceutical composition is a composition for intranasal or intravenous administration, more preferably, the composition for intranasal administration comprises between 0.8 and 8 mg of total proteins and the composition for intravenous administration comprises between 0, 8 and 8 mg of total proteins.

In another preferred embodiment, mesenchymal stem cells are obtained from adipose tissue.

A third object of the present invention relates to a pharmaceutical composition comprising between 0.05 to 50 mg of total exosome proteins of preconditioned mesenchymal stem cells during their cultivation, for use in a method of treating addiction to psychoactive substances. and / or to prevent relapse to addiction to psychoactive substances, where, preferably, said psychoactive substances are selected from the group consisting of alcohol, nicotine, opioids and cocaine.

Preferably, said pharmaceutical composition for use in a method of treating addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances is a composition for intranasal or intravenous administration, more preferably, the composition for intranasal administration comprises between 0.5 and 5 mg of total proteins and the composition for intravenous administration comprises between 0.5 and 5 mg of total proteins. In a preferred embodiment, mesenchymal stem cells are obtained from adipose tissue.

A fourth object of the present invention relates to a pharmaceutical composition comprising between 0.08 to 80 mg of total secreted proteins of preconditioned mesenchymal stem cells during culture, for use in a method of treating addiction to psychoactive substances. and / or to prevent relapse to addiction to psychoactive substances, where, preferably, said psychoactive substances are selected from the group consisting of alcohol, nicotine, opioids and cocaine.

Preferably, said pharmaceutical composition for use in a method of treating addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances is a composition for intranasal or intravenous administration, more preferably, the composition for intranasal administration comprises between 0.8 and 8 mg of total proteins and the composition for intravenous administration comprises between 0.8 and 8 mg of total proteins.

In a preferred embodiment, mesenchymal stem cells are obtained from adipose tissue.

A fifth object of the present invention relates to a method for treating a subject with addiction to psychoactive substances and / or for preventing a subject from falling into addiction to psychoactive substances, which comprises administering a pharmaceutical composition comprising between 0.05 to 50 mg of total proteins of pre-conditioned mesenchymal stem cell exosomes during their culture, where said psychoactive substances are selected from the group consisting of alcohol, nicotine, opioids and cocaine.

Preferably, said method for treating a subject with addiction to psychoactive substances and / or for preventing a subject from falling into addiction to psychoactive substances comprises administering the pharmaceutical composition intranasally or intravenously, where preferably the composition for intranasal administration comprises between 0.5 and 5 mg of proteins totals and the composition for intravenous administration comprises between 0.5 and 5 mg of total proteins.

A sixth object of the present invention relates to a method for treating a subject with addiction to psychoactive substances and / or for preventing a subject from falling into addiction to psychoactive substances comprising administering a pharmaceutical composition comprising between 0.08 a 80 mg of total secreted proteins from preconditioned mesenchymal stem cells during their culture, where said psychoactive substances are selected from the group consisting of alcohol, nicotine, opioids and cocaine.

Preferably, said method for treating a subject with addiction to psychoactive substances and / or for preventing a subject from falling into addiction to psychoactive substances comprises administering the pharmaceutical composition intranasally or intravenously, where preferably the composition for intranasal administration comprises between 0.8 and 8 mg of total proteins and the composition for intravenous administration comprises between 0.8 and 8 mg of total proteins.

A seventh object of the present invention relates to the use of a pharmaceutical composition comprising between 0.05 to 50 mg of total proteins of pre-conditioned mesenchymal stem cell exosomes during their cultivation, for the manufacture of a medicament useful for the treatment of addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances, where preferably, said psychoactive substances are selected from the group consisting of alcohol, nicotine, opioids and cocaine.

Preferably, the use of the pharmaceutical composition for the manufacture of a medicament for the treatment of addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances refers to the use of a composition for intranasal or intravenous administration, where preferably the composition for intranasal administration comprises between 0.5 and 5 mg of total proteins and The composition for intravenous administration comprises between 0.5 and 5 mg of total proteins.

An eighth object of the present invention relates to the use of a pharmaceutical composition comprising between 0.08 to 80 mg of total secreted proteins of preconditioned mesenchymal stem cells during their cultivation, for the manufacture of a medicament useful for the treatment of addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances, where preferably, said psychoactive substances are selected from the group consisting of alcohol, nicotine, opioids and cocaine.

Preferably, the use of the pharmaceutical composition for the manufacture of a medicament for the treatment of addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances comprises the use of a composition for intranasal or intravenous administration. , wherein preferably the composition for intranasal administration comprises between 0.8 and 8 mg of total proteins and the composition for intravenous administration comprises between 0.8 and 8 mg of total proteins.

Brief Description of the Figures

Figure 1 shows in (A) a distribution chart of microvesicle sizes that were isolated from mesenchymal stem cells obtained from adipose tissue (AD-MSC) preconditioned during culture; (B) sample a transmission electron microscopy image in which the integrity of the exosome sample is displayed (100 nm scale bar); (C) shows a Western Blot with classic molecular markers of exosomes (EXO lane), compared to the complete cell lysate (Cell lane).

Figure 2 shows in (A) a graph that summarizes the results obtained with exosomes in the study of the evaluation on chronic consumption, for which the voluntary ethanol consumption of animals that consumed ethanol for 13 weeks and received 3 doses was determined intranasal exosomes derived from AD-MSC preconditioned during cultivation, where ethanol consumption was expressed as g of ethanol consumed / kg of weight / day; (B) a graph of water consumption in the same animals of (A), where the data were expressed as mi of water consumed / kg weight / day; (C) a bar graph with the result of alcohol consumption during the 60 minutes of re-access to ethanol after the period of deprivation (relapse evaluation), where ethanol consumption was expressed in g of ethanol consumed / kg weight /60 minutes; (D) ethanol levels in the blood after 60 minutes of re-access where alcohol relapse was assessed.

Figure 3 shows in (A) dorsal, ventral and sagittal views of the brain of animals that consumed ethanol for 13 weeks, 2, 6, 12 and 24 hours after the administration of an intranasal dose of exosomes derived from AD-MSC labeled with PKH26 (fluorescent marker), where the administration of saline with PKH26 was used as a control; (B) Representative microphotographs showing fluorescence of labeled exosomes in the perinuclear cytoplasm of a cell and in the extracellular space. Scale bar = 25 pm.

Figure 4 shows in (A) a graph of the voluntary ethanol consumption of animals that consumed ethanol for 10 weeks and received 3 intranasal doses of AD-MSC-derived secretoma during their culture (evaluation of chronic consumption), where the Ethanol consumption was expressed as g of ethanol consumed / kg of weight / day; (B) a bar chart with the result of alcohol consumption during the 60 minutes of re-access to ethanol after the period of deprivation (relapse evaluation), where the Ethanol consumption was expressed in g of ethanol consumed / kg weight / 60 minutes; (C) blood ethanol levels after 60 minutes of re-access where alcohol relapse was assessed.

Figure 5 shows in (A) a graph of voluntary ethanol consumption of animals that consumed ethanol for 13 weeks and received 3 intranasal doses of spheroid-derived exosomes from AD-MSC (assessment of chronic consumption), where consumption of ethanol was expressed as g of ethanol consumed / kg of weight / day; (B) a bar graph with the result of alcohol consumption during the 60 minutes of re-access to ethanol after the period of deprivation (relapse evaluation), where ethanol consumption was expressed in g of ethanol consumed / kg weight /60 minutes.

Figure 6 shows in (A) a graph of the voluntary nicotine consumption of the animals that consumed nicotine for 19 weeks and received 3 intranasal doses of AD-MSC-derived secretoma pre-conditioned during their culture (evaluation of chronic consumption), where the Nicotine consumption was expressed as mg of nicotine consumed / kg of weight / day; (B) a bar graph with the result of nicotine consumption during the 60 minutes of re-access to nicotine after the period of deprivation (relapse evaluation), where nicotine consumption was expressed in mg of nicotine consumed / kg weight /60 minutes.

Detailed description of the invention

The present invention provides an effective pharmaceutical composition for the treatment of addiction to psychoactive substances and / or to prevent the relapse of a subject to said addiction. The present invention also provides the composition for use in the treatment of these diseases, a specific treatment method for addiction to psychoactive substances and / or to prevent relapse to said addiction, and the use of the composition for the manufacture of a Useful medicine in the treatment of these diseases.

The pharmaceutical composition of the invention comprises the secrecy of preconditioned mesenchymal stem cells or exosomes derived from Mesenchymal stem cells preconditioned for pharmacological treatment during all or part of the therapy, that is, for overcoming addiction to psychoactive substances.

The composition object of the present application can be used both for the treatment of chronic addiction to drugs of a subject, and during the relapse stage of the subject to said addiction, or during complete therapy. Specifically, the composition of the invention contains a concentration between 0.08 to 80 mg of total secretoma proteins or between 0.05 to 50 mg of total exosome proteins, both obtained from preconditioned mesenchymal stem cells during their cultivation, and a pharmaceutically acceptable excipient. The range of total protein concentrations described for both cases corresponds to an effective concentration for the treatment of addiction to psychoactive substances and / or to prevent relapses to addiction to said substances.

All technical and scientific terms used to describe the present invention have the same meaning understood for a person with basic knowledge in the technical field in question. However, to more clearly define the scope of the invention, a list of the terminology used in this description and its meaning is included below.

The term "psychoactive substances" or "drug" should be understood as a chemical that alters brain functions, generating changes in the perception, mood, consciousness, cognitive faculties or behavior of an individual. Said psychoactive substances may be consumed by medical prescription or for recreational purposes, and may be natural or synthetic chemicals. Among some examples we can mention central nervous system stimulants, hallucinogens, hypnotics, analgesics, among others.

The term "treatment" should be understood as the set of means used to cure or alleviate a disease or condition, and that may be part of the therapy for it. The term "mesenchymal stem cells" should be understood as pluripotential stromal cells that have the ability to differentiate into various cell types. These cells are responsible for maintaining the normal turnover of tissue cells and favor regeneration processes after damage. In addition, these cells are recognized for their ability to reduce oxidative stress and secrete anti-inflammatory cytokines.

The term "exosome" should be understood as the membranous vesicles of size 150 nm or less, which are formed in the endocytic compartments and are secreted to the extracellular space by several cell types. These exosomes contain mainly proteins and RNA of different types.

It should be understood by the term "secretoma", the set of soluble molecules, vesicles and exosomes that secrete different cell types, that is, everything that a cell secretes to the extracellular environment.

The term "preconditioned cells" should be understood as the cells that during the cultivation process are subjected to certain growth and incubation conditions, which allow inducing and / or increasing, and / or changing the composition in the secretion of metabolites, factors of growth, proteins, miRNA, vesicles and exosomes to the culture medium. Enriched culture medium, after preconditioning, is called conditioned medium. The present invention relates to a pharmaceutical composition for the treatment of addiction to psychoactive substances and / or to prevent relapses to said addiction, which comprises between 0.08 to 80 mg of total secreted proteins of preconditioned mesenchymal stem cells; and a pharmaceutically acceptable excipient. The present invention also relates to a pharmaceutical composition for the treatment of addiction to psychoactive substances and / or to prevent relapses to said addiction, which comprises between 0.05 to 50 mg of total proteins of pre-conditioned mesenchymal stem cell exosomes; and a pharmaceutically acceptable excipient. Psychoactive substances or drugs that usually generate addiction and dependence include caffeine, theophylline, theobromine, alcohol, barbiturants (eg amobarbital, pentobarbital, secobarbital, phenobarbital), ether, hydroxybutyrate range (GBH), benzodiazepines (eg lorazepam, diazepam, alpra, alpra , clonazepam, midazolam), opioids (eg codeine, morphine, heroin, oxycodone, pethidine, methadone, buprenorphine), cannabinoids, nicotine, amphetamine, dextroamptamine, methamphetamine, dimethoxyanfetamine, tenamfetamine, methylephethamine bimethalamine (ethylamine phenylamine) (b) ), ephedrine, pseudoephedrine, mezcalin, phenylethylamine, methylphenidate, cocaine, cocaine hydrochloride and sodium bicarbonate (crack), 3,4-Methylenedioxymethamphetamine (MDMA), lysergic acid diethylamide (LSD), psilocybin (psilocipta), alpha-methyl (alpha-methyl) AMT), dimethyltriptamine (DMT), ibogaine, dextromethorphan (DXM), ketamine, phencyclidine (PCP), nitric oxide, toluene, ibothenic acid and muscimol (Am anita muscaria), salvorina (Salvia divinorum), belladonna (Atropa belladonna), estramonium (Datura stramonium), phenothiazines (ex. chlorpromazine), butyrophenones (eg haloperidol, benperidol, droperidol), risperidone, quetiapine, alkyl nitrites (eg isopropyl nitrite, 2-propyl nitrite, isobutyl nitrite, amyl nitrite, butyl nitrite), inhibitors of monoamine oxidase (MAOls), tricyclic antidepressants (eg imipramine), serotonin reuptake inhibitors (eg fluoxetine), pentalenetetrazole, bicuculin, among others. In a particular embodiment of the present invention, the pharmaceutical composition is useful for the treatment of addiction to psychoactive substances and / or for the prevention of relapse to said addiction to alcohol, nicotine, opioids and cocaine, but is not limited to these , being able to be useful for any of the substances previously mentioned or others that do not appear explicitly in said list.

Mesenchymal stem cells can be obtained from a variety of sources such as veins, in particular aortic vein, adipose tissue, amniotic fluid, bone marrow, blood, brain, cartilage, umbilical cord, dental pulp (eg SHED, DFSC, DPSC, PDSC , STDPSC, CD105 < ⁺ >, STRO-1 <- ^{/ +} >, Nestin-1 ⁽⁺⁾ ), menstrual fluid, Warton jelly, endometrium, placenta, eye tissue, intestines, heart tissue, kidneys, liver, lungs, muscle tissue, pancreas, perichondrium, salivary glands, dermis, spleen, synovial membrane, tendons, thymus, among other tissues and organs. In a particular embodiment of the present invention, the pharmaceutical composition comprises secretoma or exosomes derived from mesenchymal stem cells obtained from adipose tissue, but is not limited thereto, and can be obtained from any of the previously mentioned or other organs and tissues that do not appear explicitly. in this listing.

The preconditioning of mesenchymal stem cells can be carried out by the growth of said cells under particular conditions that can be by adding chemicals and / or mechanical stimuli, such as subjecting the cells to hypoxia, incubating the cells with factors such as cytokines, subjecting the cells to radiation, subject the cells to the hanging drop method to form spheroids, or any other method known in the state of the art for this purpose. Preferably, the preconditioned mesenchymal stem cells of the present invention are incubated with a cytokine or a mixture thereof, a mixture of TNF-alpha and IFN-gamma is even more preferred. Any pro-inflammatory cytokine such as IL1a, II_1b, IL6, IL17A, MCP1, any combination of these, or any other cytokine known in the state of the art could also be used. The concentrations of cytokines that can be used are also variable, which can be between 1 ng / ml to 50 ng / ml, and the incubation periods in which the cells are grown can also vary between 24 to 72 hours.

From these preconditioned mesenchymal stem cells, the secretoma is obtained and exosomes are also obtained, which are effective both for the treatment of addiction to psychoactive substances and to prevent relapse to the addiction of a subject. The preconditioning of the mesenchymal stem cells to obtain the secretoma or to obtain the exosomes are slightly different, since these cells are incubated under specific conditions for each. Obtaining said secrets and exosomes can be performed by different techniques known in the state of the art. In a preferred embodiment, the secrets are obtained by centrifugation for the elimination of the cells, followed by filtration for the elimination of the cellular debris, obtaining in the supernatant the secretoma purified. On the other hand, exosomes are preferably obtained by ultracentrifugation, to obtain exosomes in the precipitate.

The present invention comprises a concentration between 0.08 to 80 mg of total secreted proteins of preconditioned mesenchymal stem cells, corresponding to a pharmaceutically effective concentration for the treatment of addiction to psychoactive substances and / or to prevent relapses of subjects subject to addiction. . In turn, the invention also comprises a concentration between 0.05 to 50 mg of total proteins of pre-conditioned mesenchymal stem cell exosomes, corresponding to a pharmaceutically effective concentration for the treatment of addiction to psychoactive substances and / or to prevent relapses of subject to addiction However, it is known in the related field that these concentrations may vary according to the particular conditions of each subject and the stage of the disease. In that sense, it is anticipated that the pharmaceutical composition comprises an effective amount of secrecy or exosomes that can achieve a decrease in the consumption of psychoactive substances by the subject within a certain period of time.

The pharmaceutically acceptable excipients present in the composition are vehicles that allow to administer the secretoma or exosomes derived from mesenchymal stem cells, to an individual. Said excipients will depend directly on the route of administration that is used and on the pharmacodynamic and pharmacokinetic properties that the composition is to be granted. As such, a pharmaceutically acceptable excipient can be a binder diluent, disintegrant, lubricant or a coating compound. In this sense and according to the route of administration used, the composition may be a soluble pharmaceutical form, gel, powder, transdermal patches, etc.

Any of the pharmaceutical compositions of the present invention are preferably a composition for intranasal or intravenous administration. But it is not limited to these pharmaceutical forms, other possible routes of administration being, for example, oral, subcutaneous, intramuscular, intraperitoneal, intrathecal, percutaneous, etc. In a particular embodiment of the pharmaceutical compositions intranasally or intravenously, these preferably comprise secrets or exosomes derived from mesenchymal stem cells and an excipient selected from the group consisting of saline solution (0.9% NaCl), water bi-distilled, phosphate buffered saline (PBS), ethanol, chitosan, alginate, polysorbate 80, 5% glucose, polyols, or mixtures thereof, but not limited to these, being able to select any other pharmaceutically acceptable excipient, such as those They are mentioned previously. In another preferred embodiment, the concentration of total exosome proteins present in the pharmaceutical composition intranasally and intravenously is between 0.05 to 5 mg, while the concentration of total secreome proteins present in the pharmaceutical composition intranasally. and intravenously it is between 0.08 to 8 mg.

Another object of the present invention relates to a formulation comprising between 0.05 to 50 mg of total exosome proteins or 0.08 to 80 mg of total secretoma proteins, both obtained from preconditioned mesenchymal stem cells, for use in a method of treating addiction to psychoactive substances and / or to prevent relapse of the subject to addiction. Said treatment method is useful for subjects addicted to psychoactive substances such as any of those mentioned previously, although it is preferably useful as a treatment method against addiction to alcohol, nicotine, opioids and cocaine, and to prevent relapse of the subject to addiction. to these substances.

Another object of the present invention relates to a method for treating a subject with addiction to psychoactive substances and / or for preventing the relapse of a subject to addiction to psychoactive substances which comprises administering between 0.05 to 50 mg of total proteins. of exosomes derived from preconditioned mesenchymal stem cells or administer between 0.08 to 80 mg of total secreoma proteins derived from preconditioned mesenchymal stem cells.

This method to treat a subject with addiction to psychoactive substances and / or to prevent their relapse to such substances is useful for subjects addicted to psychoactive substances such as any of those mentioned previously, although it is preferably useful as a method of treatment against addiction to alcohol, nicotine, opioids and cocaine, and / or as a method of treatment to prevent the relapse of the subject to the addiction of said drugs.

In another object, the present invention also relates to the use of a formulation comprising between 0.05 to 50 mg of total exosome proteins derived from preconditioned mesenchymal stem cells or 0.08 to 80 mg of total cell derived secretoma proteins preconditioned mesenchymal mother, for the manufacture of a drug for addiction to psychoactive substances and / or for the prevention of relapse to addiction to said psychoactive substances.

The use of these formulations for the manufacture of a medicament for treating a subject with addiction to psychoactive substances and / or for preventing their relapse to said substances is useful for subjects addicted to psychoactive substances such as any of those mentioned previously, although it is preferably useful for the manufacture of a drug against addiction to alcohol, nicotine, opioids and cocaine, and as a method of treatment to prevent the relapse of the subject to the addiction of such drugs.

The pharmaceutical compositions of the present invention have a dosage regimen that depends on medical practice and varies according to parameters of each individual subject, such as type of addiction, age, sex, body weight, site and method of administration of the composition, etc. The regimen may correspond to the administration of one or multiple doses, for a period of several days, weeks, months or even years. The treatment generally has a proportional duration depending on whether it corresponds to a treatment for addiction, for the prevention of relapse or for complete therapy, the type of psychoactive substance to which the subject is addicted, to the type of subject, stage of the disease and its adherence to treatment and therapy.

The present invention also relates to a pharmaceutical composition comprising between 0.05 to 50 mg of total exosome proteins or between 0.08 to 80 mg of total secrecy proteins of mesenchymal stem cells. preconditioned during cultivation, for use in a method of treating addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances, where, preferably, said psychoactive substances are selected from the group consisting of alcohol, nicotine , opioids and cocaine.

Preferably, said pharmaceutical composition comprising exosomes of preconditioned mesenchymal stem cells during their cultivation for use in a method of treating addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances is a composition for administration by intranasally or intravenously, more preferably, the composition for intranasal administration comprises between 0.5 and 5 mg of total exosome proteins and the composition for intravenous administration comprises between 0.5 and 5 mg of total exosome proteins.

Preferably, said pharmaceutical composition comprising secretion of preconditioned mesenchymal stem cells during their cultivation for use in a method of treatment of psychoactive substance addiction and / or for use in a treatment method to prevent relapse to substance addiction Psychoactive is a composition for intranasal or intravenous administration, more preferably, the composition for intranasal administration comprises between 0.8 and 8 mg of total secretoma proteins and the composition for intravenous administration comprises between 0.8 and 8 mg total secretoma proteins.

Another object of the present invention relates to a method for treating a subject with addiction to psychoactive substances and / or a method to prevent a subject from falling into addiction to psychoactive substances comprising administering a pharmaceutical composition comprising between 0.05 to 50 mg of total exosome proteins or between 0.08 to 80 mg of secretion of preconditioned mesenchymal stem cells during their culture, where said psychoactive substances are selected from the group consisting of alcohol, nicotine, opioids and cocaine. Preferably, said method for treating a subject with addiction to psychoactive substances and / or for preventing a subject from falling into addiction to psychoactive substances comprises administering the pharmaceutical composition comprising exosomes of preconditioned mesenchymal stem cells during their intranasal or intravenous culture. , wherein preferably the composition for intranasal administration comprises between 0.5 and 5 mg of total proteins and the composition for intravenous administration comprises between 0.5 and 5 mg of total proteins.

Preferably, said method for treating a subject with addiction to psychoactive substances and / or for preventing a subject from falling into addiction to psychoactive substances comprises administering the pharmaceutical composition comprising secretion of preconditioned mesenchymal stem cells during their intranasal or intravenous culture. , wherein preferably the composition for intranasal administration comprises between 0.8 and 8 mg of total proteins and the composition for intravenous administration comprises between 0.8 and 8 mg of total proteins.

Another object of the present invention relates to the use of a pharmaceutical composition comprising between 0.05 to 50 mg of total exosome proteins or 0.08 to 80 mg of total secreted proteins of preconditioned mesenchymal stem cells during their cultivation, for the manufacture of a medicament for the treatment of addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances, where preferably, said psychoactive substances are selected from the group consisting of alcohol, nicotine, opioids and cocaine.

Preferably, the use of the pharmaceutical composition for the manufacture of a medicament for the treatment of addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances comprises a pharmaceutical composition containing exosomes of preconditioned mesenchymal stem cells during its culture which is for intranasal or intravenous administration, where preferably the Composition for intranasal administration comprises between 0.5 and 5 mg of total proteins and the composition for intravenous administration comprises between 0.5 and 5 mg of total proteins.

Preferably, the use of the pharmaceutical composition for the manufacture of a medicament for the treatment of addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances comprises a pharmaceutical composition containing secretion of preconditioned mesenchymal stem cells during its culture, which is a composition for intranasal or intravenous administration, where preferably the composition for intranasal administration comprises between 0.8 and 8 mg of total proteins and the composition for intravenous administration comprises between 0.8 and 8 mg total protein.

The following examples are intended to illustrate the invention and its preferred embodiments, but under no circumstances should they be considered to restrict the scope of the invention, which will be defined by the wording of the appended claims.

Note that although these tests have been performed on animals, the models used have been scientifically validated for the pathologies evaluated, and the doses that are included for use in humans are based on extrapolation of the dose / brain weight ratio from the results obtained in adult rats.

EXAMPLES OF REALIZATION

Example 1. Administration of an intranasal composition with exosomes derived from MSC preconditioned during cultivation reduces alcohol consumption and prevents relapse.

Animal model of chronic alcohol consumption

Two month old female rats of the Wistar strain that were selectively bred as alcohol consumers were used in the experiments (University of Chile Bibulous; UChB). The animals were fed regularly with a diet of soy protein rodents. The experimental animal procedures were approved by the Ethics Committee for Laboratory Animal Studies of the Faculty of Medicine, University of Chile. Animals weighing between 180 and 250 g were housed in individual cages in a room with temperature and humidity control under cycles of 12 light / dark hours for a week to acclimatize the animals to the test conditions. During this period there was food and water freely available.

For the induction of chronic alcohol consumption, the animals had access to free choice 24 hours a day at 10% v / v ethanol and water for 14 weeks. In the last two weeks the animals were able to choose between 10% v / v ethanol, 20% v / v ethanol and water from separate tubes. Animals were deprived of alcohol consumption for 14 days to assess relapse to alcohol consumption, and then again allowed access to 10% v / v and 20% v / v ethanol solutions for only 60 minutes.

Isolation, expansion and characterization of mesenchymal stem cells (MSC) obtained from adipose tissue

MSCs from human adipose tissue were isolated from samples of subcutaneous adipose tissue (from the abdominal region), after written informed consent from the patients. All protocols were approved by the Ethics Committee of the Faculty of Medicine, German Clinic - University of Development. MSCs from adipose tissue were isolated and expanded according to the following protocol: 200 ml aliquots of fat were washed twice with an equal volume of phosphate buffered saline (PBS) and cut into small pieces. Samples were digested with 3 mg / ml type II collagenase in PBS, and incubated for 30 minutes at 37 ° C. At the end of digestion, 10% fetal bovine serum (FBS) was added to neutralize collagenase. The mixture was then centrifuged at 400 g for 10 minutes to remove floating adipocytes. The precipitate was re-suspended in a-MEM culture medium which was supplemented with 10% FBS and 0.16 mg / ml gentamicin. The cells were seeded at a density of 7000 cells / cm ² and were grown at 37 ° C in a 5% CO2 atmosphere. When the cells reached a confluence of 90%, they were detached with 0.25% trypsin, 2.65 mM EDTA, and centrifuged and subcultured at 7000 cells / cm ² . After two subcultures, the cells were characterized according to their potential for adipogenic and osteogenic differentiation. For this, the MSCs were incubated with adipogenic differentiation medium consisting of a-MEM which was supplemented with 10% FBS, 1 mM dexamethasone and 10 mM rosiglitazone for 15 days, and osteogenic differentiation medium consisting of a-MEM which was supplemented with 10% FBS, 0.1 mM dexamethasone and 50 pg / ml ascorbate-2-phosphate and 10 mM beta-glycerol phosphate for 21 days. In both cases, the medium containing the differentiating stimulus was replaced every 3 days. To assess the adipogenic potential, the cultures were stained with Oil Red-O. To assess the osteogenic potential, the cultures were fixed with 10% ethanol and stained with Alizarin Red-S. The cells were also characterized by immunophenotyping, for this the presence of putative human MSC markers (CD13, CD29, CD73, CD90 and CD105) and the absence of characteristic markers of other cell lineages (CD31, CD45 and CD235a) were evaluated. For this, immunophenotyping was performed by flow cytometry after immunostaining with monoclonal antibodies for said mentioned markers.

MSC preconditioning of human adipose tissue (AD-MSC) and obtaining exosomes

AD-MSC of human adipose tissue was preconditioned at a confluence of 70% by incubation in a-MEM which was supplemented with 10% fetal bovine serum free of exosomes, 10 ng / ml TNF-a and 15 ng / ml IFN -g for 48 hours. 48 hours after the preconditioning of the MSCs began, the culture medium (secretoma) was collected. The medium was centrifuged at 400 g for 10 minutes to remove entire cells. The supernatant was recovered, centrifuged again at 5,000 g and filtered on 0.22 pm syringe filters to remove the cellular debris. For the isolation of the exosomes, the medium was ultracentrifuged at 170,000 g for 1 hour to precipitate the exosomes. The precipitate was washed with PBS and re-ultracentrifuged. The exosome precipitate was diluted in filtered saline and the protein concentration was determined with the MicroBCA Protein Assay kit. The size and concentration of the exosomes was analyzed using NanoTracking System Analysis (NTA) in the NanoSight NS500 and by transmission electron microscopy with a Phillips Morgagni electron microscope. Gel electrophoresis and Western Blots were performed to confirm the enrichment of the classic exosome markers (GM130, ALIX, Flotilina and EpCAM) in exosome samples compared to the entire cell lysate. Calnexin and HSP60 markers were used to rule out contamination with endoplasmic reticulum.

Figure 1A shows the distribution of microvesicle sizes that was determined by the NanoTracking Analysis system. The main peak confirms that the microvesicles have a size range between 70 to 200 nm, with a main concentration in the range between 100 to 120 nm, size previously described for exosomes. Figure 1B shows exosomes in the order of 80 to 100 nm, which were visualized by transmission electron microscopy. The dark area surrounding the exosomes is indicative of membrane associated proteins. Figure 1C shows a Western blot with classic GM130, ALIX, Flotilin, and EpCAM exosome markers, which is compared to the complete MSC lysate. The absence of the calnexin and HSP60 markers in the exosomal fraction rules out contamination with cellular endoplasmic reticulum in exosomal samples.

Intranasal administration of the composition with pre-conditioned AD-MSC derived exosomes during culture, and determination of alcohol consumption in rats

After approximately 100 days of chronic alcohol consumption, the rats were anesthetized with doral hydrate (280 mg / kg) intraperitoneally (ip) and placed supine. 20 ml of exosome solution was administered intranasally as drops from a small pipette every 5 minutes (4 times in each nostril) on alternate sides of the nasal cavity for a total 20 minutes A total volume of 160 ml of saline solution containing 1.5 x 10 ⁹ exosomal particles (7.5 pg of exosomal proteins) was administered into the nasal cavity. The intranasal administration was repeated once a week for 5 weeks. The animals of the control group received 160 ml of saline solution with the same administration scheme previously mentioned.

The consumption of ethanol and water was determined every 24 hours by difference in volume of inverted graduated cylinders of 100 ml. Ethanol consumption was expressed as g of ethanol consumed / kg of weight / day. Water consumption was expressed as mi of water consumed / kg of weight / day. To determine the effect of the treatment on ethanol consumption after the condition of alcohol deprivation (relapse evaluation), 17 days after the administration of the first dose of the composition with exosomes or saline solution, a period of 14 days of withdrawal, followed by 60 minutes of re-access to 10% ethanol and 20% ethanol, as previously mentioned. Ethanol consumption was expressed as g of ethanol consumed / kg of weight / 60 minutes. During the period of alcohol deprivation, the animals received 2 doses of exosome composition or saline solution (vehicle) at weekly intervals. Re-access to ethanol was allowed 3 days after the last intranasal administration of the composition with exosomes or saline.

The concentration of ethanol in the blood was also determined as follows. After the rats had 60 minutes of re-access to ethanol and water, 100 ml of blood samples without anticoagulants were collected from the tip of the tail under moderate sedation with acepromazine (1 mg / kg i.p). Blood samples were immediately added to 0.9 ml of distilled water at 4 ° C in a sealed glass vial with Mininert valves. The samples were analyzed by gas chromatography using the headspace technique (Perkin Elmer SRI 8610).

Figure 2A shows the inhibitory effects on chronic ethanol consumption of exosomes that were administered intranasally. The inhibitory effect was very fast. Statistical analysis of two-way variance (ANOVA) of alcohol consumption indicated a significant effect of the dose of 1.5 x 10 ⁹ exosomal particles (7.5 pg of total proteins) that were administered intranasally once a week for 3 weeks compared to the group that was treated with vehicle only [Fttreatment (i, i92) ^'. 924.6; p <0.001]. Post hoc tests revealed that the rats that were treated with intranasal doses of 1.5 x 10 ⁹ exosomal particles (7.5 pg total protein) once a week for 3 weeks showed a significant inhibition of alcohol consumption during all the days that were recorded versus the rats that were treated with vehicle. Moreover, a single dose of 7.5 pg of total exosome proteins inhibited alcohol consumption by 61% after 48 hours of its administration. The initial effect on alcohol consumption had a half-life in the order of 5 to 7 days. The second intranasal administration of exosomes after 7 days of the first administration decreased alcohol consumption by 75% below the control levels. Compared to the ethanol consumption that resulted from the first intranasal dose, the second dose induced a significant reduction in ethanol consumption [F _{(i, i3)} : 26.89; p <0.007]. The third intranasal administration of exosomes inhibited ethanol consumption by 84% versus the control group, but there was no significant difference compared to the second dose of exosomes. Inhibition of alcohol consumption induced by exosomes was offset by increases in water consumption (Figure 2B [Ftreatment (i, i 92): 283.1; p <0.0007]), which indicates that the animals maintained their water balance. Four days after the third weekly administration, the animals were deprived of ethanol while the administration of a fourth and fifth dose of exosomes was continued under the same weekly schedule. Three days after the last intranasal administration of exosomes, the animals were offered 10% and 20% ethanol solutions (water tube also available) for only 60 minutes. Animals in the control group ingested 1.65 ± 0.09 g of ethanol / 60 minutes, while animals that were treated with exosomes ingested significantly less than 0.75 ± 0.24 g ethanol / 60 min (Figure 2C [f-test = 3,355; p <0.01 \ n \ 6 rats / group]). The concentration of ethanol in the blood that was determined immediately after 60 minutes of re-access to ethanol was 115 ± 20 mg / dl for control animals (which in humans would be considered a compulsive consumption of alcohol), while blood alcohol levels of animals treated with exosomes reached 58.6 ± 14 mg / dl [f-test = 2.318; p <0.049; n \ 6 rats / group].

These results show that the administration of exosomes derived from preconditioned mesenchymal stem cells during their culture significantly reduces ethanol consumption, reaching an inhibition of chronic alcohol consumption of 75-80% in the animals tested. Intranasal administration of exosomes also inhibited the relapse of alcohol consumption by 55% after ethanol deprivation, and prevented the increase in blood ethanol levels that in humans (> 80 mg / dl) would be considered a “ compulsive consumption ”.

Evaluation of the distribution of exosomes in the brain of rats after intranasal administration

To determine the ability to reach the brain of exosomes that were administered intranasally, the exosomes were stained with the PKH26 membrane fluorescent dye according to the manufacturer's instructions. The stained exosomes were washed with 4 ml of saline solution and concentrated again by ultracentrifugation. 160 ml of saline solution containing 1.5 x 10 ⁹ labeled exosomal particles or the same volume of control PKH26 saline solution was administered intranasally, as previously described. After 2, 6, 12 and 24 hours of exosomal administration, the animals were anesthetized and perfused intracardially with 100 ml of 0.1 M PBS (pH 7.4), followed by 200 ml of formalin solution. Brains were carefully removed and pKH26 fluorescence was evaluated in dorsal, ventral and sagittal views of the brain with the use of the In-Vivo MS FX PRO (Brunker, In Vivo Imaging System).

Additionally, sagittal sections of the brain of 30 pm thick were obtained by using a cryostat. Sections were mounted on silanized slides, washed with 0.1 M PBS and incubated with 4.6 diamino-2-phenylindole (DAPI; 0.02 M) for 2 hours. After washing them, the Samples were mounted with Fluoromount, and examined with confocal microscopy.

As shown in Figure 2A, there is a clear inhibition of ethanol consumption (60 to 70%) that was observed within the first 24 hours after intranasal administration of exosomes. In line with this observation, Figure 3A shows that fluorescent exosomes (by the fluorescent brand PKH26) are clearly found in the brain at 6, 12 and 24 hours after intranasal administration. Figure 3B shows that PKH26 positive exosomes (red fluorescence) were found in the cell cytoplasm (white arrow), with a reticular distribution, suggesting the cellular uptake of exosomes by neurons (fluorescent exosome uptake was also observed by glial and endothelial cells). PKH26 positive exosomes can also be seen in the extracellular space. These results can be observed in the olfactory bulb, cerebral cortex, dorsal and ventral striatum, thalamus, hypothalamus and brainstem.

Example 2. The administration of an intranasal composition with secrecy derived from preconditioned MSCs during their cultivation reduces alcohol consumption and prevents relapse.

The animal model of chronic alcohol consumption that was used to evaluate the intranasal composition with preconditioned MSC-derived secretoma during its cultivation was the same as that described in example 1.

MSC preconditioning of human adipose tissue (AD-MSC) and obtaining secrecy

Human AD-MSC was preconditioned at a confluence of 70% by incubation in a-MEM which was supplemented with 10% fetal bovine serum, 10 ng / ml TNF-and 15 ng / ml IFN-g for 40 hours. After 40 hours of preconditioning, the cells were washed 3 times with PBS and incubated for 48 hours with a-MEM. After 48 hours the culture medium (secretoma) was collected and centrifuged at 400 g for 10 minutes to remove the whole cells. The supernatant was again centrifuged at 5,000 g for 20 minutes and filtered by 0.22um filters to remove debris. cell phones. The secrets were concentrated 30 times (v / v) by 3 kDa threshold filters. The concentrates were washed with 15 ml of PBS and reconcentrated again with the same filters, the protein concentration was determined and frozen at -80 ° C until use.

Intranasal administration of the secretoma-derived composition of AD-MSC preconditioned during culture, and determination of alcohol consumption in rats

After approximately 75 days of chronic alcohol consumption, the rats were anesthetized with doral hydrate (280 mg / kg i.p.) and placed supine. 20 ml of secret solution was administered intranasally as drops from a small pipette every 5 minutes (4 times in each nostril) on alternate sides of the nasal cavity for a total of 20 minutes. A total volume of 160 ml of saline solution containing 25 pg of total secretoma proteins was administered into the nasal cavity. The intranasal administration was repeated once a week for 5 weeks. Control animals received 160 ml of saline solution with the same administration scheme previously mentioned.

The consumption of ethanol and water was determined every 24 hours by volume difference of inverted graduated cylinders of 100 ml. Ethanol consumption was expressed as g of ethanol consumed / kg of weight / day. Water consumption was expressed as mi of water consumed / kg of weight / day. To determine the effect of the treatment on ethanol consumption after the condition of alcohol deprivation (relapse to consumption), 17 days after the administration of the first dose of the composition with secretoma or saline solution, a period of 14 days of withdrawal, followed by 60 minutes of re-access to 10% ethanol and 20% ethanol, as previously mentioned. Ethanol consumption was expressed as g of ethanol consumed / kg of weight / 60 minutes. During the period of alcohol deprivation, the animals received 2 doses of secretory composition or saline solution (vehicle) at weekly intervals. Re-access to ethanol was allowed 3 days after the last intranasal administration of the secretoma or saline solution. The concentration of ethanol in the blood was also determined as follows. After the rats had 60 minutes of re-access to ethanol and water, 100 ml of blood samples without anticoagulants were collected from the tip of the tail under moderate sedation with acepromazine (1 mg / kg ip). Blood samples were immediately added to 0.9 ml of distilled water at 4 ° C in a sealed glass vial with Mininert valves. The samples were analyzed by gas chromatography using the headspace technique (Perkin Elmer SRI 8610).

As shown in Figure 4A, it was observed that intranasal administration of secretoma significantly reduced chronic alcohol consumption compared to control rats (82% reduction after the first intranasal dose). This effect was maintained by the administration of additional doses of secretoma at weekly intervals. Four days after the third weekly administration, the animals were deprived of ethanol while the administration of a fourth and fifth dose of secretoma was continued under the same weekly schedule. Three days after the last intranasal administration of secretoma, the animals were offered 10% and 20% ethanol solutions (water tube also available) for only 60 minutes. As shown in Figure 4B, animals in the control group ingested 1.7 ± 0.3 g of ethanol / 60 minutes, while animals that were treated with secretoma ingested significantly less than 0.4 ± 0, 15 g ethanol / 60 min. The concentration of ethanol in the blood that was determined immediately after 60 minutes of re-access to ethanol was 110 ± 15 mg / dl for control animals (which in humans would be considered a compulsive consumption of alcohol), while levels of alcohol in blood of animals treated with secretoma reached 25.6 ± 19 mg / dl (Figure 4C). Consequently, intranasal administration of secrecy significantly reduced relapse in alcohol consumption (80% reduction) after the period of deprivation. Example 3. The administration of an intranasal composition with exosomes derived from a 3D culture of MSC (spheroids) reduces alcohol consumption and prevents relapse.

The animal model of chronic alcohol consumption that was used to evaluate the intranasal composition with secretoma derived from a 3D culture of MSC (spheroids) was the same as described above.

3D MSC culture of human adipose tissue (AD-MSC) (spheroids) and obtaining exosomes

MSCs from human adipose tissue were collected, in the third culture passage, by trypsin digestion, and resuspended at a cell density of 7.5 x 10 ⁵ cells / ml in a-MEM which is supplemented with 10% of Fetal bovine serum without exosomes. 35 ml of solution was seeded with cells per drop (containing 20,000 cells) in the lid of a culture plate. Cell drops were cultured in reverse for 3 days. After this time, the secrets were obtained by collecting the culture medium. The medium was centrifuged at 400 g for 10 minutes to remove the entire cells. Supernatants were recovered, centrifuged again at 5,000 g for 20 minutes and filtered on 0.22 pm syringe filters to remove the cellular debris.

For exosome isolation, the culture medium was ultracentrifuged at 170,000 g for 1 hour to precipitate the exosomes. The precipitates were washed with PBS and ultracentrifuged again. The exosome precipitate was diluted in a filtered saline solution and the exosome protein concentration was determined by the MicroBCA Protein Assay kit. The size and concentration of the exosomes was analyzed using NanoTracking System Analysis (NTA) for the NanoSight NS500 and by transmission electron microscopy with a Phillips Morgagni electron microscope. Gel electrophoresis and Western Blots were performed to confirm the enrichment of the classic exosome markers (GM130, ALIX, Flotilina and EpCAM) in exosome samples compared to the entire cell lysate. He used the Calnexin and HSP60 markers to rule out contamination with endoplasmic reticulum.

Intranasal administration of the composition with exosomes derived from spheroids of AD-MSC, and determination of alcohol consumption in rats

After approximately 100 days of chronic alcohol consumption, the rats were anesthetized with doral hydrate (280 mg / kg ip) and placed supine. 20 ml of exosome solution was administered intranasally as drops from a small pipette every 5 minutes (4 times in each nostril) on alternate sides of the nasal cavity for a total of 20 minutes. A total volume of 160 ml of saline solution containing 1.5 x 10 ⁹ exosomal particles (7.5 pg of exosomal proteins) was administered into the nasal cavity. The intranasal administration was repeated once a week for 5 weeks. Control animals received 160 ml of saline solution with the same administration scheme previously mentioned.

As shown in Figure 5A, intranasal administration of exosomes significantly reduced chronic alcohol consumption compared to alcohol consumption before treatment (60% reduction after first dose of intranasal administration and 78% reduction after the second dose of intranasal administration). In addition, intranasal administration of exosomes significantly reduced alcohol relapse (43% reduction) after the period of deprivation (Figure 5B).

Example 4. The administration of an intranasal composition with secretion derived from MSC preconditioned during its cultivation reduces nicotine consumption and prevents relapse.

Animal model of nicotine addiction

Adult female Wistar rats were housed in individual cages with water and food ad libitum, who were given an initial dose of 0.5 mg / kg i.p. of nicotine once a week for 14 consecutive days. After the suspension of nicotine injections on day 15, a second drinking tube containing a nicotine solution (without added sweeteners) was placed in each cage with an incremental concentration on successive days. Initially, nicotine was 5 mg / l during the initial 2 days of exposure to nicotine, 10 mg / l from day 3 to day 7, and 20 mg / l from day 8 to day 13. From day 14 to day 20 the concentration of nicotine was 30 mg / l and finally from day 21 the concentration of nicotine was increased to 40 mg / l and remained constant for the rest of the experiment. Water and nicotine consumption was recorded daily.

Intranasal administration of the secretoma-derived composition of AD-MSC preconditioned during culture, and determination of nicotine consumption in rats

Obtaining secretion derived from preconditioned AD-MSC during its cultivation was carried out in the same manner as described in example 2.

After approximately 19 weeks of oral self-administration of nicotine, the rats were anesthetized with doral hydrate (280 mg / kg ip) and placed supine. 20 ml of secret solution was administered intranasally as drops from a small pipette every 5 minutes (4 times in each nostril) on alternate sides of the nasal cavity for a total of 20 minutes A total volume of 160 ml of saline solution containing 25 pg of total secretoma proteins was administered into the nasal cavity. The intranasal administration was repeated once a week for 5 weeks. Control animals received 160 ml of saline solution with the same administration scheme previously mentioned. Water and nicotine consumption was recorded daily. The consumption of nicotine was expressed as mg of nicotine consumed / kg weight / day.

To determine the effects of treatment on nicotine use followed by deprivation (relapse), 17 days after the administration of the first intranasal dose of secretoma or saline solution, a withdrawal period of 14 days was imposed, followed by 60 minutes of re-access to nicotine. During the period of deprivation, the animals received, at weekly intervals, 2 doses of secretoma that were administered intranasally or saline dose. Re-access to nicotine was allowed 3 days after the last administration of intranasal secretoma.

Figure 6A shows that intranasal administration of secretoma significantly reduced chronic nicotine consumption (66% reduction) compared to control rats. The effect was maintained by the administration of additional doses of secretoma at weekly intervals. Intranasal administration of secretoma significantly reduced the relapse to nicotine consumption (80% reduction) after the period of deprivation (Figure 6B).

Example 5. The administration of an intranasal composition with secretion derived from preconditioned MSCs during their cultivation reduces the search for opioid consumption.

Sprague-Dawley rats were used for morphine search experiments, using the CPP paradigm. The CPP device consists of a conveyor box divided into two compartments of equal size. The internal surface of one of the compartments was black with a uniform floor; the other was white with a textured floor to create an equally preferred compartment. Infrared ray sensors were positioned on each deck, and they measured the rat's time in each compartment using a computer system over a period of 900 seconds.

The experimental process consisted of 3 different sessions: a preconditioning session, conditioning session and a test session. On days 1 and 2 (preconditioning session), the partition that separated the two compartments was raised 7 cm above the ground, and a neutral platform (3 cm x 1 cm x 7 cm) was inserted along the grain that separated the compartments. The rats were placed individually on the platform and positioned on the horizontal floor. The rats were allowed to freely explore the two compartments for 900 seconds and acclimatize to the apparatus. There were no significant differences between the time they spent in the black compartment with a uniform floor and the white compartment with textured floor. Rats that spent more than 600 seconds on one side of the box were excluded from the analysis. The conditioning session was conducted once a day for 6 consecutive days (days 3 to 8). Rats were injected subcutaneously with morphine (5 mg / kg) or saline and confined in one of the compartments for 1 hour. The next day, the rats that were injected with morphine were injected with saline, and those that were injected with saline were injected with morphine, respectively, and confined to the other compartment for 1 hour. From days 3 to 8, the conditioning was repeated 3 times. Control groups received saline instead of the drug. On day 3 and 7 of the conditioning session, an intranasal dose of preconditioned MSC secretoma was administered during culture (25 pg total protein) or saline. On day 9 (test session), the time spent in each compartment for 900 seconds in a drug-free state was measured. Animals treated with secretoma spent significantly less time in the drug-associated compartment compared to rats treated with saline solution, indicating that intranasal administration of preconditioned MSC secrets reduces the desire to search for opioids.

Claims

1. A pharmaceutical composition for the treatment of addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances, characterized in that it comprises between 0.05 to 50 mg of total exosome proteins derived from preconditioned mesenchymal stem cells during its cultivation; and a pharmaceutically acceptable excipient.

2. The pharmaceutical composition according to claim 1, characterized in that it is a composition for intranasal or intravenous administration.

3. The pharmaceutical composition according to claim 2, characterized in that the composition for intranasal administration comprises between 0.5 and 5 mg of total exosome proteins.

4. The pharmaceutical composition according to claim 2, characterized in that the composition for intravenous administration comprises between 0.5 and

5 mg of total exosome proteins.

5. The pharmaceutical composition according to claim 1, characterized in that the psychoactive substance is selected from the group consisting of alcohol, nicotine, opioids and cocaine.

6. The pharmaceutical composition according to claim 1, characterized in that the mesenchymal stem cells are obtained from adipose tissue.

7. A pharmaceutical composition for the treatment of addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances, characterized in that it comprises between 0.08 to 80 mg of total secreted proteins of preconditioned mesenchymal stem cells during their culture; and a pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7, characterized in that it is a composition for intranasal or intravenous administration.

9. The pharmaceutical composition according to claim 8, characterized in that the composition for intranasal administration comprises between 0.8 and 8 mg of total secretoma proteins.

10. The pharmaceutical composition according to claim 8, characterized in that the composition for intravenous administration comprises between 0.8 and 8 mg of total secretoma proteins.

11. The pharmaceutical composition according to claim 7, characterized in that the psychoactive substance is selected from the group consisting of alcohol, nicotine, opioids and cocaine.

12. The pharmaceutical composition according to claim 7, characterized in that the mesenchymal stem cells are obtained from adipose tissue.

13. A pharmaceutical composition comprising between 0.05 to 50 mg of total protein exosomes from preconditioned mesenchymal stem cells during their cultivation, for use in a method of treating addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances.

14. The pharmaceutical composition according to claim 13, wherein said psychoactive substances are selected from the group consisting of alcohol, nicotine, opioids and cocaine.

15. The pharmaceutical composition according to claim 13, wherein the composition is a composition for intranasal or intravenous administration.

16. The pharmaceutical composition according to claim 15, wherein the composition is administered intranasally and comprises between 0.5 and 5 mg of total exosome proteins.

17. The pharmaceutical composition according to claim 15, wherein the composition is administered intravenously and comprises between 0.5 and 5 mg of total exosome proteins.

18. The pharmaceutical composition according to claim 13, wherein mesenchymal stem cells are obtained from adipose tissue.

19. A pharmaceutical composition comprising between 0.08 to 80 mg of total secreted proteins of preconditioned mesenchymal stem cells during their cultivation, for use in a method of treating addiction to psychoactive substances and / or to prevent relapse to addiction to psychoactive substances.

20. The pharmaceutical composition according to claim 19, wherein said psychoactive substances are selected from the group consisting of alcohol, nicotine, opioids and cocaine.

21. The pharmaceutical composition according to claim 18, wherein the composition is a composition for intranasal or intravenous administration.

22. The pharmaceutical composition according to claim 21, wherein the composition is administered intranasally and comprises between 0.8 and 8 mg of total exosome proteins.

23. The pharmaceutical composition according to claim 21, wherein the composition is administered intravenously and comprises between 0.8 and 8 mg of total exosome proteins.

24. The pharmaceutical composition according to claim 18, wherein mesenchymal stem cells are obtained from adipose tissue.