ES2348887A1 - Inclusion complex formed by disulfiram and a cyclodextrin, which can be used in the treatment of alcohol and cocaine dependence - Google Patents

Abstract

The invention relates to a novel inclusion complex formed by disulfiram (DSF) and a cyclodextrin (CD), methods for obtaining same, pharmaceutical compositions and the use thereof in the treatment of alcohol and cocaine dependence.

Description

Inclusion complex formed by Disulfiram and a cyclodextrin, useful in the treatment of dependence on alcohol and cocaine

The present invention is related to a new inclusion complex formed by Disulfiram (DSF) and a cyclodextrin (CD), its preparation procedure, compositions pharmaceuticals and their use in the treatment of dependence on alcohol and cocaine

State of the art

Alcohol dependence is a pathology multicausal, so it must be addressed with a type treatment biopsychosocial, which includes pharmacological support. There are several types of treatments for alcohol dependence, in which they use various pharmacological and / or psychotherapeutic tools; however, they have in common the inconvenience of presenting low adhesion, which prevents the rehabilitation of the patient and his subsequent reintegration into society.

One of the most used drugs in the treatment of alcohol dependence is Disulfiram, (1- (diethylthio-
carbamoyldisulfanyl) -N, N-diethyl-methanethioamide; CAS No. 97-77-8), molecular weight of 296,543 g / mol, molecular formula C 10 H 20 N 2 S 4, whose chemical structure corresponds to Formula (I):

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one

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DSF is mainly used in tablets for oral use, presenting the inconvenience of registering a withdrawal adherence to treatment, because the patient simply suspends the administration and / or cheats the therapist about it.

Following the low adhesion that the DSF drug therapy, strategies have been proposed radicals that involve the administration of action DSF sustained.

The first attempt to administer DSF in this way corresponds to a sustained-release implant or pellet of 1 g of DSF ( Marie, C. Propos d'un Nouveau Mode de Traitement de I'Alolisolis Chronique par Implantation de Disulfure de Tetraethyle- Thiourane, Thése, Faculté de Medicine de I'Université de Paris, 1955. ); However, it has been suggested that these implants would not reach effective pharmacological levels and that their effectiveness would be mainly placebo type in addition to presenting erratic levels at the blood level. ( Wilson, A .; Davidson, W .; Blanchard, R. Disulfiram Implantation: A Trial Using Placebo Implants and Two Types of Controls, J. Stud. Ale, 1980; 41: 429-436 ). The necessary daily therapeutic doses by oral route are of the order of 500 mg (normally the therapy with DSF consists of the administration of 500 mg / day) reaching therapeutically effective levels; On the other hand, this has not been achieved with this implant of 1 g of DSF of sustained action, since plasma levels are not obtained for a successful pharmacological therapy.

In another order, because the solubility of DSF in water is a physicochemical limitation, being around 0.02 mg / mL, which leads to low and incomplete gastrointestinal absorption, in the case of orally administered tablets, and a Erratic release in the case of subcutaneous implants or pellets, suspensions or DSF solutions have been postulated in different vehicles. From this point of view it has been shown that the bioavailability of subcutaneous DSF can be modified by using oils ( Stromme, JH; Eldjarn, L .; Stensrud, T. Distribution and Chemical Forms of Diethyldithiocarbamatae and Tetraethyltiuram Disulfide (Disulfiram) in Mice in Relation to Radioprotection, Biochem. Pharmacol. 1966; 15: 287-297 ), dissolved in polyethylene glycol ( Phillips, M. Excretion of Carbon Disulfide in Breath Following Subcutaneous Injection of Disulfiram, Clinical Research, 1980; 28 (3), 623 A ) or suspended in water ( Phillips, Michael; Gresser, Joseph G. Sustained-Release Characetristics of a New Implantable Formulation of Disufliram, J. Pharm. Sci. Subst. Abuse, 1984; 73 (12), 1718-1720 ). However, Phillips and Greenberg, ( Phillips, M .; Greenberg, J. Dose-Ranging Study of Depot Disulfiram in Alcohol Abusers, Alcohol CU. Exp-Res. 1992; 16 (5): 964-967 ) demonstrated that the suspension in DSF water it would not act as a form of
Deposit.

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Another example of an implant is a form of subcutaneous administration of DSF in microgranules suspended in a vehicle composed of a mixture of propylene glycol / water ( Chandía R. et al, Clinical study of disulfiram in injectable suspension. A new alternative in the treatment of alcoholism. Rev. Med. Chile, 118, 855-861 ), however, it has been shown that the forms of subcutaneous administration, studied and tested to date, produce intense pain and inflammatory reactions, which is uncomfortable for patients resulting in low adherence and abandonment of treatments.

From the previous studies you can then define that the main problems in the administration of DSF they are due to its low solubility and the impossibility of obtain suspensions and parenteral solutions that facilitate their administration, since on the one hand they have not proven good results in relation to the bioavailability of the drug, to the solubility and sustained release; and on the other hand there is a great impact on the application area with signs of inflammation and pain.

The inventors have designed a new complex of inclusion with DSF, which overcomes the disadvantages and disadvantages of the DSF, presented in the state of the art, and through physicochemical, pharmacokinetic, pharmacological and histopathological, have been able to demonstrate the effectiveness of these new inclusion complexes, excellent bioavailability and minimum Inoculation point damage.

Mechanism of action of disulfiram

The first report on DSF and its possible applications in the treatment of alcohol dependence appears in 1948, by Jacobsen and Hald ( Jacobsen Erik Hald J., Larsen V. The sensitizing effect of tetraethyltiuram dysulphide (Antabuse) to ethyl alcohol, Acta Pharmacol et Toxicol, 1948, 4: 285-296 ). This drug has been, for years, widely used as ethyl alcohol rejection therapy.

DSF produces hypersensitivity to alcohol by inhibiting the enzymatic mechanism that oxidizes acetaldehyde to acetic acid, which occurs in the liver during normal ethanol catabolism. The absorbed ethyl alcohol is removed from the body primarily (> 90%) through oxidative mechanisms located mostly in the liver, and mainly involving an alcohol dehydrogenase (ADH) and to a lesser extent certain microsomal enzymes (microsomal oxidative system of alcohol) .

The first step in the metabolization of alcohol is to oxidize the substrate to acetaldehyde through the ADH enzyme system that uses the nicotinamide adenine dinucleotide (NAD +) as a hydrogen acceptor. Most acetaldehyde continues to be oxidized in the liver to acetic acid, by a family of isoforms of the enzyme aldehyde dehydrogenase (ALDH), which also uses NAD + as a hydrogen acceptor. The product, acetaldehyde, is converted to acetyl-CoA, which is then oxidized by means of the citric acid cycle to CO2 or used in the various anabolic regions that participate in the synthesis of cholesterol, fatty acids and other constituents Tissues (Hardman, Joel G .; Limbird, Lee E. Goodman & Gilman, The Pharmacological Bases of Therapeutics, 9th edition, Vol. I, McGraw-Hill Interamericana Editores SA, Mexico, 1996: 417-418 ).

Normally the ability to remove the acetaldehyde by ALDH exceeds the generating capacity of acetaldehyde by oxidation of ethanol, so that the levels Acetaldehyde circulators are quite low.

DSF competes with NAD + for the ALDH enzyme producing an apparent irreversible inhibition of enzyme activity ( McEvoy, Gerald K. AHFS Drug Information, American Society of Health System Pharmacists, USA, 2002, 3628-3630 ). There is then an accumulation of acetaldehyde in the blood, which is believed responsible for the signs and symptoms, which have been referred to as "acetaldehyde syndrome" (or Ethanol-Disulfiram Reaction, RED), which appears within 10 minutes after alcohol intake and which is mainly characterized by redness of the face, pulsatile headache, dyspnea, nausea, vomiting, perspiration, hypotension, chest pain, dizziness and blurred vision (Martindale, "The Complete Drug Referential", 32th Edition, Kathleen Parfitt, USA, 1999: 1573). The duration of the syndrome varies between 30 minutes and a few hours; subsequently there is tiredness and sleep. This syndrome can be triggered even by 7 mL of alcohol in sensitive subjects. People under treatment with DSF should be promptly prevented that the use of shaving lotions or frictions, medications such as cough syrups or even sauces or fermented vinegar, can trigger the syndrome. NET may occur up to 1 or 2 weeks after the last medication was taken ( McEvoy, Gerald K. AHFS Drug Information, American Society of Health System Pharmacists, USA, 2002, 3628-3630 ).

DSF treatment is not considered a cure for alcohol dependence, but an aversive therapy or conditioning: the therapeutic response seems to lie in fear of RED more than a pharmacological action of the medicine.

Detailed description of the invention

The present invention is focused on a new inclusion complex formed by (DSF) and a cyclodextrin (CD) (hereafter referred to as the DSF-CD Complex), its methods of obtaining, its pharmaceutical compositions and its use in the treatment of alcohol dependence and cocaine dependence, since benefit of the use of DSF has been found in patients with cocaine dependence, with or without comorbid alcohol dependence ( Carroll, KM, Ziedonis, D., O'Malley, SS, McCance-Katz, E., Gordon, L. and Rounsaville, BJ, Pharmacologic interventions for abusers of alcohol and cocaine: a pilot study of disulfiram versus naltrexone, American Journal on Addictions 2, 77-79, 1993; Carroll K. et al. Efficacy of disulfiram and cognitive behavior therapy in cocaine-dependent outpatients. Arch Gen Psychiatry, 61: 264-72, 2004. Preti A. New developments in the pharmacotherapy of cocaine abuse. Addict Biol, 12 (2): 133- 51, 2007; and Kenna GA, Nielsen DM, Mello P, Schies l A, Swift RM. Pharmacotherapy of dual substance abuse and dependence. CNS Drugs, 21 (3): 213-37, 2007. Review ).

Another important aspect of this invention is provide pharmaceutical formulations in solid and liquid state which comprise the DSF-CD Complex.

Through the use of suitable excipients, it is possible to modify and control the physicochemical properties of the molecules and / or pharmacological properties by modifying the processes involved. The inclusion of drugs in cyclodextrins (CDs), as it is in this case, is an efficient alternative to improve physicochemical properties such as stability and solubility of hydrophobic molecules, a potential tool to modify and control parameters such as bioavailability or cup of release of a drug by various routes of administration ( Duchêne, Dominique New Trends in Cyclodextrins and Derivates, Editions de la Santé, Paris, 1992 ).

It should be noted that the solubility of a drug is of special interest since although many of the factors that are involved with the bioavailability of a drug depend on its lipophilicity (such as the passage through membranes, the affinity to receptors and tissue distribution), for adequate bioavailability, regardless of the route of administration, the drug absorption process depends largely on the aqueous solubility of the active substance ( Duchêne, Dominique New Trends in Cyclodextrins and Derivates, Editions de la Santé, Paris, 1992; Hardman, Joel G .; Limbird, Lee E. Goodman & Gilman, The Pharmacological Bases of Therapeutics, 9th edition, Vol. I, McGraw-Hill Interamericana Editores SA, Mexico, 1996: 5 ).

As indicated above, the principal DSF's problem is its low water solubility, so that an excipient is required to improve said property physical chemistry. Thus the present invention demonstrates the physicochemical modifications that DSF suffers to form an inclusion complex with CDs.

CDs are a group of modified starches by fermentation processes, in the form of circular rings which may be composed of six, seven or eight monomers of glucose (glucopyranoses linked by α-type bonds 1-4), structures known as α, β and γ-cyclodextrins, respectively.

Of the CD derivatives described in scientific articles and patent documents, only a few can be used for medical purposes ( Szejtli, József, Introduction and General Overview of Cyclodextrin Chemistry, Chemical Reviews, 1998; 98 (5): 1743-1753 ) , being the pharmaceutical products those that constitute the greater part of the market of the CDs. An example is the use of Hydroxypropyl-? -CD in the new oral and parenteral itraconazole fungicide from Johnson & Johnson , Sporanox®, where it improves the solubility and bioavailability of the drug.

On the other hand, the Schwarz Pharma company uses | alpha-CD to solubilize a prostaglandin, alprostadil (Edex®), a drug used for impotence ( McCoy M. "Cyclodextrins; Great product seeks a markets". Chemical & engineering news 1999; 77 (9): 25-27 ).

CDs and their derivatives have also been used for the administration of sex hormones (particularly testosterone, progesterone and estradiol) by sublingual or oral route ( Pitha J. Administration of sex hormones in the forms of hydrophilic cyclodextrin derivatives, united states patent 4,751,094 ( Jun .14 1986 )), in transdermal release preparations ( Ueda Y., et al . " Sustained-release transdermal delivery preparations ". US Patent 4,749.74 ( June 1988 ), among others.

Additionally, formulations have been described that they comprise an association of DSF with CD in the solution form. He CN 1376463 A describes a pharmaceutical composition for use ocular topic that includes a CD and DSF, where the DSF has a enhanced solubility The association of DSF and CD is mixed in water and freeze dried This document does not demonstrate the formation of a inclusion complex

In another publication, Wang S et al. (Wang S, Li D, Ito Y, Nabekura T, (2004), Bioavailability and anticataract effects of a topical ocular drug delivery system containing disulfiram and hydroxypropyl-beta-cyclodextrin on selenite-treated rats. Current Eye Research. Vol. 29 ( 1), 51-58) , a study of eye drops containing a high concentration of DSF and a CD is described. The composition is oriented to the treatment of cataracts, and a high concentration of CD is used as a solubilizer to increase the solubility of DSF and obtain an appropriate pharmaceutical form to be applied directly to the eyes. In this document the ratio of DSF: CD used is 1: 2.5, and the formation of an inclusion complex is not demonstrated, but the increase in solubility provided by the CDs to the active substance.

CD macromolecules have the peculiarity of forming a central cavity within their spatial arrangement. Because the sugars adopt the chair conformation, the CDs take the form of a truncated cone rather than that of a perfect cylinder. The hydroxyl groups are oriented towards the outside of the cone, which makes the CD water soluble. The secondary hydroxyls are oriented towards one end and those of the primary type, towards the opposite end, which determines that, due to the free rotation of the latter, they define the edge of smaller diameter. The central cavity is delineated by the carbon skeleton, hydrogen atoms and the glycosidic oxygen atoms that give it the lipophilic character. The unpaired electrons of the oxygen bridges give Lewis base characteristics, since a high electronic density is produced towards the inner part of the ring, forming a hydrophobic surface (Figure 1), which is capable of interacting with various organic molecules (or parts thereof) to form inclusion compounds ( Duchêne, Dominique. New Trends in Cyclodextrins and Derivates, Editions de la Santé, Paris, 1992 ).

An inclusion complex is an association molecular in which a molecule called host is introduced in the cavity of a macromolecule called a host, with a well-defined stoichiometry. In the case of the present invention, the DSF (host), is encapsulated or included within another molecule, or structure made of said molecules (host), in this case, the CD

In the context of the present invention, means "physical mixture" to a mixture, in solid state, of clearly defined proportions of CD and DSF.

The inclusion complex comprises weak intermolecular interactions , commonly called hydrophobic junctions similar to those found in various biological systems ( enzyme-substrate, antigen-antibody for example). One, two or three CD molecules may contain one or more host molecules. The most frequent is the host: host (1: 1) ratio.

The interactions involved in the process of inclusion are of a weak nature and are attributed to van der forces Waals, London dispersal forces, interactions dipole-dipole and hydrophobic interactions.

In aqueous solution, water molecules are normally found within the cavity of the CDs (polar-apolar interaction); However, since they are structures of a polar nature, their presence is not thermodynamically favorable. When a host is introduced into the CD, it displaces these water molecules. Both the host and the water molecules are thermodynamically favored. The result of the complexation is, then, a thermodynamically more favorable system: the repulsive forces between high enthalpy water molecules within the CD and the repulsive forces between the apolar host within the polar solution next to the interactions of a weak type already mentioned, they favor the formation of the inclusion complex (Connors K. "The stability of cyclodextrin complexes in solution". Chem. Rev 1997; 5: 1325-1357; Szejtl J. "Utilitation of cyclodextrins in industry product and processes ". J mater. Chem 1997; 7 (4): 575-587; Duchêne D. New trends in cyclodextrins and Derivates. Editions de la Santé, Paris. 1992, 53-55).

There are different CDs that were modified chemically looking to improve the properties physicochemical of the original compounds. It's like that how does the hydroxypropyl-? -cyclodextrin (HP-? -CD) as follows structural formula:

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The present invention describes a new complex DSF inclusion with hydroxypropyl-? -cyclodextrin, in order to improve the solubility characteristics and Bioavailability of the drug.

There are different methods of preparation for the "DSF complexes in hydroxypropyl-? -cyclodextrin " (DSF-CD Complex), which are obtained in the form of solid products These methods may also require different proportions of each of its components.

The processes used in this invention for the DSF complex preparation with hydroxypropyl-? -cyclodextrin they are known in the art and can be easily reproducible by an expert in pharmaceutical technology.

The products obtained by the different methods have different characteristics and their behavior It may be different.

In order to physicochemically characterize the DSF inclusion complex with CD hydroxypropyl-? -cyclodextrin (HP-? -CD) was necessary prepare said complexes in proportions of DSF and CD (from here on later the terms CD and will be used interchangeably HP-? -CD) as 1: 1 and 1: 2 in relation to their molecular weights and by different methods of Preparation, as indicated below:

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a) DSF-CD complex obtained by Rotavapor

Mixtures of HP-? -CD and DSF to which They added the same amount by weight of absolute alcohol. The mixture is completely dissolved. Later it was taken until evaporation complete solvent, then dried in an oven at 45 ° C for 48 hours

The operational conditions for obtaining of the complex by this method were:

Bath temperature: 60ºC

Stirring speed: 160 rpm

Vacuum: 80 mmHg

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b) DSF-CD complex obtained by Sprav dryinq

The mixture was dissolved HP-? -CD and DSF in alcohol absolute as a solvent to obtain the alcoholic solution correspondent. The atomization was carried out in a Büchi apparatus B-290.

The operational conditions for obtaining of the complex by this method were:

Inlet temperature: 85ºC

Peristaltic pump speed: 35%

70% extraction pump

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c) DSF-CD complex obtained by Lyophilization

The operational conditions for obtaining of the complex by this method were:

Freezing temperature: -80ºC

Sublimation temperature: -54ºC

Vacuum: 135 * 10 ^ - 3 mbar

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The DSF was acquired in the company A. Sander & Co. GMBH, Hamburg Germany. The Hydroxypropyl-? -Cyclodextrin ENCAPSIN® is from Janssen Biotech N.V., Belgium. The rest of the chemicals were analytical grade and water was used distilled Merck Titrisol® hydrochloric acid was purchased, Germany, to prepare 0.1 N solution.

The DSF inclusion complex with CD was elaborated in the Faculty of Pharmacy of the University of Concepción, Chile, by the methods described above.

In order to evaluate the formation of inclusion complex, its characterization was performed by Differential Scanning Calorimetry (DSC), Magnetic Resonance Nuclear (NMR), X-ray Diffraction (RX) and analysis topographic (Scanning Electron Microscopy) (MEB).

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Description of the figures

Figure 1: shows the spatial model of the CD.

Figure 2: Differential scanning calorimetry from: (a) DFS; (b) HP-? -CD; (C) DSF-CD 1: 1 complex prepared in Rotavapor; (d) DSF-CD 1: 2 complex prepared in Rotavapor; (and) DSF-CD 1: 1 complex prepared by Spray Drying; (F) DSF-CD 1: 2 complex prepared by Spray Drying; (g) DSF-CD 1: 1 complex prepared by lyophilization; (h) DSF-CD 1: 2 complex prepared by lyophilization.

Figure 3: Diffraction spectra of X-rays of: (a) DFS; (b) HP-? -CD; (c) Complex DSF-CD 1: 1 prepared in Rotavapor; (d) Complex DSF-CD 1: 1 prepared by lyophilization; (e) Complex DSF-CD 1: 1 prepared by Spray Drying; (f) Complex DSF-CD 1: 2 prepared in Rotavapor; (g) Complex DSF-CD 1: 2 prepared by lyophilization; (h) Complex DSF-CD 1: 2 prepared by Spray Drying.

Figure 4: DSF infrared (IR) spectrum.

Figure 5: IR spectrum of the HP-? -CD.

Figure 6: IR spectrum of the physical mixture DSF / HP-? -CD (1: 1).

Figure 7: Complex IR spectrum DSF / HP-? -CD (Complex DSF-CD).

Figure 8: Scanning electron microscopy x 1000 of: (a) DSF-CD complex developed by Rotavapor in a 1: 1 ratio; (b) Elaborated DSF-CD complex by Rotavapor in a 1: 2 ratio; (c) Complex DSF-CD prepared by Spray drying in a proportion 1: 1; (d) DSF-CD complex developed by Spray drying in a 1: 2 ratio; (e) Elaborated DSF-CD complex by lyophilization in a 1: 1 ratio; (f) Complex DSF-CD prepared by Lyophilization in a proportion 1: 2; (g) HP-? -CD (x 300).

Figure 9: Scanning electron microscopy x 4000 of: (a) DSF-CD Complex developed by Spray drying in a 1: 1 ratio; (b) DSF-CD Complex prepared by Spray drying in a 1: 2 ratio; (C) HP-? -CD (x 300).

Figure 10: Optical microscopy with light polarized: (a) DSF and (b) DSF-CD Spray Complex drying in a 1: 1 ratio; (c) DSF-CD Complex by Rotavapor (40x) in a 1: 1 ratio.

Figure 11: Histograms of particle size: (a) DSF-CD complex developed by Spray drying in a 1: 1 ratio, (b) free DSF.

Figure 12: Maximum solubility of the DSF and the DSF-CD complex in 0.1 N HCL obtained by the three Preparation methods

Figure 13: DSF dissolution kinetics, DSF-CD complex in 1: 1 and 1: 2 ratios and mixtures Physical (MF) in 1: 1 and 1: 2 ratios in 0.1 N HCL.

Figure 14: Variations in heart rate, produced by the administration of 1 g / Kg of ethanol diluted in 50% distilled water intraperitoneally (IP) in rats injected subcutaneously (SC) with free (not complexed) DSF and C DSF-CD complex (complexed DSF), in doses of 15 mg / kg. The rats were monitored at the following times: 1 hr (A), 24 hrs (B), 48 hrs (C), 12 hrs (D), 96 hrs (E) and 144 hrs (F) post ethanol administration .

Figures 15 to 22: Histological cuts of skin of rat in areas of subcutaneous administration (SC), of DSF (A); DSF-CD complex (B); and control with physiological serum SF (C). Histological cuts were obtained at different post-administration times of each of the essays. Figure 15 corresponds to 8 hours post-administration; Figure 16 to 24 hours post-administration; Figure 17 to 48 hours post-administration; Figure 18 to 72 hours post-administration; Figure 19 at 96 hours post-administration; Figure 20 to 120 hours post-administration; Figure 21 to 144 hours post administration and Figure 22 corresponds to 168 hours post-administration

Figure 23: shows the results obtained from the area under the curve (ABC) after subcutaneous administration of free DSF (A) and DSF-CD Complex (B).

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Results 1.- Physicochemical characterization of the complex of DSF inclusion with CD (DSF-CD Complex) 1.1.- Differential scanning calorimetry

Approximately 2 mg of DSF, HP-? -CD and DSF-CD Complex prepared by the three methods and in different proportions 1: 1 and 1: 2, equivalent in DSF content, were analyzed at the Advanced Polymer Research Center from the Faculty of Chemical Sciences of the University of Concepción, Chile, using Differential Scanning Calorimetry (DSC).

The samples were heated in small semi-sealed aluminum plates, under a flow of 5 mL / minute nitrogen, in a temperature range of 50ºC at 330 ° C, at a rate of 5 ° C / minute.

In Figure 2, the DSC of the DSF (a) shows two endothermic peaks, at 73.25 ° C and 206.84 ° C. The first corresponds to the melting point of the medicine and the second to decomposition of the same.

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In the DSC of the HP-? -CD (b), a slight deflection at the height of 113.4 ° C corresponding to the presence of water in the cyclodextrin cavity, and one at 316.1 ° C indicating its decomposition.

In each of the complexes elaborated by different methods and in different molecular proportions, it observes a slight displacement at higher temperatures of the signal of decomposition of the pure drug, product of stabilizing effect of the CD ensuring a physicochemical interaction between both compounds.

Due to complex formation the peak of melting point of the DSF is displaced at higher temperatures, by above 280º C, temperature at which decomposition begins of the CD.

In the case of complexes developed in a 1: 2 ratio, the interaction between both compounds is greater due to a complete disappearance of the area and signal of fusion of the drug around 73 ° C. This area reduction means a reduction of the energy necessary for the fusion of the medicine, due to the absence of free drug and a possible complexation DSF total.

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1.2.- Nuclear Magnetic Resonance

The 1 H (Proton) spectra of the C-DSF prepared by the Spray drying method in a 1: 1 and 1: 2 ratio were obtained on an NMR Spectrophotometer Brucker AC250 (250 MHz for 1 hr) in the Department of Chemistry Organic of the Faculty of Chemical Sciences of the University of Concepcion, Chile.

Table No. 1 shows higher chemical shifts both of the HP-? -CD as of the DSF in the DSF-CD complex developed in a proportion DSF: HP-? -CD 1: 2 in relation with the DSF-CD Complex developed in a proportion 1: 1.

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TABLE No. 1 1 H-NMR spectral analysis of DSF, HP-? -CD, Complex DSF-CD (1: 1) made by Spray drying; DSF-CD complex (1: 2) made by Spray drying

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Table 2 shows a displacement of -0.270 ppm and -0.284 ppm for C-1 and carbon C-4 respectively in the Complex DSF-CD made in a 1: 2 ratio. Whereas in the case of the DSF-CD Complex elaborated in a 1: 1 ratio, displacements less than -0.009 are observed and +0.002 ppm. that are not determinant since they are in the equipment sensitivity range.

On the other hand, the greatest displacements to high field of the groups -CH_ {2} of the DSF (C-2 and C-3) in the 1: 2 ratio, may indicate a possible interaction of these groups with the HP-? -CD.

TABLE No. 2 13 C NMR DEPT 135 spectral analysis of the DSF, HP-? -CD Complex DSF-CD (1: 1) and DSF-CD Complex (1: 2)

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According to the reported results, the complex made in mixture in a 1: 2 ratio (DSF / HP-? -CD) presents a greater interaction compared to the 1: 1 ratio in all applied studies. As indicated previously in the The following figure shows the inclusion scheme corresponding to Formula (II):

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The elaboration of the Complex DSF-CD in a mixture in 1: 1 ratio DSF: HP-? -CD independent of method of elaboration, does not allow to confirm the type of stoichiometry of the complex formed, because the system in this case will tend to balance with the formation of a mixture of Complex DSF-CD 1: 1, 1: 2 and free DSF, which agrees with what was previously indicated in the DSC that demonstrated a decrease, but not a complete disappearance of heat and signal of fusion of the drug in the DSF-CD Complex prepared in this proportion.

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1.3- X-rays

X-ray diffraction of the DSF, the HP-? -CD and the DSF-CD Complex developed by different methods and in different proportions was performed with a RIGAKU GEIGERFLEX diffractometer. A copper tube with a nickel filter, a radiation of 40 KW and 20 milliamps was used at an angle between a range of 3 to 70 ° C (2)) at a speed of 1 ° / min.

In Figure 3, the diffraction spectra of the Pure DSF (a) show a wide variety of peaks product of the crystallinity of the drug, while for the HP-? -CD (b) its spectrum is It presents with the absence of peaks indicating its amorphous state.

In the spectra of the complexes elaborated by the different methods and different proportions a similarity with the spectrum of the HP-? -CD, product of the loss of crystallinity of the pure drug thanks to the complexation with cyclodextrin evidencing the formation of a inclusion complex

The presence of peaks in the spectrum of complex developed by Rotavapor in a 1: 1 ratio indicates the presence of free DSF due to lack of complete complexation of the DSF, an effect that is not observed in the complexes developed in a 1: 2 ratio

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1.4- Infrared Spectroscopy

Infrared spectroscopy of the DSF, HP-? -CD, physical mixture DSF: HP-?-CD (1: 1) (ratio 1: 1 in relation to its molecular weights) and DSF-CD Complex were performed in a range of 500 to 4,000 cm -1 by a Nicolet Bexus model spectrometer , by the direct compression method with KBr. The analysis was carried out in the Department of Organic Chemistry of the Faculty of Chemical Sciences of the University of Concepción, Chile.

IR spectra obtained experimentally for DSF, HP-? -CD, mix Physical DSF: HP-? -CD (1: 1) and DSF-CD complex are shown in Figures 4, 5, 6 and 7, respectively.

In the spectrum of the Complex DSF-CD (Figure 7) appears a shift in the DSF C-S band frequency from 1267 to 1280 cm -1. The shape of this band is wider and more intense in the DSF-CD Complex spectrum compared to the DSF physical mix: HP-? -CD (1: 1) (Figure 6), while the latter maintains a profile of the same band similar to that of the DSF with frequency values of 1266 and 1267 cm -1, respectively.

The above is also presented in the band of C = S that in the DSF is located at a frequency of 1342 cm <-1> coinciding with the physical mixture, while in the DSF-CD complex moves at 1365 cm -1.

In general, within the area of 2,000 to 1,000 cm -1 of the spectrum you can see the differences that present the DSF spectra e HP-? -CD. Modifications of the spectrum of the DSF-CD Complex are mainly under 1500 cm -1, rather in the area of the fingerprint of the molecule.

According to the above, you can point out that there are changes in the spectrum of the Complex DSF-CD, which supports an interaction phenomenon between both compounds.

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1.5- Scanning Electron Microscopy

Photomicrographs by Electron Microscopy Scanning (MEB) of the HP-? -CD and of the DSF-CD Complex prepared by different methods and in different proportions, were taken in the Laboratory of Electron Microscopy of the University of Concepción, Chile.

It is not possible to obtain the DSF micrograph and of the physical mixture, because the analysis methodology in the MEB uses temperatures above the melting temperature of the medication going into a liquid state during the process.

The dust samples were dispersed on a thin glass plate fitted on an aluminum cylinder, then given a gold bath in the Edwards 5150 Sputter Coster metallizer and subsequently photographed in the Jeol model J-SM 6380 LV .

The electron microscopy studies of scan (Figure 8) reveal a change in the morphology of particles dependent on the method of elaboration. In the photography (g) the topography of the particles of HP-? -CD powder presenting a spherical shape with an amorphous appearance and smooth surfaces. At case of DSF-CD Complexes developed by Rotavapor irregularly obtained microcrystals were obtained, at unlike those obtained by spray drying of amorphous appearance with spherical shape and rough surfaces (Figure 9). In the Complexes DSF-CD prepared by lyophilization were obtained irregularly shaped particles with scaly appearance.

The MEB demonstrates a change in conformation structural structure, indicating an interaction between the two compounds.

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1.6- Optical microscopy with polarized light

Photographs of the DSF and the DSF-CD Complex were taken, the latter produced by Spray dried and Rotavapor. Small quantities of each sample were dispersed separately on a slide and photographed using a METTLER FP 82 optical microscope fitted with an ORTHOLUXIIPOL-BK polarizer and a Cannon 42X camera.

In figure 10, photograph (a), you can appreciate that the DSF has a crystalline structure what It produces refraction by the influence of polarized light on its crystals. In the case of the DSF-CD Complex prepared by both Spray drying (b) this property is lost possibly by the formation of the inclusion complex that occurs in a state amorphous.

By way of comparison the Complex was photographed DSF-CD prepared by Rotavapor (c), which presents a behavior similar to the DSF-CD Complex made by spray drying. However this phenomenon is less probably marked by the difference in the method of elaboration.

While optical microscopy with polarized light on its own it is not able to confirm that a complex of inclusion, reveals an interaction between both compounds.

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2.- Pharmaceutical characterization of the complex of DSF inclusion with CD (DSF-CD Complex)

Particle size analysis and solubility studies. The latter includes kinetics of dissolution and determination of the maximum solubility of the DSF complexed, both quantified by a method validated by Ultraviolet (UV) spectrophotometry.

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2.1- Particle size analysis

Complex particle size analysis DSF-CD prepared by Spray drying in a proportion 1: 1 and 1: 2, was determined by means of the photographs obtained by MEB (figures 8 and 9). The diameter of 110 particles was determined through a computerized statistical program.

DSF particle size analysis is conducted at the Center for Studies for the Development of Chemistry (CEPEDEC) of the Faculty of Chemical and Pharmaceutical Sciences of the Universidad de Chile, by the analytical method of diffraction of light.

About 2 mg of DSF was dispersed in 50 mL of cyclohexane and then sonicated for 5 sec. The sample was analyzed at the Mastersizer Malvern Intruments GmbH particle counter.

As Figure 11 shows the curves of distribution of DSF-CD Complexes prepared by Spray drying have a negative asymmetry towards intervals less than 7-26 µm (Figure 11a), where it exists a shift of its particles towards smaller sizes, while that the DSF presents a distribution of its concentrated particles at much larger size intervals, 7-83 µm (Figure 11b), than the first.

The smallest particle size of the Complex DSF-CD also explains its fast speed of dissolution, because this parameter is directly proportional to the surface and inversely proportional to the size of particle.

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TABLE No. 3 Particle size distribution of different samples

6

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2.2.- Solubility study 2.2.1.- Maximum solubility of the complexed DSF

An excess of DSF was incorporated in 5 mL of 0.1 HCl N in 25 mL flasks, protected from light with aluminum foil and in constant stirring for 72 hours at 21 ± 1.5 ° C until reaching the equilibrium. Simultaneously, the same process was carried out with the DSF-CD Complex (in DSF equivalents) Prepared by different methods and in different proportions. The samples were filtered by pore size Sartorius® filters 1.2 µm and conditioned to be read at 254 nm by UV spectroscopy

The results were subjected to analysis statistically, through a t-test (t-student), through the Graph Pad software Prism 2.1. Each sample was made in triplicate with a weight of 300 mg of DSF.

The solubility of the pure drug and its complexes they are shown in table Nº 4 and in figure 12.

TABLE No. 4 Solubility of the DSF and the different Complex DSF-CD in 0.1 N HCl

7

The increase in DSF solubility using HP-? -CD in 0.1 N HCl was extremely significant.
(p <0.0001) in each of the cases studied, being always higher in the DSF-CD Complexes elaborated in a 1: 2 ratio compared to those elaborated in a 1: 1 ratio, independent of the method used in its manufacture .

The elaborated DSF-CD complexes by spray dried in a ratio both 1: 1 and 1: 2 showed a higher increased solubility (equivalent to 220.4 and 296.3 times greater than that of the pure drug, respectively), because by means of atomization systems you get amorphous structures with small particle sizes, compared to Rotavapor and Lyophilisate that maintain a certain degree of crystallinity.

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2.2.4- Dissolution Kinetics

It was developed according to the conditions indicated in USP XXIV, using a Bahama St., Northridge dissolving team equipped with 6 glasses, device 2: pallet method, at a speed of 50 revolutions per minute (r.p.m) for 60 min. and using a 900 mL volume of aqueous phase (0.1 N HCl) at 37 ± 0.5 ° C.

Independent samples were taken during period indicated above with a fixed sampling system and Automatic with manual volume reset. Sampling system it is provided with a 10 µm pore size filter attached to a stainless steel tube, which is connected to a system of Sample reception (graduated glass tubes up to 10 mL) per half of a Tygon® hose.

The medication under study was incorporated into the powder form on the stirring dissolution medium and established medium temperature, 1 minute was expected for adequate wetting, considering its term as time 0. 5 mL was sampled at intervals of 1, 3, 6, 10, 20, 30, 40, 50, 60 minutes with replacement of the medium. 7 profiles of solution, with a n = 6, with a dose equivalent to 1 g of DSF of The following samples:

?

DSF

?

C-DSF elaborated by Spray dried in a 1: 1 ratio (C-DSF (1: 1))

?

C-DSF elaborated by Spray dried in a 1: 2 ratio (C-DSF (1: 2))

?

Physical mixture prepared in a 1: 1 ratio (MF 1: 1)

?

Physical mixture made in a 1: 2 ratio (MF 1: 2)

The results of the dissolution kinetics of the different samples studied. How it can be seen, the increase in percentage and speed is remarkable of dissolution due to the complexation of DSF in cyclodextrin (C-DSF), which shows a maximum close to 44% and 100% for the C-DSF prepared in a 1: 1 and 1: 2 ratio, respectively, dissolved within 3 minutes of starting the study in contrast to 0.1% for the DSF at the same time.

Physical mixtures made both in a 1: 1 and 1: 2 ratio, have marked differences in terms of dissolution rate and percentage of drug dissolved in comparison to the complex, which supports greater interaction between both compounds when they undergo an atomization process in liquid phase, with the consequent complexation of the drug.

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3.- Pharmacological characterization of the complex of DSF inclusion with CD (DSF-CD Complex)

To demonstrate the differences in intensity and duration of the pharmacological effect of DSF in its complex form of according to the present invention, in relation to the DSF in its form free, heart rate was measured as a sign of the effect of the Ethanol-Disulfiram Reaction (RED).

Moreover, to assess whether this complex administered parenterally does not produce tissue damage, were performed Histopathological studies in rats.

Finally studies were conducted pharmacokinetics to determine plasma levels of DSF and its diethyldithiocarbamate metabolites and methyl diethylthiocarbamate (DDC and Me-DDC) which showed that with the complex Obtains greater bioavailability.

In all these studies, male rats, Sprague-Dawley, were used, weighing 250 to 300 grams, with water and food ad libitum , with light-dark cycles (12:12 hrs) and temperature of 22 ± 1 ° C.

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3.1. Reaction evaluation ethanol-disulfiram

The rats were monitored for RED, based on behavioral signs: pulmonary ventilation, presence of hiccups, locomotive activity and the signs were quantified according to the following scale:

0 = Absence of the sign

1 = mild

2 = moderate

3 = severe

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The cardiovascular effect was monitored through of an electrocardiographic record.

The following controls were performed:

- Basal Control;

- Control, carried out after subcutaneous administration of 15 mg / kg of free or complex DSF DSF-CD, dissolved in 0.5 mL of physiological serum;

- RED Control, carried out after the intraperitoneal administration of 1 g / kg of 96º alcohol, diluted 50% in water. This control was carried out in the following times: 1, 8,12,24,48, 72, 96,120 and 168 hrs, after the administration of DSF

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The behavioral results of the NETWORK are expressed as an average in Table No. 5, where it is observed that the signs are higher in rats administered with DSF at complex form (DSF-CD Complex) that to the form free, especially in the controls carried out at 24 and 96 hours. The duration and intensity of the signs were analyzed by analysis of variance, followed by the Tukey Test, resulting significant differences (p <0.05) between: SF (serum physiological) v / s DSF, SF v / s DSF-CD and DSF Complex v / s DSF-CD Complex, at 24 hours.

Figure 14 shows the effects on the heart rate observed after administration of alcohol in rats treated with DSF. Figure A shows a marked decrease in heart rate with administration of DSF-CD Complex, probably due to predominance of inhibition of dopamine-? -hydroxylase by high concentrations of DDC in blood. Free DSF does not produce major modifications In figure B the bradycardia obtained with the administration of DSF-CD Complex decreases and the Free DSF experiences a mild tachycardia. In figure C the DSF-CD complex produces tachycardia and DSF produces slight modification of heart rate. In figure D, the DSF-CD complex produces tachycardia and free DSF mild bradycardia In Figure E, the DSF-CD Complex produces an increase in heart rate and free DSF does not It presents great variations. In Figure F, both the free DSF as the DSF-CD Complex does not produce variations of heart rate, because levels would no longer exist plasma of the drug and / or its metabolites.

At all times studied the effect of DSF-CD complex is larger than in the free DSF. He Statistical analysis was performed by comparing pending between DSF and DSF-CD Complex, through the Student test, providing a p-value < 0.001 for one hour control.

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3.2. Evaluation of inflammatory intensity in the area of administration: histopathological study

Biopsies were obtained after 15 mg / kg subcutaneous administration of free and Complex DSF DSF-CD, dissolved in 0.5 mL of physiological serum and compared with the control group to which serum was administered physiological.

Skin samples were obtained at the site of the injection at 8, 24, 48, 72, 96, 120, 144 and 168 hrs post-injection These samples were submitted to the Eosin / Hematoxylin staining and the presence of hyperkeratosis, collagen fiber destructuring, presence of fibroblasts and inflammatory cells, capillary congestion of the plexus superficial and deep, edema in the dermis and hemorrhage in subcutis

These signs were quantified according to the following scale:

0 = Absence of the sign

1 = mild

2 = moderate

3 = severe

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Presence or absence of response was analyzed and intensity of response, applying Fisher's exact test.

Table No. 6 shows the effect of free DSF, C-DSF and its SF control over histopathological damage induced after subcutaneous administration.

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(Table goes to page next)

9

Figure 15 shows the histological results after 8 hours of DSF administration. Free DSF induces a severe hyperemia in the deep vascular plexus with hemorrhage in hypodermis and with the DSF-CD Complex it is observed moderate and severe hyperemia of the superficial vascular plexus and deep, respectively, with the presence of inflammatory cells. In the control group, SF injection, severe hyperemia is observed in both vascular plexuses.

Figure 16 shows the histological results after 24 hours of DSF administration. Free DSF induces severe hyperemia of both plexuses, abundant presence of cells inflammatory and hemorrhage in hypodermis and with the Complex DSF-CD shows mild hyperemia of both plexuses, mild presence of inflammatory cells, very little edema and hemorrhage in hypodermis. In the control group, injection of SF, it is observed moderate hyperemia of the deep vascular plexus and moderate presence of inflammatory cells.

Figure 17 shows the histological results after 48 hours of DSF administration. Free DSF induces severe destructuring of collagen fibers, hyperemia of deep vascular plexus and moderate bleeding in hypodermis and with the DSF-CD complex looks moderate Destructuring of collagen fibers, hyperemia of the plexus deep vascular and hypodermis hemorrhage. In the control group, SF injection, moderate presence of cells is observed inflammatory and hyperemia of the deep vascular plexus.

Figure 18 shows the histological results after 72 hours of DSF administration. Free DSF induces moderate hyperemia of both plexuses, with moderate presence of inflammatory cells and hemorrhage in hypodermis and with the Complex DSF-CD shows moderate hyperemia of the plexus Deep vascular and severe presence of inflammatory cells. At control group, injection of SF, moderate hyperemia is observed with mild presence of inflammatory cells.

Figure 19 shows the histological results after 96 hours of DSF administration. Free DSF induces moderate hyperemia of the deep vascular plexus, with hemorrhage in hypodermis and severe presence of inflammatory cells and the Complex DSF-CD shows moderate hyperemia of the plexus deep vascular with mild presence of inflammatory cells, Neutrophils in diapédesis and edema in the dermis. In the control group, SF injection, mild hyperemia of the vascular plexus is observed deep.

Figure 20 shows the histological results after 120 hours of DSF administration. Free DSF induces moderate hyperemia of the deep vascular plexus, with mild presence of inflammatory cells and collagen fiber destructuring and with the DSF-CD Complex, mild hyperemia of the deep vascular plexus, presence of inflammatory cells and hemorrhage in hypodermis. In the control group, SF injection, observes mild hyperemia of the deep vascular plexus, with poor presence of fibroblasts.

Figure 21 shows the histological results after 144 hours of DSF administration. Free DSF induces moderate hyperemia of the deep vascular plexus and poor presence of inflammatory cells and with the DSF-CD Complex it observes low hyperemia of both plexuses with moderate presence of inflammatory cells In the control group, SF injection, observes mild hyperemia of the deep vascular plexus.

Figure 22 shows the histological results after 168 hours of DSF administration. Free DSF induces moderate hyperemia of the deep vascular plexus and with the Complex DSF-CD shows hyperemia of the vascular plexus deep with moderate subcutis hemorrhage and abundant presence of fibroblasts. In the control group, injection of SF, it is observed hyperemia of the superficial and deep vascular plexus.

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3.3. Pharmacokinetic Study

Blood samples were obtained from rats, after subcutaneous administration of 15 mg / kg of free DSF and of DSF-CD Complex dissolved in 0.5 mL of SF and compared with the control group to which SF was administered.

The samples were analyzed by chromatography high resolution liquid (HPLC), using as a column RP-18 and the composition of the mobile phase was acetonitrile / water (60:40 v / v). The mobile phase was filtered (filter 0.45 µm membrane) and degassed. The flow was 1 mL / min and the injection volume 30 µL. The detection range was extended between 200 and 300 nm. All analyzes were performed at 25 ° C (± 1 ° C).

Standard solutions of Me-DDC (Methyl-Diethylthiocarbamate), Et-DDC (Ethyl-Diethylthiocarbamate) and 1 mg / mL DSF dissolving 10 mg of each compound separately, completing to 10 mL with acetonitrile. From this one was obtained concentration of 10 µg / mL. To each test solution was added 0.25 mL of 10 µg / mL of Pr-DDC (Propyl-Diethylthiocarbamate), obtaining a 0.25 µg / mL concentration of internal standard.

Plasma samples were recharged with a known amount of Me-DDC, DDC and DSF. At 500 µL of plasma was added 500 µL of a 0.01 M EDTA solution, 1% NaCl, pH 8.5 and 5 µL of ethanol iodide, vortex was applied by 30 sec and allowed to react for 30 minutes at 40 ° C. Then I know added 12.5 µL of internal standard (Pr-DDC). Subsequently they were subjected to solid phase extraction.

The columns were placed in a team 12-point vacuum distributor connected to a pump empty.

The columns were conditioned with 1,000 µL of acetonitrile, followed by 1,000 µL of nanopure water, subsequently the sample was added to the column. When the samples were passed through the columns, the Me-DDC, Et-DDC, DSF and Pr-DDC were retained. To remove impurities, the columns were washed with 3,000 µL of nanopure water. He Me-DDC, Et-DDC and Pr-DDC eluted from the column with 1,000 µL of acetonitrile. The DSF could not be extracted from the samples of plasma.

Of the eluted solution, 30 µL was injected into the chromatographic column

Figure 23 shows the results obtained after subcutaneous administration of disulfiram. Low area the curve is clearly greater after the administration of DSF-CD complex.

Claims (13)

1. An inclusion complex formed by disulfiram and hydroxypropyl-? -Cyclodextrin in the 1: 2 ratio characterized in that it has the following chemical shifts in the 1 H-NMR spectrum: 10

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2. An inclusion complex formed by disulfiram and hydroxypropyl-? -Cyclodextrin according to claim 1 characterized in that it also has the following chemical shifts in the 13 C-NMR spectrum: eleven

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3. A process for preparing an inclusion complex according to claim 1 characterized in that it is performed in a rotary evaporator and comprises the following steps:

one.

mix in physical form hydroxypropyl-? -cyclodextrin and disulfiram;

2.

dissolve said mixture in alcohol absolute;

3.

completely evaporate the solvent in a rotary evaporator with a bath temperature of 60 ° C at a speed of 160 rpm and with a vacuum of 80 mm Hg (3.83x10 -3 MPa);

Four.

stage drying in an oven at 45 ° C for 48 hours.

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4. A process for preparing an inclusion complex according to claim 1 characterized in that said complex is obtained by spray-drying and comprises the following steps:

to)

mix in physical form hydroxypropyl-? -cyclodextrin and disulfiram;

b)

dissolve said mixture in alcohol absolute;

C)

atomize the solution obtained in a Büchi B-290 device, with an inlet temperature 85ºC, 35% peristaltic pump speed, extraction pump 70%

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5. A process for preparing an inclusion complex according to claim 1 characterized in that it consists in lyophilizing said complex with a freezing temperature of -80 ° C, at a sublimation temperature of -54 ° C and a vacuum of 135x10-3 mbar (0.01 MPa). 6. Pharmaceutical composition characterized in that it comprises an inclusion complex formed by disulfiram and hydroxypropyl-? -Cyclodextrin, according to claim 1. 7. Pharmaceutical composition according to claim 6 characterized in that it is a solid composition. 8. Pharmaceutical composition according to claim 6 characterized in that it is a liquid composition. 9. Use of an inclusion complex according to claim 1 characterized in that it serves to prepare a medicament for treating alcohol dependence. 10. Use of an inclusion complex according to claim 1 characterized in that it serves to prepare a medicament intended to treat cocaine dependence.