KR101131296B1 - An inclusion complex composed of disulfiram and a cyclodextrin, useful for the treatment of alcohol and cocaine dependences - Google Patents

Abstract

The present invention relates to a novel clathrate complex consisting of disulfiram (DSF) and cyclodextrin (CD), a process for its preparation, a pharmaceutical composition thereof and specifically its use in the treatment of alcohol dependence and cocaine dependence.

Description

AN INCLUSION COMPLEX COMPOSED OF DISULFIRAM AND A CYCLODEXTRIN, USEFUL FOR THE TREATMENT OF ALCOHOL AND COCAINE DEPENDENCES} Useful to Dissolve Alcohol and Cocaine Dependence

The present invention relates to a novel clathrate complex consisting of disulfiram (DSF) and cyclodextrin (CD), a process for its preparation, a pharmaceutical composition thereof and specifically its use in the treatment of alcohol dependence and cocaine dependence.

Alcohol dependence is a multifactorial condition and should be approached through biopsychological therapies, including pharmacological support. There are several types of therapies for the treatment of alcohol dependence, although some pharmacological and / or psychotherapeutic means are used, but these generally have low treatment adherence that hinders the patient's rebirth and subsequent reintegration into society. Inconvenient treatments.

One of the most commonly used drugs to treat alcohol dependence is disulfiram (1- (diethylthiocarbamoyldisulfanyl) -N, N-diethyl-methanethioamide; CAS No. 97-77- 8), the molecular weight is 296,543 g / mol and the molecular formula is C ₁₀ H ₂₀ N ₂ S ₄ , the formula of which corresponds to formula (I)

[Formula I]

DSF is mainly used as an oral tablet, which has the inconvenience of poor compliance with treatment because the patient can simply stop the administration and / or trick the therapist.

Following the low compliance seen with DSF drug treatment, radical strategies have been proposed that include the administration of sustained release DSF.

The first attempt to administer this form of DSF corresponds to a sustained release 1 g DSF pill or implant (Marie, C. A Propos d'un Nouveau Mode de Traitement de l'Alcoolisme Chronique par Implantation de Disulfure de Tetraethyle- Thiourane.These, Facult de Medicine de l'Universite de Paris, 1955.). However, it has been found that such implants do not reach effective drug levels and that their activity exists not only at varying levels in the blood, but is also similar to placebo (Wilson, A .; Davidson, W .; Blanchard, R. Disulfiram Implantation). A Trial Using Placebo Implants and Two Types of Controls, J. Stud. Alc, 1980; 41: 429-436). The daily therapeutic dose required for oral administration reaches a therapeutically effective level of approximately 500 mg / day (usually consisting of 500 mg / day of DSF treatment). However, implants of 1 g sustained-release DSF did not reach effective levels because they did not reach plasma levels for successful drug treatment.

In another aspect, the solubility of DSF is physiochemically limited, about 0.02 mg / mL, resulting in low and incomplete gastrointestinal uptake for oral tablets and variable release for subcutaneous implants or pills. As a result, solutions or suspensions of DSF in different carriers have been proposed. In this regard, the bioavailability of subcutaneous DSF is determined through the use of oil (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), (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 by water suspension (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 Disulfiram Depot in Alcohol Abusers, Alcohol CLI.Exp-Res. 1992; 16 (5): 964-967) It has been shown that it does not act as a reservoir.

Another example of an implant is a subcutaneous administration of microgranular DSF suspended in a propylene glycol / water mixture as a carrier (Chandia R. et al, Estudio clinico de disulfiram en suspension inyectable.Una nueva alternativa en el tratamiento del alcoholismo. Med. Chile, 118, 855-861). However, the subcutaneous dosage forms that have been tested and studied to date have developed a strong pain and inflammatory response, making the patient uncomfortable, resulting in low adherence to treatment and abandonment of treatment.

Thus, through prior studies, the main problem with DSF administration was that it was impossible to obtain its low solubility and suspensions and parenteral solutions that attenuated its administration, while on the other hand aspects of bioavailability, solubility and sustained release of the drug Did not show good results, and on the other hand it was because of the large impact at the site of application of signs of inflammation and pain.

The inventors have designed a novel DSF inclusion complex that overcomes the disadvantages and disadvantages of DSFs conventionally known in the art, and through physicochemistry, pharmacokinetics, pharmacology and histopathology tests, the efficacy of these new inclusion complexes, Good bioavailability and minimal damage to the site of inoculation could be demonstrated.

Mechanism of action of disulfiram

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

DSF produces hypersensitivity to alcohol by inhibiting the enzyme mechanism of oxidizing acetalaldehyde to acetic acid, which occurs in the liver during normal ethanol degradation. Absorbed ethyl alcohol is preferentially removed (> 90%) by the oxidative mechanism, which is primarily present in the liver and is primarily involved in the dehydrogenase alcohol enzyme, and is also eliminated by a small percentage of some microsomal enzymes (alcohol oxidative microsomal systems). do.

The first stage of alcohol metabolism oxidizes the substrate to acetaldehydrogen via an alcohol dehydrogenase (ADH) system, an enzyme that uses nicotinamide adenine dinucleotide (NAD +) as the hydrogen acceptor. Most of the acetalaldehyde is oxidized to acetic acid in the liver by the isoform family of the enzyme aldehyde dehydrogenase (ALDH), which also uses NAD + as the hydrogen acceptor. The resulting acetalaldehyde becomes acetyl-CoA and is then oxidized to CO ₂ by the citric acid cycle or used in some assimilation zones involved in the synthesis of cholesterol, fatty acids and other tissue components (Hardman, Joel G .; Limbird , Lee E. Goodman & Gilman, The Pharmacological Basis of Therapeutics, 9th edition, Vol. I, McGraw-Hill Interamericana Editores SA, Mexico, p. 417-418, 1996).

Normally, the ability to remove acetalaldehyde by ALDH surpasses the ability to produce acetalaldehyde by ethanol oxidation, so the circulation level of acetalaldehyde is very low.

DSF competes with NAD + for ALDH enzymes to irreversibly inhibit the activity of the enzymes (McEvoy, Gerald. AHFS Drug Information, 44th edition, American Society of Health-System Pharmacists Inc., Bethesda, USA 3630, 2002). Thereafter, acetalaldehyde accumulates in the blood, which results in symptoms and signs called "acetalaldehyde syndrome" (ethanol-disulfiram response, RED), which appear within 10 minutes after ethanol absorption, mainly hot flashes, pulsations, headaches, Dyspnea, nausea, vomiting, sweating, hypotension, chest pain, dizziness, and blurred vision (Martindale, "The Complete Drug Referente", 32th Edition, Kathleen Parfitt, USA, 1999: 1573). The duration of these signs varies from 30 minutes to several hours, followed by fatigue and drowsiness. These signs can be caused by about 7 mL of ethanol in sensitive individuals. Those receiving DSF treatment should be warned in advance that shaving lotions or rubbing, drugs such as cold syrup or even sauces or fermented vinegar may cause this syndrome. RED can occur up to one or two weeks after the last ingestion of the drug (McEvoy, Gerald K. AHFS Drug Information, American Society of Health System Pharmacists, U.S.A., 2002, 3628-3630).

DSF treatment was considered as aversive therapy, not as a treatment for alcohol dependence, which seems to be due to the fear of RED rather than the drug action of the drug itself.

The present invention provides a novel clathrate complex consisting of disulfiram (DSF) and cyclodextrin (CD), a method for preparing the same, in order to overcome the disadvantages of disulfiram conventionally used in the treatment of alcohol and cocaine dependence as described above. It is an object to provide a pharmaceutical composition thereof and in particular its use in the treatment of alcohol dependence and cocaine dependence.

The present inventors seek to provide a novel inclusion complex consisting of a disulpyram (DSF) and a cyclodextrin (CD) to solve the conventional problems in the art.

The present invention can provide novel clathrate complexes composed of disulfiram (DSF) and cyclodextrin (CD), methods for their preparation, pharmaceutical compositions thereof and specifically their use in the treatment of alcohol dependence and cocaine dependence.

According to aspects of the present invention, as DSF has proven beneficial in patients with or without alcoholic coexistence (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; y Kenna GA, Nielsen DM, Mello P, Schiesl A, Swift RM. Pharmacotherapy of dual substance abuse and dependence.CNS Drugs, 21 (3): 213-37, 2007.Review), Inclusion of Novel Disulfiram (DSF) and Cyclodextrin (CD) Complexes (hereinafter referred to as DSF-CD complexes), methods of preparation thereof, pharmaceutical compositions thereof and alcohol and cocaine dependence It provides their use in the charge.

According to another aspect of the invention, there is provided a pharmaceutical composition of the DSF-CD complex in solid and liquid form.

Through the use of suitable excipients, it is possible to modify the processes involved to change and control the physicochemical and / or pharmaceutical properties of the molecule. In such cases, inclusions of the drug in cyclodextrins (CDs) are effective substitutes to improve physico-chemical properties such as the stability and solubility of hydrophobic molecules, and parameters such as rate of drug release or bioavailability by different routes of administration. (Duchene, Dominique New Trends in Cyclodextrins and Derivates, Editions de la Sante, Paris, 1992).

Drug availability is particularly important, as many factors involved in drug bioavailability depend on their lipophilic properties (eg transmembrane transit, receptor affinity and tissue distribution, etc.), and for proper bioavailability, correlates to the route of administration. This is because the drug absorption process is largely dependent on the water solubility of the active ingredient (Duchene, Dominique New Trends in Cyclodextrins and Derivates, Editions de la Sante, Paris, 1992; Hardman, Joel G .; Limbird, Lee E. Goodman & Gilman, Las Bases Farmacologicas de la Terapeutica, 9 ^a edicion, Vol. I, McGraw-Hill Interamericana Editores SA, Mexico, 1996: 5).

As mentioned above, the main problem that DSF presents is its low water solubility, and therefore, excipients that can improve physicochemical properties are needed. Therefore, the present invention demonstrates the physicochemical changes affecting DSF by forming inclusion complexes with DCD.

CD undergoes a fermentation process, forming a cyclic ring (α-1,4-linked glucopyranose) which may consist of 6, 7 or 8 glucose monomers, known as α, β and γ-cyclodextrins, respectively. By modified starch group.

In the CD derivatives described in scientific papers and patents, only a few of them can be used for pharmaceutical use (Szejtli, Jozsef, Introduction and General Overview of Cyclodextrin Chemistry, Chemical Reviews, 1998; 98 (5): 1743-1753), Pharmaceutical products consisting of large amounts of CDs are commercially available. Examples include the hydroxypropyl -β-CD used in the novel itraconazole ^Sporanox? Oral and parenteral fungicide sold by Johnson & Johnson that improve drug availability and bioavailability.

On the other hand, Schwarz Pharma company used α-CD to solubilize prostaglandins, which is an erectile drug, alprostadil (Edex ^? ) (McCoy M. "Cyclodextrins; Great product seeks a markets Chemical & engineering news 1999; 77 (9): 25-27).

CDs and derivatives thereof are also described by the oral or sublingual route (Pitha J. Administration of sex hormones in the forms of hydrophilic ciclodextrin derivatives, United States patent 4,751,094 (jun. 14 1986)), transdermal release preparations (Ueda Y., et al. It has been used to administer sex hormones (especially testosterone, progesterone and estradiol) through "Sustained-release transdermal delivery preparations", US patent 4,749,74 (june 1988), between others.

Also described are formulations comprising associations of CD and DSF in solution form. Document CN 1376463 A describes a pharmaceutical composition for ocular topical use comprising one CD and a DSF, wherein the DSF exhibits improved solubility. The association of DSF and CD is mixed in water and lyophilized. Formation of inclusion complexes is not described in this document.

Another publication, 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. Eye Research, Vol. 29 (1), 51-58, describes the study of eye drops containing high concentrations of DSF and CD. This composition was originally intended to treat cataracts, using high concentrations of CD as a solubilizer to increase the solubility of DSF and to obtain an appropriate formulation applied to the eye. In this document, the ratio of DSF: CD used is 1: 2.5 and although CD provided rice solubility for the active ingredient, the formation of inclusion complexes is not described.

CD macromolecules have the property of forming a central cavity inside their spatial structure. Since sugars take on a chain structure, CDs take the form of truncated cones rather than complete cylinders. The hydroxyl groups are facing outwards from the cone, allowing the CD to dissolve in water.

The secondary hydroxyls face one end, the primary hydroxyls face the opposite end, and by the latter free rotation they are bounded by smaller diameter boundaries. The central cavity is bounded by carbon backbones, hydrogen atoms and glycosidic oxygen atoms that confer lipophilic characteristics. Unbound electrons from the oxygen bridge confer Lewis base properties, with a high electron density towards the interior of the ring to form a hydrophobic face (FIG. 1) that can interact with various organic molecules (or portions thereof) to form clathrate compounds. (Duchene, Dominique.New Trends in Cyclodextrins and Derivates, Editions de la Sante, Paris, 1992).

A clathrate compound is a clear stoquiometry, in which a molecule known as a guest is a covalently introduced molecular assembly of a large molecule known as a host. In the present invention, the DSF (guest) is contained or encapsulated in another molecule or structure composed of molecules (hosts), which in the present invention is CD.

In the present invention, a 'physical mixture' is defined as a mixture of solid states in which CD and DSF are included in a definite proportion.

Inclusion complexes include weak intramolecular interactions, generally called hydrophobic bonds, similar to those found in some biological systems (eg, enzyme-substrate, antibody-antigen, etc.). One, two or three CD molecules may contain one or more guest molecules. The most frequent guest: host ratio is (1: 1).

The interactions involved in the inclusion process are actually weak, involving van der Waals forces, London dispersion forces, dipole-dipole interactions, and hydrophobic interactions.

In aqueous solutions, water molecules are generally found inside the CD cavities (nonpolar and polar interactions), and their presence is thermodynamically undesirable because the structures are in fact polar. When a guest is introduced into the CD, these water molecules are substituted. Both guest and water molecules are thermodynamically preferred. The result of complex formation becomes a thermodynamically more desirable system, and the repulsive force between high enthalpy water molecules inside the CD and the repulsive force between nonpolar guests in polar solutions, with the weak interactions already mentioned, are favorable for inclusion complex formation (Connors K. "The stability of cyclodextrin complexes in solucion ". Chem. Rev 1997; 5: 1325-1357; Szejtl J." Utilization of cyclodextrins in industria product and processes ". J. mater. Chem 1997; 7 (4): 575-587; Duchene D. New trends in cyclodextrins and Derivates. Editions de la Sante Paris. 1992, 53-55).

Different CDs are chemically modified in the study to improve the physicochemical properties of the original compounds. Accordingly, hydroxypropyl-β-cyclodextrin (HP-β-CD) having the following structure appeared.

The present invention describes a novel clathrate complex of hydroxypropyl-β-cyclodextrin with DSF for the purpose of improving the solubility and bioavailability of the drug.

There are several methods of preparing 'complex of DSF in hydroxypropyl-β-cyclodextrin' (CD-complex DSF), obtained in solid form. These methods may require different amounts of each component.

The method for preparing the complex of hydroxypropyl-β-cyclodextrin and DSF used in the present invention is a method known in the art and can be easily reproduced by those skilled in the pharmaceutical art.

The products obtained by different methods have different characteristics and the aspects may be different.

In order to physicochemically characterize the inclusion complexes of hydroxypropyl-β-cyclodextrin (HP-β-CD) and DSF, 1: 1 and 1: for their molecular weights via different preparation methods, given below. Two ratios of DSF: CD (hereinafter referred to as CD and HP-β-CD) complexes were needed to be prepared.

a) DSF-CD complex using rotavopor

A mixture of HP-β-CD and DSF was prepared by adding the same amount of anhydrous alcohol by weight. This mixture was completely dissolved. This solvent was then safely evaporated and dried in an oven at 45 ° C. for 48 hours. The working conditions for obtaining the complex through this method are:

Bath temperature: 60 ℃

Stirring Speed: 160 rpm

Vacuum: 80 mmHg

b) DSF-CD composite obtained through dry spray

The corresponding alcoholic solution was obtained by dissolving the HP-β-CD and DSF mixture in anhydrous alcohol as solvent. Spraying was performed on a Buechi B-290 apparatus. The working conditions for obtaining the composites via this method are:

Inlet temperature: 85 ℃

Peristaltic Pump Speed: 35%

Pump extraction 70%

c) DSF-CD complex obtained through lyophilization

The working conditions for obtaining the complex through this method are:

Freezing Temperature: -80 ℃

Sublimation Temperature: -54 ℃

Vacuum: 135 * 10 ^-3 mbar

DSF is A. Sander & Co. It was purchased from GMBH (Germany, Hamburg). Hydroxypropyl -β- cyclodextrin is a ^ENCAPSIN? Buy from Janssen Biotech NV (Belgium Material). The remaining chemical was analytical grade and distilled water was used. As hydrochloric acid is ^Titrisol? Of Merck (Germany material) was prepared as a 0.1 N solution.

DSF-CD inclusion complexes were generated in the Faculty of Pharmacy, University of Concepcion, Chile, using the method described above.

In order to assess the formation of clathrate compounds, their characterization was carried out via differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), X-ray diffraction (RX) and morphology analysis (scanning electron microscopy) (SEM). .

result

1. Physical and chemical characterization of CD and DSF clathrate complex (DSF-CD complex)

1.1. Differential scanning calorimeter

About 2 mg DSF, HP-β-CD, and three methods, the CD-DSF complexes with similar DSF content and differing ratios of 1: 1 and 1: 2 were prepared by differential scanning calorimetry (DSC) Analyzes were conducted at the Polymer Research Center of the Department of Chemistry, Concepcion University.

At a heating rate of 5 ° C./min, the samples were heated in a small semi-sealed aluminum plate under a nitrogen stream of 5 mL / min in a temperature range of 50 ° C. to 330 ° C.

In Fig. 2, DSC (a) of DSF showed two endothermic peaks at 206.84 ° C and 73.25 ° C. The first peak corresponds to the melting point of the drug and the second peak is the decomposition point.

DSC (b) of HP-β-CD showed a slight bias corresponding to the presence of water in the cyclodextrin cavity at 113.4 ° C. and another peak at 316.1 ° C. indicating degradation.

For each complex prepared by various methods and different molecular ratios, a slight shift in signal was observed at a temperature above the point of decomposition of the pure drug, for the product of the stabilizing effect of the CD, which ensures the physicochemical interaction between the two compounds. .

By the formation of the complex, the peak of the DSF melting point shifted to a higher temperature above 280 ° C., which is the temperature at which CD decomposition begins.

In the case of the complex made in the 1: 2 ratio, the interaction between the two compounds was higher due to the complete disappearance of the drug's signal and melting points near approximately 73 ° C. This area reduction means that the energy required for melting of the drug is reduced due to the absence of free DSF and the formation of all possible complexes of DSF.

1.2. Nuclear magnetic resonance

The 1H (quantum) spectra of CD-DSF produced by spray drying at a ratio of 1: 1 to 1: 2 were obtained using the spectrometer Brucker AC250 NMR (250 MHz for 1 hour) in the Department of Organic Chemistry at the University of Concepcion of Chile. Obtained.

Table 1 shows that the chemical substitution of HP-β-CD and DSF is higher in the DSF-CD complex prepared in the DSF: HP-β-CD 1: 2 ratio than the DSF-CD complex prepared in the 1: 1 ratio. Shows.

CD-DSF complex (1: 1) prepared by spray drying; 1H-NMR Spectrum Analysis of DSF-CD Complex (1: 2), DSF, HP-β-CD Prepared by Spray Drying quantum DSF (ppm) HP-β-CD (ppm) C-DSF (1: 1) (ppm) C-DSF (1: 2) (ppm) HP-β-CD DSF HP-β-CD DSF -CH ₃ 1,284 1,125 1,115 1,292 1,091 1,278 -0,01 * + 0,008 * -0,03 * -0,007 * -CH ₃ 1,455 1,147 1,139 1,454 1,113 1,441 -0,008 * -0,001 * -0,035 * -0,014 * *substitution

Table 2 shows that the DSF-CD complex produced in the 1: 2 ratio has substitutions of -0.270 and -0.284 ppm for carbon C-1 and C-4, respectively. In contrast, for DSF-CD complexes prepared at a 1: 1 ratio, there was no significant -0.009 and up to +0.002 ppm substitutions, as this is the sensitivity range of the device.

On the other hand, the largest substitution in the high fields (C-2 and C-3) of the —CH ₂ groups of DSF at a 1: 2 ratio indicates the possible interaction of these groups with HP-β-CD.

Spectral analysis of 13C NMR DEPT 135 of DSF, HP-β-CD, DSF-CD complex (1: 1) and DSF-CD complex (1: 2) carbon DSF (ppm) C-DSF (1: 1) (ppm) C-DSF (1: 2) (ppm) C-1 (-CH ₃ ) 14,750 14,016
-0,009 * 14,044
-0,270 * C-4 (-CH ₃ ) 12,640 11,909
+ 0,002 * 11,987
-0,284 * C-2 (-CH ₂ ) 54,022 53,087
+ 0,935 * 52,997
+ 1,025 * C-3 (-CH ₂ ) 49,656 48,662
+ 0,994 * 48,611
+ 1,045 *

According to the results reported, the complex (HC / HP-β-CD) made from the 1: 2 ratio mixture showed higher interactions compared to the 1: 1 ratio in all experiments. As mentioned above, the expected clathrate compounds corresponding to formula II are shown below:

≪ RTI ID = 0.0 &

Regardless of the preparation method, DSF-CD complexes prepared with a mixture of DSF: HP-β-CD 1: 1 ratios are unable to verify the stoichiometry type of the complexes formed, in which case the system is 1: 1, 1 This is due to the tendency to equilibrate the mixture formation of the: 2 DSF-CD complex and the free DSF, indicating that the melt and thermal signals for the drug of the DSF-CD complex prepared at this ratio are not completely lost but are biased. This is consistent with the one mentioned for DSC above.

1.3. X-ray

X-ray diffraction analysis of CD-DSF complexes, DSF and HP-β-CD prepared in different methods and at different ratios was performed using a diffractometer RIGAKU GEIGERFLEX. It used a copper pipe with a nickel filter using radiation of 40 kw and 20 milliamp at an angle in the range of 3 ° C. to 70 ° C. (2θ) at a rate of 1 ° / min.

In FIG. 3, the diffraction spectrum of pure DSF (a) showed various peaks produced by the crystalline of the drug, whereas no peak was present in the spectrum of HP-β-CD (b), meaning that it was in an amorphous state.

The spectra of the complexes prepared in different methods and at different ratios were similar to the spectra of HP-β-CD, showing that complex formation with cyclodextrin resulted in the formation of a product in which the crystallinity of pure drug was lost resulting in an inclusion complex.

The peak present in the spectrum of the composite produced by the rotary evaporator at 1: 1 ratio means that the free DSF is present because the DSF did not completely form the complex, which is observed in the composite prepared at 1: 2 ratio. It wasn't.

1.4 Infrared spectroscopy

Infrared spectroscopy is performed by direct compression using KBR, DSF, HP-β-CD, and physical mixtures DSF: HP-β-CD (1: 1) in the range 500-4000 cm ^-1 using a Nicolet Bexus model spectrometer. Infrared spectroscopy was performed for (1: 1 ratio based on these molecular weights). The analysis was performed in the Department of Organic Chemistry, Department of Chemistry, Concepcion University, Chile.

The experimentally obtained IR spectra for DSF, HP-β-CD, physical mixture DSF: HP-β-CD (1: 1) and DSF-CD complex are shown in FIGS. 4, 5, 6 and 7, respectively.

The spectrum of the DSF-CD complex (FIG. 7) showed that the CS band frequency of the DSF shifted from 1267 to 1280 cm ⁻¹ . The shape of this band was wider and stronger in the spectrum of the DSF-CD complex compared to the physical mixture DSF: HP-β-CD (1: 1), while the latter was 1266 and 1267 cm ⁻ respectively. The profile of the same band similar to the DSF with the frequency value of ¹ was maintained.

There was also a C = S band, located in the DSF at a frequency of 1342 cm ⁻¹ , consistent with the physical mixture, while the DSF-CD was replaced with 1365 cm ⁻¹ .

In general, in the 2,000-1,000 cm ⁻¹ region of the spectrum, differences between DSF and HP-β-CD could be observed. The change in the spectrum of the complex DSF-CD was mainly under 1,500 cm ⁻¹ than in the pinkerprint region of the molecule.

As mentioned previously, it can be seen that the changes were generated in the spectrum of the DSF-CD complex, supporting the interaction phenomenon between the two compounds.

1.5. Scanning electron microscope

Micrographs obtained by scanning electron microscopy (SEM) of HP-β-CD and CD-DSf complexes prepared in different methods and at different ratios were taken in an electron microscopy laboratory at the University of Concepcion, Chile.

In the SEM analysis method, it was not possible to obtain a picture of the DSF and the physical mixture because the drug used a temperature above the melting point of the drug, which became liquid during the process.

The powder samples were dispersed on a thin glass plate fixed on an aluminum cylinder and placed in a gold bath of 5150 Edwards Sputter Coster and photographed on a Jeol model J-SM 6380LV device.

The scanning electron microscope (FIG. 8) confirmed that there was a change in the shape of the particles depending on the production method. In the photograph (g), the shape of the spherical HP-β-CD particles having an amorphous and smooth surface can be confirmed. In the case of the DSF-CD composite prepared by the rotary evaporator, an irregularly shaped microcrystal was obtained. In contrast, by spray drying, an amorphous form having a spherical and rough surface was obtained (FIG. 9). In the case of DSF-CD composite prepared by freeze drying, needle-shaped irregular particles were obtained.

SEM shows the change in the structural morphology of the complex, which shows the interaction between the two compounds.

1.6. Polarizing microscope

Photos of the DSF and DSF-CD complexes were taken, the latter being prepared by spray drying and rotary evaporator. Small amounts of each sample were individually dispersed on slides and photographed using an optical microscope METTLER FP 82 under conditions of polarized ORTHOLUX II POL-BK and a Canon 42x camera.

In FIG. 10, in FIG. (A), DSFs exhibited refraction when they gave polarization to their crystals as crystal structures. In the case of DSF-CD prepared by spray drying (b), its properties appear to have been lost by the inclusion complex which is in an amorphous state.

The DSF-CD composite (c) photograph prepared by the rotary evaporator was compared and was present in a similar manner to the DSF-CD composite prepared by spray drying. However, this phenomenon is probably less noticeable due to differences in manufacturing methods.

Polarization microscopy cannot verify the inclusion of clathrate complexes, but it can prove the interaction between the two compounds.

2. Characterization of DSF and CD Inclusion Complex (DSF-CD Complex)

Particle size and solubility were analyzed. The latter includes measurements of solubility kinetics and maximum solubility of the complexed DSFs, both of which were determined by validated ultraviolet spectroscopy (UV).

2.1 Analysis of Particles

Particle size analysis of DSF-CD composites prepared by spray drying at 1: 1 and 1: 2 ratios was made through SEM photographs (FIGS. 8 and 9). The diameter of 110 particles was measured using a computerized statistical program.

Particle size analysis of the DSF was performed at the Chemical Development Research Center (DEPEDEC) of the Department of Chemistry and Pharmacy, University of Chile, using optical diffraction analysis.

About 2 mg of DSF was dispersed in 50 mL cyclohexane and sonicated for 5 seconds. This sample was analyzed on a particle counter Mastersizer Malvern instrument GmbH. As shown in FIG. 11, the distribution curve of the DSF-CD composite prepared by spray drying was negative asymmetric (FIG. 11A) at smaller intervals of 7-26 m where substitutions existed at smaller particle sizes, whereas DSF showed particle distribution concentrated at larger intervals of 7-83 m (FIG. 11B) compared to the first one.

The smaller particle sizes of the DSF-CD composites account for their high solubility because these parameters are proportional to the surface and inversely proportional to the particle size.

Particle Size Distribution of Different Samples Particle volume
10% distribution (㎛) Particle volume
90% distribution (㎛) Average distribution (μm)
Average diameter (㎛)

DSF
8,31
44,10
20,28
23,82
DSF-CD Complex
2,49
15,80
8,77
9,13

2.2 Availability Experiment

2.2.1. Maximum Solubility of DSF in Complex Form

Excess DSF in 5 mL of 0.1 N HCl was equilibrated by placing it in a 25 mL flask protected from light through aluminum foil and constantly stirring at 21 ± 1.5 ° C. for 72 hours. At the same time, the same procedure was performed using DSF-CD complexes (equivalent to DSF) prepared in different methods and in different analogies. The samples were conditioned to filtered through ^Sartorius? Filter of 1.2 m pore size and read at 254 nm with UV spectrometer.

Results were statistically analyzed using t-student with GraphPad Prism 2.1 software. Each sample was performed in three replicates using 300 mg of DSF.

Solubility of pure drugs and their complexes is shown in Table 4 below and FIG. 12.

Solubility of Different DSF-CD and DSF in 0.1 N HCl Solubility in mg NmL in 0.1 N HCl ± S.E. DSF 0.1 ± 0.001 DSF-CD Composite Rotary Evaporator 1: 1 12.96 ± 0.3 DSF-CD Composite Rotary Evaporator 1: 2 18.32 ± 0.04 DSF-CD Composite Dry Spray 1: 1 22.04 ± 0.1 DSF-CD Composite Dry Spray 1: 2 26.93 ± 0.06 DSF-CD Complex Lyophilization 1: 1 11.28 ± 0.03 DSF-CD Complex Lyophilization 1: 2 15.50 ± 0.21 S.E. : Standard Deviation

The increase in solubility of DSF with HP-β-CD in 0.1 N HCl was very significant in each case tested (p <0.0001), with a ratio of 1: 2 rather than that of 1: 1, regardless of the preparation method. It was always higher in the prepared DSF-CD.

DSF-CD complexes prepared by spray drying at ratios of 1: 1 and 1: 2 are more soluble (220.4 and 296.3 times higher than pure drugs, respectively), with rotary evaporators and lyophilization with some degree of crystallinity. Compared to the spray system, an amorphous structure having a small particle size was obtained.

2.2.4 Solubility Kinetics

USP XXIV using a detacher device (Bahama St., Northridge) with 6 cups, device 2, in a paddle method at 50 rpm for 60 minutes, using 900 mL of aqueous phase (0.1 N HCl) at 37 ± 0.5 ° C. Was carried out under specific conditions.

Individual samples were taken during the period mentioned above with a fixed automatic sampling system while manually changing volumes. The sampling system is equipped with a 10 m pore size filter attached to a stainless steel tube connected to a sample receiving system (glass tube with a scale of 10 mL or less) via a Tygon® hose.

The drug under experiment was introduced in the form of a powder on the dissolution medium at a defined temperature with stirring, waiting 1 minute for proper wetting and setting this time to 0 hours. 5 mL samples were taken at intervals of 1, 3, 6, 10, 20, 30, 40, 50, 60 minutes with medium replacement (reposition). The sample described below was subjected to 7 solubility profiles with a dose equal to 1 g DSF, n = 6:

. DSF

. C-DSF (C-DSF (1: 1)) prepared by spray drying at a 1: 1 ratio

. C-DSF (C-DSF (1: 2)) prepared by spray drying at a 1: 2 ratio

. Physical mixture prepared at 1: 1 ratio (1: 1 MF)

. Physical mixture prepared at 1: 2 ratio (1: 2 MF)

The solubility kinetics results of the different samples tested in FIG. 13 are summarized. As can be seen, it can be noted that the complex rate of cyclodextrin and DSF (C-DSF) increased the dissolution rate and dissolution rate, with a maximum of approximately for C-DSF prepared at 1: 1 and 1: 2 ratios, respectively. 44% and 100% were already dissolved three minutes after the start of the experiment, while the DSF was 0.1% for the same time.

Physical mixtures prepared in 1: 1 and 1: 2 ratios differed significantly in terms of dissolution rate and dissolution rate of the dissolved drug relative to the complex, which interacted between the two compounds when forming a drug complex after grinding in the liquid. It supports more than this.

3. Pharmacological Characterization of CD and DSF Inclusion Compounds (DSF-DSF Complex)

For free DSF, heart rate was measured as a sign of efficacy of the ethanol-disulfiram response (RED) to verify the difference in strength and duration of pharmacological efficacy of the DSF complexed according to the present invention.

On the other hand, histopathological experiments were performed in rats to evaluate whether the complex administered parenterally causes tissue damage.

Finally, pharmacokinetics to measure plasma concentrations of the metabolites diethyldithiocarbamate and methyldiethyldithiocarbamate (DDC and DDC-I) of DSF, in which DSF and the complex exhibit higher bioavailability. The experiment was performed.

In all these experiments, Sprague-Dawley male rats weighing 250-300 grams were used to arbitrarily feed and water at light and dark cycles (12:12 hours) and at a temperature of 22 ± 1 ° C.

3.1 Evaluation of the Ethanol-Disulfiram Reaction

RED was monitored in rats based on behavioral signs such as pulmonary ventilation, hiccups, and momentum, and the signs were measured on the following scales.

0 = no signs, 1 = mild, 2 = medium, 3 = severe

Cardiovascular effects were monitored via ECG substrate. The following overhaul was performed:

Basic control

Control following subcutaneous administration of 15 mg / kg free DSF or DSF-CD complex dissolved in 0.5 mL of normal physiological serum;

RED control performed after intraperitoneal administration of 1 g / kg of 96 ° alcohol diluted to 50% in water. This control was performed at the following time. 1, 8, 12, 24, 48, 72, 96, 120 and 168 hours after DSF administration.

Behavioral RED results are shown as averages in Table 5. In the free form, the signs were greater in rats receiving DSF in the complex form (DSF-CD complex), especially compared to controls performed after 24 and 96 hours. The intensity and duration of the signs were analyzed by deviation analysis by performing a Tukey test, which resulted in SF (physiological serum) v / s DSF, SF v / s CD-DSF complex and DSF v / s DSF-CD complex at 24 hours. Significant deviations in liver were shown (p <0.05).

14 shows the effect on heart rate observed after alcohol administration in rats treated with DSF. FIG. A shows that the heart rate is significantly reduced upon administration of the DSF-CD complex, presumably because inhibition of dopamine-hydroxylase is predominant by high concentrations of DDC in the blood. Free DSF did not cause significant change. FIG. B shows that bradycardia resulting from the administration of the DSF-CD complex was decreased, and free DSF suffered from mild cardiac arrest. FIG. C shows that DSF-CD causes cardiac arrest and DSF caused some changes in heart rate. In FIG. D, the CD-DSF complex caused cardiac arrest and free DSF caused mild bradycardia. In Figure E, the DSF-CD complex had an increased heart rate and no significant change in the free DSF. In Figure F, both free DSF and CD-DSF complexes did not change heart rate because the plasma concentration of the drug and / or its metabolites no longer persisted.

At all times, the effect of the DSF-CD complex was higher compared to the free DSF. Through the Student test, statistical analysis was performed by comparison between the DSF and DSF-CD complexes, and after 1 hour the p value for the control group was given as <0.001.

Table 5 below shows 1 g / kg at different times in rats pretreated with SC in free DSF (DSF) or complex form (called CDSF in Table 5) at a dose of physiological serum (PS), 15 mg / kg. Ethanol (96 ° in 50% distilled water) ip Indicates the effect administered.

3.2 Evaluation of Inflammation Intensity at the Dosing Site: Histopathological Experiments

Subcutaneous administration of 15 mg / kg of the free DSF and DSF-CD complexes dissolved in 0.5 mL normal physiological serum was taken subcutaneously and compared with controls administered physiological serum.

Skin samples were taken at the injection site at 8, 24, 48, 72, 96, 120, 144 and 168 hours after injection. Eosin / hematoclin staining was performed on these samples to evaluate the presence of corneal thickening, the deorganization of collagen fibers, the presence of fibroblasts and inflammatory cells, capillary congestion of surface and deep reticular tissues, skin edema and subcutaneous tissue bleeding. . Signs were evaluated according to the following measures: 0 = no signs, 1 = mild, 2 = moderate, 3 = severe.

The inventors analyzed the presence and intensity of the reaction using Fisher extraction.

Table 6 below shows the effects of free DSF, C-DSF and SF controls on tissue damage induced after subcutaneous administration, showing the subcutaneous effects of DSF on rat skin (DSF-CD is represented by CDSF).

Figure 15 shows tissue results 8 hours after administration of DSF. Free DSF induced severe congestion in the deep vascular network with dermal bleeding, while the DSF-CD complex showed moderate and severe congestion in the superficial and deep vascular networks, respectively, with inflammatory cells. In control group injected with SF, severe hyperemia was observed in both vascular networks.

16 Tissue results after 24 hours of DSF administration. Free DSF induced severe congestion in both vasculatures, inflammatory cells were present in the dermis, bleeding was induced in the dermis, while DSF-CD complex was weakly congested in both vessels and slightly inflamed. The dermis was slightly swollen and bleeding was present. In the control group injected with PS, moderate hyperemia was present in the deep vascular network, and moderate inflammatory cells were present.

17 shows tissue results 48 hours after DSF administration. Free DSF had severe de-organization of collagen fibers, hyperemia in the deep vascular network, moderate bleeding in the dermis, whereas DSF-CD showed moderate de-organization of collagen fibers. There was congestion and bleeding in the dermis. In the control group injected with PS, moderate bleeding and inflammatory cells were observed in the deep vascular network.

18 shows tissue results 72 hours after DSF administration. Free DSF induced moderate hyperemia in both vasculatures, moderate inflammatory cells and bleeding in the dermis, and in DSF-CD complex, moderate deep vascular hyperemia and severely inflammatory cells. Was observed. In the control group injected with SF, there was moderate hyperemia with the presence of some inflammatory cells.

19 Tissue results after 96 hours of DSF administration. Free DSF induced moderate congestion in the deep vascular network, bleeding in the dermis, severe inflammatory cells, and complex DSF-CD had moderate congestion in the deep vascular network and slightly inflammatory cells. Presence, neutrophil outflow, and skin edema. In the control group injected with SF, some hyperemia was present in the deep vascular network.

20 shows tissue results after 120 hours of DSF administration. Free DSF induced moderate hyperemia in the deep vascular network, slight inflammatory cells, deorganization of collagen fibers, and in the DSF-CD complex, slight hyperemia of the deep vascular network was observed, inflammation Cells were present and bleeding was present in the dermis. In the control group injected with SF, some hyperemia was present in the deep vascular network and some fibroblasts were present.

21 shows tissue results after 144 hours of DSF administration. Free DSF induced moderate hyperemia of the deep vascular network, some inflammatory cells were present, and some hyperemia was observed in the DSF-CD complex with moderate inflammatory cells. In the control group injected with SF, some hyperemia was present in the deep vascular network.

Figure 22 Tissue results after 168 hours of DSF administration. Free DSF induced moderate congestion in the deep vascular network, and DSF-CD complex was observed in the deep vascular network with moderate congestion and large amounts of fibroblasts in the subcutaneous tissue. In the SF injection control group, hyperemia was present in the deep and superficial vascular networks.

3.3. Pharmacokinetic (PK) Experiment

Blood samples were taken from rats after subcutaneous administration of 15 mg / kg of free DSF and complex DSF-CD dissolved in 0.5 mL SF, and compared with controls administered SF.

Samples were analyzed by high performance liquid chromatography (HPLC) using an RP-18 column with a mobile phase composition of acetonitrile / water (60:40 v / v). The mobile phase was filtered (0.45 μm membrane filter) and carbonized. The flow rate was 1 mL / min and the injection volume was 30 μl. The detection range was 200 to 300 nm. All tests were performed at 25 (± 1) ° C.

Standard solutions were acetonitrile in 1 mg / mL DDC-I (methyl-diethylthiocarbamate), Et-DDC (ethyl-diethylthiocarbamate) and DSF, each with 10 mg of each chemical dissolved separately. It was prepared by making 10 mL using. Its concentration is 10 mg / mL. Each test solution was added to 0.25 mL of 10 mg / mL Pr-DDC (propyl-diethylthiocarbamate) to obtain an internal standard having a concentration of 0.25 mg / mL.

Plasma samples were filled with known amounts of I-DDC, DDC and DSF. 500 µl of plasma, 500 µl 0.01 M EDTA, 1% NaCl, pH 8.5 solution and 5 µl of iodine ethanol were added, vortex was added for 30 seconds, and reacted at 40 ° C for 30 minutes. These were added: 12.5 μl internal standard (D-DDC). Solid phase extraction was performed on these.

The column was placed on a 12 point vacuum distributor connected to a vacuum pump. The column was charged with 1,000 μl acetonitrile, followed by 1000 μl of nanopure water, and then samples were added to the column. As the sample passed through the column, Me-DDC, DDC-Et, DSF and Pr-DDC were maintained. To remove impurities, the column was washed with 3000 μl nanopure water. DDC-I, Et-Pr-DDC and DDC were eluted from the column using 1000 μl acetonitrile. DSF could not be extracted from plasma samples. 30 μl of the eluted solution was injected into the chromatography column.

23 shows the results after subcutaneous administration of disulfiram. The area under the curve was clearly higher after administration of the complex DSF-CD.

1 is a diagram illustrating a spatial model of a CD.

FIG. 2 shows a differential scanning calorie: (a) DFS, (b) HP-β-CD, (c) complex evaporator prepared with rotary evaporator, (d) rotary evaporator Composite DSF-CD 1: 2, (E) Composite DSF prepared by spray drying, (f) Composite DSF-CD 1: 2 prepared by spray drying, (g) Composite DSF prepared by lyophilization -CD 1: 1, (h) complex DSF 1: 2-CD prepared by lyophilization.

FIG. 3 shows an X-ray diffraction spectrum: (a) DFS, (b) HP-β-CD, (c) composite evaporator prepared by rotary evaporator, (d) lyophilized Complex DSF-CD 1: 1, (e) Composite DSF prepared by spray drying, (f) Composite DSF prepared by rotary evaporator, DSF-CD 1: 2, (g) Composite DSF prepared by lyophilization -CD 1: 2, (h) composite DSF-CD 1: 2 prepared by spray drying.

4 is a diagram showing an infrared spectrum (IR) of a DSF.

5 shows the IR spectrum of HP-β-CD.

Fig. 7 shows the IR spectrum of the complex DSF / HP-β-CD (CD-composite DSF).

Figure 8 is a scanning electron microscope (x1000) picture: (a) complex DSF-CD prepared by a rotary evaporator in a 1: 1 ratio, (b) complex DSF-CD prepared by a rotary evaporator in a 1: 2 ratio, (c) Composite DSF-CD prepared by spray drying at 1: 1 ratio, (d) Composite DSF-CD prepared by powder drying at 1: 2 ratio, (e) Freeze drying at 1: 1 ratio Prepared complex DSF-CD, (f) complex DSF-CD prepared by lyophilization in a 1: 2 ratio, (g) HP-β-CD (x300).

9 is a scanning electron microscope (x4000) picture: (a) composite DSF-CD prepared by spray drying at a 1: 1 ratio, (b) composite DSF-CD prepared by spray drying at a 1: 2 ratio, (c) HP-β-CD (x300).

10 is a polarization micrograph: (a) DSF, (b) composite prepared by spray drying at 1: 1 ratio DSF-CD, (c) composite prepared via rotary evaporator (x40) at 1: 1 ratio DSF-CD.

11 is a bar graph showing particle size: (a) composite prepared by spray drying in a 1: 1 ratio, (b) glassy DSF.

FIG. 12 shows the maximum solubility of DSF and complex DSF-CD in 0.1 N HCl obtained by three preparation methods.

FIG. 13 shows the solubility kinetics of physical mixtures (PM) in 1: 1 and 1: 2 ratios in 0.1 N HCl and DSF-CD complexes and DSFs in 1: 1 and 1: 2 ratios.

FIG. 14 shows intraperitoneal injection of 1 g / kg in 50% distilled water ethanol in a rat injected with a subcutaneous (SC) injection of DSF-CD complex (complex forming DSF) and free DSF (complex forming DSF) at a 15 mg / kg dose. It is a figure which shows the heart rate change produced | generated by intra (IP) administration. Rats were periodically monitored after (A) 1 hour, (B) 24 hours, (C) 48 hours, (D) 72 hours, (E) 96 hours and (F) 144 hours after ethanol administration.

15 to 22 show tissue sections of rat skin in the subcutaneous (SC) portion of the control group of (A) DSF, (B) DSF-CD complex, and (C) physiological serum (PS). These tissue sections were obtained at different time periods after administration of each test article: FIG. 15 after 8 hours of administration, FIG. 16 after 24 hours of administration, FIG. 17 after 48 hours of administration, and FIG. 18 after 72 hours of administration. FIG. 19 is 96 hours after administration, FIG. 20 is 120 hours after administration, FIG. 21 is 144 hours after administration, and FIG. 22 is 168 hours after administration.

FIG. 23 shows the results of the area under the curve (AUC) after subcutaneous administration of (A) free DSF and (B) DSF-CD complex.

Claims (24)

Disulfiram (DSF) and hydroxypropyl-β-cyclodextrin (HP-β-CD) is characterized in that the ratio of 1: 2, characterized in that the DSF and HP-β-CD represented by the formula (II) Consisting inclusion complexes: &Lt; RTI ID = 0.0 &

The inclusion complex of claim 1, wherein the mean diameter is 9.13 μm and the solubility in 0.1 N HCl is at least 26.93 mg / ml, such as a 296.3-fold increase in solubility of pure DSF. The inclusion complex of claim 1 having the following chemical shifts in 1 H-NMR. proton Complex DSF: HP-β-CD (1: 2) ppm HP-β-CD DSF -CH ₃ 1.091 1.278 -CH ₃ 1.113 1.441 The inclusion complex of claim 1 having the following chemical shifts in 13 C-NMR. carbon Complex DSF: HP-β-CD (1: 2) ppm C-1 (-CH ₃ ) 14.044 (-0,270) C-4 (-CH ₃ ) 11.987 (+0,284) C-2 (-CH ₂ ) 52.997 (+1,025) C-3 (-CH ₂ ) 48.611 (+1,045) A method for preparing the inclusion complex according to claim 1, which is carried out by the following steps: a) physically mixing hydroxypropyl-β-cyclodextrin and DSF; b) dissolving the mixture in anhydrous alcohol; c) complete evaporation of the solvent in a rotary evaporator with a bath temperature of 60 ° C. at a speed of 160 rpm with a vacuum of 80 mm Hg (3.83 × 10 ⁻³ MPa); And d) drying in an oven at 45 ° C. for 48 hours A method of manufacturing the inclusion complex according to claim 1, a) physically mixing hydroxypropyl-β-cyclodextrin and DSF; b) dissolving the mixture in anhydrous alcohol; And c) spraying the resulting solution with a Buechi B-290 device Using spray drying at an inlet temperature of 85 ° C., a peristaltic pump speed of 35% and a pump extraction of 70%. A method of preparing a clathrate complex according to claim 1, which is carried out by freeze drying at a freezing temperature of −80 ° C., a sublimation temperature of −54 ° C., and a vacuum of 135 × 10 ⁻³ mbar (0.01 MPa). A pharmaceutical composition comprising an inclusion complex consisting of disulfiram (DSF) according to claim 1 and hydroxypropyl-β-cyclodextrin (HP-β-CD). The pharmaceutical composition of claim 8 which is a solid composition. The pharmaceutical composition of claim 8, which is a liquid composition. A pharmaceutical composition comprising the inclusion complex of claim 1 for treating alcohol dependence. A pharmaceutical composition comprising the inclusion complex according to claim 1 for treating cocaine dependency. delete delete delete delete delete delete delete delete delete delete delete delete

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