RU2592625C1 - Inclusion complex of disulfiram and cyclodextrin - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and deals with inclusion complex of disulfiram (DSF) and cyclodextrin (CD), selected from a group consisting of unsubstituted cyclodextrin (uCD) or substituted cyclodextrin, such as methylcyclodextrin (MCD), dimethylcyclodextrin (DMCD), trimethylcyclodextrin (TMCD) and hydroxypropylcyclodextrin (HPCD) produced by the method, in which CD, taken in a solid or dissolved in water or alcohol form, when stirred at a temperature from 20 to 70 °C is combined with DSF in the form of a solid substance, fusion or alcohol solution, where alcohol is selected from methanol, ethanol, n-propanol or iso-propanol, where molar ratio of CD and DSF ranges from 2.01:1 to 2.40:1. Obtained mixture is cooled to 0-25 °C, subjected to sterilising filtration and lyophilized, or solvent is evaporated.

EFFECT: reception of water-soluble inclusion complex of disulfiram with cyclodextrin, which can be extracted with high degree of purity.

1 cl, 5 dwg, 2 tbl, 8 ex

Description

FIELD OF THE INVENTION

The invention relates to the field of pharmaceutical industry and relates to a disulfiram inclusion complex with unsubstituted or substituted cyclodextrin, which can be isolated as a drug substance for the manufacture of a pharmaceutical composition.

Background of the Related Art

Cataracts are one of the common eye diseases. According to statistics, in the Russian Federation it affects about 10 million people. An effective medication for treating cataracts is disulfiram (DSF). It significantly reduces the effect of hydroxyl radicals and inhibits lipid peroxidation [Noriaki Nagai, Yoshimasa Ito and Noriko Takeuchi "Corrélation between Hyper-Sensitivity to Hydrogen Peroxide and Low Defense against Ca ²⁺ Influx in Cataractogenic Lens of Ihara Cataract Rats" Biol. Pharm. Bull. 34 (7) 1005-1010 (2011)]. Unfortunately, the possibility of using DSF in ophthalmology is limited due to its low solubility in water. In order to increase the solubility of DSF, it is proposed to use cyclodextrins (CDs), which increase the solubility in water due to the formation of host-guest inclusion complexes. Thus, the synthesis of inclusion complexes of this type is an urgent task for cataract therapy.

In the article by Noriaki Nagai, Maki Takeda, Yoshimasa Ito, Noriko Takeuchi and Akira Kamei “Delay in ICR / f Rat Lens Opacification by the Instillation of Eye Drops Containing Disulfiram and Hydroxypropyl-β-cyclodextrin Inclusion Complex” (Biol. Pharm. Bull. 30. (8) 1529-1534 (2007)) the composition is disclosed and a method for preparing eye drops is described that retard clouding of the cornea of the eye and reduce its severity in the rat cataract model. Drops contain 0.25 or 0.5% disulfiram, 3 or 5% hydroxypropyl-β-cyclodextrin and 0.1% hydroxypropyl methylcellulose. To prepare drops, hydroxypropyl-β-cyclodextrin is added to a saline solution containing 0.005% benzalkonium chloride and disulfiram, after which hydroxypropyl methylcellulose is added to the resulting solution, stirred for 24 hours in the dark at room temperature and filtered through a filter with a pore size of 0.20 μm.

In the article by Noriaki Nagai, Yoshimasa Ito and Noriko Takeuchi "Pharmacokinetic and Pharmacodynamic Evaluation of the Anti-cataract Effect of Eye Drops Containing Disulfiram and Low-Substituted Methylcellulose Using ICR / f Rats as a Hereditary Cataract Model" (Biol. Pharm. Bull. 35. (2) 239-245 (2012)) a similar composition is described, additionally containing low-substituted methyl cellulose, which is added last in the form of a solution in sterile saline solution in the manufacture of droplets. The finished drops contain 0.5% disulfiram, 5% hydroxypropyl-β-cyclodextrin, 0.1% hydroxypropyl methylcellulose and 2% methylcellulose.

Both articles describe the preparation of a finished dosage form, the composition of which, if necessary, will not be possible to change. In addition, the presence of disulfiram in it in the form of an inclusion complex with hydroxypropylcyclodextrin is not discussed and is not expected. Therefore, the technical task of the invention is to obtain a water-soluble disulfiram inclusion complex, which can be isolated as an individual compound with a purity degree that meets the requirements of the pharmacopeia, which will make it possible to prepare eye drops of various compositions by methods generally accepted in the art.

Brief Description of the Drawings

The figure 1 presents powder x-ray diffraction patterns of pure DSF (curve 1), pure GPβCC (curve 2), a physical mixture of DSF and GPβCD (curve 3) and the inclusion complex of DSF with GPβCD (curve 4).

The figure 2 presents the curves of differential scanning calorimetry for pure DSF (1), a physical mixture of DSF and GPβCD (2) and the inclusion complex of DSF with GPβCD (3).

The figure 3 presents the ¹³ C NMR spectrum of the inclusion complex of DSF with GPβCD.

The figure 4 shows the symbol of carbon atoms in the molecules of HPβCC and DSF.

The figure 5 shows the proposed structure of the inclusion complex of DSF with GPβCD.

SUMMARY OF THE INVENTION

As a result of the studies, the inventors solved the technical problem: they obtained, isolated and characterized a water-soluble inclusion complex of disulfiram (DSF) with cyclodextrin (CD) selected from the group consisting of unsubstituted cyclodextrin (NCD) or substituted cyclodextrin, such as methylcyclodextrin (MCD) , dimethylcyclodextrin (DMCD), trimethylcyclodextrin (TMCD) and hydroxypropylcyclodextrin (HCPD), 1: 2 stoichiometric composition, which is characterized by the presence of ¹³ C NMR in its spectrum Signals at 12.0, 14.0, 18.5.48.4, 52.9, 60.5, 66.7, 67.0, 72.1, 72.5, 73.6, 77.4 , 78.8, 81.4, 100.3, 101.5, 102.5 191.0 ppm, and the method of differential scanning calorimetry determines the temperature depression in the region of 220-235 ° C with a minimum at 221 ° C and shoulder at 231 ° C.

Preferably, in said inclusion complex, cyclodextrin is hydroxypropyl-β-cyclodextrin (GPβCD).

The technical result is a water-soluble inclusion complex of disulfiram with cyclodextrin - the active substance of eye drops, which can be isolated with a degree of purity that meets the requirements of the State Pharmacopoeia of the Russian Federation.

To achieve the technical result of the invention, a cyclodextrin selected from the group consisting of unsubstituted cyclodextrin (CC), methylcyclodextrin (MCD), dimethylcyclodextrin (DMCD), trimethylcyclodextrin (TCDC) and hydroxypropylcyclodextrin (HCDC), taken in solid form or dissolved in water or alcohol with stirring and a temperature of from 20 to 70 ° C combined with disulfiram (DSF) in the form of a solid, melt or alcohol solution, where the alcohol is selected from methanol, ethanol, n-propanol or iso-propanol, molar ratio GPβCD and DSF are from 2.01: 1 to 2.40: 1, the resulting mixture is cooled to 0-25 ° C, filtered and the solvent is lyophilized or evaporated.

In the context of this description, the term "cyclodextrin" includes any of the known isomers: α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin.

Similarly, each of the terms methylcyclodextrin, dimethylcyclodextrin, trimethylcyclodextrin and hydroxypropyl cyclodextrin includes any of the known isomers. For example, the term “hydroxypropylcyclodextrin” refers to hydroxypropyl-α-cyclodextrin, hydroxypropyl-β-cyclodextrin and hydroxypropyl-γ-cyclodextrin, among which hydroxypropyl-β-cyclodextrin (GPβCD) is most preferred.

Obtaining a complex can be carried out in various ways. In one embodiment, equal masses of 30 mass. % aqueous solution of ββCD and 3 wt. % alcohol solution of DSF is combined at a temperature of 65 ° C, stirred for 12 hours, cooled to 20-25 ° C, subjected to sterilizing filtration and freeze-dried.

In another embodiment, 30 mass. An aqueous solution of GPβCD and a DSF melt taken so that the molar ratio of GPβCD and DSF is 2.10: 1 are combined at 65 ° C, stirred for 12 hours, cooled to 20-25 ° C, subjected to sterilizing filtration and freeze-dried.

In the next embodiment, 30 mass. % aqueous solution of GPβCD and solid DSF, taken so that the molar ratio of GPβCD and DSF is 2.10: 1, combined at a temperature of 20-65 ° C, stirred for 24 hours, if necessary, cooled to 20-25 ° C, subjected to sterilizing filtration and freeze-dried.

In another embodiment, 30 mass. % alcohol solution of ββCD and solid DSF, taken so that the molar ratio of ββCD and DSF is 2.10: 1, combined at a temperature of 65 ° C, stirred for 0.5 hours, cooled to 20-25 ° C, subjected to sterilization filtration and evaporated solvent.

Preferably, solid ββCD and DSF are taken such that the molar ratio of ββCD and DSF is 2.10: 1, combined in alcohol, heated and stirred at 65 ° C for 0.5 hours, cooled to 20-25 ° C, subjected to sterilization filtration and evaporated solvent.

The average specialist in the field of chemistry knows that the formation of complexes in a liquid medium is equilibrium. Therefore, to obtain a inclusion complex of DSF with a 1: 2 stoichiometric CD, a small excess of HPβCD is required, for example, from 2.01 mol of CD / mol of DSF to 2.40 mol of CD / mol of DSF. Used in accordance with the invention of 30 mass. % solution of ββCD and 3 wt. % DSF solution, taken in a mass ratio of 1: 1, or appropriate weighed components provide a molar ratio of DSF and GPβCD equal to 1: 2.10, i.e. an excess of 10 mol.% GPβCD. The authors found that with an excess of DSF, a inclusion complex of 1: 2 composition also forms, and from the reaction mass, DSF that is not included in the complex spontaneously precipitates upon cooling.

The fact that the inclusion complex differs from pure disulfiram and GPβCD, as well as from their physical mixture, was established by powder x-ray diffraction (PRD) and differential scanning calorimetry (DSC). The stoichiometry of this complex was determined by ¹³ C NMR spectroscopy.

PRD studies were carried out on a X′Pert Pro MPD powder diffractometer. The samples were irradiated at a voltage of 50.0 kV in a current of 40.0 mA at a scanning speed of 2 ° C / min, the diffraction angle 2θ in the range from 5 to 60.

In the diffraction patterns (Fig. 1) for pure DSF (curve 1), a pronounced series of intense peaks is observed, which indicates the crystal structure of DSF, while in the diffraction pattern of pure GPβCD (2), intense peaks are absent, showing an amorphous halo corresponding to the amorphous state of GPβCD . The diffractogram of a physical mixture of DSF and GPβCD (3) shows both a series of intense peaks identifying the crystal structure of DSF and an amorphous halo corresponding to the amorphous nature of GPβCD. However, in the X-ray diffraction pattern of the DSF inclusion complex with GPβCD (4), intense peaks identifying the crystal structure of DSF are absent, which indicates the absence of DSF crystals and its complete incorporation into the internal cavity of GPβCD on a molecular level.

DSC experiments were carried out on a differential scanning calorimeter DSC-500 in dynamic mode at a heating rate of 10 ° C / min, in an argon stream (gas flow rate of 50 ml / min) and a temperature range of 50-330 ° C.

On the curves of differential scanning calorimetry (Fig. 2) for pure DSF (curve 1) and a physical mixture of DSF and GPβCD (curve 2), narrow peaks of temperature depression in the range 63-71 ° C characterizing the melting process of DSF are observed. In curve 3, for the inclusion complex of DSF with GPβCD, a narrow peak in this region was not registered. In the high-temperature region, the temperature depression is determined for the inclusion complex in the region of 220-235 ° C with a minimum at 221 ° C and a shoulder at 231 ° C. Pure DSF and a physical mixture of DSF and GPβCD are characterized by minima at 211 ° C and 248 ° C, respectively.

Studies using ¹³ C NMR spectroscopy were carried out on an Avance Bruker DPX-300 spectrometer at 313 K with an operating frequency of 75 MHz on carbon atoms in the Inverse Gate mode (1000 scans), in which complete broadband suppression of proton signals occurs and the nuclear Overhauser effect is absent. To take into account the effect of relaxation effects, the delay between pulses was set according to the 5T ₁ rule in 15 seconds.

The interpretation and assignment of signals in the ¹³ C NMR spectra of the inclusion complex of HPβCD with DSF was carried out using the spectral database SDBS [Spectral database for organic compounds, SDBS - http: //sdbs.db.aist.go.ip/] and are presented in the table one.

Due to the isolation of the signals, the authors use the signals of the carbon nuclei C1 of the methine group of the α-D-glucopyranose residue of GPβCD and the methyl groups of DSF (C10 *, C19 * or C12 *, C17 *) to calculate the molar ratio of HPβCD to DSF. The symbol of carbon atoms in the molecules of HPβCD and DSF is shown in Figure 5. In order to be able to determine the molar ratio of GPβCD to DSF in the inclusion complex, the following factors (1) and (2) must be taken into account.

(1) Since the solubility in water (D ₂ O) of pure DSF is extremely low (4.09 mg / L, at 25 ° C), and the calculated content of DSF in the sample for spectrum registration is about 2 g / L (the solution is clear), we can conclude that we are talking about the inclusion complex, and the content of DSF not included in the complex can be neglected.

(2) The integrated intensities of the signals of the carbon nuclei in the ¹³ C NMR spectrum recorded in the Inverse Gate mode are proportional to their molar ratios.

Based on the foregoing, the number of GPβCD molecules per one DSF molecule can be calculated by the formula:

,

,

where I _C1 is the integrated signal intensity of the carbon nuclei C1 of the methane group of the α-D-glucopyranose residue of GPβCD;

I _{C10 *, C19 *} is the integrated signal intensity of the carbon nuclei C10 *, C19 * of the methyl groups of DSF.

The values of the integrated intensities of the signals of the carbon nuclei C1 and C10 *, C19 * of the three samples of the inclusion complex of HPβCD with DSF are presented in table 2.

Based on the data presented in Table 2, the stoichiometric ratio of DSF to HPβCD in the test samples is 1: 2 (with a slight excess of free HPβCD), which suggests the structure of the inclusion complex, which is shown in FIG. 5.

GPβCD is a mixture of hydroxypropyl derivatives of CD, the average degree of substitution of which in total is from 0.1 to 0.9, preferably it is approximately 0.6. From which the authors suggest that HPβCD from this mixture with a high degree of substitution does not form an inclusion complex due to steric difficulties and exists in a free form.

Not limited to a specific theory, the authors conclusively suggest that a deviation from the stoichiometric ratio of HPβCD to DSF upward may be due to the presence of fractions with a high degree of substitution of hydroxypropyl groups in the feedstock.

Assuming that the relative determination error for this method does not exceed 15% [Koichi Hatada, Tatsuki Kitayama. NMR Spectroscopy of Polymers. Springer Science & Business Media. 222 pages. P. 57], the authors convincingly argue that as a result of the implementation of the method in accordance with the invention, a water-soluble inclusion complex of DSF with GPβCD of stoichiometric composition 1: 2 was obtained.

Information confirming the possibility of carrying out the invention

Further, the invention will be illustrated by the following examples, confirming the possibility of its implementation with the achievement of the technical result indicated in the description.

Example 1. Obtaining a complex inclusion of disulfiram with cyclodextrin homogeneous method (option 1)

With stirring at a speed of 450 rpm and a temperature of 65 ° C to 1 wt.h. 30 mass. % aqueous solution of ββCD add 1 wt.h. 3 mass. % alcohol solution of DSF. When an alcohol solution of DSF is added, turbidity is observed, which disappears with time. The total mixing time is 12 hours. After cooling to room temperature, the resulting mixture was filtered through a 0.22 μm membrane filter and freeze-dried.

Example 2. Obtaining a complex inclusion of disulfiram with cyclodextrin homogeneous method (option 2)

With stirring at a speed of 450 rpm and a temperature of 65 ° C in 30 mass. % aqueous solution of GPβCD add a DSF melt (mp. 71 ° C) in an amount sufficient to obtain a final concentration of DSF in a solution of 3 wt. % The total stirring time is 12 hours, after which the mixture is cooled to room temperature, the resulting mixture is filtered through a 0.22 μm membrane filter and freeze-dried.

Example 3. Obtaining a complex inclusion of disulfiram with cyclodextrin heterogeneous method (option 1)

With stirring at a speed of 450 rpm and a temperature from room temperature to 65 ° C to 1 mass.h. 30 mass. % of an aqueous solution of GPβCD, a portion of dry DSF is added portionwise, taken so that its final concentration in the mixture is 3 wt. % The total mixing time is 24 hours and complete dissolution of the DSF is achieved. The resulting solution is cooled if it was previously heated, filtered through a 0.22 μm membrane filter and freeze-dried.

Example 4. Obtaining a complex inclusion of disulfiram with cyclodextrin heterogeneous method (option 2)

To 30 mass. % alcohol solution of HPβCD with constant stirring portionwise add a portion of dry DSF, taken so that its final concentration in the mixture was 3 wt. % The resulting suspension is stirred at a speed of 450 rpm at a temperature of 65 ° C for 30 minutes until the DSF is completely dissolved. The mixture is cooled to room temperature over 30 minutes, as a result, an insignificant amount of DSF does not form part of the complex from the alcohol solution of the complex. The resulting mixture was filtered through a 0.22 μm membrane filter and the organic solvent was evaporated on a rotary evaporator.

Example 5. Obtaining a complex inclusion of disulfiram with cyclodextrin heterogeneous method (option 3, preferred)

A portion of dry GPβCD, taken so that its final concentration was 30 mass. %, and a sample of dry DSF, taken so that its final concentration was 3 mass. %, transferred to the reactor and add alcohol. The resulting mixture was heated and stirred at a speed of 450 rpm at a temperature of 65 ° C for 30 minutes until the DSF was completely dissolved. The mixture is cooled to room temperature, as a result of which the DSF, which is not included in the complex, drops out of the alcohol solution. The resulting mixture was filtered through a 0.22 μm membrane filter and the organic solvent was evaporated on a rotary evaporator.

Example 6. Obtaining a inclusion complex of disulfiram with cyclodextrin by any of the methods listed in examples 1-5, in which unsubstituted βCD is used as cyclodextrin.

Example 7. Obtaining a inclusion complex of disulfiram with cyclodextrin by any of the methods listed in examples 1-5, in which methyl-β-cyclodextrin (M-β-CD) is used as cyclodextrin.

Example 8. Obtaining a inclusion complex of disulfiram with cyclodextrin by any of the methods listed in examples 1-5, in which dimethyl-β-cyclodextrin (DM-β-CD) is used as cyclodextrin.

The inclusion complex, obtained as described in any of examples 1-8, in accordance with the methodology of GF XII (OFS 42-0049-07), is assessed as "readily soluble in water."

The technical result is a water-soluble inclusion complex of disulfiram with cyclodextrin - the active substance of eye drops, which can be isolated with a degree of purity that meets the requirements of the State Pharmacopoeia of the Russian Federation

Claims (2)

1. A water-soluble inclusion complex of disulfiram (DSF) with cyclodextrin (CD) selected from the group consisting of unsubstituted cyclodextrin (NCD) or substituted cyclodextrin, such as methylcyclodextrin (MCD), dimethylcyclodextrin (DMCD), trimethylcyclodextrin (TCDPCD) and ), characterized in that it is obtained by a method in which CD, taken in solid or dissolved in water or alcohol form, with stirring and a temperature of from 20 to 70 ° C is combined with DSF in the form of a solid, melt or alcohol p solution, where the alcohol is selected from methanol, ethanol, n-propanol or iso-propanol, the molar ratio of CD and DSF is from 2.01: 1 to 2.40: 1, the resulting mixture is cooled to 0-25 ° C, subjected to sterilizing filtration and lyophilize or evaporate the solvent. 2. The water-soluble complex according to claim 1, characterized in that the cyclodextrin is hydroxypropyl-β-cyclodextrin (GPβCD).

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