A clofazimine ethanol crystal compound and its preparation method and application, and pharmaceutical composition

Abstract

The present invention provides a clofazimine ethanol crystalline compound, its preparation method, application, and pharmaceutical composition, relating to the field of biomedicine. The clofazimine ethanol crystalline compound provided by the present invention has a small size and a relatively large surface area. When used as a pharmaceutical, it has a high dissolution rate and a high in vivo absorption rate, enabling faster achievement of higher blood drug concentrations and providing enhanced therapeutic effects against diseases such as leprosy and drug-resistant tuberculosis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a clofazimine ethanol crystal compound, a preparation method and application thereof, and a pharmaceutical composition. Background Art

[0002] Clofazimine (Cfz), also known as clofazimine, clofazimine, etc., has the chemical name 10-(p-chlorophenyl)-2,10-dihydro-3-(p-chlorophenylamino)-2-isopropyliminophenazine, and its structural formula is as follows:

[0003]

[0004] Clofazimine, also known as clofazimine and clofazimine, was developed by Novartis and launched in the United States in 1986. It is a relatively effective anti-leprosy drug. It was registered and launched in China in 1987 by Shanxi Liye Pharmaceutical Co., Ltd. (formerly Liye Pharmaceutical Co., Ltd.). It is used in combination with dapsone as the first-line treatment for lepromatous leprosy and can also be used with other drugs to treat drug-resistant tuberculosis. There are reports on the preparation of different crystalline forms of clofazimine. For example, Bannigan P et al. (Bannigan P, Zeglinski J, Lusi M, et al. Investigation into the Solid and Solution Properties of Known and Novel Polymorphs of the Antimicrobial Molecule Clofazimine [J]. Crystal Growth & Design, 2016.) obtained clofazimine Forms I, II, and III by recrystallization using acetone and N'N-dimethylformamide-acetone. Zhong Congshan (Zhong Congshan. Design, Synthesis, Optimization and Biological Activity of Clofazimine and Its Analogs [D]. North University of China, 2021) used ether and toluene for recrystallization to obtain clofazimine Forms I, II, and III. Chinese patent CN 108892647A used methanol, ethanol, and isopropanol to obtain clofazimine Forms A and B. However, the dissolution rate of these clofazimine crystalline compounds was not high enough, affecting their efficacy. Summary of the Invention

[0005] In view of this, the present invention aims to provide a clofazimine ethanol crystalline compound, its preparation method, application, and pharmaceutical composition. The clofazimine ethanol crystalline compound provided by the present invention has a high dissolution rate, can achieve higher blood concentrations more quickly, and thus has a stronger therapeutic effect.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a clofazimine ethanol crystalline compound, wherein X-ray diffraction angles are 7.7142±0.2°, 10.1937±0.2°, 13.3520±0.2°, 13.9383±0.2°, 15.4395±0.2°, 16.7351±0.2°, 18.0314±0.2°, 19.2897±0.2°, 20.1434±0.2°, 21.5863±0.2°, 22.1685±0.2°, 28.0734±0.2°, 30.8841±0.2° and 31.8484±0.2°.

[0008] The present invention also provides a method for preparing the clofazimine ethanol crystal compound described in the above technical solution, comprising the following steps:

[0009] Heating, keeping warm, cooling and crystallizing clofazimine and anhydrous ethanol in a sealed, protective atmosphere with stirring to obtain the clofazimine ethanol crystal compound;

[0010] The pressure of the system before heating is 0.08-0.12 MPa, and the pressure of the system during the heat preservation and cooling crystallization process is 0.18-0.32 MPa;

[0011] The stirring speed during the heating and heat preservation process is 670-730 r / min, and the stirring speed during the cooling and crystallization process is 70-130 r / min.

[0012] Preferably, the ratio of the mass of clofazimine to the volume of anhydrous ethanol is 1 g: 3 to 300 mL.

[0013] Preferably, the insulation temperature is 75-85° C. and the insulation time is 20-40 minutes.

[0014] Preferably, the cooling crystallization includes sequentially performing cooling and crystallization; the cooling includes naturally dropping to room temperature; the insulation temperature of the crystallization is -5 to 5°C, and the time is 6 to 10 hours.

[0015] Preferably, after the cooling crystallization, the method further comprises: performing solid-liquid separation on the crystallization system obtained by the cooling crystallization, and drying the obtained solid component.

[0016] The present invention also provides the use of the clofazimine ethanol crystal compound described in the above technical solution or the clofazimine ethanol crystal compound prepared by the preparation method described in the above technical solution in the preparation of medicines.

[0017] Preferably, the medicine includes medicines for treating leprosy and drug-resistant tuberculosis.

[0018] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the clofazimine ethanol crystalline compound described in the above technical solution or the clofazimine ethanol crystalline compound prepared by the preparation method described in the above technical solution.

[0019] Preferably, the active ingredient further comprises one or more of dapsone, rifampicin and ethionamide.

[0020] The clofazimine ethanol crystalline compound provided by the present invention has a small size and a relatively large surface area. When used as a drug, it has a high dissolution rate and a high absorption rate in the body. It can reach a higher blood drug concentration more quickly and has a stronger therapeutic effect on diseases such as leprosy and drug-resistant tuberculosis.

[0021] The present invention also provides a method for preparing the clofazimine ethanol crystalline compound described in the above technical solution. The present invention uses ethanol as a solvent for recrystallization and controls the pressure and speed. The obtained clofazimine ethanol crystalline compound has high purity and complies with the USP-NF2024 and EP11.0 test standards. The clofazimine ethanol crystalline compound has a small size and a relatively large surface area. When used as a drug, its dissolution rate is high and its absorption rate in the body is high, so it can reach a higher blood drug concentration faster, thereby having a stronger therapeutic effect. Moreover, the present invention is prepared under a protective atmosphere, which can prevent the clofazimine ethanol crystalline compound from being oxidized. The preparation method provided by the present invention has a simple process, simple operation, low production cost, is green and environmentally friendly, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the XRD pattern of the clofazimine ethanol crystalline compound prepared in Example 1;

[0023] Figure 2 This is the XRD pattern of the dichloromethane-methanol crystalline compound of clofazimine prepared in Comparative Example 1;

[0024] Figure 3 This is the XRD pattern of the clofazimine ether crystalline compound prepared in Comparative Example 2;

[0025] Figure 4 This is the XRD pattern of the ethyl acetate crystalline compound of clofazimine prepared in Comparative Example 3;

[0026] Figure 5 This is the XRD pattern of the clofazimine toluene crystalline compound prepared in Comparative Example 4;

[0027] Figure 6 This is the XRD pattern of the chloroform crystalline compound of clofazimine prepared in Comparative Example 5;

[0028] Figure 7This is a SEM image of the clofazimine ethanol crystal compound prepared in Example 1;

[0029] Figure 8 This is an SEM image of the dichloromethane-methanol crystalline compound of clofazimine prepared in Comparative Example 1;

[0030] Figure 9 This is an SEM image of the clofazimine ether crystalline compound prepared in Comparative Example 2;

[0031] Figure 10 This is an SEM image of the ethyl acetate crystalline compound of clofazimine prepared in Comparative Example 3;

[0032] Figure 11 This is an SEM image of the clofazimine toluene crystalline compound prepared in Comparative Example 4;

[0033] Figure 12 This is an SEM image of the chloroform crystalline compound of clofazimine prepared in Comparative Example 5;

[0034] Figure 13 is the linear curve graph of clofazimine in Test Example 3;

[0035] Figure 14 This is a blood concentration-time curve of the clofazimine ethanol crystal compound prepared in Example 1;

[0036] Figure 15 This is a blood concentration-time curve of the dichloromethane-methanol crystalline compound of clofazimine prepared in Comparative Example 1;

[0037] Figure 16 This is a blood concentration-time curve of the clofazimine ether crystalline compound prepared in Comparative Example 2;

[0038] Figure 17 This is a blood concentration-time curve of the clofazimine ethyl acetate crystalline compound prepared in Comparative Example 3;

[0039] Figure 18 This is a blood concentration-time curve of the clofazimine toluene crystalline compound prepared in Comparative Example 4;

[0040] Figure 19 This is a blood concentration-time curve of the chloroform crystalline compound of clofazimine prepared in Comparative Example 5. DETAILED DESCRIPTION

[0041] The present invention provides a clofazimine ethanol crystalline compound, wherein X-ray diffraction angles (2θ) are 7.7142±0.2°, 10.1937±0.2°, 13.3520±0.2°, 13.9383±0.2°, 15.4395±0.2°, 16.7351±0.2°, 18.0314±0.2°, 19.2897±0.2°, 20.1434±0.2°, 21.5863±0.2°, 22.1685±0.2°, 28.0734±0.2°, 30.8841±0.2° and 31.8484±0.2°.

[0042] The present invention also provides a method for preparing the clofazimine ethanol crystalline compound described in the above technical solution, comprising the following steps: heating, keeping warm, and cooling clofazimine and anhydrous ethanol in a sealed, protective atmosphere and under stirring conditions, to obtain the clofazimine ethanol crystalline compound.

[0043] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0044] In the present invention, the ratio of the mass of clofazimine to the volume of anhydrous ethanol is preferably 1g:3-300mL. In specific embodiments, it can be 1g:3mL, 1g:5mL, 1g:10mL, 1g:20mL, 1g:30mL, 1g:40mL, 1g:50mL, 1g:80mL, 1g:100mL, 1g:120mL, 1g:150mL, 1g:180mL, 1g:200mL, 1g:220mL, 1g:250mL, 1g:280mL or 1g:300mL.

[0045] In the present invention, the protective atmosphere preferably includes nitrogen, argon or helium. The present invention is prepared under a protective atmosphere to prevent the clofazimine ethanol crystal compound from being oxidized.

[0046] In the present invention, the pressure of the system before heating is preferably 0.08 to 0.12 MPa, and in specific embodiments may be 0.08 MPa, 0.09 MPa, 0.10 MPa, 0.11 MPa or 0.12 MPa.

[0047] In the present invention, the initial temperature of the heating is preferably room temperature (15 to 30° C.). The present invention has no particular limitation on the heating rate of the heating, and a heating rate well known to those skilled in the art may be used. In the present invention, the stirring speed during the heating process is 670 to 730 r / min (initial speed), and in specific embodiments, it may be 670 r / min, 680 r / min, 690 r / min, 700 r / min, 710 r / min, 720 r / min or 730 r / min. In the present invention, the pressure of the system after the heating is completed is 0.18 to 0.32 MPa, and in specific embodiments, it may be 0.18 MPa, 0.20 MPa, 0.22 MPa, 0.25 MPa, 0.28 MPa, 0.30 MPa or 0.32 MPa.

[0048] In the present invention, the holding temperature is preferably 75-85°C, and in specific embodiments, it can be 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, or 85°C; the holding time is preferably 20-40 minutes, and in specific embodiments, it can be 20 minutes, 22 minutes, 25 minutes, 28 minutes, 30 minutes, 32 minutes, 35 minutes, 38 minutes, or 40 minutes; heating is stopped after the holding is completed. In the present invention, the stirring speed during the holding process is 670-730 r / min, and in specific embodiments, it can be 670 r / min, 680 r / min, 690 r / min, 700 r / min, 710 r / min, 720 r / min, or 730 r / min. In the present invention, the pressure of the system during the insulation process is 0.18-0.32 MPa, and in specific embodiments can be 0.18 MPa, 0.20 MPa, 0.22 MPa, 0.25 MPa, 0.28 MPa, 0.30 MPa or 0.32 MPa.

[0049] In the present invention, the cooling crystallization preferably includes cooling and crystallization in sequence; in the present invention, the cooling includes naturally dropping to room temperature. In the present invention, the holding temperature of the crystallization is preferably -5 to 5°C, and in specific embodiments, it can be -5°C, -4°C, -3°C, -2°C, -1°C, 0°C, 1°C, 2°C, 3°C, 4°C, or 5°C; the crystallization time is preferably 6 to 10 hours, and in specific embodiments, it can be 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours; the crystallization is preferably carried out in an ice-water bath or an ice-salt bath. In the present invention, the pressure of the system during the cooling crystallization process is 0.18 to 0.32 MPa, and in specific embodiments, it can be 0.18 MPa, 0.20 MPa, 0.22 MPa, 0.25 MPa, 0.28 MPa, 0.30 MPa, or 0.32 MPa. In the present invention, the stirring speed during the cooling crystallization process is 70 to 130 r / min (final speed), and in specific embodiments it can be 70 r / min, 80 r / min, 90 r / min, 100 r / min, 110 r / min, 120 r / min or 130 r / min.

[0050] In the present invention, the heating, heat preservation and cooling are preferably carried out in a high-pressure reactor.

[0051] After completing the cooling crystallization, the present invention preferably further comprises: subjecting the crystallization system obtained by the cooling crystallization to solid-liquid separation, and drying the obtained solid component. In the present invention, the solid-liquid separation preferably comprises suction filtration, filtration or centrifugal separation. In the present invention, the drying temperature is preferably 50 to 60°C, and in specific embodiments it can be 50°C, 55°C or 60°C; the present invention has no particular limitation on the drying time, and drying to constant weight is sufficient; the drying preferably comprises vacuum drying.

[0052] In the present invention, if the pressure is too low, the solubility of clofazimine is poor, crystallization cannot be achieved, and the product remains in an amorphous powder form; if the pressure is too high, the purity of the clofazimine ethanol crystal compound is low. If the initial rotation speed is too low, clofazimine dissolves very slowly; if the final rotation speed is too high, the crystal form is easily broken. The present invention uses ethanol as a solvent for recrystallization and controls the pressure and rotation speed. The obtained clofazimine ethanol crystal compound has high purity and meets the USP-NF2024 and EP11.0 test standards. The clofazimine ethanol crystal compound has a small size and a relatively large surface area. When used as a drug, its dissolution rate is high and its absorption rate in the body is high, so it can reach a higher blood drug concentration faster, thereby having a stronger therapeutic effect. Moreover, the preparation method provided by the present invention is simple in process, simple in operation, low in production cost, environmentally friendly, and suitable for industrial production.

[0053] The present invention also provides the use of the clofazimine ethanol crystal compound described in the above technical solution or the clofazimine ethanol crystal compound prepared by the preparation method described in the above technical solution in the preparation of medicines.

[0054] In the present invention, the medicine preferably includes medicines for treating leprosy and drug-resistant tuberculosis; the leprosy includes lepromatous leprosy and / or lepromatous leprosy.

[0055] In the present invention, the clofazimine ethanol crystal compound is preferably used in combination with other compounds, and the other compounds preferably include one or more of dapsone, rifampicin and ethionamide.

[0056] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the clofazimine ethanol crystalline compound described in the above technical solution or the clofazimine ethanol crystalline compound prepared by the preparation method described in the above technical solution.

[0057] In the present invention, the mass content of the clofazimine ethanol crystalline compound in the pharmaceutical composition is preferably 1 to 100%. In specific embodiments, it can be 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100%.

[0058] The present invention has no particular limitation on the dosage form of the pharmaceutically acceptable excipients, and any pharmaceutically acceptable excipients well known to those skilled in the art may be used.

[0059] The present invention has no particular limitation on the dosage form of the pharmaceutical composition, and any pharmaceutical dosage form well known to those skilled in the art may be used.

[0060] In the present invention, the active ingredient preferably further comprises one or more of dapsone, rifampicin and ethionamide.

[0061] The clofazimine ethanol crystalline compound provided by the present invention has a small size and a relatively large surface area. When used as a drug, it has a high dissolution rate and a high absorption rate in the body. It can reach a higher blood drug concentration more quickly and has a stronger therapeutic effect on diseases such as leprosy and drug-resistant tuberculosis.

[0062] To further illustrate the present invention, the clofazimine ethanol crystalline compound, its preparation method and application, and pharmaceutical composition provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0063] Example 1

[0064] 5 g of clofazimine (purity of 99.504%) and 30 mL of anhydrous ethanol were added to a high-pressure reactor. The container was sealed and filled with nitrogen to a pressure of 0.10±0.02 MPa (initial pressure). Magnetic stirring was turned on at a speed of 700 r / min (initial speed). The mixture was heated to 80° C. and a final pressure of 0.20±0.02 MPa. The mixture was stirred for 30 min, the heating was turned off, the speed was reduced to 100 r / min (final speed), and the temperature was naturally cooled to room temperature. The mixture was placed in a 0±5° C. ice-salt bath for crystallization for 6 h, filtered, and the resulting filter cake was vacuum dried at 50° C. for 4 h to obtain 4.2 g of clofazimine ethanol crystalline compound with a yield of 84% and an HPLC purity of 99.836%.

[0065] Comparative Example 1

[0066] 5 g of clofazimine (purity 99.504%) and 30 mL of dichloromethane:methanol volume ratio = 3:1 were added to an autoclave. The container was sealed and filled with nitrogen to a pressure of 0.10±0.02 MPa. Magnetic stirring was turned on at a speed of 700 r / min. The mixture was heated to 47°C and the final pressure was 0.20±0.02 MPa. The mixture was stirred for 30 min, the heating was turned off, the speed was reduced to 100 r / min, and the temperature was naturally cooled to room temperature. The mixture was placed in a 0±5°C ice-salt bath for crystallization for 6 h, filtered, and the filter cake was vacuum dried at 50°C for 4 h to obtain 4.6 g of clofazimine dichloromethane-methanol crystalline compound with a yield of 92% and an HPLC purity of 99.748%.

[0067] Comparative Example 2

[0068] 5 g of clofazimine (purity 99.504%) and 30 mL of ether were added to an autoclave. The container was sealed and filled with nitrogen to a pressure of 0.10±0.02 MPa. Magnetic stirring was turned on at a speed of 700 r / min and heated to 36°C. The final pressure was 0.20±0.02 MPa and stirred for 30 min. The heating was turned off and the speed was reduced to 100 r / min. The temperature was naturally cooled to room temperature and placed in a 0±5°C ice-salt bath for crystallization for 6 h. The filter cake was vacuum dried at 50°C for 4 h to obtain 4.8 g of clofazimine ether crystal compound with a yield of 96% and an HPLC purity of 99.534%.

[0069] Comparative Example 3

[0070] 5 g of clofazimine (purity 99.504%) and 30 mL of ethyl acetate were added to a high-pressure reactor. The container was sealed and filled with nitrogen to a pressure of 0.10±0.02 MPa. Magnetic stirring was turned on at a speed of 700 r / min and heated to 80°C. The final pressure was 0.20±0.02 MPa and stirred for 30 min. The heating was turned off and the speed was reduced to 100 r / min. The temperature was naturally cooled to room temperature and placed in a 0±5°C ice-salt bath for crystallization for 6 h. The filter cake was vacuum dried at 50°C for 4 h to obtain 3.9 g of clofazimine ethyl acetate crystalline compound with a yield of 78% and an HPLC purity of 99.738%.

[0071] Comparative Example 4

[0072] 5 g of clofazimine (purity 99.504%) and 30 mL of toluene were added to a high-pressure reactor. The container was sealed and filled with nitrogen to a pressure of 0.10±0.02 MPa. Magnetic stirring was turned on at a speed of 700 r / min. The mixture was heated to 110° C. and the final pressure was 0.20±0.02 MPa. The mixture was stirred for 30 min, the heating was turned off, the speed was reduced to 100 r / min, and the temperature was naturally cooled to room temperature. The mixture was placed in a 0±5° C. ice-salt bath for crystallization for 6 h, filtered, and the filter cake was vacuum dried at 50° C. for 4 h to obtain 3.3 g of clofazimine toluene crystalline compound with a yield of 66% and an HPLC purity of 99.703%.

[0073] Comparative Example 5

[0074] 5 g of clofazimine (purity 99.504%) and 30 mL of chloroform were added to a high-pressure reactor. The container was sealed and filled with nitrogen to a pressure of 0.10±0.02 MPa. Magnetic stirring was turned on at a speed of 700 r / min and heated to 61°C. The final pressure was 0.20±0.02 MPa and stirred for 30 min. The heating was turned off and the speed was reduced to 100 r / min. The temperature was naturally cooled to room temperature and placed in a 0±5°C ice-salt bath for crystallization for 6 h. The filter cake was vacuum dried at 50°C for 4 h to obtain 3.3 g of chloroform clofazimine compound with a yield of 66% and an HPLC purity of 99.578%.

[0075] Comparative Example 6

[0076] Clofazimine ethanol crystalline compound was prepared according to the method of Example 1, with the only difference from Example 1 being that the initial pressure was 0.03±0.02 MPa and the final pressure was 0.11±0.02 MPa. If the pressure is too low, the solubility of clofazimine is poor, and crystallization fails, resulting in an amorphous powder.

[0077] Comparative Example 7

[0078] A crystalline clofazimine ethanol compound was prepared according to the method of Example 1, differing only in that the initial pressure was 0.15±0.02 MPa and the final pressure was 0.27±0.02 MPa. 4.0 g of the crystalline clofazimine ethanol compound was obtained with an 80% yield and an HPLC purity of 99.534%. The content of an unknown impurity exceeded 0.1%, indicating that the purity of the crystalline ethanol compound was low.

[0079] Comparative Example 8

[0080] The clofazimine ethanol crystal compound was prepared according to the method of Example 1, with the only difference from Example 1 being that the initial rotation speed was 400 r / min. If the initial rotation speed was too low, clofazimine would dissolve very slowly.

[0081] Comparative Example 9

[0082] A clofazimine ethanol crystalline compound was prepared according to the method of Example 1, with the only difference from Example 1 being that the final rotation speed was 300 r / min. If the final rotation speed was too high, the clofazimine crystalline compound would be broken.

[0083] Test Example 1

[0084] (1) X-ray diffraction test of the chlorfazimine crystalline compounds prepared in Example 1 and Comparative Examples 1 to 5, the radiation source was Cu-Kα, X-ray powder diffractometer.

[0085] Figure 1 This is the XRD pattern of the clofazimine ethanol crystalline compound. It can be seen that the clofazimine ethanol crystalline compound has diffraction peaks at diffraction angles of 2θ = 7.7142°, 10.1937°, 13.3520°, 13.9383°, 15.4395°, 16.7351°, 18.0314°, 19.2897°, 20.1434°, 21.5863°, 22.1685°, 28.0734°, 30.8841° and 31.8484°.

[0086] Figure 2 This is the XRD pattern of the dichloromethane-methanol crystalline compound of clofazimine. It can be seen that the dichloromethane-methanol crystalline compound of clofazimine has diffraction peaks at diffraction angles 2θ = 7.7387°, 9.2239°, 13.5178°, 15.9876°, 20.0245°, 21.8626°, 22.4351°, 23.3442° and 26.1438°.

[0087] Figure 3This is the XRD pattern of the clofazimine ether crystalline compound. It can be seen that the clofazimine ether crystalline compound has diffraction peaks at diffraction angles 2θ = 6.8811°, 7.6814°, 7.8580°, 8.5616°, 10.2542°, 13.3213°, 13.9092°, 14.3152°, 18.0148°, 19.1990°, 20.1021°, 20.7648°, 21.5456°, 22.1404°, 23.8112° and 28.8689°.

[0088] Figure 4 : This is the XRD pattern of the clofazimine ethyl acetate crystalline compound. It can be seen that the clofazimine ethyl acetate crystalline compound has diffraction peaks at diffraction angles 2θ = 7.7654°, 9.2876°, 13.5445°, 19.8801°, 21.0381°, 21.8945°, 22.6949° and 26.2223.

[0089] Figure 5 This is the XRD pattern of the clofazimine toluene crystalline compound. It can be seen that the clofazimine toluene crystalline compound has diffraction peaks at diffraction angles of 2θ = 7.7326°, 10.2169°, 13.3714°, 13.9568°, 15.4491°, 16.7589°, 18.0481°, 19.3243°, 20.1517°, 20.4603°, 20.9648°, 21.5945°, 25.2140°, 28.0695° and 31.8454°.

[0090] Figure 6 This is the XRD pattern of clofazimine chloroform crystal compound. It can be seen that clofazimine chloroform crystal compound has diffraction peaks at diffraction angles 2θ = 9.3300°, 11.5007°, 12.3134°, 13.5875°, 16.1623°, 19.9500°, 21.9321°, 23.5763°, 24.7406°, 26.5062°, 28.1616° and 29.3576°.

[0091] (2) Scanning electron microscopy test of the clofazimine crystal compounds prepared in Example 1 and Comparative Examples 1 to 5

[0092] Figure 7 This is an SEM image of the clofazimine ethanol crystal compound, which shows that it has a small columnar morphology.

[0093] Figure 8 This is an SEM image of the dichloromethane-methanol crystalline compound of clofazimine, which shows that it is in a flaky form.

[0094] Figure 9This is an SEM image of the clofazimine ether crystal compound, which shows that it has a plate-like morphology.

[0095] Figure 10 This is the SEM image of the ethyl acetate crystal compound of clofazimine, which shows that it is an irregular block-shaped compound.

[0096] Figure 11 This is the SEM image of the clofazimine toluene crystalline compound, which shows that it has a prismatic morphology.

[0097] Figure 12 This is the SEM image of the chloroform crystal compound of clofazimine, which shows that it is in a loose block shape.

[0098] Test Example 2

[0099] Solubility test of clofazimine crystalline compounds prepared in Example 1 and Comparative Examples 1 to 5

[0100] 10 mg of each clofazimine crystalline compound was placed in a centrifuge tube, 5 mL of water was added, and the mixture was shaken at 37°C for 24 h. The supernatant was taken and filtered through a 0.45 μm filter membrane. The filtrate was analyzed. The results are shown in Table 1.

[0101] 10 mg of each clofazimine crystalline compound was placed in a centrifuge tube, 5 mL of artificial gastric fluid was added, and the mixture was shaken at 37°C for 24 hours. The supernatant was collected and filtered through a 0.45 μm filter membrane. The resulting filtrate was analyzed by high performance liquid chromatography (HPLC) (method according to USP-NF2024). The standard curve equation is y = 66009657.349638x, R 2 =0.9999, x is in mg / mL, and the results are shown in Table 1. The preparation process of artificial gastric juice is as follows: 16.4 mL of 12 mol / L dilute hydrochloric acid is added to 800 mL of water and 10 g of pepsin, shaken well, and diluted with water to 1000 mL to obtain artificial gastric juice.

[0102] Table 1 Solubility of six crystalline compounds of clofazimine in artificial gastric juice and pure water (37°C)

[0103] Crystal form Water (μg / mL) Artificial gastric fluid (μg / mL) Dichloromethane-methanol crystal form 0.07748866 2.608269864 Ether crystal form 0.354948063 4.716370490 Ethyl acetate crystal form 0.09148661 2.351065075 Toluene crystal form 0 0.220679831 Chloroform crystal form 0.01925476 0.990127848 Ethanol crystal form 0.04396326 5.549657652

[0104] As shown in Table 1, the solubility of various crystal forms in water is relatively low, and the ethanol crystal form has the highest solubility in artificial gastric juice.

[0105] Test Example 3

[0106] Pharmacokinetic test of clofazimine crystalline compounds prepared in Example 1 and Comparative Examples 1 to 5

[0107] 1 Research Methods

[0108] 1.1 Preparation of clofazimine suspension

[0109] 30 mg of clofazimine of different crystal forms were accurately weighed and added into 20 mL of 0.5% sodium carboxymethyl cellulose aqueous solution to prepare a 1.5 mg / mL clofazimine suspension, which was stored at 4°C in the dark.

[0110] 1.2 Sampling

[0111] Forty-eight Sprague-Dawley rats (Sprague-Dawley) were adapted to feeding for 5 days with normal light, water, and diet. The 48 rats were divided into six groups and given six crystalline forms of clofazimine suspensions, each administered orally at a single dose of 15 mg / kg based on rat body weight. Blood was collected from the eye sockets using a 1.5 mm capillary glass tube at 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, and 216 h after administration. The blood was placed in a heparinized 1.5 mL centrifuge tube and centrifuged at 5000 rpm for 10 min at 4°C. The upper layer of plasma was collected as drug-containing plasma. Blank plasma was obtained by the same method as above, but without the addition of clofazimine suspension. Both drug-containing and blank plasma were stored at -20°C until use.

[0112] 1.3 Plasma sample pretreatment

[0113] Chlorpropamide (internal standard) was accurately weighed and dissolved in acetonitrile-water (volume ratio, 4:1) to prepare a precipitant containing 120 ng / mL chlorpropamide.

[0114] Under dark conditions, accurately aspirate 50 μL of drug-containing plasma and add 250 μL of a precipitant containing 120 ng / mL chlorsulfuronamide. Vortex for 30 seconds, sonicate for 15 minutes, and centrifuge at 15,000 g for 15 minutes at 4°C. The supernatant is the pretreated drug-containing plasma. Replace the drug-containing plasma with blank plasma as described above to obtain pretreated blank plasma.

[0115] 1.4 Methodological Investigation

[0116] (1) Linearity, detection limit, and quantification limit

[0117] 1.4.1 Preparation of standard solution

[0118] Preparation of clofazimine reference solutions: Accurately weigh clofazimine standard reference substance (purchased from the Hong Kong Institute of Standard Substances) and dissolve it in acetonitrile to prepare a 1 mg / mL clofazimine stock solution. This clofazimine stock solution was diluted with acetonitrile to obtain clofazimine standard solutions at the following concentrations: 10,000 ng / mL, 8,000 ng / mL, 2,000 ng / mL, 1,000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 625 ng / mL, 313 ng / mL, and 156 ng / mL. Store these solutions at 4°C in the dark.

[0119] Preparation of standard solution: Under light-proof conditions, take 180 μL of blank plasma, accurately add 20 μL of clofazimine series reference solution of different concentrations, accurately add 1 mL of precipitant containing 120 ng / mL chlorsulfuronamide, vortex mix for 30 seconds, sonicate for 15 minutes, and then centrifuge at 15000g for 15 minutes at 4°C to obtain standard solution.

[0120] 1.4.2 Drawing of the standard curve

[0121] The standard solution was analyzed by HPLC-MS, and the chromatographic peak area of clofazimine and the chromatographic peak area of the internal standard were recorded. A linear regression (least squares method) was performed on the corresponding concentration using the clofazimine to internal standard peak area ratio to draw a standard curve.

[0122] Conditions for HPLC-MS analysis:

[0123] Quantitative data were collected using ultra-performance liquid chromatography-triple quadrupole mass spectrometry, where the chromatographic model was U3000 and the triple quadrupole mass spectrometry model was AB SCIEX 4000QTRAP.

[0124] The mass spectrometer was used for data acquisition in the positive ion mode with the following parameters: auxiliary gas, 55 psi; nebulizer gas, 55 psi; curtain gas, 15 psi; ion source temperature, 550°C; ion spray voltage, 5500 V; the mass spectrometer acquisition mode was multiple reaction monitoring; and the ion pair monitoring information was clofazimine: 473.1 / 431.1, and the internal standard chlorpropamide: 277.0 / 175.0.

[0125] Ultra-high-performance liquid chromatography (UPLC) conditions: The chromatographic column was a BEH C18 column (50 mm × 2.1 mm, 1.7 μm), with a BEHshield RP18 column (1.7 μm). The sample compartment temperature was 4°C, and the column temperature was room temperature. Mobile phase A consisted of 0.1 v / v% formic acid in water, and mobile phase B consisted of acetonitrile. The mobile phase flow rate was 0.4 mL / min. The elution method was gradient elution; the gradient elution program is shown in Table 2.

[0126] Table 2 Gradient elution program

[0127] Time (min) Volume fraction of mobile phase A (%) Volume fraction of mobile phase B (%) 0 70 30 0.5 20 80 1 5 95 3 5 95 3.1 70 30 5 70 30

[0128] The standard curve was obtained by plotting the peak area ratio of clofazimine to internal standard as the ordinate and the concentration of the standard as the abscissa. Figure 13 As shown. Clofazimine has good linearity in the concentration range of 1.56 to 1000 ng / mL. The regression equation is y = 0.0106x + 0.0057, R 2 =0.9993.

[0129] 1.4.3 Limit of Detection and Limit of Quantitation

[0130] The clofazimine standard solution was serially diluted, with an S / N ratio of 10 as the limit of quantification and an S / N ratio of 3 as the limit of detection. The limit of quantification and the limit of detection were calculated based on the signal-to-noise ratio. The limit of detection was 0.54 ng / mL and the limit of quantification was 1.56 ng / mL. The limit of quantification and the limit of detection were 1.56 ng / mL and 0.54 ng / mL, respectively.

[0131] (2) Precision inspection

[0132] Pretreated blank plasma was taken and prepared using the standard solution preparation method in 1.4.1 to obtain precision samples with clofazimine concentrations of 400 ng / mL, 50 ng / mL, and 1.5625 ng / mL, respectively. Each precision sample was subjected to HPLC-MS detection. Each sample was measured six times on the same day to obtain intra-day precision, and the inter-day precision was obtained by measuring for three consecutive days. The results are shown in Table 3.

[0133] Table 3 Precision test results of clofazimine ethanol crystal compound

[0134]

[0135] (3) Stability investigation

[0136] Pretreated blank plasma was taken and samples were prepared using the standard solution preparation method in 1.4.1 to obtain stability samples with clofazimine concentrations of 400 ng / mL, 50 ng / mL, and 1.5625 ng / mL, respectively. Each stability sample was tested at 0 h, 1 h, 2 h, 4 h, 8 h, and 12 h to obtain intraday room temperature stability. Daytime room temperature stability was obtained for three consecutive days. The results are shown in Table 4.

[0137] Table 4 Stability test results of clofazimine ethanol crystal compound

[0138]

[0139] (4) Recovery rate

[0140] Pretreated blank plasma was taken and samples were prepared using the standard solution preparation method in 1.4.1 to obtain spiked samples with clofazimine concentrations of 400 ng / mL, 50 ng / mL, and 1.5625 ng / mL, respectively. Each spiked sample was subjected to HPLC-MS detection, and the chromatographic peak area was recorded as A. Three other portions of pretreated blank plasma were added to each of the clofazimine standard solutions, mixed, and analyzed by high-performance liquid chromatography. The chromatographic peak area of the sample was recorded as B. The extraction recovery was calculated based on the above peak area: extraction recovery = A / B × 100%. The results are shown in Table 5.

[0141] Table 5 Absolute recovery test results of clofazimine ethanol crystal compound

[0142] Concentration (ng / mL) Average recovery rate (%) RSD (%) 1.56 95.02 4.74 50 99.85 4.90 400 102.30 7.67

[0143] 1.5 Data processing and blood drug concentration-time curve (drug-time curve)

[0144] Using DAS software, the compartment model was selected as a two-compartment model based on the AIC value and F test, and the weight coefficient was selected as 1 / c / c based on the goodness of fit value. The drug-time curve was drawn and the pharmacokinetic parameters were calculated.

[0145] 2 Results and Conclusions

[0146] 2.1 Methodological Investigation

[0147] Depend on Figure 13 As shown in Tables 3 to 5, the method is considered qualified based on the inspection of linear relationship, precision, stability and absolute recovery.

[0148] 2.2 Plasma concentration-time curve

[0149] The blood concentration-time curves of the clofazimine crystal compounds of Example 1 and Comparative Examples 1 to 5 are shown in Table 1. Figures 14-19 , blood drug concentration-time curve, the blood drug concentration-time curve summary results are shown in Table 6.

[0150] Table 6 Summary of blood concentration-time curves of six crystalline compounds of clofazimine

[0151] parameter Dichloromethane-methanol crystal form Ether crystal form Ethyl acetate crystal form Toluene crystal form Chloroform crystal form Ethanol crystal form <![CDATA[T 1 / 2 (h)]]> 155.1 108.7 128.6 85.0 71.0 71.3 <![CDATA[T max (h)]]> 6.5 6.5 9.6 8.0 10.0 7.5 <![CDATA[C max (μg / L)]]> 377.5 560.125 394.143 492.125 637.875 691.5 <![CDATA[AUC 0-1 (h.ng / mL)]]> 28405.659 38424.708 37620.725 30217.591 31741.306 40662.88 Cl / F (L / h / kg) 0.282 0.237 0.254 0.303 0.391 0.302

[0152] Note: AUC 0-t Represents the total exposure of the drug in the body within a certain period of time, C max Represents the rate at which the drug is absorbed in the body.

[0153] Depend on Figures 14-19 As shown in Table 6, the pharmacokinetic results show that the AUC of clofazimine ethanol crystal compound is 0-t with C maxThe results were both higher than those of other crystalline forms. This may be primarily due to the smaller size and relatively larger surface area of the clofazimine ethanol crystalline form, which results in a higher dissolution rate. This suggests that clofazimine crystals obtained by recrystallization using ethanol as a solvent have better dissolution rates and absorption characteristics, enabling faster achievement of higher blood concentrations and thus greater efficacy.

[0154] In summary, the present invention uses six solvents, including dichloromethane-methanol volume ratio = 3:1, ether, ethyl acetate, toluene, chloroform and ethanol, to improve the product purity of clofazimine crystals by recrystallization, and screens the pharmacokinetic optimal crystal form, which is the clofazimine ethanol crystal form.

[0155] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A clofazimine ethanol crystal compound, characterized in that: The X-ray diffraction angles were 7.7142±0.2°, 10.1937±0.2°, 13.3520±0.2°, 13.9383±0.2°, 15.4395±0.2°, 16.7351±0.2°, 18.0314±0.2°, 19.2897±0.2°, 20.1434±0.2°, 21.5863±0.2°, 22.1685±0.2°, 28.0734±0.2°, 30.8841±0.2° and 31.8484±0.2°.

2. The method for preparing the clofazimine ethanol crystal compound according to claim 1, characterized in that: The following steps are involved: Heating, keeping warm, cooling and crystallizing clofazimine and anhydrous ethanol in a sealed, protective atmosphere with stirring to obtain the clofazimine ethanol crystal compound; The pressure of the system before heating is 0.08~0.12MPa, and the pressure of the system during the insulation and cooling crystallization process is 0.18~0.32MPa; The stirring speed during the heating and heat preservation process is 670-730 r / min, and the stirring speed during the cooling and crystallization process is 70-130 r / min.

3. The preparation method according to claim 2, characterized in that The ratio of the mass of clofazimine to the volume of anhydrous ethanol is 1 g: 3-300 mL.

4. The preparation method according to claim 2, characterized in that The temperature of the insulation is 75-85° C., and the time is 20-40 minutes.

5. The preparation method according to claim 2, characterized in that The cooling crystallization includes sequentially performing cooling and crystallization; the cooling includes naturally dropping to room temperature; the crystallization is carried out at a holding temperature of -5 to 5°C for 6 to 10 hours.

6. The preparation method according to claim 2 or 5, characterized in that After the cooling and crystallization, the method further comprises: performing solid-liquid separation on the crystallization system obtained by the cooling and crystallization, and drying the obtained solid component.

7. Use of the clofazimine ethanol crystalline compound according to claim 1 or the clofazimine ethanol crystalline compound prepared by the preparation method according to any one of claims 2 to 6 in the preparation of medicines; The medicine is a medicine for treating leprosy or drug-resistant tuberculosis.

8. A pharmaceutical composition, characterized in that The invention comprises an active component and a pharmaceutically acceptable excipient, wherein the active component is the clofazimine ethanol crystalline compound according to claim 1 or the clofazimine ethanol crystalline compound prepared by the preparation method according to any one of claims 2 to 6.

9. The pharmaceutical composition according to claim 8, characterized in that The active component is a composition of a clofazimine ethanol crystalline compound and one or more of dapsone, rifampicin and ethionamide, and the clofazimine ethanol crystalline compound is the clofazimine ethanol crystalline compound according to claim 1 or the clofazimine ethanol crystalline compound prepared by the preparation method according to any one of claims 2 to 6.

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