COCRYSTAL COMPRISING CAMOSTAT AND NICLOSAMIDE, PHARMACEUTICAL COMPOSITION COMPRISING SAME AND PREPARATION METHOD THEREFOR

Abstract

The present invention relates to a novel cocrystal, a pharmaceutical composition comprising same and a preparation method therefor. By using the cocrystal of the present invention, cancers, inflammatory diseases, or viral infection diseases may be effectively prevented and/or treated.

Claims

1. A cocrystal comprising camostat or a pharmaceutically acceptable salt thereof, and niclosamide.

2. The cocrystal of claim 1, wherein the cocrystal consists of camostat or a pharmaceutically acceptable salt thereof, and niclosamide.

3. The cocrystal of claim 1, wherein a molar ratio of the camostat or the pharmaceutically acceptable salt thereof and the niclosamide is 1:4 to 4:1.

4. The cocrystal of claim 1, wherein the molar ratio of the camostat or the pharmaceutically acceptable salt thereof and the niclosamide is 1:1.

5. The cocrystal of claim 1, wherein a powder X-ray diffraction (XRD) pattern includes diffraction peaks represented at diffraction angle 2θ (±0.2°) values of 5.37715°, 15.1122°, 18.2258°, 18.7579°, 20.3344°, 25.596° and 26.069°; and wherein when the heating rate is 10° C./min, a differential scanning calorimetry (DSC) endothermic peak is shown at 144.38±3° C.

6. (canceled)

7. The cocrystal of claim 1, wherein the powder X-ray diffraction (XRD) pattern includes diffraction peaks represented at diffraction angle 2θ (±0.2°) values of 6.55954°, 10.7176°, 18.147°, 19.5855°, 21.3591°, and 26.8178° and wherein when the heating rate is 10° C./min a differential scanning calorimetry (DSC) endothermic peak is shown at 126.35±3° C.

8. (canceled)

9. The cocrystal of claim 1, wherein the powder X-ray diffraction (XRD) pattern includes diffraction peaks represented at diffraction angle 2θ (±0.2°) values of 11.3876°, 16.0975°, 16.6493°, 18.679°, 23.0539°, 23.9013°, 24.4333°, and 29.7344°; and wherein when the heating rate is 10° C./min, a differential scanning calorimetry (DSC) endothermic peak is shown at 182.74±3° C.

10. (canceled)

11. The cocrystal of claim 1, wherein the powder X-ray diffraction (XRD) pattern includes diffraction peaks represented at diffraction angle 2θ (±0.2°) values of 6.81572°, 7.46604°, 9.87023°, 12.3532°, 13.24°, and 18.6396°; and wherein when the heating rate is 10° C./min a differential scanning calorimetry (DSC) endothermic peak is shown at 151.69±3° C.

12. (canceled)

13. The cocrystal of claim 1 further comprising a co-former.

14. The cocrystal of claim 13, wherein the cocrystal consists of camostat or a pharmaceutically acceptable salt thereof, niclosamide, and a co-former.

15. The cocrystal of claim 13, wherein a molar ratio of the camostat or the pharmaceutically acceptable salt thereof, the niclosamide, and the co-former is 1:1:1 to 1:1:6.

16. The cocrystal of claim 13, wherein the co-former is at least one selected from meglumine, histidine, arginine, nicotinamide, benzoate, formic acid, sorbic acid, citric acid, malic acid, caffeine, theophylline and urea.

17. The cocrystal of claim 13, wherein a powder X-ray diffraction (XRD) pattern includes diffraction peaks represented at diffraction angle 2θ (±0.2°) values of 7.0522°, 7.6239°, 9.06226°, 12.4912°, 18.009°, and 21.9897°; and wherein when the heating rate is 10° C./min, a differential scanning calorimetry (DSC) endothermic peak is shown at 126.03° C.

18-23. (canceled)

24. A pharmaceutical composition for the prevention or treatment of cancers, inflammatory diseases or viral infection diseases, comprising the cocrystal according to claim 1 as an active ingredient.

25. The pharmaceutical composition of claim 24, wherein the cancer is at least one selected from pancreatic cancer, breast cancer, liver cancer and lung cancer.

26. The pharmaceutical composition of claim 24, wherein the viral infection disease is at least one selected from coronavirus infectious disease, SARS virus infection, influenza virus infection and murder mite-borne infection.

27. The pharmaceutical composition of claim 24, wherein the inflammatory disease is at least one selected from allergy, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, ankylosing spondylitis, fibromyalgia, psoriatic arthritis, osteoarthritis, tendonitis, tenosynovitis, peritendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, acute inflammatory disease, and chronic inflammatory disease.

28. A method for the prevention or treatment of cancers, inflammatory diseases or viral infection diseases, comprising administering the cocrystal according to claim 1 to a subject.

29-30. (canceled)

Description

DESCRIPTION OF DRAWINGS

[0130] FIG. 1 is a powder X-ray diffraction (PXRD) result of niclosamide, camostat mesylate, camostat, and cocrystals α, β, γ, δ, and ε of the present invention.

[0131] FIG. 2 is a differential scanning calorimetry (DSC) analysis result of niclosamide, camostat mesylate, camostat, and cocrystals α, β, γ, δ, and ε of the present invention.

[0132] FIG. 3 is a result of evaluating the anticancer efficacy of cocrystals of Examples 1 and 17 and a positive control drug in a paclitaxel-resistant breast cancer cell line.

[0133] FIG. 4 is a result of evaluating the SARS-CoV-2 antiviral efficacy of cocrystals of Examples 1 and 17 and positive control drugs.

MODES OF THE INVENTION

[0134] Hereinafter, the best mode for implementing the present invention will be described in detail.

[0135] The present inventors of the present invention have developed a cocrystal comprising camostat or a pharmaceutically acceptable salt thereof, and niclosamide; and a cocrystal comprising camostat or a pharmaceutically acceptable salt thereof, niclosamide and a co-former, and analyzed the prepared cocrystals by the following various experimental methods to confirm the formation of cocrystals.

[0136] In the cocrystals of the present invention, as described above, a single cocrystal having a constant quality was obtained with good reproducibility. In addition, the cocrystals of the present invention may be stably supplied as crystals of raw medicines (pharmaceutical raw materials) used in the manufacture of medicine and has excellent storage stability. A difference in crystal type between a simple mixture and the cocrystal may be clear from the results of differential scanning calorimetry (DSC) analysis and powder X-ray diffraction (PXRD) analysis.

[0137] Niclosamide and camostat mesylate salts are specified by PXRD diffraction angles, relative intensities and DSC endothermic peaks listed in Tables 8 and 10 and FIG. 1. As the cocrystal of the present invention, a cocrystal α, a cocrystal β, a cocrystal γ, a cocrystal δ, and a cocrystal ε are specified by PXRD diffraction angles, relative intensities and DSC endothermic peaks listed in Tables 9 and 10 and FIG. 1.

[0138] In XRD diffraction, due to the nature of the data, the crystal lattice spacing or overall pattern is important in determining the identity of crystals, and the heat flow measurement results should not be interpreted too strictly because the results may change somewhat depending on a direction of crystal growth, a particle size, and measurement conditions.

[0139] The preparation method for the cocrystal according to the present invention includes mixing and cocrystallizing the camostat or the pharmaceutically acceptable salt thereof, and the niclosamide, and at this time, the cocrystallizing may be performed by various methods known as the preparation method for the cocrystal.

[0140] For example, the cocrystals α, β, γ, δ, and ε according to the present invention may be each independently prepared by crystallizing methods such as a liquid-assisted grinding method, a slurry method, and a solvent cooling method.

[0141] In the case of using the liquid-assisted grinding method, camostat or a pharmaceutically acceptable salt thereof and an alkalizing agent were ground and reacted with a solvent such as a small amount of distilled water using an instrument such as a pestle and a bowl and then continuously ground by adding a small amount of distilled water, an organic solvent such as acetone, acetonitrile, tetrahydrofuran or alcohols, and niclosamide to prepare the cocrystal according to the present invention.

[0142] In the case of using the slurry method, camostat or a pharmaceutically acceptable salt thereof and the alkalizing agent were added with distilled water, alcohol organic solvents such as acetone, acetonitrile, tetrahydrofuran or methanol, ethanol, and isopropyl alcohol, and niclosamide to make a supersaturated solution and then continuously stirred to prepare the cocrystal according to the present invention. In addition, the dry cocrystal was added in a specific organic solvent to make a slurry to prepare a cocrystal having a different crystal type.

[0143] In the case of using the solvent cooling method, camostat or a pharmaceutically acceptable salt thereof and the alkalizing agent were stirred and reacted in a first solvent such as distilled water and then added with a niclosamide solution heated and completely dissolved in a second solvent such as acetone or alcohols, and the mixture was stirred while cooling the solvent to synthesize the cocrystal according to the present invention. A volume ratio of the first solvent and the second solvent may be about 10:1 to 1:10. For example, the volume ratio of the first solvent and the second solvent may be about 4:1 to 5:1.

[0144] In an anti-solvent method, camostat mesylate and an alkalizing agent were stirred and reacted in a first solvent such as distilled water, and then filtered and dried to obtain dry powered camostat, and the dry camostat was mixed and stirred with a niclosamide solution heated and completely dissolved in a second solvent such as acetone or alcohols. In addition, the mixture was added with distilled water as an anti-solvent to prepare the cocrystal according to the present invention.

[0145] The alkalizing agent used in the liquid-assisted grinding method, the slurry method, the solvent cooling method or the anti-solvent method may be at least one selected from basic materials such as sodium bicarbonate (NaHCO.sub.3), sodium carbonate (Na.sub.2CO.sub.3), sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca(OH).sub.2) and magnesium hydroxide (Mg(OH).sub.2), but is not limited thereto.

[0146] Hereinafter, the present invention will be specifically described by Examples. That is, the cocrystal including the camostat or the pharmaceutically acceptable salt thereof and the niclosamide of the present invention may be prepared through methods to be described below, but any examples provided herein, or use of exemplary language, are intended merely to better illustrate the present invention and do not limit the scope of the present invention to be claimed.

EXAMPLES

[0147] Niclosamide from Hengcheng Pharmaceutical Co., Ltd.; Camostat mesylate from MFC Co., Ltd.; alkalizing agents such as sodium hydroxide, sodium carbonate, and sodium bicarbonate from Samchun Co., Ltd.; ethanol from Samchun Co., Ltd.; acetonitrile and acetone from Daejung Co., Ltd.; and a co-former from Merck Co., Ltd. were purchased and used in Examples.

<Examples 1 to 8> Preparation of Cocrystal α

[0148] In one Example of the present invention, a cocrystal α according to the present invention was prepared through a slurry method. The contents of niclosamide and camostat mesylate, the type and content of alkalizing agent, and the types and contents of first and second solvents were applied as shown in Table 1 below.

Example 1

[0149] 49.45 g of camostat mesylate was added in 2.5 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. 8.4 g of sodium bicarbonate (alkalizing agent) was added in 0.1 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. In addition, 32.71 g of niclosamide was added in 2.0 kg of 30° C. anhydrous ethanol (second solvent) and completely dissolved while stirring at 100 rpm. The camostat mesylate solution and the niclosamide solution were filtered using a 0.45 μm filter to remove possible foreign substances. The filtered camostat mesylate solution was added and mixed with a sodium bicarbonate solution, and stirred at 100 rpm for 30 minutes. When the mixed solution became sufficiently opaque, the filtered niclosamide solution was added and stirred at 200 rpm for 3 hours. Thereafter, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 30° C. for one day using a vacuum dryer to obtain a cocrystal α in the form of dry powder.

Examples 2 to 8

[0150] Cocrystals α of Examples 2 to 8 were prepared using substantially the same method as the preparation method of Example 1, but using components and contents shown in Table 1 below.

TABLE-US-00001 TABLE 1 Classification Niclosamide Camostat mesylate Alkalizing agent First solvent Second solvent Example 1 32.71 g 49.45 g NaHCO.sub.3 Distilled Anhydrous  8.4 g water 2.6 kg ethanol 2.0 kg Example 2 32.71 g 49.45 g NaOH Distilled Anhydrous  3.9 g water 2.6 kg ethanol 2.0 kg Example 3 32.71 g 49.45 g KOH Distilled Anhydrous  5.6 g water 2.6 kg ethanol 2.0 kg Example 4 32.71 g 49.45 g Na.sub.2CO.sub.3 Distilled Anhydrous 10.5 g water 2.6 kg ethanol 2.0 kg Example 5 32.71 g 49.45 g NaOH Distilled Acetone  3.9 g water 2.6 kg 2.0 kg Example 6 32.71 g 49.45 g KOH Distilled Acetone  5.6 g water 2.6 kg 2.0 kg Example 7 32.71 g 49.45 g NaHCO.sub.3 Distilled Acetone  8.4 g water 2.6 kg 2.0 kg Example 8 32.71 g 49.45 g Na.sub.2CO.sub.3 Distilled Acetone 10.5 g water 2.6 kg 2.0 kg

<Examples 9 and 16> Preparation of Cocrystal β

[0151] In one Example of the present invention, a cocrystal β according to the present invention was prepared through a slurry method. The contents of niclosamide and camostat mesylate, the type and content of alkalizing agent, and the types and contents of first solvent, second solvent and third solvent were applied as shown in Table 2 below.

Example 9

[0152] 49.45 g of camostat mesylate was added in 2.5 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. 8.4 g of sodium bicarbonate (alkalizing agent) was added in 0.1 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. In addition, 32.71 g of niclosamide was added in 2.0 kg of 30° C. ethanol (second solvent) and completely dissolved while stirring at 100 rpm. The camostat mesylate solution and the niclosamide solution were filtered using a 0.45 μm filter to remove possible foreign substances. The filtered camostat mesylate solution was added and mixed with a sodium bicarbonate solution, and stirred at 100 rpm for 30 minutes. When the mixed solution became sufficiently opaque, the filtered niclosamide solution was added and stirred at 100 rpm for 3 hours. Thereafter, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 30° C. for one day using a vacuum dryer to obtain a cocrystal raw material in the form of dry powder. The obtained cocrystal raw material was added in 1.6 kg of 25° C. anhydrous ethanol (third solvent) and stirred at 100 rpm for 3 hours. Thereafter, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 25° C. for one day using a vacuum dryer to obtain a cocrystal β in the form of a dry powder.

Examples 10 to 16

[0153] Cocrystals β of Examples 10 to 16 were prepared using substantially the same method as the preparation method of Example 9, but using components and contents shown in Table 2 below.

TABLE-US-00002 TABLE 2 Camostat Alkalizing First Second Third Classification Niclosamide mesylate agent solvent solvent solvent Example 9 32.71 g 49.45 g NaHCO.sub.3 Distilled Anhydrous Anhydrous 8.4 g water ethanol ethanol 2.6 kg 2.0 kg 1.6 kg Example 10 32.71 g 49.45 g NaOH Distilled Anhydrous Anhydrous 3.9 g water ethanol ethanol 2.6 kg 2.0 kg 1.6 kg Example 11 32.71 g 49.45 g KOH Distilled Anhydrous Anhydrous 5.6 g water ethanol ethanol 2.6 kg 2.0 kg 1.6 kg Example 12 32.71 g 49.45 g Na.sub.2CO.sub.3 Distilled Anhydrous Anhydrous 10.5 g  water ethanol ethanol 2.6 kg 2.0 kg 1.6 kg Example 13 32.71 g 49.45 g NaOH Distilled Acetone Anhydrous 3.9 g water 2.0 kg ethanol 2.6 kg 1.6 kg Example 14 32.71 g 49.45 g KOH Distilled Acetone Anhydrous 5.6 g water 2.0 kg ethanol 2.6 kg 1.6 kg Example 15 32.71 g 49.45 g Na.sub.2CO.sub.3 Distilled Acetone Anhydrous 8.4 g water 2.0 kg ethanol 2.6 kg 1.6 kg Example 16 32.71 g 49.45 g Na.sub.2CO.sub.3 Distilled Acetone Anhydrous 10.5 g  water 2.0 kg ethanol 2.6 kg 1.6 kg

<Examples 17 to 20> Preparation of Cocrystal β

[0154] In one Example of the present invention, a cocrystal Q according to the present invention was prepared through a slurry method. The contents of niclosamide and camostat mesylate, the type and content of alkalizing agent, and the types and contents of first and second solvents were applied as shown in Table 3 below.

Example 17

[0155] 49.45 g of camostat mesylate was added in 2.5 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. 10.6 g of sodium carbonate (alkalizing agent) was added in 0.1 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. The camostat mesylate solution was filtered using a 0.45 μm filter to remove possible foreign substances. The filtered camostat mesylate solution was added and mixed with a sodium carbonate solution, and stirred at 100 rpm for 30 minutes. When the mixed solution became sufficiently opaque, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 25° C. for one day using a vacuum dryer to obtain camostat in the form of dry powder. In addition, 32.71 g of niclosamide was added in 1.6 kg of 30° C. anhydrous ethanol (second solvent) and completely dissolved while stirring at 100 rpm and then filtered using a 0.45 μm filter to remove possible foreign substances. The obtained camostat was added to the filtered niclosamide solution and stirred at 100 rpm for 3 hours at 25° C. Thereafter, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 25° C. for one day using a vacuum dryer to obtain a cocrystal β in the form of dry powder.

Examples 18 to 20

[0156] Cocrystals β of Examples 18 to 20 were prepared using substantially the same method as the preparation method of Example 17, but using components and contents shown in Table 3 below.

TABLE-US-00003 TABLE 3 Classification Niclosamide Camostat mesylate Alkalizing agent First solvent Second solvent Example 17 32.71 g 49.45 g Na.sub.2CO.sub.3 Distilled water Anhydrous 10.6 g 2.6 kg ethanol 1.6 kg Example 18 32.71 g 49.45 g NaOH Distilled water Anhydrous  3.9 g 2.6 kg ethanol 1.6 kg Example 19 32.71 g 49.45 g KOH Distilled water Anhydrous  5.6 g 2.6 kg ethanol 1.6 kg Example 20 32.71 g 49.45 g NaHCO.sub.3 Distilled water Anhydrous  8.4 g 2.6 kg ethanol 1.6 kg

<Examples 21 to 24> Preparation of Cocrystal γ

[0157] In one Example of the present invention, a cocrystal γ according to the present invention was prepared through a slurry method. The contents of niclosamide and camostat mesylate, the type and content of alkalizing agent, and the types and contents of first and second solvents were applied as shown in Table 4 below.

Example 21

[0158] 49.45 g of camostat mesylate was added in 2.5 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. 8.4 g of sodium bicarbonate was added in 0.1 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. The camostat mesylate solution was filtered using a 0.45 μm filter to remove possible foreign substances. The filtered camostat mesylate solution was added and mixed with a sodium bicarbonate solution, and stirred at 100 rpm for 30 minutes. When the mixed solution became sufficiently opaque, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 25° C. for one day using a vacuum dryer to obtain camostat in the form of dry powder. In addition, 32.71 g of niclosamide was added in 1.6 kg of 30° C. acetone (second solvent) and completely dissolved while stirring at 100 rpm and then filtered using a 0.45 μm filter to remove possible foreign substances. The obtained camostat was added to the filtered niclosamide solution and stirred at 100 rpm for 3 hours at 25° C. Thereafter, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 25° C. for one day using a vacuum dryer to obtain a cocrystal γ in the form of dry powder.

Examples 22 to 24

[0159] Cocrystals γ of Examples 22 to 24 were prepared using substantially the same method as the preparation method of Example 21, but using components and contents shown in Table 4 below.

TABLE-US-00004 TABLE 4 Classification Niclosamide Camostat mesylate Alkalizing agent First solvent Second solvent Example 21 32.71 g 49.45 g NaHCO.sub.3 Distilled Acetone 8.4 g water 2.6 kg 1.6 kg Example 22 32.71 g 49.45 g NaOH Distilled Acetone 3.9 g water 2.6 kg 1.6 kg Example 23 32.71 g 49.45 g KOH Distilled Acetone 5.6 g water 2.6 kg 1.6 kg Example 24 32.71 g 49.45 g Na.sub.2CO.sub.3 Distilled Acetone 8.4 g water 2.6 kg 1.6 kg

<Examples 25 to 28> Preparation of Cocrystal δ

[0160] In one Example of the present invention, a cocrystal δ according to the present invention was prepared through a slurry method. The contents of niclosamide and camostat mesylate, the type and content of alkalizing agent, and the types and contents of first solvent, second solvent, third solvent, and fourth solvent were applied as shown in Table 5 below.

Example 25

[0161] 49.45 g of camostat mesylate was added in 2.5 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. 8.4 g of sodium bicarbonate (alkalizing agent) was added in 0.1 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. In addition, 32.71 g of niclosamide was added in 1.6 kg of 30° C. acetone (second solvent) and completely dissolved while stirring at 100 rpm. The camostat mesylate solution and the niclosamide solution were filtered using a 0.45 μm filter to remove possible foreign substances. The filtered camostat mesylate solution was added and mixed with a sodium carbonate solution, and stirred at 100 rpm for 30 minutes. When the mixed solution became sufficiently opaque, the filtered niclosamide solution was added and stirred at 100 rpm for 3 hours. Thereafter, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 30° C. for one day using a vacuum dryer to obtain a cocrystal raw material in the form of dry powder. The obtained cocrystal raw material was added in 1.6 kg of 25° C. anhydrous ethanol (third solvent) and stirred at 100 rpm for 3 hours. Thereafter, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 25° C. for one day using a vacuum dryer to obtain a cocrystal raw material in the form of dry powder. The obtained cocrystal raw material was added in 1.6 kg of 25° C. acetonitrile (fourth solvent) and stirred at 100 rpm for 3 hours. Thereafter, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 25° C. for one day using a vacuum dryer to obtain a cocrystal S in the form of dry powder.

Examples 26 to 28

[0162] Cocrystals δ of Examples 26 to 28 were prepared using substantially the same method as the preparation method of Example 25, but using components and contents shown in Table 5 below.

TABLE-US-00005 TABLE 5 Camostat Alkalizing First Second Third Fourth Classification Niclosamide mesylate agent solvent solvent solvent solvent Example 25 32.71 g 49.45 g NaHCO.sub.3 Distilled Acetone Anhydrous Acetonitrile  8.4 g water 2.0 kg ethanol 1.6 kg 2.6 kg 1.6 kg Example 26 32.71 g 49.45 g Na.sub.2CO3 Distilled Acetone Anhydrous Acetonitrile 10.5 g water 2.0 kg ethanol 1.6 kg 2.6 kg 1.6 kg Example 27 32.71 g 49.45 g NaHCO.sub.3 Distilled Anhydrous Anhydrous Acetonitrile  8.4 g water ethanol ethanol 1.6 kg 2.6 kg 2.0 kg 1.6 kg Example 28 32.71 g 49.45 g Na.sub.2CO.sub.3 Distilled Anhydrous Anhydrous Acetonitrile 10.5 g water ethanol ethanol 1.6 kg 2.6 kg 2.0 kg 1.6 kg

<Examples 29 to 32> Preparation of Cocrystal δ

[0163] In one Example of the present invention, a cocrystal S according to the present invention was prepared through a slurry method. The contents of niclosamide and camostat mesylate, the type and content of alkalizing agent, and the types and contents of first and second solvents were applied as shown in Table 6 below.

Example 29

[0164] 49.45 g of camostat mesylate was added in 2.5 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. 8.4 g of sodium carbonate (alkalizing agent) was added in 0.1 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. The camostat mesylate solution was filtered using a 0.45 μm filter to remove possible foreign substances. The filtered camostat mesylate solution was added and mixed with a sodium carbonate solution, and stirred at 100 rpm for 30 minutes. When the mixed solution became sufficiently opaque, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 25° C. for one day using a vacuum dryer to obtain camostat in the form of dry powder. Then, 32.71 g of niclosamide was added to 1.6 kg of 30° C. acetonitrile (second solvent), and added with the obtained camostat, and stirred at 100 rpm for 3 hours. Thereafter, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 30° C. for one day using a vacuum dryer to obtain a cocrystal S in the form of dry powder.

Examples 30 to 32

[0165] Cocrystals δ of Examples 30 to 32 were prepared using substantially the same method as the preparation method of Example 29, but using components and contents shown in Table 6 below.

TABLE-US-00006 TABLE 6 Classification Niclosamide Camostat mesylate Alkalizing agent First solvent Second solvent Example 29 32.71 g 49.45 g Na.sub.2CO.sub.3 Distilled water Acetonitrile 8.4 g 2.6 kg 1.6 kg Example 30 32.71 g 49.45 g NaOH Distilled water Acetonitrile 3.9 g 2.6 kg 1.6 kg Example 31 32.71 g 49.45 g KOH Distilled water Acetonitrile 5.6 g 2.6 kg 1.6 kg Example 32 32.71 g 49.45 g NaHCO.sub.3 Distilled water Acetonitrile 8.4 g 2.6 kg 1.6 kg

<Examples 33 to 40> Preparation of Cocrystal ε

[0166] In one Example of the present invention, a cocrystal ε according to the present invention was prepared through a slurry method. The contents of niclosamide, camostat mesylate, and meglumine, the type and content of alkalizing agent, and the types and contents of first solvent, second solvent and third solvent were applied as shown in Table 7 below.

Example 33

[0167] 49.45 g of camostat mesylate was added in 2.5 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. 10.5 g of sodium carbonate was added in 0.1 kg of 25° C. distilled water (first solvent) and completely dissolved while stirring at 100 rpm. In addition, 32.71 g of niclosamide was added in 2.0 kg of 30° C. anhydrous ethanol (second solvent) and completely dissolved while stirring at 100 rpm. The camostat mesylate solution and the niclosamide solution were filtered using a 0.45 μm filter to remove possible foreign substances. The filtered camostat mesylate solution was added and mixed with a sodium carbonate solution, and stirred at 100 rpm for 30 minutes. When the mixed solution became sufficiently opaque, the filtered niclosamide solution was added and stirred at 100 rpm for 3 hours. Thereafter, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 30° C. for one day using a vacuum dryer to obtain a cocrystal raw material in the form of dry powder. The obtained cocrystal raw material was added to 1.6 kg of 25° C. acetonitrile (third solvent) together with 97.6 g of meglumine (co-former), and then stirred at 100 rpm for 3 hours. Thereafter, the reactant was filtered using a vacuum pump and a paper filter to remove the solvent, and then dried at 20° C. for one day using a vacuum dryer to obtain a cocrystal & in the form of dry powder.

Examples 34 to 40

[0168] Cocrystals ε of Examples 34 to 40 were prepared using substantially the same method as the preparation method of Example 33, but using components and contents shown in Table 7 below.

TABLE-US-00007 TABLE 7 Camostat Co- Alkalizing First Second Third Classification Niclosamide mesylate former agent solvent solvent solvent Example 33 32.71 g 49.45 g Meglumine Na.sub.2CO.sub.3 Distilled Anhydrous Acetonitrile 97.6 g 10.5 g  water ethanol 1.6 kg 2.6 kg 2.0 kg Example 34 32.71 g 49.45 g Meglumine NaOH Distilled Anhydrous Acetonitrile 97.6 g 3.9 g water ethanol 1.6 kg 2.6 kg 2.0 kg Example 35 32.71 g 49.45 g Meglumine KOH Distilled Anhydrous Acetonitrile 39.0 g 5.6 g water ethanol 1.6 kg 2.6 kg 2.0 kg Example 36 32.71 g 49.45 g Nicotinamide NaHCO.sub.3 Distilled Anhydrous Acetonitrile 12.2 g 8.4 g water ethanol 1.6 kg 2.6 kg 2.0 kg Example 37 32.71 g 49.45 g Nicotinamide NaHCO.sub.3 Distilled Anhydrous Acetonitrile 36.6 g 8.4 g water ethanol 1.6 kg 2.6 kg 2.0 kg Example 38 32.71 g 49.45 g Caffeine NaHCO.sub.3 Distilled Anhydrous Acetonitrile 19.4 g 8.4 g water ethanol 1.6 kg 2.6 kg 2.0 kg Example 39 32.71 g 49.45 g Arginine NaHCO.sub.3 Distilled Anhydrous Acetonitrile 17.4 g 8.4 g water ethanol 1.6 kg 2.6 kg 2.0 kg Example 40 32.71 g 49.45 g Citric acid NaHCO.sub.3 Distilled Anhydrous Acetonitrile 19.2 g 8.4 g water ethanol 1.6 kg 2.6 kg 2.0 kg

[0169] The production yields of the cocrystals of the present invention prepared in Examples 1 to 40 were all 70% or more, and the purities thereof were at least 95% or more.

[0170] Hereinafter, analysis experiments and results of the cocrystals α, β, γ, δ, and ε prepared in Examples 1, 17, 21, 25, and 33 will be described in more detail.

EXPERIMENTAL EXAMPLES

[0171] Each cell line used in an experiment was purchased and used from the American Type Culture Collection (ATCC) or Korea Cell Line Bank (KCLB).

Experimental Example 1. Powder X-Ray Diffraction (PXRD) Analysis

[0172] With respect to cocrystal samples according to Examples 1, 17, 21, 25 and 33 of the present invention and comparative samples (niclosamide, camostat, and camostat mesylate), PXRD analysis was performed under the following conditions using D8 ADVANCE with Davinci (trade name, Bruker AXS Inc, GmbH, Germany) as a powder X-ray diffraction (PXRD) analyzer.

[0173] Detector: High-speed LynxEye detector

[0174] Tube: Cu

[0175] Tube current: 40 mA

[0176] Tube voltage: 40 kV

[0177] Sampling width: 0.020°

[0178] Scanning rate: 0.1 sec/step

[0179] Wavelength: 1.54056 Å

[0180] Measured diffraction angle range (2θ): 2.5 to 40°

[0181] The cocrystal samples of the present invention were cocrystals in a dry powder form obtained in Examples 1, 17, 21, 25 and 33, respectively, and the comparative samples were niclosamide, camostat and camostat mesylate, which were the raw materials used to form the cocrystals. The diffraction angles 2θ of main X-ray diffraction patterns through PXRD analysis of the cocrystal samples according to Examples 1, 17, 21, 25 and 33 of the present invention and the comparative samples represented relative intensities in Tables 8 and 9 below.

TABLE-US-00008 TABLE 8 Niclosamide Camostat mesylate Relative Relative Camostat Diffraction intensity Diffraction intensity Diffraction Relative intensity angle (2θ) (intensity) angle (2θ) (intensity) angle (2θ) (intensity) 12.9444 227 9.61405 550 7.30839 725 13.6933 282 12.2153 289 12.4715 565 19.7629 204 13.2992 107 12.905 267 22.1277 115 14.383 139 14.7377 187 23.3298 156 15.1122 161 15.8216 179 25.596 493 16.531 244 16.5507 314 26.2266 236 17.1222 648 17.4769 395 26.6799 452 19.9403 287 19.6447 664 20.4329 1041 22.0686 399 20.7482 1065 23.6254 306 21.6547 369 24.0983 475 23.5268 201 24.8669 241 24.1574 303 26.1675 206 28.3155 406 26.5813 436 27.6258 201

TABLE-US-00009 TABLE 9 Cocrystal α Cocrystal β Cocrystal γ Cocrystal δ (Example 1) (Example 17) (Example 21) (Example 25) Relative Relative Relative Relative Diffraction intensity Diffraction intensity Diffraction intensity Diffraction intensity angle (2θ) (intensity) angle (2θ) (intensity) angle (2θ) (intensity) angle (2θ) (intensity) 5.37715 907 6.55954 1746 7.82075 145 6.81572 454 7.38721 138 10.7176 193 8.35283 97 7.46604 613 9.41698 131 13.8509 184 8.80608 68 9.87023 249 10.6979 177 15.2501 171 11.3876 331 12.3532 558 13.1612 173 16.6296 151 16.0975 228 12.8262 149 15.1122 270 18.147 290 16.6493 397 13.24 423 17.7134 150 18.9943 317 18.679 185 18.6396 404 18.2258 236 19.5855 493 19.0929 125 22.6795 185 18.7579 235 20.9059 156 22.6992 134 23.259 162 20.0782 146 21.3591 191 23.0539 272 24.7881 194 20.3344 222 22.3642 160 23.9013 436 25.6946 227 21.0044 126 24.3151 170 24.4333 399 27.5667 191 21.97 144 24.8866 165 25.4975 141 24.3939 126 26.8178 425 26.9755 132 25.1034 139 27.7637 156 29.2417 111 25.596 261 29.7344 200 26.069 306 30.3256 132 26.4434 178 27.882 156 Crystal ε (Example 33) Relative Diffraction intensity angle (2θ) (intensity) 7.0522 330 7.6239 340 9.06226 4975 12.4912 370 18.009 644 21.9897 361 24.2166 213 27.1134 187

[0182] In FIG. 1, an x-axis was 2θ (Bragg angle, unit: °) and a y-axis was an X-ray intensity (cps).

[0183] Referring to Tables 8 and 9 and FIG. 1 together, in a process of forming cocrystals of co-formers, there was non-stoichiometric hydrate, and as a result, it was confirmed that a discrepancy occurred at a far distance, and a phenomenon in which the diffraction angle 2θ was shifted occurred. That is, as compared with each of niclosamide, camostat mesylate and camostat in FIG. 1, it was confirmed that the cocrystals of the present invention exhibit novel crystals showing diffraction patterns different from those of the raw materials.

Experimental Example 2. Differential Scanning Calorimetry (DSC) Analysis

[0184] Differential scanning calorimetry was performed on niclosamide, camostat mesylate, camostat, and cocrystals prepared in Examples 1, 17, 21, 25 and 33.

[0185] For the analysis according to temperature differential scanning calorimetry, a DSC Q2000 System (trade name, TA Instrument, USA) was used as a DSC analysis device, and the measurement was performed while the temperature was increased from 0° C. to a melting point at a heating rate of 10° C./min. During the measurement, N.sub.2 gas was supplied at a rate of 50 mL/min, and the measurement was performed in an aluminum sample pan. Data were analyzed using Universal Analysis 2000 software (trade name, TA instruments, USA). The maximum endothermic peak temperatures of monocomponents niclosamide and camostat, and fusion crystals thereof obtained through DSC analysis were shown in Table 10 and FIG. 2 below.

TABLE-US-00010 TABLE 10 Maximum endothermic peak temperature (° C.) Maximum endothermic peak temperature (°C) of of niclosamide camostat mesylate 230.06 194.85 Maximum endothermic peak temperature (° C.) Example 1 - Maximum endothermic peak of camostat free base temperature (° C.) of Cocrystal α 176.13 144.38 Example 17 - Maximum endothermic peak Example 21 - Maximum endothermic peak temperature (° C.) of Cocrystal β temperature (° C.) of Cocrystal γ 126.35 182.74 Example 25 - Maximum endothermic peak Example 33 - Maximum endothermic peak temperature (° C.) of Cocrystal δ temperature (° C.) of Cocrystal ε 151.69 126.03

[0186] In FIG. 2, an x-axis represents a temperature (unit: ° C.), and a y-axis represents a heat flow (unit: W/g).

[0187] Referring to Table 10 together with FIG. 2, when the heating rate is 10° C./min, it may be confirmed that the cocrystal α shows a DSC endothermic peak at 144.38° C., the cocrystal β shows a DSC endothermic peak at 126.35° C., the cocrystal γ shows a DSC endothermic peak at 182.74° C., the cocrystal S shows a DSC endothermic peak at 151.69° C., and the cocrystal ε shows a DSC endothermic peak at 126.03° C.

[0188] That is, as compared with each of niclosamide, camostat mesylate and camostat, it was confirmed that the cocrystals of the present invention exhibit novel crystals exhibiting new thermodynamic characteristics different from those of the raw materials.

Experimental Example 3. Evaluation of Solubility of Niclosamide in Cocrystal of the Present Invention

[0189] The solubility of a niclosamide single material and the solubility of niclosamide included in the cocrystals prepared in Examples 17 and 33 of the present invention were measured in a pH 7 buffer solution and compared with each other. A pH 7 solution prepared by mixing 0.1 N potassium phosphate and 0.1 N sodium hydroxide was used as the pH 7 buffer solution. At room temperature, 5 mg of the niclosamide single material, 5 mg of the cocrystal β of Example 17, and 5 mg of the cocrystal ε of Example 33 were added to 25 ml of the prepared pH solution, respectively, and stirred at 600 rpm. After 1 hour of stirring, 5 ml of a supernatant was filtered with a 0.45 μm PVDF filter, and then the amount of dissolved niclosamide was evaluated using the filtered solution as a sample.

[0190] HPLC instrument conditions were a flow rate of 1.5 ml/min, an injection volume of 100 μl, a detection wavelength of 287 nm, and a column oven temperature of 25° C., and a mobile phase A was a pH 6 buffer (potassium phosphate 2 g/L, disodium phosphate 1 g/L, tetrabutylammonium hydrogen sulfate 2 g/L, adjusted to pH 6.0±0.05 with 1 M NaOH), and a mobile phase B was acetonitrile. Mobile phase conditions were shown in Table 11 below.

TABLE-US-00011 TABLE II Time Mobile Mobile (min) phase A (%) phase B (%)  0 90 10 13 90 10 18 45 55 25 45 55 30 90 10 40 90 10

[0191] Referring to Table 12 below, niclosamide was hardly detected due to its low solubility at pH 7, and in the cocrystal of Example 17, it was confirmed that 55.2 μg/ml of niclosamide was dissolved, so that the solubility of niclosamide was significantly improved compared to the niclosamide single material. In addition, even in the cocrystal of Example 33, it was confirmed that 0.4 μg/ml of niclosamide was dissolved, and the solubility of niclosamide was improved compared to the niclosamide single material.

TABLE-US-00012 TABLE 12 Drug Niclosamide Example 17 Example 33 pH 7 solubility <0.3 μg/ml 55.2 μg/ml 0.4 μg/ml (μg/ml)

Experimental Example 4. Evaluation of Artificial Membrane Permeability of Camostat in Cocrystal of the Present Invention

[0192] The artificial membrane permeability of camostat in the cocrystal prepared in Example 21 of the present invention was evaluated using a side-bi-side cell system and compared with that of a camostat single material. As an artificial membrane, 200 μl of a GIT-0-Lipid solution was used by dropping on the center of a 25 mm hydrophobic PVDF membrane. 5 ml of a 37° C. pH 5.0 FeSSIF solution was taken and contained in a donor cell, and 2θ mg of the sample was weighed and administered to the donor cell. The test was started after adding 5 ml of an ethyl alcohol. pH 7.4 PBS buffer (1:9, v/v) solution to an acceptor cell, and 200 μl of the solution was taken five times from the acceptor cell every predetermined time and used as a test solution. Then, the acceptor cell was filled with 1 ml of the ethyl alcohol: pH 7.4 PBS buffer (1:9, v/v) solution to maintain a total volume of 5 ml.

TABLE-US-00013 TABLE 13 Drug Camostat Example 21 Cumulative permeation 561.89 876.26 amount J (μg/ml/cm.sup.2/h)

[0193] Referring to Table 13, it was confirmed that a cumulative permeation amount of camostat single material was 561.89, and the cumulative permeation amount of camostat in the cocrystal of Example 21 was 876.29, so that the cumulative permeation amount of camostat was 1.56-fold better.

[0194] That is, it was confirmed that the cocrystal of the present invention had excellent membrane permeability to have excellent absorption in vivo.

Experimental Example 5. Evaluation of Artificial Membrane Permeability of Niclosamide in Cocrystal of the Present Invention

[0195] The permeability of an artificial membrane (barrier membrane from PermeaPad, 25 mm) to niclosamide of the cocrystal of Example 17 of the present invention was evaluated using the side-bi-side cell system, and compared with that of the niclosamide single material. 5 ml of an FeSSIF solution (37° C.) at pH 6.5 added with 3% (w/w) Kolliphor ELP was taken and contained in a donor cell, and 2θ mg of each sample was weighed and administered to the donor cell. 5 ml of a PBS solution of pH 7.4 added with 20% (w/w) HP-β-CD was added to the acceptor cell, the test was started, and 200 μl of the solution was taken 5 times from the acceptor cell every predetermined time and used as the test solution. Then, the acceptor cell was filled with the PBS solution of pH 7.4 added with 1 ml of 20% (w/w) HP-p-CD, and the total volume was maintained at 5 ml.

TABLE-US-00014 TABLE 14 Drug Niclosamide Example 17 Cumulative permeation 1.18 2.03 amount of drug (μg/ml/cm.sup.2/h)

[0196] Referring to Table 14, it was confirmed that a cumulative permeation amount of niclosamide single material was 1.18, and the cumulative permeation amount of niclosamide in the cocrystal of Example 17 was 2.03, so that the cumulative permeation amount of niclosamide of the cocrystal of the present invention was 1.72-fold better.

[0197] That is, it was confirmed that the cocrystal of the present invention had excellent membrane permeability to have excellent absorption in vivo.

Experimental Example 6. Evaluation of Inhibition of Cancer Cell Proliferation

[0198] Cell proliferation inhibition was evaluated to verify the anticancer efficacy of the cocrystals of the present invention in pancreatic cancer, breast cancer, non-small cell lung cancer, and liver cancer cell lines.

[0199] The conditions of an anticancer in vitro drug efficacy test for the evaluation of proliferation inhibition by the cocrystals of the present invention in each cancer cell line were shown in Table 15 below. Hereinafter, the cocrystal α was the cocrystal prepared in Example 1.

[0200] 2×10.sup.3 to 4×10.sup.3 cells per well were inoculated in a 96-tissue culture plate using total 8 types of cell lines including two pancreatic cancer cell lines PANC-1 and MIAPACA-2, two breast cancer cell lines MCF-7 and MDA-MB-231, two non-small cell lung cancer cell lines A-549 and H-1299, and two liver cancer cell lines Hep-3B and Huh-7. The cells were incubated in a cell incubator for 24 hours, and then treated with each cocrystal drug at a total of six concentrations of 0.3, 3, 3°, 30°, 3000, and 30000 ng/mL. After 48 hours of drug treatment, a reagent was reacted for 1 to 4 hours using a CCK-8 assay kit to observe color changes, and the cell viability was measured by measuring absorbance at a wavelength of 450 nm with a microreader.

TABLE-US-00015 TABLE 15 Cell line Pancreatic cancer cell lines PANC-1, MIAPACA-2 Breast cancer cell line MCF-7, MDA-MB-231 Non-small cell lung cancer cell lines A-549, H-1299 Liver cancer cell line Hep-3B, Hub-7 Cell number 2 × 10.sup.3 to 4 × 10.sup.3 cells/well, 96 well plate Incubation condition 96 well plate, 24 hr incubation Drug concentration 0.3 to 30000 ng/mL (10 serial dilution in DMSO, 6 point) Medium DMEM (Dulbecco Modified Eagle Medium), RPMI (Roswell Park Memorial Institute) 1640, MEM (Minimum Essential Media)

[0201] In order to verify the anticancer efficacy of cocrystals for various types of cancer cells, the cell viability was measured in pancreatic cancer, breast cancer, non-small cell lung cancer, and liver cancer cells to derive IC.sub.50. The results were shown in Table 16 below.

[0202] The IC.sub.50 values of positive controls were based on facts described in the literatures below, respectively.

[0203] Gemcitabine: HONGGANG WANG, BEVERLY R. WORD and BEVERLY D. LYN-COOK: Enhanced Efficacy of Gemcitabine by Indole-3-carbinol in Pancreatic Cell Lines: The Role of Human Equilibrative Nucleoside Transporter 1. Anticancer research 31 (10): 3171-3180, 2011.

[0204] Gefitinib: CHI PAN, HUUIE DUAN, YINAN WU, CHUNPENG ZHU, CHENGHAO YI, YIN DUAN, DEMIN LU, CHENG GUO, DEQI WU, YANYAN WANG, XIANHUA FU, JING XU, YLDING CHEN, MENG LUO, WEI TIAN, TAO PAN, WENHONG XU, SUZHAN ZHANG and JIANJIN HUANG: Inhibition of DNA-PK by gefitinib causes synergism between gefitinib and cisplatin in NSCLC. International journal of oncology 57:939-955, 2020

[0205] Docetaxel: Aliakbar Taherian, 1Tahereh Mazoochi: Different Expression of Extracellular Signal-Regulated Kinases (ERK) 1/2 and Phospho-Erk Proteins in MBA-MB-231 and MCF-7 Cells after Chemotherapy with Doxorubicin or Docetaxel. Iranian Journal of basic Medical sciences 15, 669-677, 2012

[0206] Sorafenib: Y-C Shen 1, D-L Ou, C Hsu, K-L Lin, C-Y Chang, C-Y Lin, S-H Liu, A-L Cheng: Activating oxidative phosphorylation by a pyruvate dehydrogenase kinase inhibitor overcomes sorafenib resistance of hepatocellular carcinoma. BJC 108, 72-81, 2013

TABLE-US-00016 TABLE 16 IC.sub.50 (nM) cell line Example 1 Positive control PANC-1 99.30 Gemcitabine 42900.00 MiaPaCa-2 729.32 92700.00 A-549 975.81 Gefitinib 13200.00 H-1299 381.13 17500.00 MCF-7 427.86 Docetaxel 763.00 MDA-MB-231 126.31 635.00 Hep-3B 41.33 Sorafenib 12300.00 Huh-7 134.45 6000.00

[0207] Referring to Table 16 above, it was confirmed that the IC.sub.50 of the cocrystal of Example 1 according to the present invention was 99.30 nM in PANC-1 cells as a pancreatic cancer cell line; 729.32 nM in MIAPACA-2 cells; 427.86 nM in MCF-7 cells and 126.31 nM in MDA-MB-231 cells as a breast cancer cell line; 975.81 nM in A-549 cells and 381.13 nM in H-1299 cells as a non-small cell lung cancer cell line; and 41.33 nM in Hep-3B cells and 134.45 nM in Huh-7 cells as a liver cancer cell line.

[0208] Through this, the IC.sub.50 value of the cocrystal of Example 1 was confirmed in a total of 8 types of cell lines, every two types of pancreatic cancer, breast cancer, non-small cell lung cancer, and liver cancer cell lines, and in a pancreatic cancer cell line that has been classified as incurable carcinoma and has no therapeutic agent, a low IC.sub.50 value was confirmed to be effectively applied to inhibit the growth of pancreatic cancer cells. In addition, under the corresponding test conditions, low IC.sub.50 values were also confirmed in breast cancer, non-small cell lung cancer and liver cancer cell lines, and as a result, the cancer cell growth inhibitory effect of the cocrystal of the present invention was confirmed.

[0209] In addition, in order to compare the IC.sub.50 of the positive control and the cocrystal of Example 1, with respect to a drug, gemcitabine used as a therapeutic agent for pancreatic cancer patients, a drug, gefitinib used as a therapeutic agent for non-small cell lung cancer patients, a drug, docetaxel used as a therapeutic agent for breast cancer patients, and a drug, sorafenib used as a therapeutic agent for liver cancer patients, the IC.sub.50 was confirmed through the literatures. As compared with the positive control drugs, it was confirmed that the cocrystal of Example 1 of the present invention showed a significantly lower IC.sub.50 value than the positive control drug in all cell lines. The IC.sub.50 of the cocrystal of the present invention was 432-fold lower in the PANC-1 cell line and 127-fold lower in the MIAPACA-2 cell line than gemcitabine. In addition, the IC.sub.50 of the cocrystal was 13-fold lower in the A-549 cells and 45-fold lower in the H-1299 cells than gefitinib. In addition, the IC.sub.50 of the cocrystal was 1.7-fold lower in the MCF-7 cells and 5-fold lower in the MDA-MB-231 cells than docetaxel. In addition, the IC.sub.50 of the cocrystal was 297-fold lower in the Hep-3B cells and 44-fold lower in the Huh-7 cells than Sorafenib.

[0210] That is, it was confirmed that the cocrystal of the present invention had an excellent cancer cell proliferation inhibitory effect compared to conventionally used cancer therapeutic agents.

Experimental Example 7. Comparison and Verification of Anticancer Efficacy of Cocrystal of the Present Invention and Positive Control in Subtype-Specific Breast Cancer Cell Lines

[0211] In subtype-specific breast cancer cell lines, the cell proliferation inhibition of the cocrystal of the present invention was evaluated, and the conditions of the anticancer in vitro drug efficacy evaluation test for evaluation were shown in Table 17 below.

[0212] A total of 19 types of breast cancer cell lines were verified for the cocrystal of the present invention. As a positive control, drugs shown Table 17 below were used. Luminescent cell viability assay was performed using CellTiter glo by setting the cocrystals prepared in Examples 1 and 17 in 8 concentration ranges serially diluted by ½ fold for each cell line. A CellTiter-Glo substrate and a CellTiter-Glo buffer were mixed to make a CellTiter-Glo reagent, and then a CellTiter-Glo reagent was added in the same amount as a cell culture medium (40 μl volume) and left at room temperature for 10 minutes, and then the sensitivity to the formulation was confirmed by a method for measuring ATP using a luminometer (GloMax® Discover Microplate Reader). IC.sub.50 values were calculated and compared and evaluated by nonlinear regression analysis of GraphPad Prism 9 software.

TABLE-US-00017 TABLE 17 Cell line <19 types of breast cancer cell lines> Hormone-receptor positive breast cancer: MCF7, ZR-75-1, T47D, BT474 HER2-positive breast cancer: SK-BR-3, MDA-MB-453, HCC1954, HCC1419, JMIT1 Triple negative breast cancer: HCC1937, HCC1143, MDA-MB- 157, MDA-MB-231, Hs578T, HCC38, BT-549 Drug-resistant breast cancer: MCF7/ADR; Doxorubicin resistant MCF7 cell line, MCF7/PR; Paclitaxel resistant MCF7 cell line, MCF7/TAMR; Tamoxifen resistant MCF7 cell line Positive control Hormone-receptor positive breast cancer: Tamoxifen, Docetaxel HER2-positive breast cancer: Herceptin, Docetaxel Triple negative breast cancer: Cisplatin, Docetaxel Drug-resistant breast cancer: Docetaxel Cell number 0.5 to 1 × 10.sup.3 cells/well. 384 well plate 1 × 10.sup.4 cells/well, 96 well plate Incubation condition 10% FBS, 1% PS (including 0.023 U/ml insulin in the case of DMEM culture medium) 384 well plate, 24 hr incubation Drug concentration 0 to 25 μM (1/2 serial dilution in Vehicle, 8 point) Medium DMEM (Dulbecco Modified Eagle Medium), RPMI (Roswell Park Memorial Institute) 1640, MEM (Minimum Essential Media)

[0213] In order to verify the anticancer efficacy of the cocrystal of the present invention for subtype-specific breast cancer cell lines, a dose-response curve test was conducted in a total of 19 types of breast cancer cells, and IC.sub.50 was derived by measuring the cell viability. The representative results were shown in FIG. 3 and Tables 18 to 21 below.

TABLE-US-00018 TABLE 18 Hormone-positive breast cancer cell line Drug IC.sub.50 (nM) Cells Example 1 Example 17 Tamoxifen Docetaxel ZR-75-1 5366.0 6293.0 9198.0 24242.0

TABLE-US-00019 TABLE 19 HER2-positive breast cancer cell line Drug IC.sub.50 (nM) Cells Example 1 Example 17 Herceptin Docetaxel MDA-MB-453 7465.0 7484.0 25000.0 25000.0 HCC1419 1272.0 1500.0 25000.0 25000.0

TABLE-US-00020 TABLE 20 Triple negative breast cancer cell line Drug IC.sub.50 (nM) Cells Example 1 Example 17 Cisplatin Docetaxel HCC1937 4979.0 4546.0 12296.0 25000.0 MDA-MB-231 1747.0 2248.0 25000.0 25000.0

TABLE-US-00021 TABLE 21 Drug-resistant breast cancer cell line Drug IC.sub.50 (nM) Cells Example 1 Example 17 Paclitaxel Docetaxel MCF7/PR 5691.0 5824.0 13680.0 11406.0 * PR (Paclitaxel Resistance)

[0214] The drug response was varied for each cell, but in all cell lines, IC.sub.50 values of Examples 1 and Example 17, and the drugs used as control drugs were secured.

[0215] In the case of hormone-positive breast cancer cell lines, it was confirmed that Example 1 and Example 17 exhibited lower IC.sub.50 values than positive controls Tamoxifen and Docetaxel. In particular, in the case of ZR-75-1 cells, it was confirmed that the IC.sub.50 value of Example 1 was 1.7-fold lower than Tamoxifen, and the IC.sub.50 value of Example 17 was 1.4-fold lower than Tamoxifen. As a result, it was confirmed that the cocrystals of Example 1 and Example 17, which were the cocrystals of the present invention, exhibited a better anticancer effect than the positive control drug currently used for hormone-positive breast cancer patients.

[0216] In the case of the HER2-positive breast cancer cell line, HCC1419 cells, it was confirmed that Example 1 showed a significantly 19-fold lower IC.sub.50 value and Example 17 showed a significantly 16-fold lower IC.sub.50 value than a positive control drug Herceptin. Even in MDA-MB-453 cells, it was confirmed that the cocrystal of the present invention had a significantly low IC.sub.50 value. That is, it was confirmed that a cell viability reduction effect of the cocrystal of the present invention in HER2-positive breast cancer cell lines was very superior to that of the positive control.

[0217] Even in the case of a triple-negative breast cancer cell line, MDA-MB-231 cells, which are most commonly known, showed IC.sub.50 values 14-fold and 11-fold lower than those of Cisplatin, a positive control drug, in both Examples 1 and 17. Even in the triple-negative breast cancer cell line, which was the most difficult treatment among breast cancers, the anticancer effect of the cocrystal of the present invention, which was very superior to existing therapeutic agents, was confirmed.

[0218] IC.sub.50 values for Examples 1 and 17 and the positive control drugs paclitaxel and docetaxel were confirmed using drug-resistant breast cancer cell lines resistant to the corresponding drugs by long-term exposure to Doxorubicin, Paclitaxel, Tamoxifen, or Herceptin. In the drug-resistant breast cancer cell line, a low IC.sub.50 value of the cocrystal of Example was confirmed compared to the positive control drug Docetaxel, and particularly, in Paclitaxel-resistant breast cancer cell lines, a representative drug used as a cytotoxic therapeutic agent, Examples 1 and 17 were able to secure 2-fold or more low IC.sub.50 values.

Experimental Example 8. Comparison and Evaluation of Antiviral Efficacy of Cocrystal of the Present Invention Against SARS-CoV-2 with Positive Control Drugs

[0219] An experiment was conducted to verify the antiviral efficacy of the cocrystal of the present invention against SARS-CoV-2 using a SARS-CoV-2 cell infection model, and the experimental conditions were shown in Table 22 below. A group treated with each drug, chloroquine, lopinavir, or remdesivir, was used as a positive control.

[0220] To verify the antiviral efficacy of the cocrystal of the present invention against SARS-CoV-2, 1.2×10.sup.4 Vero cells per well were inoculated in a 384-tissue culture plate. After 24 hours, the cells were treated with a cocrystal prepared by serial dilution 2-fold in DMSO at 10 points or a positive control drug at the highest concentration of 50 μM. After about 1 hour of the cocrystal or positive control treatment, the cells were infected with SARS-CoV-2 (provided by the Korea Centers for Disease Control and Prevention (KCDC), 0.125 MOI) in a Biosafety level 3 (BSL3) facility, and incubated at 37° C. for 24 hours. Thereafter, the cells were fixed with 4% paraformaldehyde (PFA) and permeabilized. Then, the cells were stained by treating an anti-SARS-CoV-2 nucleocapsid (N) primary antibody, an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and Hoechst 33342. Fluorescent images of the infected cells were obtained using Operetta (Perkin Elmer), a large-capacity image analysis device.

TABLE-US-00022 TABLE 22 Cell line VeroE6 Cell number 1.2 × 10.sup.4 cells/well, 384 well plate Incubation condition DMEM, 2% FBS, IX Antibiotic-Antimycotic sol in black 384 well, clear plate, 24 hr prior to the experiment Drug concentration 0.1 to 50 μM (2X serial dilution in DMSO, 10 point) Stock concentration 10 mM (working top cone. 50 μM Viral infection titration 0.0125 (multiplicity of infection; MOI) 4% PFA fixation .fwdarw. permeabilization .fwdarw. 1.sup.st Ab anti-SARS-CoV-2 nucleocapsid (N) .fwdarw. 2.sup.nd Ab Alexa Fluor 488-conjugated goat anti-rabbit IgG/Hoechst 33342 staining .fwdarw. Perkin Elmer Immunofluorescence analysis

[0221] In order to verify the antiviral efficacy of the cocrystal against SARS-CoV-2, a dose-response curve test was conducted in a SARS-CoV-2 cell infection model, and the results were shown in Table 23 below. Selectivity index (SI) values were calculated as CC.sub.50/IC.sub.50.

TABLE-US-00023 TABLE 23 IC.sub.50 (μM) CC.sub.50 (μM) SI Example 1 0.16 >50 306 Example 17 0.16 >50 313 Remdesivir 3.48 >50 14.35 Lopinavir 10.30 >50 4.85 Chloroquine 7.47 >150 20.08

[0222] Referring to Table 23, it was confirmed that the cocrystals of Example 1 and Example 17 had IC.sub.50 of 0.16 μM, CC.sub.50 of 50 μM or more, and the SI was 306 for the cocrystal of Example 1 and 313 for the cocrystal of Example 17. It was confirmed that the IC.sub.50 of remdesivir, a positive control drug currently used as a corona therapeutic agent, was 3.48 μM, which was about 21.75-fold higher than the IC.sub.50 of the cocrystal. In addition, in the case of an AIDS therapeutic agent, lopinavir, which had been noted as a corona therapeutic agent, the IC.sub.50 was 10.30 μM, which was about 64.38-fold higher than the IC.sub.50 of the cocrystal of the present invention. In addition, in the case of a malaria therapeutic agent, chloroquine, the IC.sub.50 was 7.47 μM, which was about 46.69-fold higher than the IC.sub.50 of the cocrystal of the present invention.

[0223] As a result, it was confirmed that the cocrystal of the present invention had a significantly low IC.sub.50 value and an excellent antiviral effect compared to the drug, remdesivir currently used as a corona therapeutic agent.

[0224] In addition, it was confirmed that the cocrystal of the present invention had a significantly high selective index (SI) value for the virus compared to the positive control. The SI is an index showing a ratio of cytotoxicity to the antiviral activity, and the higher the value, the more effective and safe it is. Therefore, it was confirmed that the proliferation of SARS-CoV-2 virus may be selectively inhibited.

[0225] The present invention has been described with reference to the preferred embodiments of the present invention, but those skilled in the art will understand that the present invention may be variously modified and changed without departing from the spirit and the scope of the present invention which are defined in the appended claims. Therefore, the substantial scope of the present disclosure will be defined by the appended claims and their equivalents.