PHARMACEUTICAL COMPOSITION COMPRISING TIZOXANIDE AND PHARMACEUTICAL USE THEREOF

Abstract

A pharmaceutical composition contains tizoxanide and choline hydroxide, a choline salt of tizoxanide and crystal form thereof. A mixture of tizoxanide and choline hydroxide, or a salt thereof and crystal form of the salt significantly improve the solubility and bioavailability of tizoxanide, and exhibits favorable effects in drugs for treating viruses, fibrosis, bacteria, tumors and intestinal parasites.

Claims

1. A pharmaceutical composition, containing tizoxanide and choline hydroxide; preferably, a molar ratio of tizoxanide to choline hydroxide therein is 1:4 to 1:0.25, more preferably 1:2 to 1:0.5, such as 1:1, 1:0.5, 1:0.25, 1:2, 1:3 or 1:4.

2. The pharmaceutical composition as claimed in claim 1, wherein tizoxanide and choline hydroxide are in a molar ratio of 1:1 to form a tizoxanide choline salt of formula (I): ##STR00008## preferably, the tizoxanide choline salt of formula (I) is in an amorphous form or in a crystalline form, preferably in a crystalline form; preferably, the tizoxanide choline salt of formula (I) is in a crystalline form having characteristic peaks at the following 2θ angles (°) in a powder X-ray diffraction pattern obtained using Cu-Kα radiation: 9.92±0.10, 15.10±0.10, 19.72±0.10, 20.69±0.10 and 25.66±0.10, wherein the peak at 9.92±0.10 is the strongest peak; preferably, the tizoxanide choline salt of formula (I) is in a crystalline form having characteristic peaks at the following 2θ angles (°)in a powder X-ray diffraction pattern obtained using Cu-Kα radiation: 9.92±0.10, 15.10±0.10, 16.16±0.10, 19.72±0.10, 19.88±0.10, 20.69±0.10, 25.66±0.10 and 27.59±0.10, wherein the peak at 9.92±0.10 is the strongest peak; preferably, the tizoxanide choline salt of formula (I) is in a crystalline form having characteristic peaks at the following 2θ angles (°) in a powder X-ray diffraction pattern obtained using Cu-Kα radiation: 9.92±0.10, 11.78±0.10, 15.10±0.10, 16.16±0.10, 19.72±0.10, 19.88±0.10, 20.69±0.10, 25.66±0.10, 27.59±0.10 and 28.88±0.10, wherein the peak at 9.92±0.10 is the strongest peak; preferably, the tizoxanide choline salt of formula (I) is in a crystalline form having a powder X-ray diffraction pattern substantially as shown in the figure.

3. The pharmaceutical composition as claimed in claim 1 , wherein the pharmaceutical composition further comprises a non-toxic pharmaceutically acceptable pharmaceutical carrier or excipient; preferably, the pharmaceutical composition is a capsule, a tablet, a granule, a powder, a suspension, a solution formulation or a lozenge; or preferably, the pharmaceutical composition is administered by an oral or injectable route.

4. A tizoxanide choline salt of formula (I), ##STR00009## preferably, the tizoxanide choline salt of formula (I) is in an amorphous form or in a crystalline form, preferably in a crystalline form; preferably, the tizoxanide choline salt of formula (I) is in a crystalline form having characteristic peaks at the following 2θ angles (°) in a powder X-ray diffraction pattern obtained using Cu-Kα radiation: 9.92±0.10, 15.10±0.10, 19.72±0.10, 20.69±0.10 and 25.66±0.10, wherein the peak at 9.92±0.10 is the strongest peak; preferably, the tizoxanide choline salt of formula (I) is in a crystalline form having characteristic peaks at the following 2θ angles (°) in a powder X-ray diffraction pattern obtained using Cu-Kα radiation: 9.92±0.10, 15.10±0.10, 16.16±0.10, 19.72±0.10, 19.88±0.10, 20.69±0.10, 25.66±0.10 and 27.59±0.10, wherein the peak at 9.92±0.10 is the strongest peak; preferably, the tizoxanide choline salt of formula (I) is in a crystalline form having characteristic peaks at the following 2θ angles (°) in a powder X-ray diffraction pattern obtained using Cu-Kα radiation: 9.92±0.10, 11.78±0.10, 15.10±0.10, 16.16±0.10, 19.72±0.10, 19.88±0.10, 20.69±0.10, 25.66±0.10, 27.59±0.10 and 28.88±0.10, wherein the peak at 9.92±0.10 is the strongest peak; preferably, the tizoxanide choline salt of formula (I) is in a crystalline form having a powder X-ray diffraction pattern substantially as shown in the figure.

5. A crystalline form of a tizoxanide choline salt of formula (I), ##STR00010## the crystalline form has characteristic peaks at the following 2θ angles (°) in a powder X-ray diffraction pattern obtained using Cu-Kα radiation: 9.92±0.10, 15.10±0.10, 19.72±0.10, 20.69±0.10 and 25.66±0.10, wherein the peak at 9.92±0.10 is the strongest peak; preferably, the crystalline form has characteristic peaks at the following 2θ angles (°) in a powder X-ray diffraction pattern obtained using Cu-Kα radiation: 9.92±0.10, 15.10±0.10, 16.16±0.10, 19.72±0.10, 19.88±0.10, 20.69±0.10, 25.66±0.10 and 27.59±0.10, wherein the peak at 9.92±0.10 is the strongest peak; preferably, the crystalline form has characteristic peaks at the following 2θ angles (°) in a powder X-ray diffraction pattern obtained using Cu-Kα radiation: 9.92±0.10, 11.78±0.10, 15.10±0.10, 16.16±0.10, 19.72±0.10, 19.88±0.10, 20.69±0.10, 25.66±0.10, 27.59±0.10 and 28.88±0.10, wherein the peak at 9.92±0.10 is the strongest peak; preferably, the crystalline form has a powder X-ray diffraction pattern substantially as shown in the figure.

6-10. (canceled)

11. A medicament comprising the pharmaceutical composition of claim 1, wherein the medicament is effective in inhibiting growth of hepatitis virus, respiratory virus, bacteria, tumor, fibrosis, and/or intestinal parasite.

12. The medicament of claim 11, wherein the hepatitis virus is a hepatits B virus or a hepatitis C virus, the respiratory virus is influenza virus or coronavirus, the bacteria is helicobacter pylori or tubercle bacillus, the tumor is ovarian cancer or colon cancer, the fibrosis is hepatic fibrosis or pulmonary fibrosis, and the intestinal parasite is -cryptosporidium parvum medicament or giardia lamblia.

13. A medicament comprising the tizoxanide choline salt of formula (I) of claim 4.

14. The medicament of claim 13, wherein the hepatitis virus is a hepatits B virus or a hepatitis C virus, the respiratory virus is influenza virus or coronavirus, the bacteria is helicobacter pylori or tubercle bacillus, the tumor is ovarian cancer or colon cancer, the fibrosis is hepatic fibrosis or pulmonary fibrosis, and the intestinal parasite is -cryptosporidium parvum medicament or giardia lamblia.

15. A medicament comprising the crystalline form of the tizoxanide choline salt of formula (I) of claim 5.

16. The medicament of claim 15, wherein the hepatitis virus is a hepatits B virus or a hepatitis C virus, the respiratory virus is influenza virus or coronavirus, the bacteria is helicobacter pylori or tubercle bacillus, the tumor is ovarian cancer or colon cancer, the fibrosis is hepatic fibrosis or pulmonary fibrosis, and the intestinal parasite is -cryptosporidium parvum medicament or giardia lamblia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG. 1 shows a powder X-ray diffraction pattern of tizoxanide choline salt of formula (I) determined in Example 3, wherein the abscissa is 2θ angles (°) and the ordinate is the intensity of the absorption peak.

DETAILED DESCRIPTION

[0059] The technical solutions of the present disclosure will be further illustrated in detail with reference to the following specific examples. It should be understood that the following examples are merely exemplary illustration and explanation of the present disclosure, and should not be construed as limiting the protection scope of the present disclosure. All techniques implemented based on the content of the present disclosure described above are encompassed within the protection scope of the present disclosure.

[0060] Unless otherwise stated, the starting materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

[0061] Unless otherwise specified, “%” in the examples is defined as mass percentage.

[0062] The tizoxanide choline salt of formula (I) can be prepared according to the following synthetic route:

##STR00005##

##STR00006##

##STR00007##

[0063] Nitazoxanide is hydrolyzed under the action of hydrochloric acid to remove acetyl to obtain deacetylated nitazoxanide, namely tizoxanide; tizoxanide is reacted with choline hydroxide to obtain the tizoxanide choline salt of formula (I).

Example 1: Preparation of Tizoxanide

[0064] To a 500 mL three-necked flask was added 300 mL of concentrated hydrochloric acid, followed by addition of 50 g of nitazoxanide under stirring; after the addition was completed, the reaction mixture was stirred and reacted at 50° C. for 20 h, cooled to room temperature, and filtered, and the filter cake was washed with a small amount of water and acetone in sequence, filtered under suction to dryness, and dried at 45° C. to obtain 42.6 g of light yellow powder, namely tizoxanide.

Example 2: Preparation of Tizoxanide Choline Salt of Formula (I)

[0065] To a 1000 mL three-necked flask was added 500 mL of methanol, followed by addition of 50 g of tizoxanide under stirring, and the mixture was stirred homogeneously; the reaction mixture was stirred at room temperature, and added dropwise with 57.2 g of 40% aqueous choline hydroxide solution; after the addition was completed, the system was warmed to 45° C. and stirred and reacted for 1 h, and filtered while hot. The filtrate was placed at -5° C. overnight and then filtered. The filter cake was rinsed with 100 mL of methanol, filtered under suction to dryness, and dried to obtain 49.03 g of yellow crystalline powder (purity: 98%), namely the tizoxanide choline salt of formula (I). δ (ppm, DMSO-d.sub.6, 600 MHz) of the tizoxanide choline salt of formula (I) in .sup.1H NMR spectroscopy: 3.10 (9H, s); 3.38-3.40 (2H, dd); 3.81-3.85 (2H, m); 5.26-5.27 (1H, t); 6.79-6.82 (2H, m); 7.29-7.32 (1H, m); 7.90-7.91 (1H, m); 8.50 (1H, s); 14.70 (1H, s).

Example 3: Characterization of Crystalline Form

[0066] The crystalline form of tizoxanide choline salt of formula (I) prepared in Example 2 was characterized by powder X-ray diffraction.

[0067] Test conditions and method for powder X-ray diffraction were as follows: the instrument model was DMAX-2500; the experimental method was as follows: the test sample was ground into 200-300 meshes, with the scanning angle range being 3.0-60.0 degrees and the scanning rate being 0.15 degree/second for counting.

[0068] FIG. 1 showed a powder X-ray diffraction pattern of tizoxanide choline salt of formula (I), wherein the abscissa was 2θ angles and the ordinate was the intensity of the absorption peak. The characterization results were as follows: the tizoxanide choline salt of formula (I) had characteristic peaks at the following 2θ angles (°) in the X-ray powder diffraction pattern: 9.92±0.10, 11.78±0.10, 15.10±0.10, 16.16±0.10, 19.72±0.10, 19.88±0.10, 20.69±0.10, 25.66±0.10, 27.59±0.10 and 28.88±0.10, wherein the peak at 9.92±0.10 was the strongest peak.

Example 4: Pharmacokinetic Evaluation of Drug in Rat by Oral Administration

[0069] 34.75 mg of nitazoxanide was added to 10 mL of 0.5% sodium carboxymethylcellulose solution, and the mixture was mixing homogeneously for later use;

[0070] 41.67 mg of tizoxanide choline salt of formula (I) prepared in Example 2 was added to 10 mL of 0.5% sodium carboxymethylcellulose solution, and the mixture was mixed homogeneously for later use;

[0071] 30 mg of tizoxanide was added to 5 mL of 0.5% sodium carboxymethylcellulose solution, followed by addition of 40% aqueous choline hydroxide solution, and the mixture was mixed homogeneously, and then added with 0.5% sodium carboxymethyl cellulose solution to a final volume of 10 mL to obtain a mixture of tizoxanide and choline for later use. The addition amount of the aqueous choline hydroxide solution was adjusted to obtain mixtures of tizoxanide and choline with molar ratios of tizoxanide to choline hydroxide of 1:2, 1:0.5 and 1:1, respectively.

[0072] Provided were male SD rats weighing 180 g to 220 g, 3 rats each group; all animals were fasted for 12 h prior to administration. The rats were separately administered intragastrically with the solution of the nitazoxanide, the solution of the tizoxanide choline salt of formula (I) or the mixture of tizoxanide and choline described above at a dose of 10 mL/kg each. Blood was collected via jugular veins at 0.2 mL/time prior to administration and 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h after administration. The collected blood was added with sodium heparin for anticoagulation, followed by centrifugation to separate plasma, and the plasma was storing at -80° C. for determination. The content of tizoxanide in plasma was determined by LC-MS/MS (instrument: LC-MS/MS-19, TQ5500 Triple quad); internal Standard (IS) was tolbutamide; mass spectrum conditions: an electrospray ion source and a negative ion multiple reaction detection (Tizoxanide: Q1/Q3 Masses: 263.80/217.00 Da, and internal standard: Q1/Q3 Masses: 269.20/106.10 Da); liquid phase chromatography conditions: mobile phase A was 0.3% aqueous formic acid solution, and mobile phase B was 0.1% formic acid in acetonitrile; chromatographic column: ACQUITY UPLC CORTECS T3, 2.7 .Math.m (2.1 × 100 mm); column temperature was 40° C.; flow rate was 0.6 mL/min.

[0073] The pharmacokinetic parameters were calculated by utilizing WinNonlin according to the plasma concentration data at different time points. The results are shown in Table 1:

TABLE-US-00001 Results of pharmacokinetic evaluation of drug in rat by oral administration Group T.sub.½ (h) T.sub.max (h) C.sub.max (ng/mL) AUC.sub.(0-t) (h*ng/mL) AUC.sub.(0-∞) (h*ng/mL) Nitazoxanide 0.65 2.00 380.55 1325.50 1327.91 Mixture of tizoxanide + choline (molar ratio 1:2) 0.69 1.00 909.94 2490.09 2492.64 Mixture of tizoxanide + choline (molar ratio 1:0.5) 1.10 2.33 587.30 1739.98 1741.39 Mixture of tizoxanide + choline (molar ratio 1:1) 1.68 1.08 602.42 2478.67 2197.56 Tizoxanide choline salt of formula (I) 2.53 0.25 837.16 2070.01 2280.30

Example 5: Evaluation of In Vivo Anti-Hepatitis B Virus Effect

[0074] The sheld ducks (Tadorna) with vertical transmission infection and positive DHBV DNA were randomly grouped, 8 sheld ducks each group. The test compounds, i.e., the tizoxanide choline salt of formula (I) prepared in Example 2 and nitazoxanide, were each formulated into suspensions with 0.1% sodium carboxymethylcellulose, and administered intragastrically once every day. Blood was collected intravenously prior to administration (T.sub.0) and 7 days (T.sub.7), 14 days (T.sub.14), 21 days (T.sub.21) and 28 days (T.sub.28) after administration, and the serum was separated and frozen at -70° C. for determination.

[0075] Determination of DHBV DNA content in serum by using dot-blot molecular hybridization was as follows: the duck serum described above was taken, spots were simultaneously formed on detection films for each batch, the change of the DHBV DNA level in the duck serum was determined, the DHBV DNA probe was labeled by .sup.32P according to the method of the instruction of the nick translation kit, and a duck serum dot-blot experiment was performed. After the autoradiography, the spectral absorbance (filter wavelength was 490 nm) OD.sub.490 of the spots on the autoradiography X-film was determined with an enzyme-linked immunoassay instrument, a higher OD.sub.490 value indicates a higher virus content.

[0076] The determination results are shown in Table 2:

TABLE-US-00002 Results of evaluation of in vivo anti-hepatitis B virus effect Group Dose (mg/kg, active ingredie nt basis) OD.sub.490 value T.sub.0 T.sub.7 T.sub.14 T.sub.21 T.sub.28 Vehicle group 1.59±0.17 1.62±0.14 1.73±0.20 1.86±0.60 1.83±0.23 Tizoxanide choline salt 200 0.96±0.47 0.15±0.08**.sub.ΔΔ 0.10±0.05**.sub.Δ.sub.Δ 0.07±0.04**.sub.Δ.sub.Δ 0.27±0.17**.sub.Δ.sub.Δ of formula (I) 100 1.47±0.20 0.43±0.18**.sub.ΔΔ 0.39±0.34**.sub.Δ.sub.Δ 0.34±0.25**.sub.Δ.sub.Δ 0.53±0.37**.sub.Δ.sub.Δ Nitazoxanid e 200 1.67±0.11 1.37±0.27 1.19±0.51 0.72±0.38**.sub.Δ.sub.Δ 0.72±0.48**.sub.Δ.sub.Δ Note: the drug groups administered at different time points were compared with those groups prior to administration (T.sub.0): **P < 0.01, *P < 0.05; The drug groups and the control group were compared at the same time points of administration: .sub.ΔΔP < 0.01, .sub.ΔP < 0.05.

Example 6: Evaluation of Anti-Influenza Virus Effect in Mouse Model

[0077] Male Kunming mice weighing 18 g to 22 g were randomly divided into a normal group, a model group and an administration group, 10 mice each group. The mice in the model group and the administration group were infected with influenza A virus mouse lung adaptive strain A/PR/8/34 via nasal instillation at 10 folds of LD.sub.50 dose; the test compounds, i.e., nitazoxanide and the tizoxanide choline salt of formula (I) prepared in Example 2, were each formulated as suspensions with 0.1% sodium carboxymethylcellulose, and administered intragastrically 1 hour after infection, 2 times every day, for 5 consecutive days. The mice were observed for mortality and recorded for 14 consecutive days for examination of their survival rates.

[0078] The experimental results are shown in Table 3:

TABLE-US-00003 Protective effect of target compounds on influenza virus infected mice Drug Dose (mg/kg, active ingredient basis) Survival rates (%) Normal group 0 10/10 Nitazoxanide 100 7/10 50 2/10 Tizoxanide choline salt of formula (I) 50 9/10 25 3/10

[0079] The embodiments of the present disclosure have been described above. However, the present disclosure is not limited thereto. Any modification, equivalent, improvement and the like made without departing from the spirit and principle of the present disclosure shall fall within the protection scope of the present disclosure.