CRYSTAL OF POLYMYXIN B1 SULFATE, POLYMYXIN B2 SULFATE OR THEIR MIXTURE AND PREPARATION METHOD THEREOF

Abstract

The present invention provides an anhydrous crystal of polymyxin B1 sulfate, polymyxin B2 sulfate or a mixture thereof and a preparation method thereof. The preparation method comprises using an organic solvent to precipitate a solid from a saturated solution of polymyxin B1 sulfate, polymyxin B2 sulfate or a mixture thereof, drying it under vacuum to obtain an anhydrous crystal of polymyxin B1 sulfate, polymyxin B2 sulfate or a mixture thereof.

Claims

1. An anhydrous crystal 1 of polymyxin B1 sulfate, characterized in that, said anhydrous crystal 1 has an X-ray powder diffraction pattern having diffraction peaks at 3.396, 4.895 and 6.903 expressed by 2θ degree using Cu-Ka radiation; and preferably, said crystal 1 has an X-ray powder diffraction pattern expressed by 2θ degree using Cu-Ka radiation as shown in FIG. 2A; preferably, said anhydrous crystal 1 has an infrared absorption spectrum having characteristic bands at 1071.93 cm.sup.−1, 1242.91 cm.sup.−1, 1384.25 cm.sup.−1, 1457.69 cm.sup.−1, 1524.29 cm.sup.−1, 1639.38 cm.sup.−1, 2957.69 cm.sup.−1, 3064.55 cm.sup.−1 and 3270.53 cm.sup.−1 as measured by KBr tableting method; and more preferably, said anhydrous crystal 1 has an infrared absorption spectrum as measured by KBr tableting method as shown in FIG. 3A; and more preferably, said anhydrous crystal 1 has a melting point of 226.97° C., and has a differential scanning calorimetry pattern as shown in FIG. 4.

2. An anhydrous crystal A of polymyxin B1 sulfate, characterized in that, said anhydrous crystal A has an X-ray powder diffraction pattern having a diffraction peak at 3.401 expressed by 2θ degree using Cu-Ka radiation; and preferably, said anhydrous crystal A has an X-ray powder diffraction pattern expressed by 2θ degree using Cu-Ka radiation as shown in FIG. 8A; preferably, said anhydrous crystal A has an infrared absorption spectrum having characteristic bands at 1071.93 cm.sup.−1, 1242.91 cm.sup.−1, 1384.25 cm.sup.−1, 1457.69 cm.sup.−1, 1524.29 cm.sup.−1, 1639.38 cm.sup.−1, 2957.69 cm.sup.−1, 3064.55 cm.sup.−1 and 3270.53 cm.sup.−1 as measured by KBr tableting method; and more preferably, said anhydrous crystal A has an infrared absorption spectrum as measured by KBr tableting method as shown in FIG. 9A; and more preferably, said anhydrous crystal A has a melting point of 225.00° C., and a differential scanning calorimetry pattern as shown in FIG. 10.

3. An anhydrous crystal of polymyxin B2 sulfate, characterized in that, said anhydrous crystal of polymyxin B2 sulfate has an X-ray powder diffraction pattern expressed by 2θ degree using Cu-Ka radiation as shown in FIG. 17A.

4. A method for preparing an anhydrous crystal of polymyxin B1 sulfate, polymyxin B2 sulfate or a mixture thereof, said method comprises the following steps of: (1) adding water to polymyxin B1 sulfate, polymyxin B2 sulfate or a mixture thereof to just completely dissolve the solid to obtain a saturated solution; (2) slowly adding an organic solvent dropwise into said saturated solution, or slowly adding said saturated solution dropwise into an organic solvent at a controlled temperature within the range of 0-60° C. to precipitate a solid; wherein, said organic solvent is selected from one or more of C1-C4 alcohol, C3-C4 ketone, ethyl acetate or butyl acetate; preferably, said C1-C4 alcohol is selected from one or more of methanol, ethanol, isopropanol, n-propanol or n-butanol; and still preferably, said C3-C4 ketone is selected from one or more of acetone or 2-butanone; and (3) filtering off the solid and drying it under vacuum to obtain an anhydrous crystal of polymyxin B1 sulfate, polymyxin B2 sulfate or a mixture thereof.

5. A method for preparing the anhydrous crystal 1 of polymyxin B1 sulfate according to claim 1, said method comprises the following steps of: (1) adding water to polymyxin B1 sulfate to just completely dissolve the solid to obtain a saturated solution; (2) slowly adding an organic solvent dropwise into said saturated solution, or slowly adding said saturated solution dropwise into an organic solvent at a controlled temperature within the range of 0-60° C. to precipitate a solid; wherein, said organic solvent is selected from n-butanol, isopropanol, n-propanol, or 2-butanol; and (3) filtering off the solid and drying it under vacuum to obtain an anhydrous crystal 1 of polymyxin B1 sulfate.

6. A method for preparing the anhydrous crystal A of polymyxin B1 sulfate according to claim 2, said method comprises the following steps of: (1) adding water to polymyxin B1 sulfate to just completely dissolve the solid to obtain a saturated solution; (2) slowly adding an organic solvent dropwise into said saturated solution, or slowly adding said saturated solution dropwise into an organic solvent at a controlled temperature within the range of 0-60° C. to precipitate a solid; wherein, said organic solvent is selected from ethanol, ethanol-n-butanol, n-butanol-isopropanol, methanol, acetone, butanone, or ethanol-ethyl acetate; and (3) filtering off the solid and drying it under vacuum to obtain an anhydrous crystal A of polymyxin B1 sulfate.

7. The method according to any one of claims 4 to 6, characterized in that, in step (1), after adding water to polymyxin B1 sulfate, polymyxin B2 sulfate or a mixture thereof, the solid is just completely dissolved by heating it at a temperature below 60° C.; preferably, in step (2), said organic solvent is used in an amount of 0.5-20 volumes in terms of the volume of said saturated solution; and more preferably, in step (2), after the solid is precipitated, stirring is continued for 0-8 hours.

8. A pharmaceutical composition comprising the anhydrous crystal 1 of polymyxin B1 sulfate according to claim 1, the anhydrous crystal A of polymyxin B1 sulfate according to claim 2, the anhydrous crystal of polymyxin B2 sulfate according to claim 3, or the anhydrous crystal of polymyxin B1 sulfate, polymyxin B2 sulfate or a mixture thereof prepared according to the method according to any one of claims 4 to 7.

9. The pharmaceutical composition according to claim 8, comprising a pharmaceutically acceptable carrier or excipient, and optionally an antibacterial active ingredient other than the anhydrous crystal of polymyxin B1 sulfate, polymyxin B2 sulfate or a mixture thereof.

10. Use of the anhydrous crystal 1 of polymyxin B1 sulfate according to claim 1, the anhydrous crystal A of polymyxin B1 sulfate according to claim 2, the anhydrous crystal of polymyxin B2 sulfate according to claim 3, or the anhydrous crystal of polymyxin B1 sulfate, polymyxin B2 sulfate or a mixture thereof prepared according to the method according to any one of claims 4 to 7 in the preparation of a medicament for preventing and/or treating a disease caused by bacteria, in particular Gram-negative bacteria.

11. The use according to claim 10, wherein said disease is selected from various infections caused by Gram-negative bacteria, in particular Pseudomonas aeruginosa and Escherichia coli; and preferably, said disease is respiratory system infection, peritonitis, bile duct infection, urinary tract infection, burn infection, corneal infection and sepsis.

12. A method for preventing and/or treating a disease caused by bacteria, in particular Gram-negative bacteria, comprising administering to a subject a prophylactically and/or therapeutically effective amount of the anhydrous crystal 1 of polymyxin B1 sulfate according to claim 1, the anhydrous crystal A of polymyxin B1 sulfate according to claim 2, the anhydrous crystal of polymyxin B2 sulfate according to claim 3, or the anhydrous crystal of polymyxin B1 sulfate, polymyxin B2 sulfate or a mixture thereof prepared according to the method according to any one of claims 4 to 7.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1A shows the crystal of polymyxin B1 sulfate in an ethanol-n-butanol aqueous solution;

[0040] FIG. 1B shows a sample of crystal of polymyxin B1 sulfate after drying;

[0041] FIG. 2A is an XRD pattern of the crystal 1 of polymyxin B1 sulfate of the present invention;

[0042] FIG. 2B is a peak list of the XRD pattern of the crystal 1 of polymyxin B1 sulfate of the present invention;

[0043] FIG. 2C is a hot stage XRD pattern of the crystal 1 of polymyxin B1 sulfate of the present invention;

[0044] FIG. 3A is an IR spectrum of the crystal 1 of polymyxin B1 sulfate of the present invention;

[0045] FIG. 3B is a peak list of the IR spectrum of the crystal 1 of polymyxin B1 sulfate of the present invention;

[0046] FIG. 4 is a DSC pattern of the crystal 1 of polymyxin B1 sulfate of the present invention;

[0047] FIG. 5 is a DVS pattern of the crystal 1 of polymyxin B1 sulfate of the present invention;

[0048] FIG. 6 is a TGA spectrum of the crystal 1 of polymyxin B1 sulfate of the present invention;

[0049] FIG. 7 is an isotherm diagram of the crystal 1 of polymyxin B1 sulfate of the present invention;

[0050] FIG. 8A is an XRD pattern of the crystal A of polymyxin B1 sulfate of the present invention;

[0051] FIG. 8B is a peak list of the XRD pattern of the crystal A of polymyxin B1 sulfate of the present invention;

[0052] FIG. 8C is a hot stage XRD pattern of the crystal A of polymyxin B1 sulfate of the present invention;

[0053] FIG. 9A is an IR spectrum of the crystal A of polymyxin B1 sulfate of the present invention;

[0054] FIG. 9B is a peak list of the IR spectrum of the crystal A of polymyxin B1 sulfate of the present invention;

[0055] FIG. 10 is a DSC pattern of the crystal A of polymyxin B1 sulfate of the present invention;

[0056] FIG. 11 is a DVS pattern of the crystal A of polymyxin B1 sulfate of the present invention;

[0057] FIG. 12 is a TGA spectrum of the crystal A of polymyxin B1 sulfate of the present invention;

[0058] FIG. 13 is an isotherm diagram of the crystal A of polymyxin B1 sulfate of the present invention;

[0059] FIG. 14 is an XRD pattern of an initial mixed sample of polymyxin B1 sulfate crystal 1 and crystal A in a crystal form competitive experiment of the present invention;

[0060] FIG. 15 is an XRD pattern of the polymyxin B1 sulfate crystal 1 and crystal A of the present invention in ethanol in a competitive experiment;

[0061] FIG. 16 is an XRD pattern of the polymyxin B1 sulfate crystal 1 and crystal A of the present invention in ethanol/water mixed solvent (v/v=5/1) in a competitive experiment;

[0062] FIG. 17A is an XRD pattern of the crystal of polymyxin B2 sulfate of the present invention; and

[0063] FIG. 17B is a peak list of the XRD pattern of the crystal of polymyxin B2 sulfate of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0064] The present invention will be further described in conjunction with the accompanying drawings and the embodiments to achieve better illustration of the present invention and to facilitate understanding of the technical solution of the present invention. It is to be understood that the specific examples of the present invention are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1

[0065] Ethanol-water system: 100 ml aqueous solution of polymyxin B1 sulfate at a concentration of 50 g/L was taken and stirred at room temperature, to which was added 3 volumes of a 95% (v/v) ethanol aqueous solution to precipitate a polymyxin B1 sulfate crystal. After stirring at a temperature of 0-5° C. for 3-5 h, the mixture was filtered, and dried under vacuum for 8 hours to obtain a crystalline polymyxin B1 sulfate, and the crystal form was crystal form A.

Example 2

[0066] Isopropanol-water: 26 ml aqueous solution of polymyxin B1 sulfate and polymyxin B2 sulfate at a concentration of 50 g/L was taken, and slowly added dropwise to 260 ml (10 volumes) isopropanol under stirring. The temperature of isopropanol was always controlled within the range of 5-10° C. The crystals of polymyxin B1 sulfate and polymyxin B2 sulfate precipitated during the dropwise adding gradually increased, which was in a uniform dispersion state without adhesion. Stirring was continued for 1 hour, the mixture was filtered to obtain a solid, which was dried under vacuum for 10 hours to obtain a crystal powder of polymyxin B1 sulfate and polymyxin B2 sulfate (1.23 g), crystal yield: 94.6%. The crystal form of polymyxin B1 sulfate was crystal form 1.

Example 3

[0067] N-butanol-water: 13.5 g polymyxin B2 sulfate was dissolved in 150 ml pure water, and slowly added dropwise to 1500 ml (10 volumes) n-butanol under stirring with the temperature being always controlled within the range of 25-30° C. The crystal of polymyxin B2 sulfate precipitated during the dropwise adding gradually increased, which was in a uniform dispersion state without adhesion and had a good granularity. After the dropwise addition of the aqueous solution of polymyxin B2 sulfate was completed, stirring was continued for 30 min, the mixture was filtered, and the filtered solid was dried under vacuum for 20 hours to obtain a crystal powder of polymyxin B2 sulfate (13.2 g), crystal yield: 97.8%.

[0068] From the XRD pattern of polymyxin B2 sulfate and polymyxin B1 sulfate, it can be seen that in the XRD pattern of polymyxin B2 sulfate, a weak small peak appears at 2θ=31.8°.

Example 4

[0069] Ethanol-n-butanol-water: 100 ml aqueous solution of polymyxin B1 sulfate at a concentration of 50 g/L was taken and slowly added dropwise to 1000 ml (10 volumes) ethanol (500 ml)+n-butanol (500 ml) under stirring, and the temperature during the dropwise adding was always controlled within the range of 0-5° C. The crystal of polymyxin B1 sulfate precipitated during the dropwise adding gradually increased, which was in a uniform dispersion state and had a good granularity. After the dropwise addition of the aqueous solution of polymyxin B1 sulfate was completed, stirring was continued for 2 hours, a solid was filtered off and was dried under vacuum for 5 hours to obtain a crystal powder of polymyxin B1 sulfate (4.6 g), crystal yield: 92.0%. The crystal form was crystal form A.

Example 5

[0070] Ethanol-isopropanol-water: 10 ml aqueous solution of polymyxin B1 sulfate and polymyxin B2 sulfate at a concentration of 50 g/L was taken and slowly added dropwise to 100 ml (10 volumes) ethanol (50 ml)+isopropanol (50 ml) under stirring, and the temperature during the dropwise adding was always controlled within the range of 15-20° C. The crystal of polymyxin B1 sulfate and polymyxin B2 sulfate precipitated during the dropwise adding gradually increased, which was in a uniform dispersion state and had a granularity, with solid-liquid separation being quickly achieved after standing (30 min). After the dropwise addition of the aqueous solution of polymyxin B1 sulfate and polymyxin B2 sulfate was completed, stirring was continued for 30 min, a solid was filtered off and was dried under vacuum for 15 hours to obtain a crystal powder of polymyxin B1 sulfate and polymyxin B2 sulfate (0.43 g), crystal yield: 86.0%. The crystal form was crystal form A.

Example 6

[0071] N-butanol-isopropanol-water: 50 ml aqueous solution of polymyxin B1 sulfate at a concentration of 50 g/L was taken and slowly added dropwise to 250 ml (5BV) n-butanol (125 ml)+isopropanol (125 ml) under stirring at a controlled temperature within the range of 25-30° C. After the crystal precipitated, the remaining solution was added completely to obtain a crystal, which was in a uniform dispersion state and had a good granularity, with solid-liquid separation being quickly achieved after standing (30 min). After the dropwise addition of the aqueous solution of polymyxin B1 sulfate was completed, stirring was continued for 60 min, a solid was filtered off and was dried under vacuum for 8 hours to obtain a crystal powder of polymyxin B1 sulfate (2.3 g), crystal yield: 92.0%. The crystal form was crystal form 1.

Example 7

[0072] Characterization and analysis of crystal form 1 and crystal form A of polymyxin B1 sulfate

[0073] Crystal Form 1 of Polymyxin B1 Sulfate

TABLE-US-00001 Characterization mode Characterization results and analysis XRD Weak crystalline state, numbered as crystal form 1 Hot stage XRD The crystal form has not changed when heating up to 170° C. DSC/TGA DSC pattern shows that the sample of crystal form 1 has a broad endothermic peak at 50-200° C., which is due to the removal of the surface solvent, and the melting point is 226.97° C., accompanied by decomposition; TGA spectrum shows a 16% slow weight loss before 150° C., which was confirmed to be a surface solvent in conjunction with hot stage XRD, and the decomposition temperature was 222° C. DVS Weight change between 0% RH-80% RH is about 15.9%, with an extremely strong hygroscopicity. FTIR The absorption peaks in detail are shown in FIG. 3A. HPLC The purity is 98.43%. Description of crystal form Property of Anhydrate crystal form Preparation It can be obtained by crystal mushing a crystal form A of polymyxin B1 sulfate in a mixed solution of isopropyl alcohol and water or a mixed solution of n-propanol and water, or it can be obtained by crystal mushing a crystal form A of polymyxin B1 sulfate at a high temperature in butanol or 2-butanol solution. Typical 50 mg crystal form A of polymyxin B1 sulfate was taken and added preparation into 1 mL mixed solvent of isopropanol/water = 4/1 to crystal mush method at room temperature for more than 5 days and centrifuge. Stability Unstable, the crystal form changed to crystal form A when placed evaluation in a dry state at room temperature for 3 days.

[0074] Crystal Form A of Polymyxin B1 Sulfate

TABLE-US-00002 Characterization mode Characterization results and analysis XRD Weak crystalline state, numbered as crystal form A Hot stage XRD The crystal form has not changed when heating up to 150° C. and keeping for 5 min. DSC/TGA DSC pattern shows that the sample of crystal form A has a broad endothermic peak before 200° C., which is due to the removal of the surface solvent, and the melting point is 225.00° C., accompanied by decomposition; TGA spectrum shows an about 11.8% slow weight loss before 150° C., which was confirmed to be a surface solvent in conjunction with hot stage XRD, and the decomposition temperature was 213° C. DVS/Isothermal Weight change between 0% RH-80% RH is about 17.3%, with an adsorption extremely strong hygroscopicity. diagram FT-IR The absorption peaks in detail are shown in FIG. 9A. HPLC The purity is 98.43%.

Example 8

[0075] A competitive experiment between crystal form 1 and crystal form A of polymyxin sulfate was carried out to investigate crystal form stability of the two crystal forms in the solvent, water and ethanol, which are commonly used in the preparation, and to evaluate the conversion of the two crystal forms, in order to provide reference for subsequent preparation operations such as granulation. In this experiment, a mixed solvent of ethanol and ethanol/water=5/1 (v/v) was used as the solvent for the competitive experiment, to confirm the most stable crystal form at room temperature in the corresponding solvent in combination with crystal mush test at room temperature.

[0076] Crystal Form Competitive Experiment

[0077] 1. Equal amounts of samples of crystal form 1 and form A were taken and mixed uniformly to obtain an initial mixed sample, which was used for XRD detection (FIG. 14).

[0078] 2. The uniformly-mixed sample was divided into two portions, to one of which was added 200 μL ethanol, and to another of which was added 300 μL mixed solvent of ethanol/water=5/1 (v/v), to form a suspension. The suspension was stirred at room temperature, and centrifuged and sampled at different times for XRD detection. The results showed that after crystal mushing in ethanol at room temperature for one day, the crystal form converted into crystal form A (FIG. 15), and after crystal mushing in ethanol/water at room temperature for six days, the crystal form converted into crystal form A (FIG. 16).

[0079] According to the results of this competitive experiment, it was confirmed that among the two crystal forms of polymyxin B1 sulfate, the most stable crystal form in ethanol and in a mixed solvent of ethanol/water at room temperature is crystal form A.

[0080] According to the results of crystal mush experiment at room temperature, it was confirmed that the most stable crystal form of polymyxin B1 sulfate in ethanol and in a mixed solvent of ethanol/water at room temperature is crystal form A.

[0081] Based on the above results, when polymyxin B1 sulfate is handled at room temperature, polymyxin B1 sulfate can be stably present as the crystal form A using ethanol or a mixed solvent of ethanol/water.