Composition composed of ceftriaxone sodium and sulbactum sodium

Abstract

The present disclosure provides a composition consisting of ceftriaxone sodium and sulbactam sodium, a pharmaceutical formulation comprising the same and the application thereof. The composition is characterized by having an X-ray powder diffraction pattern with peaks at specific angles. The pharmaceutical formulation according to the present disclosure have better antibacterial activity and stability compared with known compositions, and are thus very suitable for the treatment of bacterial infections, especially for the treatment of refractory urogenital system infections caused by Neisseria gonorrhoeae which has drug-resistance to a variety of antibiotics (-lactams, tetracyclines, macrolides, fluoroquinolones and aminoglycosides).

Claims

1. A crystalline composition consisting essentially of ceftriaxone sodium and sulbactam sodium and having an X-ray powder diffraction pattern with peaks at 2-values of 11.20.2, 14.30.2, 17.80.2, 19.30.2, 21.20.2, 22.80.2 and 23.80.2.

2. The crystalline composition according to claim 1, wherein said composition further has an X-ray powder diffraction pattern with peaks at 2-values of 12.60.2, 16.70.2, 18.40.2, 20.00.2, 20.40.2 and 28.00.2.

3. The crystalline composition according to claim 1, wherein said composition further has an X-ray powder diffraction pattern with peaks at the following interplanar crystal spacing values of 7.90.2 , 6.20.2 , 5.00.2 , 4.60.2 , 4.20.2 , 3.90.2 and 3.70.2 .

4. The crystalline composition according to claim 3, wherein said composition further has an X-ray powder diffraction pattern with peaks at the following interplanar crystal spacing values of 7.10.2 , 5.30.2 , 4.80.2 , 4.40.2 , 4.30.2 and 3.20.2 .

5. The crystalline composition according to claim 1, wherein said composition has an infrared absorption spectrum with peaks at wavenumbers of 32555 cm.sup.1, 17425 cm.sup.1, 16045 cm.sup.1, 15395 cm.sup.1, 13985 cm.sup.1, 13025 cm.sup.1, 11985 cm.sup.1, 11245 cm.sup.1, 10325 cm.sup.1, 8975 cm.sup.1, 8045 cm.sup.1, 6005 cm.sup.1 and 4795 cm.sup.1.

6. The crystalline composition according to claim 3, wherein said composition further has an infrared absorption spectrum with peaks at wavenumbers of 32555 cm.sup.1, 17425 cm.sup.1, 16045 cm.sup.1, 15395 cm.sup.1, 13985 cm.sup.1, 13025 cm.sup.1, 11985 cm.sup.1, 11245 cm.sup.1, 10325 cm.sup.1, 8975 cm.sup.1, 8045 cm.sup.1, 6005 cm.sup.1 and 4795 cm.sup.1.

7. The crystalline composition according to claim 5, wherein said composition further has an infrared absorption spectrum with peaks at wavenumbers of 34415 cm.sup.1, 31165 cm.sup.1, 29385 cm.sup.1, 15005 cm.sup.1 and 10995 cm.sup.1.

8. The crystalline composition according to claim 1, wherein said composition has a differential scanning calorimetry pattern with an exothermic peak at 269.60.5 C.

9. The crystalline composition according to claim 1, wherein said composition is prepared by a process comprising the following steps of: providing ceftriaxone sodium raw material and sulbactam sodium raw material; separately grinding each of said raw materials to form particles having a median particle diameter of 25-88 m; and mixing said grinded raw materials to give said composition.

10. The crystalline composition according to claim 9, wherein said median particle diameter has a values of 25-47 m, 38-62 m, or 58-88 m.

11. A pharmaceutical formulation comprising the crystalline composition according to claim 1.

12. A pharmaceutical formulation comprising the crystalline composition according to claim 9.

13. The pharmaceutical formulation according to claim 11, wherein said pharmaceutical formulation is a powder for injection or an injection solution.

14. The pharmaceutical formulation according to claim 13, wherein said pharmaceutical formulation is a sterile powder for injection or a lyophilized powder for injection.

15. The pharmaceutical formulation according to claim 11, wherein said pharmaceutical formulation has a mass ratio of ceftriaxone-to-sulbactam of from 1:1 to 8:1.

16. The pharmaceutical formulation according to claim 15, wherein said pharmaceutical formulation has a mass ratio of ceftriaxone-to-sulbactam of from 1:1 to 4:1.

17. A method for treatment of a bacterial infection disease comprising administering a therapeutically effective amount of the pharmaceutical formulation according to claim 11 to a subject in need thereof.

18. The method according to claim 17, wherein said bacterial infection is caused by Neisseria gonorrhoeae having drug-resistance.

19. The method according to claim 18, wherein said drug-resistance is to a -lactam antibacterial drug, a tetracycline antibacterial drug, a macrolide antibacterial drug, a fluoroquinolone antibacterial drug or an aminoglycoside antibacterial drug.

20. The method according to claim 17, wherein said bacterial infection disease is a urogenital system infection disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the X-ray powder diffraction analysis spectrum for Lyophilized Powder for Injection A in Comparative Example 1.

(2) FIG. 2 shows the X-ray powder diffraction analysis spectrum for mixed powder in Comparative Example 2.

(3) FIG. 3 shows the X-ray powder diffraction analysis spectrum for Composition I in Example 1.

(4) FIG. 4 shows the infrared absorption analysis spectrum for Composition I in Example 1.

(5) FIG. 5 shows the X-ray powder diffraction analysis spectrum for Composition II in Example 2.

(6) FIG. 6 shows the infrared absorption analysis spectrum for Composition II in Example 2.

(7) FIG. 7 shows the X-ray powder diffraction analysis spectrum for Composition III in Example 3.

(8) FIG. 8 shows the infrared absorption analysis spectrum for Composition III in Example 3.

DETAILED DESCRIPTION

(9) Hereinafter, Comparative Examples, Examples and Test Examples are used to further explain the present disclosure, but are not intended to limit the technical solutions and the technical effects of the present disclosure.

(10) Explanations for the following Comparative Examples, Examples and Test Examples:

(11) (1) The amount of ceftriaxone sodium/ceftriaxone sodium crystals is calculated by ceftriaxone; the amount of sulbactam sodium/sulbactam sodium crystals is calculated by sulbactam, for example, take 500 g ceftriaxone sodium means that the amount of ceftriaxone in the taken ceftriaxone sodium is 500 g.

(12) (2) The crystal morphology is measured by X-ray powder diffraction method using BRUKER D8 ADVANCE X-ray powder diffraction instrument under the following measurement conditions: CuK radiation, tube voltage of 40 kV, scanning range of from 5 to 60, scanning speed of 17.7 s/step, step-length of 0.02.

(13) (3) In the infrared absorption spectrum analysis, the samples are pressed into tablets with KBr and then measured;

(14) (4) STA409 integrated thermal analyzer is purchased from a Germany company NETZSCH and is used in the differential scanning calorimetry method to perform measurement. Measurement conditions: heating rate of 10 K/min, flow rate of protective gas N.sub.2 of 30 mL/min, flow rate of purging gas N.sub.2 of 20 mL/min, temperature of from 35 to 300 C.

(15) (5) The crystallinity is measured using the US Pharmacopoeia USP35-NF30 method.

(16) (6) The content of impurity is measured using HPLC method according to the European Pharmacopoeia EP8.0 method.

(17) (7) The clarity of solutions is measured according to the method described in Chinese Pharmacopoeia 2015.

Comparative Example 1

(18) A lyophilized powder was prepared according to the following process. In a sterile, dark condition and under nitrogen stream, 100 g of ceftriaxone sodium, 50 g of sulbactam sodium and 25 g of mannitol were added into 90 ml of pretreated 70% ethanol solution which was cooled to below 10 C. and was placed in a non-metallic container, then dissolved completely with stirring. The obtained solution was adjusted to a pH of 6.5 with 0.1 mol/L aqueous solution of sodium bicarbonate, sterile-filtered, subpackaged, placed in a lyophilizer filled with inert gas for pre-freeze (from 48 C. to 20 C.), and then subjected to pressure reduction, sublimation and drying to give the lyophilized powder named Lyophilized Powder for Injection A. The Lyophilized Powder for Injection A was sampled and determined for crystal morphology, and the X-ray powder diffraction spectrum was showed in FIG. 1.

Comparative Example 2

(19) Ceftriaxone sodium was prepared according to the following process: condensing 7-ACT and AE-active ester to give crude product of ceftriaxone sodium; taking 5 g of the crude product and dissolving them by adding 40 ml of water; adding 4.1 g of sodium iso-octoate into the obtained solution at 13 C.; stirring until the solution was clear; adjusting the solution to a pH of 7.0 with 10% diluted hydrochloric acid; stirring for additional 30 minutes to give a mother liquor; adding anhydrous ethanol slowly into the mother liquor under stirring, until turbidity occurred; after standing for 25 minutes, slowly adding anhydrous ethanol into the solution until a large amount of crystals were formed, in which 150 ml of anhydrous ethanol was used in total; filtering the resulted crystals; washing the filter cake to neutral with a mixed solvent of anhydrous ethanol:water=4:1; washing with anhydrous ethanol again; and drying at 35 C. under ambient pressure.

(20) Sulbactam sodium was prepared according to the following process: taking crude sulbactam sodium; adding it into a mixed solvent of N,N-dimethylformamide and water (3:1) which was 5 times (ml/g) the amount of the crude sulbactam sodium; heating to 70 C.; stirring until the crude sulbactam sodium was completely dissolved; keeping the temperature; adjusting the solution to a pH of 5.5; and making the solution flow at a speed of 5 m/s through a direct current magnetic field of 0.5 T, the direction of which was perpendicular to the flow direction of the solution; after magnetic treatment, adding activated carbon in an amount 0.3 times (ml/g) of the mixed solvent into the solution to decolor; stirring for 30 minutes and filtering to give a clear solution; adding ethanol in an amount 5 times (ml/ml) of the mixed solvent into the clear solution; filtering to give the filter cake; washing the filter cake with distilled water 3 times; and then drying for 4 hours under reduced pressure to give sulbactam sodium.

(21) 1,000 g of ceftriaxone sodium and 500 g of sulbactam sodium prepared according to the above-mentioned method were taken, mixed well by a trough mixer under sterile condition to give a mixed powder, which was sampled and subjected to crystal morphology measurement. The X-ray powder diffraction spectrum was showed in FIG. 2. The mixed powder was subpackaged under a sterile condition to give a sterile powder for injection, named sterile powder for injection A.

Comparative Example 3

(22) 2,000 g of commercially available ceftriaxone sodium and 1,000 g of commercially available sulbactam sodium were taken, mixed well through a trough mixer under sterile condition to give a mixed powder, which was sampled and measured for crystal morphology. The X-ray powder diffraction spectrum pattern of the powder was similar to that of FIG. 2. The mixed powder was subpackaged under a sterile condition to give a sterile powder for injection, named sterile powder for injection B.

Example 1

(23) 1200 g of ceftriaxone sodium and 600 g of sulbactam sodium both prepared according to the method in Comparative Example 2 were taken and ground together by a ball mill until the particle size D.sub.50 was from 25 m to 47 m, and then placed in a single-cone ribbon mixer (HF1600 type) and mixed well, with rotating speed of 30 rpm and mixing for 25 minutes. After the operation was completed, the material was taken out and a crystalline substance was obtained and named Composition I.

(24) The Composition I was sampled and measured for crystal morphology. The X-ray powder diffraction spectrum pattern was showed in FIG. 3. The main data of the spectrum pattern were listed in Table 1.

(25) Its infrared absorption was measured by infrared absorption spectrum analysis. The spectrum pattern was showed in FIG. 4. The main data of the infrared absorption peaks were listed in Table 2.

(26) The Composition I was analyzed by the differential scanning calorimetry, and was found to have an exothermic peak at 269.51 C.

(27) TABLE-US-00001 TABLE 1 Main Data of X-ray Powder Diffraction Spectrum for Composition I Interplanar 2 Crystal Peak Peak No. Angle () Spacing() Height Aera 1 11.138 7.9372 1502 16530 2 12.521 7.0634 437 4363 3 14.289 6.1934 11014 68918 4 16.638 5.3238 1798 12883 5 17.835 4.9692 1569 11493 6 18.432 4.8096 883 7497 7 19.276 4.6008 2908 21910 8 19.978 4.4406 1034 12897 9 20.453 4.3386 1342 20418 10 21.171 4.1930 1651 17080 11 22.736 3.9079 1889 25822 12 23.769 3.7403 1299 15065 13 27.985 3.1856 2612 25393

(28) TABLE-US-00002 TABLE 2 Main Data of Infrared Absorption Spectrum for Composition I No. Wave Number(cm.sup.1) 1 3441 2 3254 3 3117 4 2937 5 1742 6 1605 7 1539 8 1501 9 1398 10 1302 11 1198 12 1124 13 1099 14 1032 15 897 16 804 17 600 18 480

Example 2

(29) Commercially available ceftriaxone sodium 500 g and sulbactam sodium 500 g were taken, and ground together by a ball mill until the particle size D.sub.50 was from 38 m to 62 m, then placed in a single-cone ribbon mixer (HF1600 type) and mixed well, with rotating speed of 40 rpm and mixing for 20 minutes. After the operation was completed, the material was taken out, and a crystalline substance was obtained and named Composition II.

(30) The Composition II was sampled and measured for crystal morphology. The X-ray powder diffraction spectrum was showed in FIG. 5. The main data of the spectrum pattern were listed in Table 3.

(31) Its infrared absorption was measured by infrared absorption spectrum analysis. The spectrum pattern was showed in FIG. 6. The main data of infrared absorption peaks were listed in Table 4.

(32) The Composition II was analyzed by differential scanning calorimetry analysis, and it was found to have an exothermic peak at 269.67 C.

(33) TABLE-US-00003 TABLE 3 Main Data of X-ray Powder Diffraction for Composition II Interplanar 2 Crystal Peak Peak No. Angle() Spacing() Height Aera 1 11.120 7.9500 1250 15628 2 12.521 7.0635 413 3990 3 14.272 6.2007 8409 51004 4 16.656 5.3181 1282 10862 5 17.815 4.9746 1514 10864 6 18.431 4.8097 733 6124 7 19.292 4.5970 2475 16754 8 19.923 4.4528 537 18373 9 20.453 4.3386 1604 15208 10 21.171 4.1931 1308 13632 11 22.735 3.9080 1626 23393 12 23.825 3.7317 947 14460 13 27.966 3.1878 2090 21313

(34) TABLE-US-00004 TABLE 4 Main Data of Infrared Absorption Spectrum for Composition II No. Wave Number(cm.sup.1) 1 3441 2 3254 3 3115 4 2939 5 1742 6 1605 7 1539 8 1499 9 1398 10 1302 11 1198 12 1124 13 1099 14 1032 15 897 16 804 17 600 18 478

Example 3

(35) 930 g of commercially available ceftriaxone sodium was taken and ground; 310 g of commercially available sulbactam sodium was taken and ground; and then these two raw materials were ground together by a ball mill until the particle size D.sub.50 was from 58 m to 88 m. The obtained powder was placed in a single-cone ribbon mixer (HF1600 type) and mixed well, with rotating speed of 25 rpm and mixing for 10 minutes. After the operation was completed, the material was taken out, and a crystalline substance was obtained and named Composition III.

(36) The Composition III was sampled and measured for crystal morphology. The X-ray powder diffraction spectrum pattern was showed in FIG. 7. The main data of the spectrum pattern were listed in Table 5.

(37) Its infrared absorption was measured by infrared absorption spectrum analysis. The spectrum pattern was showed in FIG. 8. The main data of infrared absorption peaks were listed in Table 6.

(38) The Composition III was analyzed by differential scanning calorimetry, and it was found to have an exothermic peak at 269.69 C.

(39) TABLE-US-00005 TABLE 5 Main Data of X-ray Powder Diffraction for Composition III 2 interplanar crystal Peak Peak No. Angle() spacing() Height Aera 1 11.159 7.9227 1519 16847 2 12.576 7.0326 458 4586 3 14.294 6.1913 13132 70185 4 16.658 5.3176 1786 11679 5 17.851 4.9647 1873 10087 6 18.450 4.8049 910 7192 7 19.315 4.5917 3940 24924 8 19.998 4.4362 859 11627 9 20.476 4.3338 1901 13788 10 21.208 4.1858 1446 16328 11 22.701 3.9138 1468 21912 12 23.807 3.7345 1261 15779 13 27.973 3.1870 1935 23623

(40) TABLE-US-00006 TABLE 6 Main Data of Infrared Absorption Spectrum for Composition III No. Wave Number(cm.sup.1) 1 3441 2 3256 3 3117 4 2937 5 1742 6 1603 7 1539 8 1499 9 1398 10 1302 11 1198 12 1124 13 1099 14 1032 15 897 16 804 17 600 18 480

Examples 4 to 8

(41) 800 g of ceftriaxone sodium and 200 g of sulbactam sodium, 1,150 g of ceftriaxone sodium and 230 g of sulbactam sodium, 2,700 g of ceftriaxone sodium and 450 g of sulbactam sodium, 1,330 g of ceftriaxone sodium and 190 g of sulbactam sodium, 2,200 g of ceftriaxone sodium and 275 g of sulbactam sodium, all of which were commercially available, were taken respectively and then processed respectively according to the method in Example 2 to get Compositions IV to VIII respectively. It was found that the spectrum patterns of these five compositions were basically the same as those obtained in Examples 1 to 3 through X-ray diffraction analysis.

Example 9

(42) 500 g of ceftriaxone sodium and 500 g of sulbactam sodium, both of which were commercially available, were taken and ground together by a ball mill until the particle size D.sub.50 was from 104 m to 175 m. The materials were mixed according to the same process as that of Example 2. After the operations were completed, the material was taken out and subpackaged under sterile condition to give Sterile Powder for Injection C.

(43) 500 g of ceftriaxone sodium and 500 g of sulbactam sodium, both of which were commercially available, were taken again and ground together by a ball mill until the particle size D.sub.50 was below 10 m. The materials were mixed according to the same process as that of Example 2. After the operations were completed, the material was taken out and subpackaged under a sterile condition to give Sterile Powder for Injection D.

Example 10

(44) The compositions of Examples 1 to 8 were taken respectively, and subpackaged under sterile condition to give Sterile Powder for Injections I to VIII.

(45) The compositions of Examples 1 to 8 were taken respectively and processed according to the conventional process of lyophilized agents to give Lyophilized Powder for Injections I to VIII.

(46) The compositions of Examples 1 to 8 were taken respectively and dissolved with 50 times amount of 0.9% sodium chloride aqueous solution and subpackaged to give Injections I to VIII.

Test Example 1: Stability Test

(47) Stability test was performed for Compositions I to VIII, Sterile Powder for Injection I, Sterile Powder for Injection II, Sterile Powder for Injection IV, Lyophilized Powder for Injection I, Lyophilized Powder for Injection II, Sterile Powder for Injection A, Sterile Powder for Injection B, Sterile Powder for Injection C, Sterile Powder for Injection D and Lyophilized Powder for Injection A obtained in the above-mentioned Comparative Examples or Examples.

(48) Test environment: placing under 25 C.2 C. and 60%10% relative humidity condition for 30 months.

(49) Test results: clarity of solution; contents of impurities 1, 2, 3 and 4 (wherein, impurities 1, 2 and 3 were respectively impurities A, B and E of ceftriaxone sodium described in EP8.0, and impurity 4 was impurity A of sulbactam sodium described in EP8.0), crystallinity and the like were measured and main test results were shown in Tables 7 to 10.

(50) ##STR00001##

(51) TABLE-US-00007 TABLE 7 Situations of Various Samples at 0.sup.th Month in Stability Test Con- Con- Con- Con- tent tent tent tent of of of of Impu - Impu - Impu - Impu - Clarity Crys- rity rity rity rity of tallinity 1 2 3 4 Solu- Samples (%) (%) (%) (%) (%) tion Composition I 90.5 0.12 0.08 0.09 0.11 Clear Composition II 92.7 0.15 0.10 0.13 0.12 Clear Composition III 91.2 0.12 0.14 0.15 0.14 Clear Composition IV 92.1 0.11 0.20 0.12 0.10 Clear Composition V 90.4 0.18 0.27 0.19 0.10 Clear Composition VI 91.7 0.09 0.15 0.16 0.18 Clear Composition VII 92.5 0.21 0.10 0.15 0.13 Clear Composition 92.1 0.29 0.27 0.18 0.22 Clear VIII Sterile Powder 90.6 0.14 0.10 0.15 0.15 Clear for Injection I Sterile Powder 92.3 0.14 0.11 0.14 0.14 Clear for Injection II Sterile Powder 90.4 0.17 0.16 0.15 0.12 Clear for Injection IV Lyophilized 87.6 0.12 0.20 0.16 0.17 Clear Powder for Injection I Lyophilized 85.3 0.16 0.20 0.21 0.18 Clear Powder for Injection II Lyophilized / 0.32 0.14 0.22 0.11 Clear Powder for Injection A Sterile Powder / 0.18 0.13 0.26 0.18 Clear for Injection A Sterile Powder / 0.33 0.19 0.18 0.19 Clear for Injection B Sterile Powder 78.2 0.25 0.21 0.20 0.18 Clear for Injection C Sterile Powder 72.1 0.22 0.31 0.23 0.16 Clear for Injection D

(52) TABLE-US-00008 TABLE 8 Situations of Various Samples at 12.sup.th Month in Stability Test Con- Con- Con- Con- tent tent tent tent of of of of Impu - Impu - Impu - Impu - Clarity Crys- rity rity rity rity of tallinity A B E G Solu- Samples (%) (%) (%) (%) (%) tion Composition I 90.4 0.14 0.10 0.11 0.14 Clear Composition II 91.6 0.18 0.11 0.15 0.14 Clear Composition III 91.3 0.19 0.16 0.17 0.17 Clear Composition IV 92.0 0.21 0.25 0.17 0.14 Clear Composition V 89.2 0.16 0.30 0.24 0.13 Clear Composition VI 91.1 0.15 0.21 0.18 0.22 Clear Composition VII 92.4 0.26 0.24 0.21 0.14 Clear Composition 91.9 0.31 0.29 0.19 0.22 Clear VIII Sterile Powder 90.8 0.25 0.14 0.23 0.18 Clear for Injection I Sterile Powder 92.7 0.19 0.17 0.24 0.15 Clear for Injection II Sterile Powder 91.7 0.22 0.19 0.20 0.15 Clear for Injection IV Lyophilized 85.8 0.16 0.23 0.19 0.18 Clear Powder for Injection I Lyophilized 85.7 0.22 0.21 0.26 0.19 Clear Powder for Injection II Lyophilized / 0.53 0.38 0.46 0.54 Clear Powder for Injection A Sterile Powder / 0.69 0.52 0.32 0.41 Clear for Injection A Sterile Powder / 0.53 0.48 0.37 0.43 Clear for Injection B Sterile Powder 75.1 0.34 0.31 0.29 0.34 Clear for Injection C Sterile Powder 73.2 0.29 0.42 0.33 0.30 Clear for Injection D

(53) TABLE-US-00009 TABLE 9 Situations of Various Samples at 24.sup.th Month in Stability Test Con- Con- Con- Con- tent tent tent tent of of of of Impu - Impu - Impu - Impu - Clarity Crys- rity rity rity rity of tallinity A B E G Solu- Samples (%) (%) (%) (%) (%) tion Composition I 89.2 0.18 0.21 0.17 0.25 Clear Composition II 91.0 0.20 0.10 0.19 0.23 Clear Composition III 90.4 0.23 0.20 0.21 0.26 Clear Composition IV 91.7 0.21 0.29 0.23 0.17 Clear Composition V 88.2 0.28 0.33 0.29 0.30 Clear Composition VI 91.0 0.19 0.27 0.28 0.23 Clear Composition VII 91.6 0.33 0.27 0.32 0.22 Clear Composition 91.7 0.35 0.35 0.23 0.29 Clear VIII Sterile Powder 90.1 0.34 0.26 0.37 0.26 Clear for Injection I Sterile Powder 91.9 0.21 0.20 0.35 0.22 Clear for Injection II Sterile Powder 91.4 0.23 0.38 0.24 0.22 Clear for Injection IV Lyophilized 85.2 0.23 0.29 0.33 0.22 Clear Powder for Injection I Lyophilized 84.1 0.30 0.32 0.29 0.30 Clear Powder for Injection II Lyophilized / 0.83 0.77 0.88 0.62 Clear Powder for Injection A Sterile Powder / 0.99 0.82 0.79 0.70 Slightly for Injection A turbid Sterile Powder / 0.78 0.97 0.85 0.75 Slightly for Injection B turbid Sterile Powder 64.0 0.38 0.47 0.59 0.41 Clear for Injection C Sterile Powder 62.2 0.39 0.56 0.44 0.33 Clear for Injection D

(54) TABLE-US-00010 TABLE 10 Situations of Various Samples at 30.sup.th Month in Stability Test Con- Con- Con- Con- tent tent tent tent of of of of Impu - Impu - Impu - Impu - Clarity Crys- rity rity rity rity of tallinity A B E G Solu- Samples (%) (%) (%) (%) (%) tion Composition I 88.1 0.32 0.31 0.33 0.31 Clear Composition II 90.5 0.31 0.29 0.34 0.30 Clear Composition III 88.2 0.38 0.37 0.28 0.34 Clear Composition IV 90.0 0.43 0.32 0.32 0.31 Clear Composition V 87.1 0.37 0.45 0.32 0.33 Clear Composition VI 89.9 0.25 0.33 0.33 0.30 Clear Composition VII 90.5 0.38 0.41 0.39 0.33 Clear Composition 90.2 0.45 0.41 0.31 0.31 Clear VIII Sterile Powder 88.5 0.47 0.45 0.42 0.38 Clear for Injection I Sterile Powder 89.7 0.37 0.39 0.39 0.33 Clear for Injection II Sterile Powder 90.2 0.43 0.42 0.37 0.36 Clear for Injection IV Lyophilized 84.9 0.39 0.39 0.43 0.38 Clear Powder for Injection I Lyophilized 83.7 0.47 0.38 0.42 0.31 Clear Powder for Injection II Lyophilized / 1.23 1.05 1.12 0.78 Slightly Powder for turbid Injection A Sterile Powder / 1.36 1.12 0.94 1.19 Slightly for Injection A turbid Sterile Powder / 1.13 0.99 1.27 1.06 Slightly for Injection B turbid Sterile Powder 60.6 0.57 0.62 0.70 0.47 Clear for Injection C Sterile Powder 53.5 0.65 0.71 0.58 0.42 Clear for Injection D

(55) It can be seen from the results of the stability test that after 12 months, the compositions and the formulations according to the present disclosure, as compared with Comparative Examples, had differences in the following aspects such as crystallinity, impurities, clarity of solution and the like; and after 30 months, such differences were more significant.

(56) The formulations of Comparative Examples did not show a crystalline form and there was no data of crystallinity; however, the compositions and formulations according to the present disclosure had a crystallinity of up to 90%, which kept substantially unchanged within 30 months and showed very good physical stability.

(57) At 24.sup.th-30.sup.th month, the formulations of Comparative Examples showed slight turbidity, and the compositions and the formulations according to the present disclosure did not show turbidity.

(58) Although, the contents of four impurities in the formulations of Comparative Examples were equivalent to those in the compositions according to the present disclosure at 0.sup.th month, the increase of impurities was very significant as time passed. Especially, the contents of impurity 1 (an isomer impurity of ceftriaxone) and impurity 4 (an open-loop degraded impurity of sulbactam) ware greater than 1% and greater than 0.5% respectively at 24.sup.th-30.sup.th month, and failed to meet the requirements of EP 8.0. However, the contents of the four impurities in the compositions and the formulations according to the present disclosure were not significantly increased and in a defined scope, indicating very good chemical stability. This finding revealed that there may be a close relation between the generation of specific impurities and crystal morphology.

Test Example 2: In Vivo Antibacterial Test

(59) The stability test samples in test example 1 were sampled at 0.sup.th month and subjected to in vivo antibacterial test; and the stability test samples were sampled at 30th month again, and subjected to in vivo antibacterial test. The in vivo antibacterial tests were performed according to the following method.

(60) 2.1 Test Materials

(61) Test Samples: Compositions I to VIII, Sterile Powder for Injection I, Sterile Powder for Injection II, Sterile Powder for Injection IV, Lyophilized Powder for Injection I, Lyophilized Powder for Injection II, Lyophilized Powder for Injection A, Sterile Powder for Injection A and Sterile Powder for Injection B, all of which were obtained from the test example 1 at 0.sup.th and 30.sup.th month; and control drugs: ceftriaxone sodium, levofloxacin hydrochloride, azithromycin, doxycycline hydrochloride and spectinomycin hydrochloride, wherein, a batch of the control drugs were purchased at 0.sup.th month of the stability test so as to match with the test samples at 0.sup.th month, and another batch of control drugs were purchased at 30th month of the stability test so as to match with the test samples at 30th month, and the time when the two batches were purchased was within one month from the date of their production.

(62) Animals: ICR mice weighed 18-22 g, half male and half female.

(63) Strains: ATCC700603 (Klebsiella pneumoniae), ATCC43069 (Neisseria gonorrhoeae), Q-R (quinolone-resistant Neisseria gonorrhoeae), T-R (tetracycline-resistant Neisseria gonorrhoeae), PP-R (-lactam-resistant Neisseria gonorrhoeae), MAC-R (macrolide-resistant Neisseria gonorrhoeae), SPE-R (spectinomycin-resistant Neisseria gonorrhoeae) and QS-R (ceftriaxone-resistant Neisseria gonorrhoeae), wherein, ATCC700603, and ATCC43069 were purchased from ATCC; QS-R was prepared by in vitro inducing Neisseria gonorrhoeae, which was sensitive to ceftriaxone, with a subinhibitory concentration of ceftriaxone, culturing at 36 C., 5% CO.sub.2, passaging once every 24 hours, and then transferring to a higher concentration of ceftriaxone on GC medium until the strain was resistant to ceftriaxone; and other strains were clinical isolates.

(64) Protective agent: highly active dry yeast.

(65) 2.2 Test Method

(66) Enhancing Cytotoxicity:

(67) After incubating for 18 hours, the experimental strain was diluted to 10.sup.2 and 10.sup.3 with 5% highly active dry yeast; 0.5 ml of the strain was injected intraperitoneally into mice to induce systemic infection, resulting in a septicemia mouse model. No protective measures was provided and thus mice died of infection. Aseptic thoracotomy was performed timely on the mice died of infection, the heart blood or intracavity effusion was taken and evenly coated on corresponding agar plates, and incubated for 18 hours at 37 C. The strain was identified to be consistent with the administrated infectious bacteria. The obtained strain was used to infect mice again. By repeating this method 1-2 times, the cytotoxicity of the strain could be enhanced and was relatively stable.

(68) Determining Minimum Lethal Dose (MLD):

(69) the experimental strain was diluted with 5% highly active dry yeast to give diluted bacteria solutions at different concentrations: 10.sup.1, 10.sup.2, 10.sup.3 and 10.sup.4, which were injected intraperitoneally into experimental animals (0.5 ml per mouse) respectively to establish a septicemia model. After infection, the number of dead mice was recorded. The minimum dose causing 100% death of mice was 1 MLD, and was used as the dose in animal protection experiments. The bacterial concentration, which was one log lower than 1 MLD and infected the animals without mortality, was named 0.1 MLD. In experiments, a bacteria concentration of 1 MLD was used as the positive control for the infection experiments; and a bacteria concentration of 0.1 MLD was used as the negative control for the infection. In both positive and negative control groups, only the bacteria were administrated and no antibiotic treatment was provided. The mortality of the positive group should be 70% or more, and the mortality of the negative group should be 30% or less.

(70) Pre-Test for Determining the Dose Range of Test Samples:

(71) 1 MLD of bacteria was formulated with 5% highly active dry yeast and used to infect mice Immediately and after 6 hours, pre-tests were performed using test samples at three different concentrations (high concentration, medium concentration and low concentration), 4 mice per dose group. The survival number of mice was recorded after infection, and based on this result, the administration dose in the animal protection test was designed. When the administration dosage was investigated first, there may be relatively great differences between the dose concentrations of the groups. When the range of the administration dosage was determined, it may be further investigated by reducing the differences until the suitable administration dosage (that is, for the group administrated with the highest concentration, 70% or more infected animals survived; and for the group administrated with the lowest concentration, no less than 70% of infected animals died) was found.

(72) Animal Protection Test:

(73) In the pre-tests, the drug dosages at which 100% of mice died or no death occurred were obtained for the mice infected with the test strains. Then, the mice were fasted from food other than water for 18 hours before experiment, weighted 18-22 g; half male and half female. The mice were randomly divided into the following groups: (1) ATCC700603 group, (2) ATCC43069 group, (3) Q-R group, (4) T-R group, (5) PP-R group, (6) MAC-R group, (7) SPE-R group and (8) QS-R group; with five test sample concentrations per group, and 10 animals per group. 10 animals were used as blank controls (equal volume of sterile water for injection was administrated). Each mouse was injected intraperitoneally with 0.5 ml of 1 MLD bacteria solution to establish an infection model Immediately and after 6 hours, test sample solutions at different concentrations were injected subcutaneously respectively at 0.2 ml per mouse. The mice were observed for 7 consecutive days, and the death status of animals in each group were recorded. The dead mice were investigated with autopsy and observed with naked eye. The 50% effective dose (ED.sub.50) and 95% confidence intervals were calculated by Bliss method; t-test was performed to determine the significant difference between groups. The lower the ED.sub.50 was, the better the protection for animals infected by bacteria the test sample provided.

(74) 2.3 Test Results

(75) Main results of the animal protection tests were listed in Table 11 and Table 12, wherein, Table 11 showed the in vivo antibacterial ED.sub.50 values of the samples at 0.sup.th month of the stability test, and Table 12 showed the in vivo antibacterial ED.sub.50 values of the samples at 30.sup.th month of the stability test.

(76) TABLE-US-00011 TABLE 11 In Vivo Antibacterial ED.sub.50 Values of Samples at 0.sup.th Month of the Stability Test (mg/kg) Strains Test ATCC ATCC Q- PP- MAC- SPE- QS Samples 700603 43069 R T-R R R R -R Cef- 35 16 31 46 208 34 52 337 triaxone Sodium Levo- 25 573 48 37 36 67 72 floxacin Hydro- chloride Azithro- 24 43 62 49 487 59 63 mycin Doxy- 37 55 456 52 58 52 105 cycline Hydro- chloride Spectino- 19 40 61 56 38 621 41 mycin Hydro- chloride Com- 21 11 24 34 47 27 42 62 position I Com- 23 13 21 37 50 23 42 65 position II Com- 19 14 22 35 45 25 46 59 position III Com- 22 15 20 32 44 26 47 62 position IV Com- 27 13 26 37 56 27 49 78 position V Com- 30 16 25 38 68 27 48 85 position VI Com- 31 17 25 38 60 29 52 82 position VII Com- 28 15 27 39 66 28 50 84 position VIII Sterile 25 13 24 32 42 25 46 64 Powder for Injection I Sterile 27 10 23 35 48 27 43 67 Powder for Injection II Sterile 24 14 25 34 46 26 44 66 Powder for Injection IV Lyo- 27 14 22 35 43 24 46 62 philized Powder for Injection I Lyo- 28 12 23 37 41 22 45 60 philized Powder for Injection II Lyo- 31 16 27 39 62 26 50 95 philized Powder for Injection A Sterile 32 15 30 40 56 29 48 92 Powder for Injection A Sterile 33 16 32 38 49 24 46 94 Powder for Injection B Note: represents Not Performed.

(77) TABLE-US-00012 TABLE 12 In Vivo Antibacterial ED.sub.50 Values of Samples at 30.sup.th Month of the Stability Test (mg/kg) Strains Test ATCC ATCC PP- MAC- SPE- QS- Samples 700603 43069 Q-R T-R R R R R Cef- 37 21 30 57 224 37 66 373 triaxone Sodium Levo- 30 564 43 42 41 72 82 floxacin Hydro- chloride Azithro- 31 40 59 55 497 65 69 mycin Doxy- 32 51 432 47 62 51 99 cycline Hydro- chloride Spectino- 22 42 68 67 42 571 56 mycin Hydro- chloride Com- 23 16 22 42 50 29 52 71 position I Com- 25 15 20 46 53 27 49 69 position II Com- 19 14 20 45 56 29 48 68 position III Com- 25 17 24 42 51 25 57 70 position IV Com- 31 20 27 48 68 31 55 87 position V Com- 37 20 28 51 75 28 59 88 position VI Com- 38 22 28 47 65 33 57 92 position VII Com- 36 23 26 49 71 31 59 94 position VIII Sterile 26 16 21 41 51 28 48 75 Powder for Injection I Sterile 30 17 20 43 52 28 47 69 Powder for Injection II Sterile 28 16 22 39 56 27 46 71 Powder for Injection IV Lyo- 30 18 26 42 53 25 47 70 philized Powder for Injection I Lyo- 29 17 24 48 61 25 48 67 philized Powder for Injection II Lyo- 35 26 41 78 93 53 79 153 philized Powder for Injection A Sterile 36 28 47 75 102 48 87 174 Powder for Injection A Sterile 35 28 46 77 106 47 91 188 Powder for Injection B Note: represents Not Performed.

(78) The test results showed that the compositions and the formulations according to the present disclosure had better antibacterial activity, and especially exhibited significantly higher inhibitory activity against various drug-resistant Neisseria gonorrhoeae bacteria and in some cases the activity may increase 3-4 times as compared with the formulations in Comparative Examples.

(79) In addition, the compositions and the formulations according to the present disclosure had relatively good stability of biological activity, and particularly had stable inhibitory activity against various non-drug-resistant and drug-resistant Neisseria gonorrhoeae. The compositions and the formulations according to the present disclosure had basically unchanged biological activity after 30 months. In contrast, the formulations in the comparative examples showed significant reduction in the activity after 30 months.

(80) Although the present disclosure has been described above in detail with general description, specific embodiments and tests, a person skilled in the art may make suitable modifications or improvements thereon based on the present disclosure. Thus, those modifications or improvements made without departing from the spirits of the present disclosure all belong to the content of the present disclosure. The background, summary of the disclosure, and specific modes for carrying out the present disclosure are only for illustrative purpose, but not used as evidence for identifying the prior art of the present disclosure.