Crystal characteristics, preparation processes and anticancer applications of 17beta-neriifolin crystal forms
11091510 · 2021-08-17
Assignee
- ENKANG PHARMACEUTICALS (GUANGZHOU), LTD. (Guangzhou, CN)
- ENZHI (GUANGZHOU) PHARMACEUTICALS LIMITED (Guangzhou, CN)
- ENZYNOMICS (H.K.) LIMITED (Hong Kong, CN)
- FOSHAN INTELGEN PHARMACEUTICAL CO. LTD. (Foshan, CN)
Inventors
- Chun LIANG (Guangzhou, CN)
- Haibin Liu (Guangzhou, CN)
- Zeming Zhang (Guangzhou, CN)
- Guoqiang Song (Guangzhou, CN)
- Yushan Li (Guangzhou, CN)
- Weiqiang Zhang (Guangzhou, CN)
Cpc classification
International classification
Abstract
The present invention provides the preparation processes for several crystal forms of the compound 17beta-Neriifolin (17bNF) and use thereof in anti-tumor applications. The present invention provides preparation processes for four kinds of non-solvated crystal forms, amorphous powders and eleven kinds of solvated crystalline solids, including ethanol-solvated ones, of 17bNF that are not present in nature, and the identification and characterization of these crystal forms by crystallographic research methods such as X-ray powder diffraction. In addition, through vivo anti-tumor experiments with five of the crystal forms in nude mice, we provide anti-tumor and anti-cancer applications of the crystal forms of 17bNF.
Claims
1. Crystal Form of 17bNF is 17β-neriifolin, comprising: i) a non-solvated Crystal Form 17bNF-I which has a powder X-ray diffraction pattern comprising characteristic peaks with 2θ values of 6.1°±0.2°, 11.2°±0.2°, 11.4°±0.2°, 12.2°±0.2°, 15.5°±0.2°, 16.4°±0.2°, 17.5°±0.2°, 19.7°±0.2°, and 20.4°±0.2°; ii) a non-solvated Crystal Form 17bNF-II which has a powder X-ray diffraction pattern comprising characteristic peaks with 2θ values of 6.4°±0.2°, 10.1°±0.2°, 12.6°±0.2°, 12.8°±0.2°, 13.3°±0.2°, 14.8°±0.2°, 15.5°±0.2°, 16.5°±0.2°, 17.8°±0.2°, 18.1°±0.2°, 18.4°±0.2°, 18.7°±0.2°, 19.3°±0.2°, 20.2°±0.2°, 21.9°±0.2°, 22.4°±0.2°, 23.4°±0.2°, 24.3°±0.2°, 25.8°±0.2°, 26.4°±0.2°, 27.0°±0.2° and 27.5°±0.2°; or iii) a non-solvated Crystal Form 17bNF-IV which has a powder X-ray diffraction pattern comprising characteristic peaks with 2θ values of 5.6°±0.2°, 12.6°±0.2°, 13.5°±0.2°, 14.2°±0.2°, 14.5°±0.2°, 15.3°±0.2°, 16.5°±0.2°, 18.1°±0.2°, 18.8°±0.2°, 19.6°±0.2°, 19.9°±0.2°, 20.6°±0.2°, 22.0°±0.2°, 24.2°±0.2°, 24.9°±0.2° and 25.4°±0.2°.
2. The Crystal Form of 17bNF according to claim 1, wherein said Crystal Form 17bNF-I, further has at least one of following characteristics: a differential scanning calorimetry spectrum has an endothermic peak at 218-238° C.; and a thermogravimetric analysis spectrum has a weight loss of ˜0.6% before 250° C.; the corresponding characteristic spectra are shown in
3. The Crystal Form of 17bNF according to claim 1, wherein said Crystal Form 17bNF-II, further has at least one of following characteristics: a differential scanning calorimetry spectrum has endothermic peaks at ˜82-92° C., ˜145-155° C. and ˜225-235° C., and has exothermic peak at ˜144-164° C. and a thermogravimetric analysis spectrum has a weight loss of ˜2.9% before 100° C., the corresponding characteristic spectra are shown in
4. The Crystal Form of 17bNF according to claim 1, wherein said Crystal Form 17bNF-IV, further has at least one of following characteristics: a differential scanning calorimetry spectrum has endothermic peaks at 180-200° C. and 222-242° C.; it will be changed into Crystal Form 17bNF-I after being heated at 180-200° C.; the corresponding characteristic spectra are shown in
5. A preparation process for the Crystal Form of 17bNF according to claim 1, comprising: i) the preparation of the non-solvated Crystal Form 17bNF-I, comprising the following steps: a. dissolving or suspending 17bNF powder or crystals including 17bNF-0 in methanol, ethanol, tetrahydrofuran, acetone, isobutanol, isopropyl acetate, 2-butanone, isopropyl alcohol, ethyl acetate, acetonitrile, toluene, methyl tert-butyl ether or water, separately; b. separately filtering the solutions or suspensions from Step a, and then taking the filtrates; c. taking the 17bNF methanol solution, or mixing the 17bNF methanol solution with the filtrate of the 17bNF isopropyl alcohol suspension, 17bNF tetrahydrofuran solution, 17bNF acetone solution, 17bNF isobutyl alcohol solution, 17bNF isopropyl acetate solution, the filtrate of 17bNF ethyl acetate suspension, the filtrate of 17bNF toluene suspension, or the filtrate of the 17bNF methyl tert-butyl ether suspension, at ratios of 30%:70% to 70%:30%; or taking the 17bNF ethanol solution, or mixing the 17bNF ethanol solution with the 17bNF tetrahydrofuran solution, 17bNF acetone solution, 17bNF isobutyl alcohol solution, 17bNF isopropyl acetate solution, 17bNF 2-butanone solution, the filtrate of the 17bNF isopropyl alcohol suspension, the filtrate of the 17bNF ethyl acetate suspension, the filtrate of the 17bNF toluene suspension, or the filtrate of the 17bNF methyl tert-butyl ether suspension, at ratios of 30%:70% to 70%:30%; or mixing the filtrate of the 17bNF isopropyl alcohol suspension with 17bNF tetrahydrofuran solution, 17bNF acetone solution, 17bNF isobutyl alcohol solution, 17bNF isopropyl acetate solution, 17bNF 2-butanone solution, the filtrate of the 17bNF isopropyl alcohol suspension, the filtrate of the 17bNF ethyl acetate suspension, the filtrate of the 17bNF acetonitrile suspension, or the filtrate of the 17bNF toluene suspension, at ratios of 30%:70% to 70%:30%; or taking the 17bNF tetrahydrofuran solution, or mixing the 17bNF tetrahydrofuran solution with the 17bNF acetone solution, 17bNF isobutyl alcohol solution, 17bNF isopropyl acetate solution, 17bNF 2-butanone solution, the filtrate of the 17bNF ethyl acetate suspension, the filtrate of the 17bNF acetonitrile suspension, the filtrate of the 17bNF toluene suspension, or the filtrate of the 17bNF methyl tert-butyl ether suspension, at ratios of 30%:70% to 70%:30%; or taking filtrate of the 17bNF ethyl acetate suspension, or mixing the filtrate of the 17bNF ethyl acetate suspension with the 17bNF acetone solution, 17bNF 2-butanone solution, or the filtrate of the 17bNF acetonitrile suspension, at ratios of 30%:70% to 70%:3070%:30%; or taking the 17bNF acetone solution, or mixing the 17bNF acetone solution with the 17bNF isobutyl alcohol solution, 17bNF isopropyl acetate solution, 17bNF 2-butanone solution, or the filtrate of the 17bNF toluene suspension, at ratios of 30%:70% to 70%:30%; or taking the 17bNF isobutyl alcohol solution, or mixing the 17bNF isobutyl alcohol solution with the 17bNF isopropyl acetate solution, the filtrate of the 17bNF acetonitrile suspension, 17bNF 2-butanone solution, the filtrate of the 17bNF toluene suspension, the filtrate of the 17bNF water suspension or the filtrate of the 17bNF methyl tert-butyl ether suspension, at ratios of 30%:70% to 70%:30%; or mixing the 17bNF isopropyl acetate solution with the 17bNF 2-butanone solution, at ratios of 30%:70% to 70%:30%; or mixing the filtrate of the 17bNF acetonitrile suspension with the 17bNF 2-butanone solution, or the filtrate of the 17bNF methyl tert-butyl ether suspension, at ratios of 30%:70% to 70%:30%; or taking the 17bNF 2-butanone solution, or mixing the 17bNF 2-butanone solution with the filtrate of the 17bNF toluene suspension, the filtrate of the 17bNF water suspension or the filtrate of the 17bNF methyl tert-butyl ether suspension, at ratio of 30%:70% to 70%:30%; d. volatilizing any one of the solutions of 17bNF in a single solvent or two mixed solvents obtained from Step c at an environmental condition to obtain the non-solvated Crystal Form 17bNF; ii) the preparation process of the non-solvated Crystal Form 17bNF-II, comprising the following steps: a. suspending the 17bNF-0 solid in water, and keeping stirring; b. filtering the suspension, collecting and drying the solid, and obtaining the non-solvated Crystal Form 17bNF-II; or iii) the preparation process of the non-solvated Crystal Form 17bNF-IV, comprising the following steps: a. suspending the 17bNF-0 solid in methyl tert-butyl ether to produce a suspension; b. stirring and beating the suspension; and adding some seed crystal 17bNF-1V; c. filtering the suspension, collecting and drying the solid, and obtaining the non-solvated Crystal Form 17bNF-IV.
6. A preparation process for the non-solvated Crystal Form 17bNF-I according to claim 1, comprising the following steps: a. suspending the ethanol-solvated Crystal Form 17bNF-0 solid in ethyl ether, and keeping stirring to obtain a suspension; b. filtering the suspension, collecting and drying the solid, and obtaining the non-solvated Crystal Form 17bNF-I.
7. The preparation process for the Crystal Form of 17bNF according to claim 5, wherein two 17bNF solutions are mixed at ratio of 40%:60% to 60%:40% in Step i) c.
8. The preparation process for the Crystal Form of 17bNF according to claim 5, wherein two 17bNF solutions are mixed at ratio of 50%:50% in Step i) c.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE EMBODIMENTS
(53) Instrument
(54) X-Ray Powder Diffraction (XRPD)
(55) The methods used for determining the X-ray diffraction patterns of the crystalline forms are known in the art. We used Bruker D8 advance X-ray powder diffraction instrument equipped with the LynxEye detector. The scanning range was from 3° to 400 2θ with the scanning step of 0.02°. The power supply setting was 40 kV and 40 mA. The sample tray used for sample measurement was a zero background tray.
(56) Thermogravimetric Analysis (TGA)
(57) Thermogravimetric analysis instrument TA TGA Q500 was used to analyze the samples in aluminum sample trays at a heating rate of 10° C./min. The weight of the samples ranged from 2 mg to 3 mg, the protective gas was N.sub.2, and the flow rates of N.sub.2 to the balance chamber and sample chamber were 40 mL/min and 60 mL/min, respectively.
(58) The Differential Scanning Calorimetry (DSC)
(59) TA QSC Q200 differential scanning calorimeter was used to analyze the samples at a heating rate of 10° C./min. The standard sample used for calibration was Indium. The weight of the samples ranged from 2 mg to 3 mg, the protective gas was N.sub.2, and the flow rate of N.sub.2 was 50 mL/min.
(60) The following examples further illustrate this invention:
EXAMPLES
Example 1—Preparation of the Crystal Form 0 of 17bNF (17bNF-0)
(61) 17bNF powder or solids were completely dissolved in ethanol solution under heating and refluxing conditions, followed by cooling statically at room temperature until crystallisation. The solids were filtered, collected and dried to obtain the Crystal Form 0 of 17bNF (17bNF-0).
Example 2—Preparation of the Crystal Form I of 17bNF (17bNF-I)
(62) 17bNF-0 powder or crystalline solids were dissolved or suspended in solvents to produce solutions or suspensions as stated in Table 1.
(63) TABLE-US-00001 TABLE 1 The mass and volume of materials used for the liquid sample preparation Number Solvent Volume (mL) 17bNF Mass (mg) 1 Methanol 3 59.8 2 Ethanol 3 60.0 3 Isopropyl alcohol 3 60.3 4 Tetrahydrofuran 3 60.2 5 Ethyl acetate 3 59.8 6 Acetone 3 59.8 7 Isobutyl alcohol 3 59.9 8 isopropyl acetate 3 60.2 9 Acetonitrile 3 60.1 10 2-Butanone 3 60.3 11 Toluene 3 60.1 12 Water 3 60.1 13 Methyl tert-butyl ether 3 60.2
(64) The solutions or suspensions were filtrated separately to obtain filtrates. 100 μl of the 17bNF methanol solution were mixed with 100 μl of the 17bNF methanol solution, filtrate of the 17bNF tetrahydrofuran solution, 17bNF acetone solution, 17bNF isobutyl alcohol solution, 17bNF isopropyl acetate solution, 17bNF isopropyl alcohol suspension, the filtrate of the 17bNF ethyl acetate suspension, the filtrate of the 17bNF toluene suspension, or the filtrate of the 17bNF methyl tert-butyl ether suspension, separately.
(65) 100 μl of the 17bNF ethanol solution were mixed with 100 μl of the filtrate of the 17bNF ethanol solution, 17bNF isopropyl alcohol suspension, 17bNF tetrahydrofuran solution, 17bNF ethyl acetate suspension, 17bNF acetone solution, 17bNF isobutyl alcohol solution, 17bNF isopropyl acetate solution, 17bNF 2-butanone solution, the filtrate of the 17bNF toluene suspension or the filtrate of the 17bNF methyl tert-butyl ether suspension separately.
(66) 100 μl of the 17bNF isopropyl alcohol suspension were mixed with 100 μl of the filtrate of the 17bNF ethanol solution, 17bNF tetrahydrofuran solution, 17bNF acetone solution, 17bNF isobutyl alcohol solution, 17bNF isopropyl acetate solution, 17bNF 2-butanone solution, 17bNF isopropyl alcohol suspension, the filtrate of the 17bNF ethyl acetate suspension, the filtrate of the 17bNF toluene suspension, or the filtrate of the 17bNF methyl tert-butyl ether suspension 100 μl separately.
(67) 100 μl of the filtrate of the 17bNF isopropyl alcohol suspension were mixed with 100 μl of the 17bNF tetrahydrofuran solution, 17bNF acetone solution, 17bNF isobutyl alcohol solution, 17bNF isopropyl acetate solution, 17bNF 2-butanone solution, the filtrate of the 17bNF isopropyl alcohol suspension, the filtrate of the 17bNF ethyl acetate suspension, the filtrate of the 17bNF acetonitrile suspension, or the filtrate of the 17bNF toluene suspension, separately.
(68) 100 μl of the filtrate of the 17bNF tetrahydrofuran solution were mixed with 100 μl of the filtrate of the 17bNF tetrahydrofuran solution, 17bNF acetone solution, 17bNF isobutyl alcohol solution, 17bNF isopropyl acetate solution, 17bNF 2-butanone solution, the filtrate of the 17bNF ethyl acetate suspension, the filtrate of the 17bNF acetonitrile suspension, the filtrate of the 17bNF toluene suspension, or the filtrate of the 17bNF methyl tert-butyl ether suspension, separately.
(69) 100 μl of the filtrate the 17bNF ethyl acetate suspension were mixed with 100 μl of the filtrate of the 17bNF ethyl acetate suspension, 17bNF acetone solution, 17bNF 2-butanone solution, or the filtrate of the 17bNF acetonitrile suspension, separately.
(70) 100 μl of the filtrate of the 17bNF acetone solution were mixed with 100 μl of the 17bNF acetone solution, 17bNF isobutyl alcohol solution, 17bNF isopropyl acetate solution, 17bNF 2-butanone solution, or the filtrate of the 17bNF toluene suspension, separately.
(71) 100 μl of the filtrate of the 17bNF isobutyl alcohol solution were mixed with 100 μl of the 17bNF isobutyl alcohol solution, 17bNF isopropyl acetate solution, 17bNF isopropyl acetate solution, the filtrate of the 17bNF acetonitrile suspension, 17bNF 2-butanone solution, the filtrate of the 17bNF toluene suspension, the filtrate of the 17bNF water suspension, or the filtrate of the 17bNF methyl tert-butyl ether suspension, separately.
(72) 100 μl of the filtrate of the 17bNF isopropyl acetate solution were mixed with 100 μl of the 17bNF 2-butanone solution.
(73) 100 μl of the filtrate of the 17bNF acetonitrile suspension were mixed with 100 μl of the 17bNF 2-butanone solution, or the filtrate of the 17bNF methyl tert-butyl ether suspension, separately.
(74) 100 μl of the filtrate of the 17bNF 2-butanone solution were mixed with 100 μl of the 17bNF 2-butanone solution, the filtrate of the 17bNF toluene suspension, the filtrate of the 17bNF water suspension, or the filtrate of the 17bNF methyl tert-butyl ether suspension, separately.
(75) Each mixed solution or suspension was put into a 96-well plate and the 96-well plate was sealed with a sealing membrane with some small pinholes on it. The plate was then placed in a fume hood under atmospheric conditions to evaporate to obtain the Crystal Form I of 17bNF (17bNF-I).
Example 3—Preparation of 17bNF Crystal Form I (17bNF-I)
(76) As described in Table 1, 0.5 mL of filtrates of the solutions of 17bNF in methanol, ethanol, tetrahydrofuran, ethyl acetate, acetone or 2-butanone, separately, was placed into test tubes, and the test tubes were placed in a fume hood to dry the filtrates at room temperature and obtain 17bNF Crystal Form I (17bNF-I).
Example 4—Preparation of 17bNF Crystal Form I (17bNF-I)
(77) As described in Table 1, 1 mL of suspension of 17bNF-0 in diethyl ether was stirred and beat for 3 days at room temperature. The suspension was then filtered, and the solids were dried to obtain 17bNF Crystal Form I (17bNF-I).
Example 5—Preparation of 17bNF Crystal Form II (17bNF-II)
(78) As described in Table 1, 1 mL of 17bNF-0 suspension in water was stirred and beat for 3 days at room temperature. The suspension was then filtered, and the solids were dried to obtain 17bNF Crystal Form II (17bNF-II).
Example 6—Preparation of 17bNF Crystal Form III (17bNF-III)
(79) As described in Table 1, 1 mL of 17bNF-0 suspension in acetonitrile was stirred and beat for 3 days at room temperature. The suspension was then filtered, and the solids were dried to obtain 17bNF Crystal Form VIII. 17bNF Crystal Form VIII was heated to 160° C. by DSC to lose solvent and obtain 17bNF Crystal Form III (17bNF-III).
Example 7—Preparation of 17bNF Crystal Form IV (17bNF-IV)
(80) As described in Table 1, a suspension of 17bNF-0 (180 mg) in methyl tertiary butyl ether (3 mL) was prepared, 3 mg of 17bNF Crystal Form IV as seed crystal were added, and the suspension was stirred at room temperature. After 2 days, the suspension was then filtered to collect the solids and obtain 17bNF Crystal Form IV (17bNF-IV).
Example 8—Preparation of 17bNF Crystal Form V
(81) As described in Table 1, 1 mL of 17bNF-0 suspension in isopropanol was stirred and beat for 3 days at room temperature. The suspension was then filtered to obtain solids which were then, dried to obtain 17bNF Crystal Form V.
Example 9—Preparation of 17bNF Crystal Form VI
(82) As described in Table 1, solutions were mixed and filtered to obtain filtrates. 100 μl of the filtrate of 17bNF-0 methanol solution were mixed with 100 μl of the filtrate of 17bNF 2-butanone solution, and the mixture was placed into a 96-well plate. The 96-well plate was sealed by a sealing film with some small pinholes on it and placed in a fume hood to dry the filtrates in natural environment and obtain 17bNF Crystal Form VI.
Example 10—Preparation of 17bNF Crystal Form VII
(83) As described in Table 1, 1 mL of suspension of 17bNF-0 in ethyl acetate was stirred and beat for 3 days at room temperature. The suspension was filtered, and the solids were, dried to obtain 17bNF Crystal Form VII.
Example 11—Preparation of 17bNF Crystal Form VII
(84) 20.3 mg of 17bNF-0 were dissolved in 0.1 mL of tetrahydrofuran, and some anti-solvent (0.4 mL of diethyl ether) was added. The mixture was stirred to obtain precipitates which were then filtered to obtain 17bNF Crystal Form VII.
Example 12—Preparation of 17bNF Crystal Form VII
(85) As described in Table 1, solutions were mixed and filtered to obtain filtrates. 100 μl of the filtrates of 17bNF ethyl acetate solution were mixed with 100 μl of the filtrates of 17bNF isopropyl acetate solution. The mixture was placed into a 96-well plate which was then sealed by sealing film with small pinholes on it, and the 96-well plate was placed in a fume hood to dry the filtrates in natural environment and obtain 17bNF Crystal Form VII.
Example 13—Preparation of 17bNF Crystal Form VIII
(86) As described in Table 1, 1 mL of suspension of 17bNF-0 in acetonitrile was stirred and beat for 3 days at room temperature and then filtered to obtain solids which were then dried to obtain 17bNF Crystal Form VIII.
Example 14—Preparation of 17bNF Crystal Form IX
(87) As described in Table 1, 1 mL of the suspension of 17bNF-0 in methyl benzene was stirred and beat for 3 days at room temperature and then filtered to obtain solids which were then dried to obtain 17bNF Crystal Form IX.
Example 15—Preparation of 17bNF Crystal Form X
(88) As described in Table 1, solutions were mixed and filtered to obtain filtrates. 100 μl of the filtrates of 17bNF ethyl acetate solution were mixed with 100 μl of the filtrates of 17bNF isobutanol solution. The mixture was placed into a 96-well plate which was then sealed by sealing film with small pinholes on it. The 96-well plate was then placed in a fume hood to dry the filtrates in natural environment and obtain 17bNF Crystal Form X.
Example 16—Preparation of 17bNF Crystal Form XI
(89) As described in Table 1, solutions were mixed and filtered to obtain filtrates. 100 μl of the filtrates of 17bNF ethyl acetate solution were mixed with 100 μl of filtrates of 17bNF methyl tertiary butyl ether solution. The mixture was placed into a 96-well plate which was then sealed by sealing film with small pinholes on it. The 96-well plate was then place in a fume hood to dry the filtrates in natural environment and obtain 17bNF Crystal Form XI.
Example 17—Preparation of 17bNF Crystal Form XII
(90) As described in Table 1, solutions were mixed and filtered to obtain filtrates. 100 μl of the filtrates of 17bNF acetone solution were mixed with 100 μl of the filtrates of 17bNF methyl tertiary butyl ether solution. The mixture was placed into a 96-well plate which was then sealed by sealing film with small pinholes on it. The 96-well plate was then place in a fume hood to dry the filtrates in natural environment and obtain 17bNF Crystal Form XII.
Example 18—Preparation of 17bNF Crystal Form XIII
(91) As described in Table 1, 0.5 ml of the filtrate of the 17bNF solution in isopropyl acetate was placed into a test tube which was then put in a fume hood to dry the filtrate at room temperature and obtain 17bNF Crystal Form XIII.
Example 19—Preparation of 17bNF Crystal Form XIV
(92) Some 20.8 mg of 17bNF were dissolved in 0.3 mL of acetone, and an anti-solvent (1.54 mL of diethyl ether) was then added. The mixture was stirred to obtain precipitates which were then filtered to obtain 17bNF Crystal Form XIV.
Example 20—Preparation of Amorphous 17bNF
(93) As described in Table 1, solutions were mixed and filtered to obtain filtrates. 100 μl of the filtrates of 17bNF acetonitrile solution were mixed with 100 μl of the filtrates of 17bNF methyl benzene solution. The mixture was placed into a 96-well plate which was then sealed by a sealing film with small pinholes on it. The 96-well plate was then placed in a fume hood to dry the filtrates in natural environment and obtain amorphous 17bNF.
Example 21—In Vivo Efficacy Test of 17bNF Crystal Form 0 and Form II (17bNF-0 and 17bNF-II)
(94) Human gastric cancer cells MGC-803 were subcutaneously planted into the armpit of nude mice. The test drugs 17bNF-0 and 17bNF-II were separately administrated orally when the average tumor volume reached 1450 mm.sup.3. Drugs were given once a day, six times every week until the 15th day. CMC (drug-carrier; 0.5%) was used as the blank control. Tumor volumes were measured once about every three days (
Example 22—In Vivo Efficacy Test-1 of 17bNF Crystal Form 0 to Form IV (17bNF-0 to 17bNF-IV)
(95) Human gastric cancer cells MGC-803 were subcutaneously planted into the armpit of nude mice. The test drugs 17bNF-0 to 17bNF-IV were separately administrated orally when the average tumor volume reached ˜600 mm.sup.3. Drugs were given once a day, six times every week until the 15th day. Nude mice injected intraperitoneally with 5 mg/kg Cisplatin once every five days were as positive control. CMC (drug-carrier; 0.5%) was used as the blank control. Tumor volumes were measured once about every three days (
Example 23—In Vivo Efficacy Test-2 of 17bNF Crystal Form 0 to Form IV
(96) (17bNF-0 to 17bNF-IV)
(97) Human gastric cancer cells MGC-803 were subcutaneously planted into the armpit of nude mice. The test drugs 17bNF-0 to 17bNF-IV were separately administrated orally when the tumor average volume reach ˜100 mm.sup.3. Drugs were given once a day, six times every week until the 15th day. Nude mice injected intraperitoneally with 5 mg/kg Cisplatin once every five days were as positive control. CMC (drug-carrier; 0.5%) was used as the blank control. Tumor volumes were measured once about every three days (
Example 24—the Result of Stability Testing of Different Crystal Forms
(98) Samples of crystal forms I, III and IV were placed in an environment of 92.5% RH for 5 and 10 days, respectively, and tested with XRPD. The XRPD results show that crystal forms I and IV are stable and do not change, while some of the samples of crystal form III transform to crystal form II. The results are shown in
Example 25—the Data for Stability Testing of Crystal Form II
(99) 1. Stress Testing
(100) Six samples of crystal form II (17bNF-II) (batch number: 20161203) were tested under three separate stress conditions: at a temperature of 60° C., at a humidity of 90%, and at a light intensity of 6500Lx; each condition tested over both 5 and 10 days (6 samples total). These samples were tested by X-ray powder diffraction (XRPD) in accordance with the Chinese Pharmacopeia 2015 edition.
(101) The results are shown in
(102) 2. Accelerated Testing
(103) Samples of crystal form II (17bNF-II) (batch numbers: 20161201, 20161202, 20161203) were taken and placed at a temperature of 40° C. and humidity of 75% for 1, 2, 3 and 6 months, respectively. These samples were tested by X-ray powder diffraction (XRPD) in accordance with the Chinese Pharmacopeia of 2015 edition.
(104) The results are shown in
(105) 3. Long-Term Testing
(106) Samples of crystal form II (17bNF-II) (batch number: 20161201, 20161202, 20161203) were taken and placed at a temperature of 25° C. and humidity of 60% for 3, 6, 9, 12, 18, and 24 months, respectively. These samples were tested by X-ray powder diffraction (XRPD) in accordance with the Chinese Pharmacopeia 2015 edition.
(107) The results are shown in
(108) After 24 months of long-term testing, the results show that crystal form II(17bNF-II) is still stable at high temperature, high humidity and high light intensity.
Example 26—Suspended Crystal Transformation Testing
(109) The four different crystal forms (17bNF-I to 17bNF-IV) were mixed in equal proportion and stirred under different conditions and times. The samples were filtered and the solids were collected for XRPD testing, the results of crystal transformation testing are shown in Table 2 and
(110) TABLE-US-00002 TABLE 2 The results of suspended crystal transformation testing Testing Stirring Crystal number Solvent Temperature time Transformation 1 1 ml of water Room 1 day Crystal form II temperature 2 1 ml of water 60° C. 1 day Crystal form II 3 1 ml of MTBE + Room 9 days Crystal form IV 2 μl of water temperature 4 1 ml of MTBE 60° C. 6 hours Crystal form IV 5 1 ml of MTBE Room 1 day Crystal form IV temperature
(111) TABLE-US-00003 TABLE 3 Purity and content data for different crystal forms Crystal form Content (%) Purity (%) 17bNF-O 98.72 98.95 17bNF-I 97.09 98.83 17bNF-II 95.91 98.96 17bNF-III 95.51 95.45 17bNF-IV 98.54 98.69