Crystal forms of an antitumor agent and their preparation methods
20210047362 ยท 2021-02-18
Inventors
Cpc classification
C07J69/00
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides two crystal forms of jervine and their preparation methods, and their application as antitumor agents. The preparation methods of these two crystal forms are simple, high yield and can be easily scaled to industry scale.
Claims
1. The present invention provides crystal form I of jervine, wherein the characteristic peaks represented by 2 angle (0.2) are positioned at 13.5097, 13.7117, 17.7732, 14.5810 in the X-ray powder diffraction pattern. The structure of jervine is as follow: ##STR00002##
2. The crystal form I of claim 1, wherein the characteristic peaks represented by 2 angle (0.2) are positioned at 6.7399 8.7103
10.9690
11.0651
12.1224
15.0048
16.1113
20.2443
20.6768
23.1278
26.2035
27.1966
27.6453
41.0536 in the X-ray powder diffraction pattern.
3. The crystal form of claim 1, wherein the X-ray powder diffraction pattern of crystal form I is shown in
4. A preparation method for the crystal form I of claim 1, comprise the following steps: (1) Jervine is mixed with menthanol at room temperature for a clear solution; (2) remove menthanol till solid start to precipitate, filter to obtain crystal form I.
5. A pharmaceutical agent, wherein the agent contains a therapeutically effective amount of crystal form I of claim 1 and a pharmaceutically acceptable carrier and/or an excipient.
6. The present invention provides crystal form II of jervine, wherein the characteristic peaks represented by 2 angle (0.20) are positioned at 13.6199 13.3548
14.6803
15.0188
14.4729
20.7405
20.9599
17.1282
8.8458
16.3034 in the X-ray powder diffraction pattern.
7. The crystal form II of claim 6, wherein the characteristic peaks represented by 2 angle (0.2) are positioned at 16.0515 19.7802
15.8398
9.1034
11.6825
21.2135
20.4846
17.7607
20.2196
26.0347
23.3938
25.7750
24.0115
18.2995
44.4657
27.4530
31.6225
35.6230
30.7323
26.3009
42.2070 in the X-ray powder diffraction pattern.
8. The crystal form of chain 6, wherein the X-ray powder diffraction pattern of crystal form II is shown in
9. A preparation method for the crystal form II of claim 6, comprise the following steps: (1) Jervine is mixed with acetonitrile at room temperature for a clear solution; (2) remove acetonitrile till solid start to precipitate, filter to obtain crystal form H.
10. A pharmaceutical agent, wherein the agent contains a therapeutically effective amount of crystal form II of claim 6 and a pharmaceutically acceptable carrier and/or an excipient.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0020]
[0021]
[0022]
[0023]
[0024]
EXAMPLES
[0025] The following examples are used to describe the invention, but not to limit the invention.
Example 1: Preparation of Jervine
[0026] 2 kg Li Lu from Sichuan province was grounded to powders, followed by extraction with 95% ethanol (2 L). The liquid was concentrated by vacuum to 350 g leftover. The leftover was dissolved in ethylacetate. Distilled water was used to wash the organic phase, the resulting aqueous phase was disregarded. The organic phase was dried by anhydrous sodium sulfate. The solvent in the organic phase was removed under vacuum, lead to 80 g concentrated raw material. The raw material was loaded on to a silica gel column and CHCl3-MeOH (20:1) was used for the chromatography. 200 mg jervine was obtained at the end.
Example 2: Preparation of Crystal Form I
[0027] Jervine (300 mg) was dissolved in methanol (300 ml), solvent was removed using rotary evaporator till solid start to pericipitate, cooling in refrigerator, filter to obtaining crystal form I, its XRPD spectrum is shown in
Example 3: Preparation of Crystal Form II
[0028] Jervine (300 mg) was dissolved in acetonitrile (300 ml), solvent was removed using rotary evaporator till solid start to pericipitate, cooling in refrigerator, filter to obtaining crystal form II, its XRPD spectrum is shown in
Example 4: In Vitro Test: Experimental Method to Measure the Inhibition of Jervine on the Hedgehog Signaling Pathway
4.1 Cell Culture
[0029] Hedgehog signaling pathway Gli Reporter-NIH3T3 cells were cultured in DMEM medium with 10% Calf serum, 1% Penn-strep, 500 g/ml of Geneticin.
4.2 Measurement Method
[0030] To perform the Gli luciferase reporter assay, Gli ReporterNIH3T3 cells were seeded at 25,000 cells per well into white clear-bottom 96-well microplate in 100 l of growth medium. Cells were incubated at 37 C. and 5% CO2 for overnight. Next day remove the medium from wells and add 45 l of diluted inhibitor in assay medium (Opti-MEM Reduced Serum Medium+0.5% calf serum+1% non-essential amino acids+1 mM Na-pyruvate+10 mM HEPES+1% Pen/Strep) per well. Incubate cells at 37 C. in a CO.sub.2 incubator for 2 hours. Then add 5 l of diluted mShh in assay medium to wells (final [mShh]=1 g/ml). Add 55 l of assay medium to cell-free control wells. Cells were treated for 27 hours. After treatment, cells were lysed and luciferase assay was performed using ONE-Glo luciferase assay system: add 50 l of One-Glo Luciferase reagent per well and rock at room temperature for 20 minutes. Luminescence was measured using a luminometer (BioTek Synergy 2 microplate reader)
4.3 Data Analysis
[0031] Reporter assays in triplicate were performed at each concentration. The luminescence intensity data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the luminescence intensity (L.sub.t) in each data set was defined as 100%. In the absence of cells, the luminescence intensity (L.sub.b) in each data set was defined as 0%. The percent luminescence in the presence of each compound was calculated according to the following equation: % Luminescence=(LL.sub.b)/(L.sub.tL.sub.b), where L=the luminescence intensity in the presence of the compound, L.sub.b=the luminescence intensity in the absence of cells, and L.sub.t=the luminescence intensity in the absence of the compound.
[0032] The values of % luminescence versus a series of compound concentrations were then plotted using non-linear regression analysis of Sigmoidal dose-response curve generated with the equation Y=B+(TB)/1+10.sup.((Log EC50-X)Hill Slope), where Y=percent luminescence, B=minimum percent luminescence, T=maximum percent luminescence, X=logarithm of compound and Hill Slope=slope factor or Hill coefficient. The IC50 value was determined by the concentration causing a half-maximal percent activity.
[0033] The IC50 of jervine inhibiting the Hedgehog signal pathway is 50 nM measured by the above experiment.
Example 5: The Antitumor Effect and Toxicity Study of Jervine on NCI-H2122 and A431 Xenograft Tumor Model Using BALB/c Nude Mice Inoculated Subcutaneously
[0034] The antitumor effect and toxicity study of jervine and GDC-0449 on NCI-H2122 and A431 xenograft tumor model were studied using BALB/c nude mice inoculated subcutaneously.
1. Cell Culture
[0035] NCI-H2122 cells were cultured in RMPI1640 medium, A431 tumor cells cultured in DMEM medium, all the medium were supplemented with 10% heat inactivated fetal bovine serum, all cells used EDTA containing trypsin, passaged twice a week, incubated at 37 C. in an atmosphere of 5% CO2 in air and cultured.
2. Experimental Animals
[0036] 110 BALB/c nude mice, female, 6-8 weeks old, 18-22 g in body weight, were purchased from Shanghai Laboratory Animal Center (SLAC, Shanghai, China). The mice were kept in individually ventilated cage (IVC) systems at constant temperature and humidity, with 20.5-24.5 C. for temperature, 40-75% for humidity. With 4 animals in each cage, the size ogf the cage is 325 mm210 mm180 mm. The bedding material is corn cob, which is changed twice per week. Animals had free access to irradiation sterilized dry granule food and water during the entire study period. The food and water were changed twice per week. The identification labels for each cage contain the following information: number of animals, sex, strain, and date received treatment, study number, group number, and the starting date of the treatment. Animals were marked by ear coding. The experiments started one week after receiving the animals.
3. Cell Inoculation
[0037] Each mouse were inoculated subcutaneously at the right flank region with NCI-H2122 tumor cells (510.sup.7/ml), NCI-H1703 tumor cells (110.sup.8/ml) and A431 tumor cells (510.sup.7/ml), respectively in a mixture of phosphate-buffered saline and matrigel for tumor development via a 1 ml syringe. The treatments were started when the mean tumor size reached about 200 (150-250) mm.sup.3. The test articles administration and the animal numbers in each study group were shown in table 1.
TABLE-US-00001 TABLE 1 Dose Dosing Group N Treatment (mg/kg) Route Schedule 1 8 Vehicle Control 0 p.o. QD 21 days 2 8 Jervine 40 p.o. QD 21 days 3 8 Jervine 60 p.o. QD 21 days 4 8 Jervine 80 p.o. QD 21 days 5 8 GDC-0449 40 p.o. QD 21 days Note: N: animal number; p.o. oral administration, QD: once every day, Administration volume: 0.1 ml/10 g, Administration time will be adjusted after 15% weights lose.
4. Randomized Grouping
[0038] To avoid errors between groups, the animals were first grouped by the tumor volumes according to the tumor volume measurements, and then randomly re-assigned to different groups.
5. Observations
[0039] During the study, the care and use of animals were conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). After tumor cell inoculation, the animals were checked daily for morbidity and mortality. At the time of routine monitoring, the animals were checked for any effects of tumor growth and treatments on normal behavior such as mobility, visual estimation of food and water consumption, body weight gain/loss, eye/hair matting and any other abnormal effect.
6. Aseptic
[0040] The preparation of the solutions of the test articles, observation, dosing, measurement of the tumor and the body weight were all done in a biological safety cabinet.
7. Data Processing
[0041] After tumor volume reached 3000 mm.sup.3 or average tumor volume reached 2000 mm.sup.3, before experimental animals emaciated or coma, anesthesia and euthanasia will be performed. During the experiment, if the weight loss of experimental animal reaches 20%, administration will be discontinued and experimental animals will be observed until the weight returned to 10% range before restart of the administration. In this experiment, when the mean tumor volume reached 2000 mm.sup.3 or at the end of 59 days after the administration, experimental animals will be anesthesiaed to collect tumor mass which will be weighed and photographed.
8. Measurement
[0042] 8.1 Tumor size was measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm.sup.3 using the formula: V=0.5 ab.sup.2 where a and b are the long and short diameters of the tumor, respectively. [0043] 8.2 T/C, percent value, T and C were the mean volume of the treated and control groups, respectively, on a given day. [0044] 8.3 TC, calculated with T as the time (in days) required for the mean tumor size of the treatment group to reach a predetermined size, and C was the time (in days) for the mean tumor size of the control group to reach the same size. [0045] 8.4 (1((TdT0)/(CdC0)))100% value (in percent) was an indication of antitumor effectiveness, Td and Cd were the mean tumor volumes of the treated and vehicle control animals on a given day and T0 and C0 were the mean tumor volumes of the treated and vehicle control animals at the start of the experiment. [0046] 8.5 Tumor mass weight was measured at the end of study.
9. Statistical Analysis
[0047] A one-way analysis of variance was performed followed by multiple comparison procedures. All data was analyzed using SPSS 18.0. *p<0.05 or **P<0.01 are generally considered statistically significant when compared with vehicle group.
[0048] Results showed jervine can effectively inhibit the tumor growth in a NCI-2122 human cancer xenograft model, the T/C value at dose levels of 40 mg/kg, 60 mg/kg and 80 mg/kg for 21 days is 0.79, 0.701 and 0.594. The T/C value for the test compound GDC-0449 is 0.794 at a dose level of 40 mg/kg. The experimental result was shown in
[0049] Results showed jervine can effectively inhibit the tumor growth in A-431 xenograft tumor model, the T/C value at dose levels of 40 mg/kg, 60 mg/kg and 80 mg/kg for 21 days is 0.52, 0.80 and 0.82. The T/C value for the test compound GDC-0449 is 0.89 at a dose level of 40 mg/kg. The result was shown in
Example 6: The Antitumor Effect and Toxicity Study of Jervine on BXPC-3 Xenograft Tumor Model Using BALB/c Nude Mice Inoculated Subcutaneously
[0050] The antitumor effect and toxicity study of jervine and GDC-0449 on BXPC-3 xenograft tumor model were studied using BALB/c nude mice inoculated subcutaneously.
Cell Culture
[0051] BXPC-3 tumor cells cultured in RMPI1640 medium supplemented with 10% heat inactivated fetal bovine serum, all cells used EDTA containing trypsin, passaged twice a week, incubated at 37 C. in an atmosphere of 5% CO2 in air and cultured.
Experimental Animal
[0052] BALB/c nude mice, female, 6-8 weeks old, 18-22 g in body weight, were purchased from Shanghai Laboratory Animal Center (SLAC, Shanghai, China).
Cell Inoculation
[0053] Each mouse was inoculated subcutaneously at the right flank region with BXPC-3 tumor cells (510.sup.7) in 0.1 ml of phosphate-buffered saline with matrigel via a 1 ml syringe for tumor development. The treatments were started when the mean tumor size reaches approximately 200 (150-250) mm.sup.3 at 9 days after inoculation. The test article administration and the animal numbers in each study group are shown in table 2.
TABLE-US-00002 TABLE 2 Dose Dosing Group N Treatment (mg/kg) Route Schedule 1 8 Vehicle Control 0 iv QD 21 days 2 8 Jervine 20 iv QD 21 days 3 8 Jervine 40 iv. QD 21 days 4 8 Jervine 80 iv QD 21 days 5 8 GDC-0449 40 iv QD 21 days Note: N: animal number; p.o. oral administration, QD: once every day, Administration volume: 0.1 ml/10 g, Administration time will be adjusted after 15% weights lose.
Randomized grouping, observations, aseptic, data processing, measurement and statistical analysis are as described in Example 3.
[0054] Results showed jervine can effectively inhibit the tumor growth in a BXPC-3 human pancreatic carcinoma xenograft model, the T/C value at dose levels of 20 mg/kg, 40 mg/kg and 80 mg/kg is 25.8%, 59.1% and 54.2%. The T/C value for the test compound GDC-0449 is 80.7% at a dose level of 40 mg/kg. Result was shown in
Example 7: Pharmacokinetic Study of Jervine
[0055] Orally dosed: A total of 3 male experimental CD-1 mice, approximately 8 weeks of age at receipt was administered jervine via oral gavage at 10 mg/kg. Blood samples (300 L) were collected via cardiac puncture predose and at 2 minute, 5 minute, 15 minute, 0.5 hour, 1 hour, 2 hour, 4 hour, 8 hour, 24 hours postdose. Plasma was made form the blood samples in order to measure the concentration of jervine. The PK data is shown in table 3, average bioavailability is 41.2%. The experimental data suggests jervine can maintain a certain concentration inside the body, which will help its antitumor effect.
TABLE-US-00003 TABLE 3 pharmacokinetic result of jervine in mice PK Cmax T1/2 CL Vz AUC(0-t) AUC(0-inf) F Parameters ng/mL hr L/hr L ng*hr/mL ng*hr/mL % PO 368 11.8 0.13 0.68 1794 2260 41.2 10 mg/Kg
Example 8: Study the MTD of Jervine on BALB/c Nude Mice
[0056] 9 BALB/c nude mice were divided equally into 3 groups. The mice were orally dosed at 50 mg/kg, 80 mg/kg and 150 mg/kg by groups. Two mice in the 150 mg/kg treatment group were dead, there was no animal dead in the other two groups after 14 days treatment. It was initially determined that the MTD is higher than 80 mg/kg for BALB/c nude mice.
Example 9: Preparation of Jervine Hydrochloride Acid Salt
[0057] Jervine (1 g) was dissolved in anhydrous ethanol (30 ml) by heating. After the solution cooled to room temperature, hydrochloride acid was added drop by drop till white precipitate was formed. Jervine hydrochloride acid salt (1 g) was obtained after filtration and heat drying.