WO2023221419A1 - 沃诺拉赞焦谷氨酸盐的晶型及其制备方法 - Google Patents

沃诺拉赞焦谷氨酸盐的晶型及其制备方法 Download PDF

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WO2023221419A1
WO2023221419A1 PCT/CN2022/131030 CN2022131030W WO2023221419A1 WO 2023221419 A1 WO2023221419 A1 WO 2023221419A1 CN 2022131030 W CN2022131030 W CN 2022131030W WO 2023221419 A1 WO2023221419 A1 WO 2023221419A1
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crystal form
pyroglutamate
vonoprazan
gastric
pharmaceutical composition
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PCT/CN2022/131030
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English (en)
French (fr)
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宋伟国
王西龙
甲宗青
李法辉
宋成刚
徐明杰
乔玉晶
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山东道合药业有限公司
道合(潍坊)医药技术有限公司
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Priority to CN202280046217.9A priority Critical patent/CN117597338A/zh
Priority to CN202311001894.1A priority patent/CN116966180B/zh
Publication of WO2023221419A1 publication Critical patent/WO2023221419A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the invention belongs to the field of pharmaceutical crystal forms, and specifically relates to the crystal form of vonorazan pyroglutamate and its preparation method.
  • Vonoprazan Fumarate (TAK-438) was developed by Takeda of Japan and obtained marketing approval in Japan on December 26, 2014.
  • the drug is a potassium-competitive acid blocker (Potassium-competitive acid blocker).
  • P-CABs have the characteristics of lipophilicity, weak alkalinity, high dissociation constant and stability at low pH value. Therefore, vonoprazan fumarate has a quick-acting, strong and lasting inhibitory effect on gastric acid secretion.
  • the invention provides a crystalline form I of vonolazan pyroglutamate, which is characterized in that: using Cu/K ⁇ radiation, the X-ray powder diffraction pattern expressed in 2 ⁇ diffraction angle is 9.000 ⁇ 0.200, 10.280 ⁇ 0.200, 11.340 ⁇ 0.200, 12.440 ⁇ 0.200, 13.480 ⁇ 0.200, 14.360 ⁇ 0.200, 15.640 ⁇ 0.200, 17.100 ⁇ 0.200, 18.000 ⁇ 0.200, 18.500 ⁇ 0.200, 19.240 ⁇ 0.200, 19.7 20 ⁇ 0.200, 20.820 ⁇ 0.200, 21.660 ⁇ 0.200, 22.500 ⁇ 0.200, 24.040 ⁇ 0.200, 24.860 ⁇ 0.200, 25.720 ⁇ 0.200, 26.400 ⁇ 0.200, 27.220 ⁇ 0.200, 28.600 ⁇ 0.200, 31.320 ⁇ 0.200, 33.200 ⁇ 0.200, 34.100 ⁇ 0.20 0, 34.600 ⁇ 0.200, 36.480 ⁇ 0.200, 43.460 ⁇ 0.200 There are characteristic peaks. Form I may also have characteristics represented by an X-ray powder
  • Another embodiment of the present invention provides a crystalline form I of vonolazan pyroglutamate, which is characterized in that: using Cu/K ⁇ radiation, the crystalline form I has 27 characteristic peaks, and its X-ray powder diffraction
  • the diagram is basically as shown in Figure 2.
  • the infrared spectrum of Vonoprazanpyroglutamate crystal form I according to the present invention has characteristic absorption peaks at approximately 3255 ⁇ 5, 2738 ⁇ 5, 1690 ⁇ 5, 1657 ⁇ 5, and 1573 ⁇ 5cm -1 .
  • Form I may also have characteristics substantially represented by an infrared spectrum as shown in Figure 3.
  • thermogravimetric analysis the Vonoprazan pyroglutamate crystal form I of the present invention loses weight for the first time at 297.9 ⁇ 1.0°C, with a weight loss rate of 50.3 ⁇ 2.0%, which is caused by material decomposition.
  • the thermogravimetric analysis TGA chart of Form I is basically shown in Figure 4.
  • Another embodiment of the present invention provides a method for preparing the above-mentioned vonorazan pyroglutamate crystal form I, which is characterized in that it includes the following steps: adding 5-(2-fluorophenyl)-N-methyl-1- Dissolve (3-pyridylsulfonyl)-1H-pyrrole-3-methylamine pyroglutamate in methanol, add ketone solvent dropwise, stir for crystallization, filter and dry to obtain crystal form I.
  • Methanol dissolution can be carried out at room temperature, or artificially heated to 20-35°C.
  • Ketone solvents are preferably acetone, methyl ethyl ketone, etc.
  • the crystallization process can be carried out at room temperature, or artificially cooled to 0-5°C. Parameters such as the amount of methanol and ketone solvent, temperature, etc. can be reasonably selected by those skilled in the art based on the actual situation of recrystallization (such as experimental phenomena).
  • Another embodiment of the present invention provides the use of the above-mentioned vonoprazan pyroglutamate crystal form I or a pharmaceutically acceptable salt thereof in the preparation of a potassium ion competitive acid blocker.
  • Another embodiment of the present invention provides the use of the above-mentioned vonoprazan pyroglutamate crystal form I or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting gastric acid secretion.
  • the medicine is used to treat and/or prevent gastric ulcer, duodenal ulcer, reflux esophagitis, erosive esophagitis, gastroesophageal reflux disease, Helicobacter infection, peptic ulcer and other diseases.
  • Another embodiment of the present invention provides a use of a pharmaceutical composition in the treatment and/or prevention of diseases, including gastric ulcer, duodenal ulcer, reflux esophagitis, erosive esophagitis, gastroesophageal reflux One or more diseases, Helicobacter infection, peptic ulcer, etc.; the pharmaceutical composition uses vonolazan pyroglutamate crystal form I or its pharmaceutically acceptable salt as an active ingredient; the pharmaceutical combination
  • the pharmaceutical composition may also include other potassium ion competitive acid blockers or drugs that inhibit gastric acid secretion; the pharmaceutical composition may also include pharmaceutically acceptable pharmaceutical excipients (such as pharmaceutically acceptable carriers, diluents or excipients).
  • the dosage form of the pharmaceutical composition can be a solid preparation, a liquid preparation or a semi-solid preparation, preferably tablets, capsules, injections (including powder injections), microemulsions, sub-microemulsions, including sustained-release tablets, sustained-release capsules, sustained-release capsules, etc. release injection.
  • pharmaceutically acceptable salt in the present invention refers to non-toxic addition salts of inorganic or organic acids and/or bases. See “Salt selection for basic drugs”, Int.J.Pharm. (1986), 33, 201-217.
  • the various dosage forms involved in the pharmaceutical composition of the present invention can be prepared in accordance with the technical specifications and requirements in the pharmaceutical field (for example, the requirements and specifications of the General Preparation Chapters of the "Chinese Pharmacopoeia” (2015 Edition), textbooks or other methods in the prior art) to meet clinical treatment requirements. and/or preparations that prevent the need to inhibit gastric acid secretion and their various suitable specifications and dosage forms (including solid preparations, liquid preparations and semi-solid preparations) such as sustained-release, controlled-release and enteric-coated capsules, tablets, microemulsions, submicron Emulsions may contain the same single and multi-component combinations of various injections.
  • the X-ray powder diffraction (XRPD) analysis of Vonoprazan pyroglutamate crystal form I according to the present invention is carried out under ambient temperature and ambient humidity using a Rigaku MiniFlex600 powder X-ray diffractometer, Rigaku (3- 80°, step: 0.0200°, speed: 10°/min), Cu/K ⁇ source (40kV, 40mA) measurement completed.
  • the infrared spectrum analysis of the present invention is measured by the KBr tableting method using an American Thermo Nicolet 6700 infrared spectrophotometer under ambient temperature and ambient humidity.
  • thermogravimetric analysis (TGA) of the present invention is measured by a Swiss Mettler TGA/DSC1/1100LF synchronous thermal analyzer under ambient temperature and ambient humidity.
  • the scanning rate is 10.00°C/min, the maximum temperature is 800.00°C, and the protective gas is nitrogen.
  • "Ambient temperature” is generally 0-40°C;
  • “ambient humidity” is generally 30%-80% relative humidity.
  • the 2 ⁇ value of the X-ray powder diffraction pattern may vary slightly between machines or samples, and the experimental error of the 2 ⁇ value is generally ⁇ 0.2°; the experimental error of the absorption peak in the infrared spectrum is generally ⁇ 5cm -1 ; thermogravimetry The experimental error of the weight loss temperature in the analysis is generally ⁇ 1°C, and the experimental error of the weight loss percentage is generally ⁇ 2.0%.
  • Vonoprazan pyroglutamate refers to the fact that one molecule of Vonoprazan corresponds to 1 molecule of L-pyroglutamic acid, that is, the 5-(2- The molar ratio of fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methanamine and L-pyroglutamic acid is 1:1.
  • Mobile phase A acetonitrile-0.02mol/L potassium dihydrogen phosphate (use phosphoric acid to adjust the pH to 2.5) (volume ratio 5:95)
  • Mobile phase B methanol-acetonitrile-0.02mol/L potassium dihydrogen phosphate (use phosphoric acid to adjust the pH to 2.5) (volume ratio 7:63:30)
  • the advantages of the present invention are: (1) The present invention obtains a new crystal form of vonolazan pyroglutamate, namely crystal form I, which not only has good solubility, but also has excellent Storage stability; (2) In vitro activity experiments show that the inhibitory effect of crystalline form I of the present invention on H + K + -ATPase is equivalent to that of TAK-438, which is better than that of the prior art (Chinese patent application number: 201410154778.8) Wonuo Lazen pyroglutamate (another crystal form, Figure 5); (3) Animal experiments show that intravenous administration of crystal form I of the present invention can significantly inhibit histamine-induced gastric acid secretion in rats, and is more effective than TAK- 438 is administered into the duodenum and exerts its efficacy faster.
  • Figure 1 is an X-ray powder diffraction pattern of Vonoprazan pyroglutamate crystal form I of the present invention
  • Figure 2 shows the 27 characteristic peaks of X-ray powder diffraction of Form I and their diffraction angle 2 ⁇ and crystal plane spacing d. Relative intensity (%) and other specific parameter charts;
  • Figure 3 is the infrared spectrum of crystal form I
  • Figure 4 is a thermogravimetric analysis diagram of Form I
  • Figure 5 is the X-ray powder diffraction pattern of Product A
  • Figure 6 is a graph showing the inhibition rate of TAK438, product A and crystal form I on microsomal vesicle H + K + -ATPase in pig gastric mucosa;
  • Figure 7 is a comparison chart of the body weight of rats in each group.
  • Figure 8 is a graph showing the effects of each group of test substances on histamine-induced gastric acid secretion
  • Figure 9 is a graph showing the effects of each group of test substances on the inhibition rate of gastric acid secretion in rats induced by histamine.
  • Deliquescence test Store product A, crystal form I, and TAK-438 under the conditions of 40°C and 75% relative humidity, and observe whether there is deliquescence phenomenon. The test time is 30 days, and the results are recorded every 10 days. See table below for details.
  • No deliquescence means that no moisture absorption is observed on the surface of the sample, slight deliquescence means that moisture absorption on the surface of the sample is observed, and significant deliquescence means that the surface of the sample can be observed to be smooth.
  • the crystalline form I of the present invention has good stability and is convenient for long-term storage.
  • the basic method of melting point testing Grind an appropriate amount of the sample to be tested into fine powder, and put it into a capillary tube with one end sealed, so that the powder is tightly gathered at the sealed end of the capillary tube.
  • the height of the loaded sample is about 3mm, and the heating rate of the melting point analyzer is 1°C/min. Repeat the measurement three times and take the average value. The results are shown in the table below.
  • H + K + -ATPase is an ATPase located in gastric mucosal parietal cells and can be specifically activated by K + .
  • H + K + -ATPase was extracted from the microsomal vesicles of pig gastric mucosa. ATPase can decompose ATP to generate ADP and inorganic phosphorus.
  • the test substance binds to H + K + -ATPase and inhibits its activity. By measuring the content of inorganic phosphorus amount, determine the level of ATPase activity, and determine the inhibitory effect of the test substance.
  • the specific method is as follows:
  • the pig gastric mucosal microsomal vesicle H + K + ATPase used in this experiment was extracted by Beijing Huizhi Taikang.
  • Preparation of BCA working solution According to the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), and mix thoroughly. For example, add 5 mL BCA reagent A to 100 ⁇ L BCA reagent B, mix well, and prepare 5.1 mL BCA working solution. BCA working solution is stable at room temperature for 24 hours.
  • TRIS-HCL 100mM TRIS-HCL (PH6.8): Add 100ul of 1M TRIS-HCL to 900ul of ddH 2 O and mix evenly.
  • MgCl 2 37.5mM MgCl 2 : Weigh 50 mg of MgCl 2, add 14 ml of ddH 2 O, and mix well.
  • 10mM nigericin and valinomycin add 669ul and 890ul DMSO respectively, mix well, divide into 1ul per tube of nigericin and 5ul per tube of valinomycin, and store at -20°C.
  • microsomal vesicles After arrival, measure the protein concentration and prepare separately for use. Store at -80°C and dilute with 5mM TRIS-HCL before use.
  • Control compound TAK438 was prepared into a 10mM stock solution with DMSO, and 1ul was dispensed into each tube.
  • Test compound Product A and crystal form I were prepared into 10mM stock solutions with ddH 2 O, and 1ul was dispensed into each tube. With 20 ⁇ M (or 40 ⁇ M) as the highest concentration, the control compound and the test compound are gradually diluted 3 times with DMSO and ddH 2 O respectively, to obtain 6 (or 7 or 8) concentration gradient test samples.
  • Control tube 100mM TRIS-HCL 60 60 37.5mM MgCl 2 20 20 200mM KCl 15 / ddH 2 O / 15 nigericin 1.5 1.5 Valinomycin 1.5 1.5 0.1mg/mL microsomal vesicles 50 50 compound 1.5 1.5
  • Inhibition rate (%) (1-(RLU compound -RLU blank )/(RLU DMSO -RLU blank )) ⁇ 100%.
  • BCA protein quantification According to the BSA standard curve, the average protein concentration was calculated to be 3mg/mL.
  • Rats were anesthetized by intraperitoneal injection of 1.5g/kg Ule sugar. After complete anesthesia, the hair on the neck and abdomen of the rats was shaved. In order to keep the airway open during the operation, the rats were given tracheal intubation. Insert the cardia perfusion tube approximately 1cm into the stomach of the rat through the mouth, and connect the other end of the cardia perfusion tube to the constant flow pump.
  • Histamine-induced gastric acid secretion Histamine was continuously administered to rats through the tail vein using a microsyringe pump at a dose of 20 ⁇ mol/kg/hr. Collect gastric acid after 80 minutes. After collecting three points, the rats' histamine secretion reaches a stable state. Rats are divided into groups according to the value of gastric acid secretion after administration of histamine. Candidate drugs or solvent controls are administered according to the protocol. Continue to collect gastric acid, and collect 9 points after administration, when the drug effect reaches a stable state.
  • vonoprazan fumarate (TAK-438), preparation method: the entire preparation process is carried out under light-proof conditions. Weigh the required amount of vonoprazan fumarate, add an appropriate amount of 0.5% CMC-Na, stir, and sonicate to obtain a uniform suspension. Dilute to the corresponding concentration in sequence according to the concentration required in the plan.
  • Test substance Crystal Form I (represented as HW-N2001 in the experiment), preparation method: The entire preparation process is carried out under light-proof conditions. Weigh the required amount of HW-N2001, add appropriate physiological saline, mix thoroughly to obtain a clear solution, and adjust the pH value to the range of 3.2 to 3.8. According to the concentration required by the protocol, dilute to the corresponding concentration in sequence.
  • Type of litter corn cob litter (Dezhou Gumei Agricultural Technology Co., Ltd., batch number: GMCC202102170-2).
  • Feed type SPF low-protein rat maintenance feed (Jiangsu Synergy Pharmaceutical Bioengineering Co., Ltd., 20210225(X)).
  • Feeding method Free intake.
  • Type of drinking water High-pressure sterilized drinking water for experimental animals.
  • Water supply method Drinking water bottle, free intake.
  • mice such as mice, rats, dogs and monkeys
  • mice are commonly used to conduct studies on pharmacokinetic parameters, pharmacodynamics and toxicology.
  • the number of animals used is the minimum required to determine inter-individual variability among experimental animals. Animals used in experiments must be healthy and adapted to the environment, with free access to food and water. This experiment used bisex SD rats for testing.
  • 118 SD rats in good health were selected from 130 SD rats. After the adaptation period, they were randomly divided into 8 groups according to their body weight and baseline value after histamine administration. See the table below for details.
  • Histamine dihydrochloride Freshly prepare histamine dihydrochloride (0.6mg/mL, dissolved in normal saline).
  • Ulay sugar Weigh an appropriate amount of Ulay sugar, add physiological saline, dissolve with ultrasound, and prepare to a final concentration of 0.3g/mL, set aside.
  • Phenolphthalein Weigh an appropriate amount of phenolphthalein, add absolute ethanol, and prepare to a final concentration of 0.5%. Set aside.
  • Basic titrant Take an appropriate amount of 0.1mol/L NaOH solution, add fresh deionized water, dilute to 0.01mol/L, and set aside.
  • Heparin sodium Weigh an appropriate amount of heparin sodium, add physiological saline, dissolve to a final concentration of 100U/mL, and set aside.
  • Gastric cardia perfusion tube Take about 20cm of medical polyethylene hose (outer diameter 2mm, inner diameter 1.5mm) as a spare cardia-gastric tube.
  • Gastric pylorus sample collection tube Take a medical polyethylene hose (outer diameter 4mm, inner diameter 3mm) about 30cm, and use it as the pylorus intubation end for later use.
  • Histamine perfusion tube draw 10 mL of histamine dihydrochloride into a 10 mL syringe, connect it to the infusion extension tube, wrap it in tin foil to protect it from light, and fix it in the syringe pump for later use.
  • Acid secretion amount ( ⁇ Eq) NaOH volume (mL) ⁇ 10 -3 ⁇ 0.01mol/L ⁇ 10 6
  • Tail vein indwelling needle Disinfect the tail vein with alcohol, insert the indwelling needle into the tail vein, pull out the needle core of the indwelling needle, push in 0.5ml of heparin sodium solution, remove the syringe and screw on the heparin sodium cap.
  • Collect 2 basic gastric juice samples After the basic gastric acid level is stable, connect the infusion extension tube for inhaling histamine in advance to the tail vein indwelling needle of the rat. Turn on the syringe pump. Calculate and set the injection according to the amount of histamine of 20 ⁇ mol/kg/hr. Pump the intravenous infusion speed of each animal. After continuously injecting the histamine solution for 80 minutes, the gastric acid samples after histamine administration can be collected. The gastric juice after three administrations of histamine is collected. When the gastric acid secretion reaches a stable state, the test substance is administered intravenously according to the grouping. Or give positive drug or solvent control to the duodenum, and continue to collect gastric juice. About 3 hours after administration, the alkali consumption reaches a steady state, and a total of about 14 points are collected.
  • Body weight Weigh before fasting the day before the experiment and before anesthesia on the day of the experiment.
  • Total acidity of gastric juice Determine the total acidity of gastric juice using the acid-base titration method.
  • the color indicator is 0.5% phenolphthalein, and the titration endpoint is pH7.0.
  • the experimental data are expressed as mean plus or minus standard deviation (Mean ⁇ SD), and the One-Sample Kolmogorov-Smirnov Test (One-Sample Kolmogorov-Smirnov Test) method is used to test the normal distribution.
  • One-Sample Kolmogorov-Smirnov Test One-Sample Kolmogorov-Smirnov Test
  • one-way ANOVA is used.
  • the LSD test is used for multiple comparisons between the means of multiple samples.
  • P ⁇ 0.05 is considered to be a significant difference; the variances are uneven.
  • the Games-Howell test was used for multiple comparisons between multiple sample means.
  • the Independent samples test of non-parametric test is used. P ⁇ 0.05 is considered to be a significant difference between multiple samples. All data statistical analysis is carried out using SPSS16.0 statistical software.
  • Vehicle control group Gastric acid was collected at two points before rats were given histamine. The basic values of gastric acid secretion were: 6.43 ⁇ 2.37 and 8.83 ⁇ 5.1 ( ⁇ Eq HCl/20min), respectively. There was no significant difference in the group ratio; after rats were given histamine for 80 minutes, gastric acid secretion increased significantly at three points and reached steady state, which were 42.3 ⁇ 14.58, 45.96 ⁇ 16.61, and 46.65 ⁇ 16.96 ( ⁇ Eq HCl/20min), respectively. and There were significant differences in group ratios; after intravenous administration of solvent control, each time point was compared with There is a significant difference in the group ratio.
  • Vonoprazan fumarate (1mg/kg) group After the animals were given histamine, the gastric acid secretion reached a steady state. After administration, the gastric acid secretion of rats did not significantly decrease, and there was no statistical difference compared with the Vehicle group.
  • Vonoprazan fumarate (2mg/kg) group After the animals were given histamine, the gastric acid secretion reached a steady state. The gastric acid secretion of rats gradually decreased between 60min and 180min after administration. The gastric acid secretion of rats gradually decreased 60min after administration. The secretion amount was 33.21 ⁇ 15.63 ( ⁇ Eq HCl/20min), and the gastric acid secretion inhibition rate was 13.71% ⁇ 2.12, which was statistically different from the solvent group. The gastric acid secretion amount of rats 180 minutes after administration was 16.21 ⁇ 14.46 ( ⁇ Eq HCl/20min). 20min), the inhibition rate of gastric acid secretion was 57.48% ⁇ 2.27, which was significantly different from the blank control group.
  • Vonoprazan fumarate (4mg/kg) group After the animals were given histamine, the gastric acid secretion reached a steady state. The gastric acid secretion of rats gradually decreased between 40min and 180min after administration. The gastric acid secretion of rats gradually decreased 40min after administration. The secretion amount was 33.18 ⁇ 11.15 ( ⁇ Eq HCl/20min), which was not statistically different from the solvent group. The inhibition rate of gastric acid secretion was 12.47% ⁇ 1.13, which was statistically different from the solvent control group. Rats 180 minutes after administration The amount of gastric acid secretion was 4.41 ⁇ 4.02 ( ⁇ Eq HCl/20min), and the inhibition rate of gastric acid secretion was 87.03% ⁇ 0.81, which was significantly different from the blank control group.
  • HW-N2001 (0.375mg/kg) group After the animals were given histamine, the gastric acid secretion reached a steady state. The gastric acid secretion of rats gradually decreased between 60min and 180min after administration. At 60min after administration, the gastric acid secretion of rats was 39.15 ⁇ 8.54 ( ⁇ Eq HCl/20min), the inhibition rate of gastric acid secretion was 4.34% ⁇ 1.28, and there was no statistical difference compared with the solvent control group. The gastric acid secretion of rats 180 minutes after administration was 28.46 ⁇ 5.71 ( ⁇ Eq HCl/20min) ), the gastric acid secretion inhibition rate was 27.52% ⁇ 1.43, which was significantly different from the blank control group.
  • HW-N2001 (0.75mg/kg) group After the animals were given histamine, the gastric acid secretion reached a steady state. The gastric acid secretion of rats gradually decreased between 20min and 180min after administration. At 20min after administration, the gastric acid secretion of rats was 37.53 ⁇ 9.87 ( ⁇ Eq HCl/20min), there is no significant difference compared with the blank control group, the gastric acid secretion inhibition rate is 11.6% ⁇ 0.82, there is a statistical difference compared with the solvent control group, the gastric acid secretion of rats 180 minutes after administration The amount was 10 ⁇ 5.32 ( ⁇ Eq HCl/20min), and the gastric acid secretion inhibition rate was 73.68% ⁇ 1.47, which was significantly different from the blank control group.
  • HW-N2001 (1.5mg/kg) group After the animals were given histamine, the gastric acid secretion reached a steady state. 20min-80min after administration, the gastric acid secretion of rats gradually decreased, and the drug effect reached a steady state. 20min after administration, The gastric acid secretion of rats was 25.67 ⁇ 10.15 ( ⁇ Eq HCl/20min), and the inhibition rate of gastric acid secretion was 33.28% ⁇ 0. There was a statistical difference compared with the solvent control group. The gastric acid secretion of rats 80 minutes after administration was 3.4 ⁇ 0. 1.18 ( ⁇ Eq HCl/20min), the gastric acid secretion inhibition rate is 89.64% ⁇ 0, which is significantly different from the blank control group. The gastric acid secretion of rats 180 minutes after administration was 2.2 ⁇ 0.56 ( ⁇ Eq HCl/20min), and the gastric acid secretion inhibition rate was 93.56% ⁇ 0.09, which was significantly different from the blank control group.
  • the present invention adopts a gastric acid secretion model induced by histamine in SD rats by continuously detecting it to study the pharmacodynamic effect of intravenous administration of Vonoprazan pyroglutamate crystal form I (HW-N2001) on gastric acid secretion in rats.
  • HW-N2001 (0.375mg/kg, 0.75mg/kg, 1.5mg/kg) was administered intravenously.
  • the gastric acid secretion in the administration group was significantly lower than that in the solvent control group, and there was a certain dose-effect relationship.
  • 0.75 and In the 1.5 mg/kg dosage group the drug gradually began to exert its efficacy 20 minutes after administration, and reached a steady state 60-100 minutes after administration.
  • the ID 50 was 0.53 mg/kg 180 minutes after administration.
  • the intravenous administration of vonoprazan pyroglutamate crystal form I (HW-N2001) of the present invention can significantly inhibit histamine-induced gastric acid secretion in rats, and is more effective than vonoprazan fumarate.
  • Duodenal administration results in faster pharmacological effects.

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Abstract

本发明涉及沃诺拉赞焦谷氨酸盐的晶型及其制备方法。本发明获得的沃诺拉赞焦谷氨酸盐晶型，不仅具有良好的溶解性，而且具备优异的存储稳定性；体外活性实验表明，本发明晶型I对H +K +-ATP酶的抑制作用与TAK-438相当；动物实验表明，本发明晶型经静脉给药能够显著抑制组胺诱导的大鼠胃酸分泌，且能够较TAK-438十二指肠给药更快发挥药效作用。

Description

沃诺拉赞焦谷氨酸盐的晶型及其制备方法 技术领域
本发明属于药物晶型领域，具体涉及沃诺拉赞焦谷氨酸盐的晶型及其制备方法。
背景技术
沃诺拉赞(Vonoprazan)，化学名为5-(2-氟苯基)-N-甲基-1-(3-吡啶基磺酰基)-1H-吡咯-3-甲胺，其化学结构式如下：
Figure PCTCN2022131030-appb-000001
富马酸沃诺拉赞(Vonoprazan Fumarate，TAK-438)是由日本武田开发，2014年12月26日在日本获得上市许可，该药属于钾离子竞争性酸阻滞剂(Potassium-competitive acid blocker，P-CAB)的新一类抑制剂。P-CABs具有亲脂性、弱碱性、解离常数高和在低pH值时稳定的特点，因此富马酸沃诺拉赞具有速效、强劲、持久的胃酸分泌抑制作用。同时，在胃壁细胞胃酸分泌的最后一步中，通过抑制K +对H +-K +-ATP酶(质子泵)的结合作用，对胃酸分泌也具有提前终止作用，但该化合物的水溶性较差。本发明对水溶性较好的沃诺拉赞焦谷氨酸盐进行晶型研究及体内外生物活性研究，获得一种溶解性、稳定性、体内生物活性均较好的沃诺拉赞焦谷氨酸盐晶型I。
发明内容
本发明提供一种沃诺拉赞焦谷氨酸盐的晶型I，其特征在于：使用Cu/Kα辐射，以2θ衍射角表示的X-射线粉末衍射图在9.000±0.200，10.280±0.200，11.340±0.200，12.440±0.200，13.480±0.200，14.360±0.200，15.640±0.200，17.100±0.200，18.000±0.200，18.500±0.200，19.240±0.200，19.720±0.200，20.820±0.200，21.660±0.200，22.500±0.200，24.040±0.200，24.860±0.200，25.720±0.200，26.400±0.200，27.220±0.200，28.600±0.200，31.320±0.200，33.200±0.200，34.100±0.200，34.600±0.200，36.480±0.200，43.460±0.200处有特征峰。晶型I还可具有基本如图1所示的X-射线粉末衍射图所代表的特征。
本发明的另一实施方案提供一种沃诺拉赞焦谷氨酸盐的晶型I，其特征在于：使用Cu/Kα辐射，该晶型I具有27个特征峰，其X-射线粉末衍射图基本如图2所示。
本领域的技术人员应当理解的是，当就基本上相同的晶体形式进行测量时，XRPD图谱中2θ特征峰的峰高相对强度、可能出于各种原因发生变化。
本发明所述的沃诺拉赞焦谷氨酸盐晶型I的红外图谱在约3255±5、2738±5、1690±5、1657±5、1573±5cm -1的位置有特征吸收峰。晶型I还可具有基本如图3所示的红外图谱所代表的特征。
本发明所述的沃诺拉赞焦谷氨酸盐晶型I经热重分析，其在297.9±1.0℃第一次失重，失重率为50.3±2.0％，由物质分解所致。晶型I的热重分析TGA图基本如图4所示。
本发明的另一实施方案提供上述沃诺拉赞焦谷氨酸盐晶型I的制备方法，其特征在于包括如下步骤：将5-(2-氟苯基)-N-甲基-1-(3-吡啶基磺酰基)-1H-吡咯-3-甲胺焦谷氨酸盐用甲醇溶解，再滴加酮类溶剂，搅拌析晶，过滤，干燥即得晶型I。
甲醇溶解可选择室温进行，或人工加热至20～35℃，酮类溶剂优选丙酮、甲基乙基酮等，析晶过程可室温进行，或人工降温至0～5℃。甲醇、酮溶剂的用量、温度等参数，本领域技术人员可以根据重结晶的实际情况(如实验现象)进行合理选择。
本发明的另一实施方案提供上述沃诺拉赞焦谷氨酸盐晶型I或其药学上可接受的盐在制备钾离子竞争性酸阻滞剂中的应用。
本发明的另一实施方案提供上述沃诺拉赞焦谷氨酸盐晶型I或其药学上可接受的盐在制备抑制胃酸分泌的药物中的应用。所述药物用于治疗和/或预防胃溃疡、十二指肠溃疡、反流性食管炎、糜烂性食管炎、胃食管反流疾病、螺杆菌感染和消化性溃疡等疾病。
本发明的另一实施方案提供一种药物组合物在治疗和/或预防疾病中的应用，所述胃溃疡、十二指肠溃疡、反流性食管炎、糜烂性食管炎、胃食管反流疾病、螺杆菌感染和消化性溃疡等中一种或几种；所述药物组合物以沃诺拉赞焦谷氨酸盐晶型I或其药学上可接受的盐作为有效成分；该药物组合物还可包括其他钾离子竞争性酸阻滞剂或抑制胃酸分泌的药物；该药物组合物还可包括药学上可接受的药用辅料(例如药学上可接受的载体、稀释剂或赋形剂，包括增溶剂、表面活性剂、成膜剂、抗氧剂、稳定剂、粘合剂、润滑剂等)。所述药物组合物的剂型可以是固体制剂、液体制剂或半固体制剂，优选片剂、胶囊、注射剂(含粉针剂)、微乳剂、亚微乳剂，包括缓释片剂、缓释胶囊、缓释注射剂。
本发明中术语“药学上可接受的盐”是指非毒性的无机或有机酸和/或碱的加成盐。可参见“Salt selection for basic drugs”,Int.J.Pharm.(1986),33,201-217。
本发明药物组合物涉及的各种剂型可按照制药领域技术规范和要求(例如《中国药典》(2015年版)制剂通则要求和规范、教科书或其他现有技术中的方法，分别制备成符合临床治疗和/或预防抑制胃酸分泌需求的制剂及其适宜的各种规格与剂型(包括固体制剂、液体制剂和半固体制剂)如缓释、控释和肠溶性胶囊、片剂、微乳剂、亚微乳剂或含有与之相同单一及多成分组合性各种注射剂。
本发明所述的沃诺拉赞焦谷氨酸盐晶型I的X-射线粉末衍射(XRPD)分析是在环境温度及环境湿度下，经日本理学MiniFlex600粉末X射线衍射仪，Rigaku(3-80°，歩进：0.0200°，速度：10°/min)，Cu/Kα源(40kV,40mA)测定完成的。本发明的红外光谱分析是在环境温度及环境湿度下，由美国Thermo Nicolet 6700型红外分光光度计经KBr压片法测定完成的。本发明的热重分析(TGA)是在环境温度及环境湿度下，经瑞士Mettler TGA/DSC1/1100LF型同步热分析仪测定，扫描速率10.00℃/min，最高温度800.00℃，保 护气体：氮气。“环境温度”一般是0-40℃；“环境湿度”一般是30％-80％的相对湿度。
本发明所述的沃诺拉赞焦谷氨酸盐晶型I代表性的X-射线粉末衍射图谱、红外图谱及热重分析图列于说明书附图中。“代表性的X-射线粉末衍射图谱或红外图谱”是指该晶型的X-射线粉末衍射特征或红外特征基本符合本图谱显示的整体形貌，可以理解的是在测试过程中，由于受到多种因素(如测试样品的粒度、测试样品的处理方法、仪器、测试参数、测试操作等)的影响，同一种晶型所测得的X-射线粉末衍射图谱或红外图谱的出峰位置或峰强度会有一定的差异。X-射线粉末衍射图的2θ值可在机器之间或样品之间稍有变化，其2θ值的实验误差一般为±0.2°；红外图谱中吸收峰的实验误差一般为±5cm -1；热重分析中失重温度的实验误差一般为±1℃，失重百分比的实验误差一般为±2.0％。
本发明所述“沃诺拉赞焦谷氨酸盐”指的是1分子沃诺拉赞对应1分子L-焦谷氨酸，即沃诺拉赞焦谷氨酸盐中5-(2-氟苯基)-N-甲基-1-(3-吡啶基磺酰基)-1H-吡咯-3-甲胺与L-焦谷氨酸的摩尔比为1:1。可通过商业途径购买或按照现有技术(例如中国专利申请号：201410154778.8)中的方法由5-(2-氟苯基)-N-甲基-1-(3-吡啶基磺酰基)-1H-吡咯-3-甲胺与L-焦谷氨酸进行制备。本发明所述的“富马酸沃诺拉赞”即5-(2-氟苯基)-N-甲基-1-(3-吡啶基磺酰基)-1H-吡咯-3-甲胺富马酸盐(Vonoprazan Fumarate，简称TAK-438)，通过商业途径购买，纯度99.5％。
本发明中涉及产品HPLC纯度检测的色谱条件如下：
色谱柱：Ultimate XB-C18，柱长：150mm，内径：4.6mm，粒径：5μm
流速：1.2ml/min；柱温：30℃；波长：210nm；进样量：20μl；溶剂：乙腈-水(体积比5：95)
流动相A：乙腈-0.02mol/L磷酸二氢钾(用磷酸调节pH值至2.5)(体积比5:95)
流动相B：甲醇-乙腈-0.02mol/L磷酸二氢钾(用磷酸调节pH值至2.5)(体积比7:63:30)
流动相A、B随时间的比例关系如下：
Figure PCTCN2022131030-appb-000002
与现有技术相比，本发明的优点在于：(1)本发明获得一种沃诺拉赞焦谷氨酸盐新晶型，即晶型I其不仅具有良好的溶解性，而且具备优异的存储稳定性；(2)体外活性实验表明，本发明晶型I对H +K +-ATP酶的抑制作用与TAK-438相当，优于现有技术中(中国专利申请号：201410154778.8)沃诺拉赞焦谷氨酸盐(另一种晶型，图5)；(3)动物实验表明，本发明晶型I静脉给药能够显著抑制组胺诱导的大鼠胃酸分泌，且能够较TAK-438十二指肠给药更快发挥药效作用。
附图说明
图1是本发明沃诺拉赞焦谷氨酸盐晶型I的X-射线粉末衍射图；
图2是晶型I的X-射线粉末衍射的27个特征峰及其衍射角2θ、晶面间距d
Figure PCTCN2022131030-appb-000003
相对强度(％)等具体参数图；
图3是晶型I的红外图谱；
图4是晶型I的热重分析图；
图5是产品A的X-射线粉末衍射图；
图6是TAK438、产品A和晶型I对猪胃黏膜的微粒体泡H +K +-ATP酶的抑制率曲线图；
图7是各组大鼠体重比较图；
图8是各组受试物对组胺诱导胃酸分泌的影响图；
图9是各组受试物对组胺诱导大鼠胃酸分泌量抑制率的影响图。
具体实施方式
为了便于对本发明的进一步理解，下面提供的实施例对其做了更详细的说明。但是这些实施例仅供更好的理解发明而并非用来限定本发明的范围或实施原则，本发明的实施方式不限于以下内容。
实施例1
按照中国专利申请号：201410154778.8中实施例1记载的方法，获得5-(2-氟苯基)-N-甲基-1-(3-吡啶基磺酰基)-1H-吡咯-3-甲胺焦谷氨酸盐(10.0g，HPLC纯度98.0％，即沃诺拉赞焦谷氨酸盐，以下简称产品A，其X-射线粉末衍射图谱如图5所示)。
称取5-(2-氟苯基)-N-甲基-1-(3-吡啶基磺酰基)-1H-吡咯-3-甲胺焦谷氨酸盐样品2.00克，加入到100毫升烧瓶中，加入甲醇(3.0毫升)，20～25℃搅拌10分钟，溶解，于20～25℃滴加丙酮(约10.0毫升)，在滴加丙酮过程中有固体析出，搅拌30分钟，降温至0～5℃，搅拌1小时，抽滤，50℃真空烘干，得到1.54克白色固体，即为沃诺拉赞焦谷氨酸盐晶型I(以下简称晶型I)；其X-射线粉末衍射图谱如图1所示，具体衍射角2θ、晶面间距d
Figure PCTCN2022131030-appb-000004
相对强度(％)如图2所示，其红外图谱如图3所示，热重分析图如图4所示，HPLC纯度为99.8％。
实施例2稳定性试验
(1)潮解试验：在40℃、75％相对湿度的条件下，对产品A、晶型I、TAK-438进行存储，观察是否存在潮解现象，试验时间30天，每10天记录一次结果，详见下表。
测试样品 第10天 第20天 第30天
产品A 轻微潮解 明显潮解 明显潮解
晶型I 无潮解 轻微潮解 明显潮解
TAK-438 无潮解 无潮解 无潮解
无潮解指没有观察到样品表面吸潮现象，轻微潮解指观察到样品表面吸潮现象，明显潮解指能够观察到样品表面滑润。
(2)热稳定性试验：60℃敞口容器中，分别对产品A、晶型I进行储存，30天后(第31天)检测储存稳定性。通过HPLC对初始时和储存30天后的样品进行纯度检测，每种样品设置3个平行组，取平均值，结果如下表。
Figure PCTCN2022131030-appb-000005
由潮解及热稳定性试验可以看出，本发明晶型I稳定性好，便于长期储存。
实施例3溶解性试验及熔点测试
溶解度测试基本方法：取适量待测样品研成细粉，置适宜容器中，适量水，于37℃±2℃下，每隔5分钟强力振摇30秒钟，观察30分钟内的溶解情况，如不溶解，继续加入定量的水，在37℃±2℃放置，每隔5分钟强力振摇30秒钟，观察溶解情况，计算37℃±2℃下的溶解度。重复测定3次，取其平均值。
熔点测试基本方法：取适量待测样品研成细粉，装入一端熔封的毛细管，使粉末紧密集结在毛细管的熔封端。装入样品的高度约为3mm，熔点测定仪的升温速率为1℃/min。重复测定3次，取其平均值。结果见下表。
测试样品 溶解度(mg/mL) 熔点(℃)
产品A 1018 174-175
晶型I 901 167-168
实施例4对猪胃黏膜微粒体泡H +K +-ATP酶的抑制作用
试验方法：H +K +-ATP酶是一种定位于胃粘膜壁细胞，能被K +专一激活的ATP酶。本实验提取猪胃黏膜的微粒体泡H +K +-ATP酶，ATP酶可以分解ATP生成ADP及无机磷，受试物结合H +K +-ATP酶，抑制其活性，通过测定无机磷的量，判断ATP酶活的高低，并测定受试物的抑制效果。具体方法如下：
本试验用猪胃粘膜微粒体泡H +K +ATP酶由北京汇智泰康代为提取。
(1)BCA蛋白定量
BCA工作液的配制：根据样品数量，按50体积BCA试剂A加1体积BCA试剂B(50:1)配制适量BCA工作液，充分混匀。例如5mLBCA试剂A加100μL BCA试剂B，混匀，配制成5.1mL BCA工作液。BCA工作液室温24小时内稳定。
(2)蛋白浓度检测
1)将标准品按0、1、2、4、8、12、16、20μL加到96孔板的标准品孔中，加标准品稀释液补足到20μL，相当于标准品浓度分别为0、0.025、0.05、0.1、0.2、0.3、0.4、0.5mg/mL，1.25mg/mL，2.5mg/mL。
2)将样品分别以2x，5x，10x稀释好，加20ul样品到96孔板的样品孔中。请注意记录好样品体积和稀释倍数。
3)各孔加入200μL BCA工作液，37℃放置20-30分钟。
注:也可以室温放置2小时，或60℃放置30分钟。BCA法测定蛋白浓度时，颜色会随着时间的延长不断加深。并且显色反应会因温度升高而加快。如果浓度较低，适合在 较高温度孵育，或适当延长孵育时间。
4)用酶标仪测定A562，或540-595nm之间的波长的吸光度。
5)根据标准曲线和使用的样品体积计算出样品的蛋白浓度。
(3)反应体系的配置
1)酶反应体系的成分及配置
100mM TRIS-HCL(PH6.8)：取100ul 1M TRIS-HCL加到900ul ddH 2O中，混匀。
37.5mM MgCl 2：称取50mg的MgCl 2，加入14ml ddH 2O，混匀。
200mM KCl：称取500mg的KCl，加入33.5ml ddH 2O，混匀。
10mM的尼日利亚菌素和缬氨霉素：分别加入669ul和890ul DMSO混匀，分装成1ul每管尼日利亚菌素和5ul每管缬氨霉素，-20℃保存。
0.1mg/ml微粒体泡：到货测定蛋白浓度后分装备用，保存于-80℃，用时5mM TRIS-HCL稀释。
2)待测样品的配制
对照化合物：TAK438用DMSO配制成10mM的储存液，1ul每管分装。
受试化合物：产品A和晶型I分别用ddH 2O配制成10mM的储存液，1ul每管分装。以20μM(或40μM)为最高浓度，对照化合物和受试化合物分别用DMSO和ddH 2O逐步3倍稀释，得到6(或7个或8)个浓度梯度的待测样品。
(4)反应的进行
酶促反应
  测定管(uL) 对照管(uL)
100mM TRIS-HCL 60 60
37.5mM MgCl 2 20 20
200mM KCl 15 /
ddH 2O / 15
尼日利亚菌素 1.5 1.5
缬氨霉素 1.5 1.5
0.1mg/mL微粒体泡 50 50
化合物 1.5 1.5
37℃水浴30min，加入16.5ul ATP混匀，37℃水浴30min
(5)显色反应
按照*H +K +ATP试剂盒说明书(南京建成)的要求，配置显色剂。
(6)读板
按照每150uL酶促反应体积加入45uL显色剂混匀，静置2min，酶标仪读取660nm处的吸收光信号。
(7)数据统计
1)按下列公式计算抑制率：抑制率(％)＝(1-(RLU compound-RLU blank)/(RLU DMSO–RLU blank))×100％。
2)利用Graphpad Prism5绘制药效抑制率曲线并计算IC 50值，利用4参数模型[fit＝(A+((B-A)/(1+((C/x)^D))))].
(8)实验结果
1)BCA蛋白定量：根据BSA标准曲线，计算出蛋白平均浓度为3mg/mL。
2)TAK438、产品A和晶型I对猪胃黏膜的微粒体泡H +K +-ATP酶的抑制作用见下表及图6。
测试样品 IC 50(nM)
TAK438 41.5
产品A 53.4
晶型I 40.4
实施例5静脉注射给药对组胺诱导大鼠胃酸分泌模型的药效研究
手术造模：大鼠腹腔注射乌来糖1.5g/kg麻醉。完全麻醉后的大鼠剃除颈部、腹部的毛，为保持手术过程中气路畅通，给予大鼠气管插管。经口将贲门灌流管插入大鼠胃部约1cm，将贲门灌流管的另一端连接恒流泵。剑状软骨下方开小口，找到十二指肠，横向开一个切口，将胃幽门样本采集管端通过创口沿十二指肠进入胃部约1cm，用缝合线将十二指肠与胃幽门样本采集管扎紧，十二指肠切口的另一端扎紧。以150mL 37℃预热的生理盐水沿食道-贲门插管快速冲洗胃内残余物，经胃幽门样本采集管流出，缝合腹腔壁与皮肤，继续用生理盐水以1mL/min的速度灌流30分钟，将胃幽门样本采集管另一端留于体外，以锥形瓶收集胃液样本(每收集20分钟为一份样本)，收集2个基础胃液样本，此时基础胃酸分泌达到稳定状态。
组胺诱导胃酸分泌：用微量注射泵持续尾静脉给予大鼠组胺，剂量为20μmol/kg/hr。80min后收集胃酸，收集三个点后，大鼠组胺分泌达到稳定状态，依据大鼠给予组胺后胃酸分泌量的值分组，根据方案给药给予候选药物或溶剂对照。继续收集胃酸，给药后收集9个点，此时药物药效达到了稳定状态。
具体如下：
(1)测试药物
阳性药：富马酸沃诺拉赞(TAK-438)，配置方法：整个配制过程在避光条件下进行。称取需要量的富马酸沃诺拉赞，加入合适量的0.5％CMC-Na，搅拌，超声，得到均一的混悬液，根据方案所需的浓度，依次稀释到相应浓度。
受试物：晶型I(实验中以HW-N2001表示)，配制方法：整个配制过程在避光条件下进行。称取需要量的HW-N2001，加入合适的生理盐水，充分混匀，得到澄清的溶液， 调节pH值至3.2～3.8范围。根据方案所需的浓度，依次稀释到相应浓度。
(2)实验动物饲养及说明
1)动物信息
品种和品系：SD大鼠，级别：SPF级，性别：雌性、雄性，来源：上海吉辉实验动物饲养有限公司(许可证号码：SCXK(沪)2017-0012)，合格证号：雌性：20170012019182、20170012019726；雄性：20170012017891、20170012018177、20170012018680、20170012018969、20170012019726。动物数量：订购130只，选择健康状况良好的118只(雌性51只，雄性67只)用于实验，实验开始时动物年龄：6-9周龄，实验开始时动物体重：雌性160-220g，雄性180-260g，适应环境时间：5-7天。
2)饲养环境
SPF级饲养室，符合中华人民共和国国标GB14925-2010标准，温度：23±2℃，湿度：湿度40～70％，光照：人工照明，12小时明暗交替。垫料种类：玉米芯垫料(德州谷美农业科技有限公司，批号：GMCC202102170-2)。饲料种类：SPF低蛋白大小鼠维持饲料(江苏协同医药生物工程有限责任公司，20210225(X))。给料方法：自由摄取。饮水种类：试验动物高压灭菌饮用水，供水方法：饮水瓶盛装，自由摄取。
3)动物选择和禁食
用于实验的动物将保持健康状况。实验前禁食不禁水24h。
4)动物使用伦理批准
本实验项目已经获得中国科学院上海药物研究所苏州药物创新研究院实验动物伦理委员会许可，IACUC号为2021-06-WZC-10。
5)实验动物选择说明
实验应用普通的哺乳动物，像小鼠、大鼠、犬和猴子等，作为实验动物。这些动物通常用来进行药代参数、药效学和毒理学的研究。所用动物的数量为判定实验动物个体间变异性所需的最小数量。实验所用动物必须健康并适应环境，自由饮食饮水。本实验采用双性别SD大鼠进行试验。
(3)实验设计
1)实验分组
130只SD大鼠选择健康状况良好的118只，适应期结束后，根据体重和给予组胺后基值随机分成8组，详情见下表。
Figure PCTCN2022131030-appb-000006
2)动物模型制备和实验过程
仪器和试剂准备：
烧杯内放置生理盐水，置于水浴箱内，保持水温为37℃。
调整恒流泵流速(1mL/min)备用。
组胺二盐酸盐：新鲜配置组胺二盐酸盐(0.6mg/mL，生理盐水溶解)。
乌来糖：称取适量乌来糖，加入生理盐水，超声溶解，配置成终浓度0.3g/mL，备用。
酚酞：称取适量酚酞，加入无水乙醇，配置成终浓度0.5％，备用。
碱式滴定液：取适量0.1mol/L的NaOH溶液，加入新鲜去离子水，稀释至0.01mol/L，备用。
肝素钠：称取适量肝素钠，加入生理盐水，溶解至终浓度100U/mL，备用。
动物准备：
适应期过后，118只SD大鼠(雌雄)禁食不禁水24小时。随机分为8组(雌雄)。
插管的准备与制作：
气管插管：取医用聚乙烯塑料管(外直径2mm，内直径1.5mm)2-3cm，备用。
胃贲门灌流管：取医用聚乙烯软管约20cm(外直径2mm，内直径1.5mm)，作为贲门胃管备用。
胃幽门样本采集管：取医用聚乙烯软管(外直径4mm,内直径3mm)约30cm，作为幽门插管端备用。
组胺灌流管：10mL注射器抽取10mL组胺二盐酸盐，连接输液延长管，以锡纸包裹避光，固定在注射泵内备用。
动物麻醉与护理:乌来糖给药剂量1.5g/kg腹腔注射麻醉大鼠。剃除颈部、腹部的毛。
气管插管：
为保持手术过程中气路畅通，在颈部(会厌软骨处)纵开2cm创口，分离肌肉暴露会厌软骨与气管，分离气管将一段缝合线从气管下穿过。在气管上做一个“T”字形切口，轻柔的将气管插管插入气管切口近心端约1cm，并用缝合线扎紧。检查气管插管的通畅性，缝合伤口，气管插管另一端暴露在皮肤切口外。
胃贲门灌流管插管：
量取口到胃部的距离(大约15cm)在胃贲门灌流管上做好标记，将胃贲门灌流管经食道插入至标记处停止，确保胃贲门灌流管穿过贲门括约肌抵达胃部。将胃贲门灌流管与恒流泵上的硅胶管连接。
胃幽门样本采集管插管：
剑状软骨下方沿腹中白线开一个小口，开腹腔，检查胃贲门灌流管位置。在幽门下端1cm处十二指肠上横向开一个切口，将胃幽门样本采集管端通过创口沿十二指肠进入胃部约1cm，用一根缝合线将十二指肠与胃幽门样本采集管扎紧，十二指肠切口的另一端扎紧。以150ml 37℃预热的生理盐水沿食道-贲门插管快速冲洗胃内残余物，经胃幽门样本采集管流出，缝合腹腔壁与皮肤，继续用生理盐水以1ml/min的速度灌流30分钟，将胃幽门样本采集管另一端留于体外，以锥形瓶收集胃液样本(每收集20分钟为一份样本)，收集2个基础胃液样本，此时基础胃酸分泌达到稳定状态。
样品收集与处理：
取收集的胃液样本，用碱式滴定管以0.01N氢氧化钠溶液滴定，酚酞指示剂为颜色指示剂，滴定至PH7为终点，计量滴定体积，计算酸分泌量。
酸分泌量(μEq)＝NaOH体积(mL)×10 -3×0.01mol/L×10 6
尾静脉留置针：尾静脉用酒精消毒，将留置针打入尾静脉，拔出留置针针芯，推入肝素钠溶液0.5ml，拔下注射器拧上肝素钠帽。
收集2个基础胃液样本，基础胃酸水平稳定后，将提前吸入组胺的输液延长管与大鼠的尾静脉留置针连接，打开注射泵，组胺按照20μmol/kg/hr的量计算并设置注射泵每只动物静脉滴注速度，连续注射组胺溶液80min后，即可收集给组胺后胃酸样本，收集3次给予组胺后的胃液，胃酸分泌达到稳定状态，根据分组静脉给予受试物或十二指肠给予阳性药或溶剂对照，继续收集胃液，大约在给药后3个小时，碱消耗量达到稳定状态，总共收集约14个点。
体重：实验前一天禁食前和实验当天在麻醉之前分别称重。
胃液总酸度：用酸碱滴定法测定胃液总酸度，颜色指示剂为0.5％的酚酞，滴定终点为pH7.0。
(4)数据统计
实验数据如无特别注明采用均数加减标准差(Mean±SD)表示，采用柯尔莫诺夫-斯米尔诺夫检验(One-Sample Kolmogorov-Smirnov Test)方法进行正态分布性检验，当组内数据满足正态分布，采用单因素方差分析(one way ANOVA)，方差齐时，多个样本均数间的多重比较采用LSD检验，P<0.05认为是有显著性差异；方差不齐时，多个样本均数间的多重比较采用Games-Howell检验。当组内数据不满足正态分布采用非参数检验的Independent samples检验，P<0.05认为是多样本间有显著性差异，所有数据统计分析均采用SPSS16.0统计软件进行。
(4)实验结果
1)大鼠体重各组比较
整个实验过程中，各组实验动物状态未见异常，禁食前后体重无显著性差异。(图7)
2)受试物对组胺诱导胃酸分泌的影响
各组SD大鼠在给予组胺前基值没有显著差异，给予组胺后，Vehicle组与
Figure PCTCN2022131030-appb-000007
组有显著差异，与阳性药(富马酸沃诺拉赞)或受试物(HW-N2001)各剂量组比内有显著性差异。十二指肠给予阳性药或静脉给予受试物后各组大鼠的胃酸分泌量、胃酸分泌量抑制率与溶剂对照(Vehicle)组比较有不同程度差异。具体如下(图8、9)。
溶剂对照(Vehicle)组：大鼠给予组胺前收集胃酸两个点，胃酸分泌量基值分别为：6.43±2.37、8.83±5.1(μEq HCl/20min)，与
Figure PCTCN2022131030-appb-000008
组比没有显著差异；大鼠给予组胺80min后收取胃酸三个点，胃酸分泌量显著升高并达到稳态，分别为42.3±14.58、45.96±16.61、46.65±16.96(μEq HCl/20min)，与
Figure PCTCN2022131030-appb-000009
组比均有显著性差异；静脉给予溶剂对照后各时间点与
Figure PCTCN2022131030-appb-000010
组比有显著性差异。
富马酸沃诺拉赞(1mg/kg)组：动物给予组胺后，胃酸分泌量达到稳态，给药后，大鼠胃酸分泌量没有显著降低，与Vehicle组比较没有统计差异。
富马酸沃诺拉赞(2mg/kg)组：动物给予组胺后，胃酸分泌量达到稳态，给药后60min-180min，大鼠胃酸分泌量逐渐降低，给药后60min，大鼠胃酸分泌量为33.21±15.63(μEq HCl/20min)，胃酸分泌量抑制率为13.71％±2.12，与溶剂组比较有统计性差异，给药后180min大鼠胃酸分泌量为16.21±14.46(μEq HCl/20min)，胃酸分泌量抑制率为57.48％±2.27，与空白对照组比较有显著性差异。
富马酸沃诺拉赞(4mg/kg)组：动物给予组胺后，胃酸分泌量达到稳态，给药后40min-180min，大鼠胃酸分泌量逐渐降低，给药后40min，大鼠胃酸分泌量为33.18±11.15 (μEq HCl/20min)，与溶剂组比较没有统计性差异，胃酸分泌量抑制率为12.47％±1.13，与溶剂比较照组比较有统计性差异，给药后180min大鼠胃酸分泌量为4.41±4.02(μEq HCl/20min)，胃酸分泌量抑制率为87.03％±0.81，与空白对照组比较有显著性差异。
HW-N2001(0.375mg/kg)组：动物给予组胺后，胃酸分泌量达到稳态，给药后60min-180min，大鼠胃酸分泌量逐渐降低，给药后60min，大鼠胃酸分泌量为39.15±8.54(μEq HCl/20min)，胃酸分泌量抑制率为4.34％±1.28，与溶剂比较照组比较没有统计性差异，给药后180min大鼠胃酸分泌量为28.46±5.71(μEq HCl/20min)，胃酸分泌量抑制率为27.52％±1.43，与空白对照组比较有显著性差异。
HW-N2001(0.75mg/kg)组：动物给予组胺后，胃酸分泌量达到稳态，给药后20min-180min，大鼠胃酸分泌量逐渐降低，给药后20min，大鼠胃酸分泌量为37.53±9.87(μEq HCl/20min)，与空白对照组比较没有显著性差异，胃酸分泌量抑制率为11.6％±0.82，与溶剂比较照组比较有统计性差异，给药后180min大鼠胃酸分泌量为10±5.32(μEq HCl/20min)，胃酸分泌量抑制率为73.68％±1.47，与空白对照组比较有显著性差异。
HW-N2001(1.5mg/kg)组：动物给予组胺后，胃酸分泌量达到稳态，给药后20min-80min，大鼠胃酸分泌量逐渐降低，药效达到稳定状态，给药后20min，大鼠胃酸分泌量为25.67±10.15(μEq HCl/20min)，胃酸分泌量抑制率为33.28％±0，与溶剂比较照组比较有统计性差异，给药后80min大鼠胃酸分泌量为3.4±1.18(μEq HCl/20min)，胃酸分泌量抑制率为89.64％±0，与空白对照组比较有显著性差异。给药后180min大鼠胃酸分泌量为2.2±0.56(μEq HCl/20min)，胃酸分泌量抑制率为93.56％±0.09，与空白对照组比较有显著性差异。
(5)结论
本发明采用连续检测组胺诱导SD大鼠的胃酸分泌模型，研究沃诺拉赞焦谷氨酸盐晶型I(HW-N2001)静脉给药对大鼠胃酸分泌的药效作用。
富马酸沃诺拉赞十二指肠给药，1mg/kg给药后效果不显著，2和4mg/kg分别在给药后60min、140min逐渐发挥药效作用，抑制大鼠胃酸分泌，且有一定的剂量-效应关系。药物起效缓慢，180min达到药效最强，ID 50为1.87mg/kg。
HW-N2001(0.375mg/kg、0.75mg/kg、1.5mg/kg)静脉给药，给药组胃酸分泌量较溶剂对照组均有显著降低，且有一定的剂量-效应关系，其中0.75和1.5mg/kg剂量组给药后20min即开始逐渐发挥药效作用，给药后60-100min药效达到稳定状态，给药后180min ID 50为0.53mg/kg。
综上所述，本发明沃诺拉赞焦谷氨酸盐晶型I(HW-N2001)静脉给药能够显著抑制组胺诱导的大鼠胃酸分泌，且能够较富马酸沃诺拉赞十二指肠给药更快发挥药效作用。

Claims (12)

  1. 一种沃诺拉赞焦谷氨酸盐的晶型I，其特征在于：使用Cu/Kα辐射，以2θ衍射角表示的X-射线粉末衍射图在9.000±0.200，10.280±0.200，11.340±0.200，12.440±0.200，13.480±0.200，14.360±0.200，15.640±0.200，17.100±0.200，18.000±0.200，18.500±0.200，19.240±0.200，19.720±0.200，20.820±0.200，21.660±0.200，22.500±0.200，24.040±0.200，24.860±0.200，25.720±0.200，26.400±0.200，27.220±0.200，28.600±0.200，31.320±0.200，33.200±0.200，34.100±0.200，34.600±0.200，36.480±0.200，43.460±0.200处有特征峰。
  2. 一种沃诺拉赞焦谷氨酸盐的晶型I，其特征在于：使用Cu/Kα辐射，以2θ衍射角表示的X-射线粉末衍射图，具有27个特征峰，其X-射线粉末衍射图基本如图1或图2所示。
  3. 权利要求1-2任一项所述的沃诺拉赞焦谷氨酸盐晶型I，其特征在于所述晶型I的红外图谱在约3255±5、2738±5、1690±5、1657±5、1573±5cm -1的位置有特征吸收峰。
  4. 权利要求1-3任一项所述的沃诺拉赞焦谷氨酸盐晶型I，其特征在于所述晶型I经热重分析，在297.9±1.0℃时第一次失重，失重率为50.3±2.0％。
  5. 权利要求1-4任一项所述的沃诺拉赞焦谷氨酸盐晶型I的制备方法，其特征在于包括如下步骤：将5-(2-氟苯基)-N-甲基-1-(3-吡啶基磺酰基)-1H-吡咯-3-甲胺焦谷氨酸盐用甲醇溶解，再滴加酮类溶剂，搅拌析晶，过滤，干燥即得晶型Ⅰ。
  6. 权利要求1-4任一项所述的沃诺拉赞焦谷氨酸盐晶型I或其药学上可接受的盐在制备钾离子竞争性酸阻滞剂中的应用。
  7. 权利要求1-4任一项所述的沃诺拉赞焦谷氨酸盐晶型I或其药学上可接受的盐在制备抑制胃酸分泌的药物中的应用。
  8. 权利要求7所述的应用，其特征在于所述药物用于治疗和/或预防胃溃疡、十二指肠溃疡、反流性食管炎、糜烂性食管炎、胃食管反流疾病、螺杆菌感染和消化性溃疡等疾病。
  9. 一种药物组合物在治疗和/或预防疾病中的应用，所述胃溃疡、十二指肠溃疡、反流性食管炎、糜烂性食管炎、胃食管反流疾病、螺杆菌感染和消化性溃疡等中一种或几种；所述药物组合物以权利要求1-4任一项所述的沃诺拉赞焦 谷氨酸盐晶型I或其药学上可接受的盐作为有效成分。
  10. 权利要求9所述的药物组合物，其特征在于该药物组合物还可包括其他钾离子竞争性酸阻滞剂或抑制胃酸分泌的药物。
  11. 权利要求9-10任一项所述的药物组合物，其特征在于该药物组合物还可包括药学上可接受的药用辅料，例如药学上可接受的载体、稀释剂或赋形剂，包括增溶剂、表面活性剂、成膜剂、抗氧剂、稳定剂、粘合剂、润滑剂等。
  12. 权利要求9-11任一项所述的药物组合物，其特征在于所述药物组合物的剂型可以是固体制剂、液体制剂或半固体制剂，优选片剂、胶囊、注射剂(含粉针剂)、微乳剂、亚微乳剂，包括缓释片剂、缓释胶囊、缓释注射剂。
PCT/CN2022/131030 2022-05-20 2022-11-10 沃诺拉赞焦谷氨酸盐的晶型及其制备方法 WO2023221419A1 (zh)

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