Crystalline form of vonoprazan pyroglutamate and its preparation method

Abstract

The present invention relates to a crystalline form of vonoprazan pyroglutamate and a method for preparing the same. The present invention provides a new crystalline form of vonoprazan pyroglutamate with good solubility and excellent storage stability. In vitro experiments have shown that the inhibitory effect of crystalline form I of the present invention on H+K+-ATP enzyme is equivalent to that of TAK-438. Animal experiments have shown that intravenous administration of crystalline form I of the present invention can significantly inhibit histamine-induced gastric acid secretion in rats, and the pharmacodynamic effect appears earlier than that of intraduodenal administration of TAK-438.

Description

[Technical field]

[0001] The present invention relates to the field of drug crystalline forms, and in particular to a crystalline form of vonoprazan pyroglutamate and a method for preparing the same. [Background technology]

[0002] Vonoprazan has the chemical name N-methyl-5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrole-3-methanamine and its chemical structure is as follows:

[0003] [ka]

[0004] Vonoprazan fumarate (TAK-438), developed by Takeda Pharmaceuticals Japan, was approved for sale in Japan on December 26, 2014. The drug belongs to a new type of inhibitor called potassium-competitive acid blocker (P-CAB). P-CABs are lipophilic, weakly alkaline, have a high dissociation constant and high stability at low pH, so Vonoprazan fumarate has a rapid, strong and sustained inhibitory effect on gastric acid secretion. At the same time, in the final step of gastric acid secretion from gastric parietal cells, K + H + -K + -By inhibiting the binding activity to ATP enzyme (proton pump), it also has an early termination effect on gastric acid secretion, but the water solubility of the compound is comparatively poor. The present invention conducts crystal form research and in vitro physiological activity research on vonoprazan pyroglutamate, which has comparatively good water solubility, to obtain vonoprazan pyroglutamate crystal form I, which has comparatively good solubility, stability, and in vivo physiological activity. Summary of the Invention [Means for solving the problem]

[0005] The present invention provides a crystalline form I of vonoprazan pyroglutamate, X-ray powder diffraction patterns expressed at 2θ diffraction angles of 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, and 21.000±0.200 were obtained using Cu / Kα radiation. 00, 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, and 43.460±0.200. Crystalline form I can also have the characteristics represented by the X-ray powder diffraction pattern shown in FIG.

[0006] Another embodiment of the present invention provides crystalline form I of vonoprazan pyroglutamate, characterized in that the crystalline form I has 27 characteristic peaks using Cu / Kα radiation, and its X-ray powder diffraction pattern is essentially as shown in FIG.

[0007] One of ordinary skill in the art should understand that the peak height relative intensities of 2θ characteristic peaks in an XRPD spectrum can vary for a variety of reasons when measuring substantially the same crystalline form.

[0008] The infrared spectrum of the crystalline form I of vonoprazan pyroglutamate according to the present invention is approximately 3255±5, 2738±5, 1690±5, 1657±5, 1573±5 cm -1 The crystalline form I can have the characteristics represented by the infrared spectrum shown in FIG.

[0009] According to the thermogravimetric analysis, the crystalline form I of vonoprazan pyroglutamate according to the present invention is initially weightless at 297.9±1.0°C through material decomposition, and the weightless rate is 50.3±2.0%. The thermogravimetric analysis TGA diagram of the crystalline form I is basically as shown in FIG.

[0010] Another embodiment of the present invention provides a method for preparing the crystalline form I of vonoprazan pyroglutamate, the characteristics of which include dissolving N-methyl-5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrole-3-methanamine pyroglutamate in methanol, adding a ketone solvent dropwise, stirring the crystals, filtering, and drying to obtain the crystalline form I.

[0011] The dissolution of methanol may be carried out at room temperature or may be artificially heated to 20-35° C. The ketone solvent is preferably acetone, methyl ethyl ketone, etc., and the crystallization process may be carried out at room temperature or may be artificially cooled to 0-5° C. Those skilled in the art can reasonably select parameters such as the amount of methanol and ketone solvent used, and temperature based on the actual situation of recrystallization (e.g., experimental phenomena).

[0012] Another embodiment of the present invention provides an application of the crystalline form I of vonoprazan pyroglutamate or a pharma- ceutically acceptable salt thereof in the preparation of a potassium ion-competitive acid blocker.

[0013] Another embodiment of the present invention provides an application of the crystalline form I of vonoprazan pyroglutamate or a pharma- ceutically acceptable salt thereof in the preparation of a gastric acid secretion inhibitor, which is used for the treatment and / or prevention of diseases such as gastric ulcer, duodenal ulcer, reflux esophagitis, erosive esophagitis, gastroesophageal reflux disease, Helicobacter pylori infection and peptic ulcer.

[0014] Another embodiment of the present invention provides the application of the pharmaceutical composition in the treatment and / or prevention of one or more diseases of gastric ulcer, duodenal ulcer, reflux esophagitis, erosive esophagitis, gastroesophageal reflux disease, Helicobacter pylori infection and peptic ulcer. The pharmaceutical composition contains crystalline form I or a pharma- ceutical acceptable salt of vonoprazan pyroglutamate as an active ingredient. The pharmaceutical composition contains a drug including a potassium ion-competitive acid blocker or a gastric acid secretion inhibitor. The pharmaceutical composition contains pharmaceutical additives such as pharma- ceutical acceptable carriers, diluents or excipients including solubilizers, surfactants, coating agents, antioxidants, stabilizers, adhesives and lubricants. The dosage form of the pharmaceutical composition may be a solid formulation, liquid formulation or semi-solid formulation, and is preferably a tablet, capsule, injection (including powder injection), microemulsion, or submicroemulsion, including sustained release tablets, sustained release capsules and sustained release injections.

[0015] The term "pharmaceutical acceptable salt" in the present invention means a non-toxic inorganic or organic acid and / or alkali addition salt. "Salt selection for basic drugs", Int. J. Pharm. (1986), 33201-217 can be referred to.

[0016] Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to the technical standards and requirements in the pharmaceutical field (e.g., the requirements and standards of the general principles for preparations in the Chinese Pharmacopoeia (2015 edition), methods in textbooks or other prior art) to meet the needs of clinical treatment and / or gastric acid secretion inhibition and various appropriate specifications and dosage forms thereof (including solid preparations, liquid preparations and semi-solid preparations), such as sustained release, controlled release and enteric soluble capsules, tablets, microemulsions, submicroemulsions, or various single and multi-component complex injections thereof).

[0017] The X-ray powder diffraction (XRPD) analysis of the crystalline form I of vonoprazan pyroglutamate according to the present invention was completed under ambient temperature and humidity using a Japan Rigaku MiniFlex600 powder X-ray diffractometer, Rigaku (3-80°, step: 0.0200°, speed: 10° / min), Cu / Kα source (40kV, 40mA). The infrared spectrum analysis of the present invention was completed under ambient temperature and humidity using a US Thermo Nicolet 6700 infrared spectrophotometer by KBr tablet method. The thermogravimetric analysis (TGA) of the present invention was measured under ambient temperature and humidity using a Swiss Mettler TGA / DSC1 / 1100 LF simultaneous thermal analyzer, with a scanning speed of 10.00°C / min, a maximum temperature of 800.00°C, and a protective gas of nitrogen gas. The "ambient temperature" is generally 0-40°C, and the "ambient humidity" is generally 30-80% relative humidity.

[0018] The representative X-ray powder diffraction pattern, infrared spectrum and thermogravimetric analysis pattern of the crystalline form I of vonoprazan pyroglutamate according to the present invention are described in the specification drawings. "Representative X-ray powder diffraction pattern or infrared spectrum" means that the X-ray powder diffraction characteristics or infrared characteristics of the crystalline form are basically consistent with the overall form shown in the spectrum, and it can be understood that the same crystalline form will have a certain difference in the peak position or peak intensity of the measured X-ray powder diffraction pattern or infrared spectrum due to the influence of various factors such as the particle size of the test sample, the processing method of the test sample, the equipment, the test parameters, the test operation, etc. The 2θ value of the X-ray powder diffraction pattern may vary slightly between machines or samples, and the experimental error of the 2θ value is generally ±0.2°, and the experimental error of the absorption peak in the infrared spectrum is generally ±5cm. ?1 In thermogravimetry, the experimental error for the weightless temperature is typically ±1°C, and the experimental error for the weightless percentage is typically ±2.0%.

[0019] "Vonoprazan pyroglutamate" according to the present invention means that one molecule of vonoprazan corresponds to one molecule of L-pyroglutamic acid, i.e., the molar ratio of N-methyl-5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrole-3-methanamine to L-pyroglutamic acid in vonoprazan pyroglutamate is 1:1. It can be purchased commercially or prepared by using N-methyl-5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrole-3-methanamine and L-pyroglutamic acid according to existing technology (e.g., the method in Chinese Patent Application No. 20140154778.8). The "vonoprazan fumarate" described in the present invention, i.e., N-methyl-5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrole-3-methanamine fumarate (Vonoprazan Fumarate, abbreviated as TAK-438), is purchased commercially and has a purity of 99.5%.

[0020] The chromatographic conditions for HPLC purity detection of the product according to the present invention are as follows: Chromatography column: Ultimate XB-C18, column length: 150 mm, inner diameter: 4.6 mm, particle size: 5 μm Flow rate: 1.2 ml / min, column temperature: 30°C, wavelength: 210 nm, injection volume: 20 μl, solvent: acetonitrile-water (volume ratio 5:95) Mobile phase A: Acetonitrile - 0.02 mol / L potassium dihydrogen phosphate (adjusted to pH 2.5 with phosphoric acid) (volume ratio 5:95) Mobile phase B: Methanol-acetonitrile-0.02 mol / L potassium dihydrogen phosphate (pH adjusted to 2.5 with phosphoric acid) (volume ratio 7:63:30) The proportional relationship of mobile phase A and B with respect to time is as follows:

[0021] [Table 1]

[0022] Compared with the prior art, the advantages of the present invention are as follows: (1) the present invention provides a new crystalline form of vonoprazan pyroglutamate with good solubility and excellent storage stability; (2) in vitro experiments have demonstrated that the crystalline form I of the present invention is H + K + It was revealed that the inhibitory effect on -ATP enzyme is equivalent to that of TAK-438 and is superior to that of vonoprazan pyroglutamate (another crystal form, Figure 5) described in the prior art (Chinese Patent Application No. 20140154778.8). (3) Animal experiments revealed that intravenous administration of the crystal form I of the present invention can significantly inhibit histamine-induced gastric acid secretion in rats, and the pharmacodynamic effect appears earlier than that of intraduodenal administration of TAK-438. [Brief description of the drawings]

[0023] [Figure 1] 1 is an X-ray powder diffraction pattern of crystalline form I of vonoprazan pyroglutamate of the present invention. [Diagram 2] FIG. 1 shows 27 characteristic peaks in the X-ray powder diffraction pattern of crystalline form I and their specific parameters such as diffraction angle 2θ, crystal plane spacing d (Angstroms), and relative intensity (%). [Diagram 3] 1 is an infrared spectrum of crystalline form I. [Figure 4] 1 is a thermogravimetric analysis pattern of crystalline form I. [Diagram 5] 1 is an X-ray powder diffraction pattern of product A. [Figure 6] FIG. 1 shows the inhibition rate curves of TAK438, product A and crystal form I against microvesicular H+K+-ATPase from porcine gastric mucosa. [Figure 7] FIG. 13 is a graph comparing rat body weights in each group. [Figure 8] 1 is a graph of the effect of subjects in each group on histamine-induced gastric acid secretion. [Figure 9] 1 is a graph showing the effect of each group of subjects on the histamine-induced gastric acid secretion inhibition rate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] In order to facilitate a further understanding of the present invention, the embodiments provided below are described in more detail. However, these embodiments are not intended to limit the scope or implementation principles of the present invention, but are intended to provide a better understanding, and the embodiments of the present invention are not limited to the following.

[0025] Example 1 According to the method described in Example 1 of Chinese Patent Application No. 201410154778.8, N-methyl-5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrole-3-methanamine pyroglutamate (10.0 g, HPLC purity 98.0%, i.e., vonoprazan pyroglutamate, hereinafter referred to as product A, and its X-ray powder diffraction pattern is shown in FIG. 5) was obtained.

[0026] Weigh out 2.00 g of N-methyl-5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrole-3-methanamine pyroglutamate sample, add it to a 100 ml flask, add methanol (3.0 ml), stir and dissolve at 20-25°C for 10 minutes, add acetone (approximately 10.0 ml) dropwise at 20-25°C, a solid precipitates during the acetone drop, stir for 30 minutes, and lower the temperature to 0-5°C. The mixture was stirred for 1 hour, filtered, extracted, and dried under vacuum at 50°C to obtain 1.54 g of a white solid, i.e., vonoprazan pyroglutamate crystal form I (hereinafter referred to as crystal form I), whose X-ray powder diffraction pattern is shown in Figure 1, whose specific diffraction angle 2θ, crystal plane spacing d (angstroms), and relative intensity (%) are shown in Figure 2, whose infrared spectrum is shown in Figure 3, and whose thermogravimetric analysis pattern is shown in Figure 4, with an HPLC purity of 99.8%.

[0027] Example 2: Stability test Deliquescence test: Product A, crystal form I, TAK-438 is stored under the conditions of 40℃ and 75% relative humidity, and the presence of deliquescence is observed. The test time is 30 days, and the results are recorded every 10 days. Please refer to the table below for details.

[0028] [Table 2]

[0029] No deliquescence means that no moisture absorption phenomenon is observed on the sample surface, slight deliquescence means that moisture absorption phenomenon is observed on the sample surface, and obvious deliquescence means that the sample surface is smooth.

[0030] Thermal stability test: Product A and Form I were stored in an open container at 60℃, and the storage stability was measured after 30 days (31st day). The purity of the initial samples and the samples after 30 days of storage was measured by HPLC, and three parallel groups were set up for each sample, and the average value was taken. The results are shown in the table below.

[0031] [Table 3]

[0032] As can be seen from the deliquescence and thermal stability tests, the crystalline form I of the present invention has good stability and is convenient for long-term storage.

[0033] (Example 3) Solubility test and melting point test The basic method of solubility testing is to take an appropriate amount of sample to be measured, grind it into fine powder, place it in a suitable container, add an appropriate amount of water, shake vigorously for 30 seconds every 5 minutes at 37°C±2°C, observe the dissolution status within 30 minutes, if it does not dissolve, continue to add a certain amount of water, leave it at 37°C±2°C, shake vigorously for 30 seconds every 5 minutes, observe the dissolution status, and calculate the solubility at 37°C±2°C. The measurement is repeated 3 times and the average value is taken.

[0034] The basic method for melting point testing is to take an appropriate amount of the sample to be measured, grind it into fine powder, and put it into a capillary tube with one end melted and sealed, so that the powder is tightly concentrated at the melted and sealed end of the capillary tube. The height of the sample put in is about 3mm, and the heating rate of the melting point tester is 1℃ / min. The measurement is repeated three times and the average value is taken. The results are shown in the table below.

[0035] [Table 4]

[0036] (Example 4) Microvesicles H of porcine gastric mucosa + K + -Inhibitory effect on ATP enzyme

[0037] Test method, H + K + The ATPase is located in the parietal cells of the gastric mucosa and is specifically activated by K+. + K + - ATP enzyme is extracted, ATP enzyme can decompose ATP to generate ADP and inorganic phosphorus, and the subject is H + K + - ATP enzyme is bound, its activity is inhibited, and the amount of inorganic phosphorus is measured to determine the level of ATP enzyme activity and to measure the inhibitory effect of the subject. The specific method is as follows: Microvesicles of porcine gastric mucosa for this study H + K + -ATP enzyme was extracted from Beijing Huizhi Taikang Co., Ltd.

[0038] BCA protein quantification Preparation of BCA working solution: Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B (50:1) to 50 volumes of BCA reagent A and mix well. For example, add 100 μL BCA reagent B to 5 mL BCA reagent A and mix well to prepare a 5.1 mL BCA working solution. The BCA working solution is stable for 24 hours at room temperature.

[0039] Protein concentration detection Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of standards to standard wells of a 96-well plate and top up to 20 μL with standard diluent, corresponding to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL, 1.25 mg / mL, and 2.5 mg / mL, respectively.

[0040] The samples were diluted 2x, 5x, and 10x, respectively, and 20ul of sample was added to the sample wells of a 96-well plate. Take care to record the sample volume and dilution factor.

[0041] Add 200 µL of BCA working solution to each well and leave at 37 °C for 20-30 min.

[0042] Note: You can leave it at room temperature for 2 hours or at 60°C for 30 minutes. When measuring the protein concentration by BCA method, the color will darken over time. Also, the color reaction will accelerate with increasing temperature. If the concentration is low, it is suitable for incubation at a higher temperature or extend the incubation time appropriately.

[0043] 4) Measure A562 or absorbance at wavelengths between 540 and 595 nm using a microplate reader.

[0044] 5) Calculate the protein concentration of the sample from the standard curve and the volume of sample used.

[0045] Reaction system layout Components and configuration of enzyme reaction system 100mM TRIS-HCL (PH 6.8): Take 100ul 1M TRIS-HCL and add it to 900ul ddH2O and mix well.

[0046] 37.5 mM MgCl2: Weigh out 50 mg MgCl2, add 14 ml ddH2O and mix well.

[0047] 200 mM KCl: Weigh out 500 mg of KCl and add 33.5 ml of ddH2O and mix well.

[0048] 10mM Nigericin and Valinomycin: Add 669ul and 890ul DMSO respectively, mix well, divide into 1ul Nigericin and 5ul Valinomycin and store at -20°C.

[0049] 0.1 mg / ml microvesicles: After receiving the product and measuring the protein concentration, the product is aliquoted and stored at -80°C. When using the product, the product is diluted with 5 mM TRIS-HCL.

[0050] 2) Preparation of the sample to be measured Reference compound: TAK438 was prepared as a 10 mM stock solution in DMSO and dispensed in 1 ul aliquots.

[0051] Test compounds: Product A and Form I were prepared in 10 mM stock solution using ddH2O, and aliquoted in 1 ul portions. The highest concentration was 20 μM (or 40 μM), and the reference and test compounds were diluted 3-fold with DMSO and ddH2O, respectively, to obtain 6 (or 7 or 8) concentration gradient samples.

[0052] Reaction progress

[0053] Enzyme reaction

[0054] [Table 5]

[0055] 37℃ water bath for 30 min, add 16.5ul ATP, mix well, and 37℃ water bath for 30 min

[0056] Color reaction *H + K + Place the color developer according to the requirements of the ATP test kit instruction manual (manufactured by Nanjing Jiancheng).

[0057] (6) Microplate reading Add 45 uL of color reagent per 150 uL of enzyme reaction volume, mix well, leave to stand for 2 minutes, and read the absorbance signal at 660 nm using a microplate reader.

[0058] (7) Data Statistics 1) The inhibition rate is calculated using the following formula.

[0059]

number

[0060] Pharmacodynamic effect inhibition curves were plotted using Graphpad Prism5, and IC 50 Calculate the values ​​of the 4-parameter model [fit=(A+((BA) / (1+((C / x)^D)))].

[0061] Experimental Results BCABCA protein quantification: The average protein concentration was calculated to be 3 mg / mL from the BSA standard curve. TAK438, product A and crystalline form I of porcine gastric mucosa microvesicles H + K + The inhibition rate against -ATP enzyme is shown in the table below and FIG.

[0062] [Table 6]

[0063] Example 5: Pharmacodynamic effect study of intravenous administration on histamine-induced gastric acid secretion in rats Surgery modeling: Rats were anesthetized by intraperitoneally injecting 1.5g / kg ethyl carbamate. The neck and abdominal hair of the fully anesthetized rats were shaved, and the rats were intubated to maintain a smooth airway during surgery. A cardia perfusion tube was orally inserted into the rat stomach about 1cm, and the other end of the cardia perfusion tube was connected to a constant flow pump. A small opening was made below the xiphoid process, the duodenum was found, an incision was made horizontally, and the end of the gastric pylorus sample collection tube was inserted into the stomach about 1cm along the duodenum through the incision, and the duodenum and gastric pylorus sample collection tube were tightly sutured, and the other end of the duodenal incision was tightly closed. 150mL of 37℃ pre-warmed saline was used to rapidly flush out the intragastric residues along the cardiac intubation, and then flowed out through the gastric pylorus sampling tube. The abdominal wall and skin were sutured, and then saline was used to perfuse for 30 minutes at a rate of 1mL / min. The other end of the gastric pylorus sampling tube was left outside the body, and gastric juice samples (one sample was collected every 20 minutes) were collected in a conical bottle, and two basic gastric juice samples were collected, at which time the basic gastric acid secretion reached a stable state.

[0064] Histamine-induced gastric acid secretion: Histamine was continuously administered to rats via the tail vein using a microinjection pump at a dose of 20 μmol / kg / hr. Gastric acid was collected after 80 min, and after collecting three points, the rats' histamine secretion reached a stable state. The rats were divided into groups based on the gastric acid secretion value after histamine administration, and the candidate drug or solvent control was administered according to the method. Gastric acid collection was continued, and after nine points were collected after administration, the pharmacodynamic effect reached a stable state.

[0065] in particular,

[0066] (1) Drug testing Positive drug: Vonoprazan fumarate (TAK-438), placement method: The entire preparation process was carried out under light-shielded conditions. The required amount of Vonoprazan fumarate was weighed out, and an appropriate amount of 0.5% CMC-Na was added, stirred, and ultrasonicated to obtain a uniform suspension, which was then diluted to the corresponding concentration according to the concentration required for the scheme.

[0067] Subject: Crystal form I (represented as HW-N2001 in the experiment), Preparation method: The entire preparation process was carried out under light-shielded conditions. The required amount of HW-N2001 was weighed out, and the appropriate saline was added, mixed thoroughly to obtain a clear solution, and the pH value was adjusted to the range of 3.2-3.8. Depending on the concentration required for the scheme, it was diluted sequentially to the corresponding concentration.

[0068] (2) Breeding and explanation of experimental animals

[0069] 1) Animal information Breed and lineage: SD rats; Grade: SPF grade; Gender: female, male; Source: Shanghai Jihui Laboratory Animal Breeding Co., Ltd. (License number: SCXK (Shanghai) 2017-0012); Certificate number: female: 20170012019182, 20170012019726, male: 20170012017891, 20170012018177, 201700120118680, 20170012018969, 20170012019726. Number of animals: 130 were ordered, and 118 animals (51 females, 67 males) in good health were used in the experiment. Animal age at the start of the experiment: 6-9 weeks. Animal weight at the start of the experiment: females 160-220g, males 180-260g. Environmental adaptation time: 5-7 days.

[0070] 2) Rearing environment SPF-grade breeding room, in compliance with the national standard GB14925-2010 of the People's Republic of China, temperature: 23±2℃, humidity: 40-70%, lighting: artificial lighting, 12-hour light / dark cycle. Filling type: corn cob filling (Dezhou Gu Mei Agricultural Science and Technology Co., Ltd., lot number: GMCC202102170-2). Feed type: SPF low-protein rat maintenance feed (Jiangsu Cooperative Pharmaceutical and Biotechnology Co., Ltd., 20210225(X)). Feeding method: ad libitum. Drinking water type: high-pressure sterilized drinking water for test animals, water supply method: drinking water bottles are provided and the animals can drink ad libitum.

[0071] 3) Animal selection and fasting The animals used in the experiment were kept in good health. They were fasted for 24 hours before the experiment and had no access to water.

[0072] 4) Ethical approval for animal experiments This experimental project has already been approved by the Experimental Animal Ethics Committee of the Suzhou Institute of Drug Innovation, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, IACUC number 2021-06-WZC-10.

[0073] 5) Explanation of experimental animal selection The experimental animals used were common mammals such as mice, rats, dogs, and monkeys. These animals are usually used to study pharmacokinetic parameters, pharmacodynamic effects, and toxicology. The number of animals used was the minimum required to determine the interindividual variability of the experimental animals. The animals used in the experiment should be healthy, adapted to the environment, and have free access to food and water. The experiment was conducted using SD rats of both sexes.

[0074] (3) Experimental design

[0075] 1) Experimental group assignment Of the 130 SD rats, 118 in good health were selected and randomly divided into 8 groups based on body weight and baseline values ​​after histamine administration after the adaptation period. For details, see the table below.

[0076] [Table 7]

[0077] 2) Preparation of animal models and experimental procedures

[0078] Preparation of equipment and reagents: A beaker containing physiological saline is placed in a water bath and the water temperature is maintained at 37°C. Adjust the flow rate of the constant flow pump (1 mL / min) to reserve. Histamine dihydrochloride: Place fresh histamine dihydrochloride (0.6 mg / mL, dissolved in saline). Ethyl carbamate: Weigh out an appropriate amount of ethyl carbamate, add saline, dissolve ultrasonically, and adjust to a final concentration of 0.3 g / mL, and keep aside. Phenolphthalein: Weigh out the appropriate amount of phenolphthalein and add absolute ethanol to make it a final concentration of 0.5%, and keep aside. Alkaline titrant: Take an appropriate amount of 0.1 mol / L NaOH solution, add fresh deionized water, dilute to 0.01 mol / L, and keep as a reserve. Heparin sodium: Weigh out an appropriate amount of heparin sodium, add saline and dissolve to a final concentration of 100 U / mL, and keep aside.

[0079] Animal preparation: After the adaptation period, 118 SD rats (male and female) were fasted for 24 hours without water deprivation and randomly divided into 8 groups (male and female).

[0080] Preparation and creation of the intubation: Tracheal intubation: 2-3 cm of medical polypropylene tube (outer diameter 2 mm, inner diameter 1.5 mm), with a spare. Gastric cardia perfusion tube: Collect approximately 20 cm of medical polypropylene tubing (outer diameter 2 mm, inner diameter 1.5 mm) to use as a spare cardia perfusion tube. Gastric pylorus sample collection tube: Collect approximately 30 cm of medical polypropylene tube (outer diameter 4 mm, inner diameter 3 mm) and use it as a spare for the pylorus intubation end. Histamine perfusion tube: A 10 mL syringe is used to extract 10 mL of histamine dihydrochloride, an infusion extension tube is connected, the syringe is shielded from light using tin foil, and the syringe is fixed in the syringe pump as a spare. Animal anesthesia and care: Rats were anesthetized by intraperitoneal injection of ethyl carbamate at a dose of 1.5 g / kg. The hair on the neck and abdomen was shaved.

[0081] Intubation: To maintain a smooth airway during surgery, a 2 cm incision was made vertically in the neck (at the perineum), the muscles were separated to expose the perineum and trachea, the trachea was separated and a suture was passed underneath the trachea. A "T" shaped incision was made in the trachea and the tracheal intubation tube was gently inserted about 1 cm into the proximal end of the tracheal incision and tightened with a suture. The patency of the tracheal intubation tube was inspected, the wound was sutured, and the other end of the tracheal intubation tube was exposed outside the skin incision.

[0082] Gastric Fundus Intubation: Measure the distance from the mouth to the stomach (about 15 cm) and make a mark on the gastric cardia perfusion tube, insert the gastric cardia perfusion tube through the esophagus to the mark and stop, ensuring that the gastric cardia perfusion tube reaches the stomach through the cardiac sphincter. Connect the gastric cardia perfusion tube to the silica gel tube on the constant flow pump.

[0083] Gastric pylorus sample collection tube intubation: A small incision was made along Monk's white line under the xiphoid cartilage, the abdominal cavity was opened, and the position of the gastric cardia perfusion tube was examined. An incision was made transversely in the duodenum 1 cm below the pylorus, and the end of the gastric pylorus sample collection tube was inserted into the stomach about 1 cm along the duodenum through the incision, and the duodenum and gastric pylorus sample collection tube were tightly sutured, and the other end of the duodenal incision was tightly closed. 150 mL of saline preheated at 37°C was used to rapidly flush out the intragastric residues along the esophagus-cardia intubation, and the gastric pylorus sample collection tube was drained out, and the abdominal wall and skin were sutured, followed by perfusion with saline at a rate of 1 mL / min for 30 minutes, and the other end of the gastric pylorus sample collection tube was left outside the body, and gastric juice samples were collected in a conical bottle (one sample was collected every 20 minutes), and two basic gastric juice samples were collected, at which time the basic gastric acid secretion reached a stable state.

[0084] Sample collection and processing: The collected gastric juice samples were taken and titrated with 0.01N sodium hydroxide solution using an alkaline burette, with phenolphthalein indicator as the color indicator and pH 7 as the end point, and the titration volume was measured to calculate the amount of acid secreted.

[0085]

number

[0086] Tail vein indwelling needle: the tail vein was disinfected with alcohol, the indwelling needle was inserted into the tail vein, the core of the needle was removed, 0.5 ml of sodium heparin solution was pushed in, the syringe was removed, and the sodium heparin cap was twisted off.

[0087] Collect two basic gastric juice samples, and after the basic gastric acid level is stable, connect the infusion extension tube that has been inhaled histamine in advance to the rat tail vein indwelling needle, open the syringe pump, calculate the amount of histamine at 20 μmol / kg / hr, set the infusion rate of intravenous injection per animal, and after 80 minutes of continuous injection of histamine solution, collect gastric acid samples after histamine administration, collect gastric juice after three histamine administrations, gastric acid secretion reaches a stable state, and administer positive drug or solvent control intravenously to the subject or duodenum based on the group, continue to collect gastric juice, about 3 hours after administration, alkali consumption reaches a stable state, and a total of about 14 points are collected.

[0088] Body weight: The animals were weighed before fasting on the day before the experiment and before anesthesia on the day of the experiment.

[0089] Total gastric acidity: The total gastric acidity was measured by acid-base titration, the color indicator was 0.5% phenolphthalein, and the titration end point was pH 7.0.

[0090] Data Statistics Unless otherwise specified, experimental data are expressed as mean ± standard deviation (Mean ± SD). The Kolmogorov-Smirnov Test method was used to test for normality. If the data within the group followed normal distribution, one-way ANOVA was used. If the variances were uniform, the multiple contrasts between the mean numbers of multiple samples were performed using the LSD test. P < 0.05 was considered to indicate a significant difference. If the variances were not uniform, the multiple contrasts between the mean numbers of multiple samples were performed using the Games-Howell test. If the data within the group did not follow normal distribution, the Independent samples test using nonparametric tests was performed. P < 0.05 was considered to indicate a significant difference between multiple samples. All data statistical analyses were performed using SPSS 16.0 statistical software.

[0091] (5) Experimental results

[0092] 1) Comparison of rat weight groups During the experiment, no abnormalities were observed in the condition of the animals in each group, and there was no significant difference in body weight before and after fasting (Figure 7).

[0093] 2) Effect of subjects on histamine-induced gastric acid secretion There was no significant difference in the baseline values ​​of SD rats in each group before histamine administration, and after histamine administration, there was a significant difference between the vehicle group and the naive group, and there was a significant difference compared to each dose group of the active drug (vonoprazan fumarate) or the subject (HW-N2001). There was a difference in the gastric acid secretion amount and the gastric acid secretion inhibition rate of rats in each group after duodenal active drug administration or subject intravenous administration compared to the solvent control (vehicle) group. Specifically, it is as follows (Figures 8 and 9).

[0094] Vehicle group: Two points of gastric acid were collected before the rats were administered histamine, and the baseline values ​​of gastric acid secretion were 6.43±2.37, 8.83±5.1 (μEq HCl / 20 min), respectively, with no significant difference compared to the naive group. After the rats were administered histamine 80 min, they received three points of gastric acid, and the gastric acid secretion increased significantly and reached a steady state, with 42.3±14.58, 45.96±16.61, 46.65±16.96 (μEq HCl / 20 min), respectively, with significant difference compared to the naive group. Each time point after intravenous administration of the vehicle control was significantly different compared to the naive group.

[0095] Vonoprazan fumarate (1 mg / kg) group: After histamine was administered to the animals, the gastric acid secretion reached a steady state. After administration, the gastric acid secretion of the rats did not decrease significantly, and there was no statistical difference compared with the vehicle group.

[0096] Vonoprazan fumarate (2mg / kg) group: After histamine was administered to the animals, the gastric acid secretion reached a steady state. From 60min to 180min after administration, the gastric acid secretion of the rats gradually decreased. At 60min after administration, the gastric acid secretion of the rats was 33.21±15.63 (μEq HCl / 20min), and the inhibition rate of gastric acid secretion was 13.71%±2.12, which was statistically different from the vehicle group. At 180min after administration, the gastric acid secretion of the rats was 16.21±14.46 (μEq HCl / 20min), and the inhibition rate of gastric acid secretion was 57.48%±2.27, which was significantly different from the blank control group.

[0097] Vonoprazan fumarate (4mg / kg) group: After histamine was administered to the animals, the gastric acid secretion reached a steady state. From 40min to 180min after administration, the gastric acid secretion of the rats gradually decreased. At 40min after administration, the gastric acid secretion of the rats was 33.18±11.15(μEq HCl / 20min), which was not statistically different from the vehicle group. The inhibition rate of gastric acid secretion was 12.47%±1.13, which was statistically different from the vehicle control group. At 180min after administration, the gastric acid secretion of the rats was 4.41±4.02(μEq HCl / 20min), and the inhibition rate of gastric acid secretion was 87.03%±0.81, which was significantly different from the blank control group.

[0098] HW-N2001 (0.375mg / kg) group: After histamine was administered to the animals, the gastric acid secretion reached a steady state. From 60min to 180min after administration, the gastric acid secretion of the rats gradually decreased. At 60min after administration, the gastric acid secretion of the rats was 39.15±8.54 (μEq HCl / 20min), and the inhibition rate of gastric acid secretion was 4.34%±1.28, which was not statistically different from the solvent control group. At 180min after administration, the gastric acid secretion of the rats was 28.46±5.71 (μEq HCl / 20min), and the inhibition rate of gastric acid secretion was 27.52%±1.43, which was significantly different from the blank control group.

[0099] HW-N2001 (0.75mg / kg) group: After histamine was administered to the animals, the gastric acid secretion reached a steady state. From 20min to 180min after administration, the gastric acid secretion of the rats gradually decreased. After 20min after administration, the gastric acid secretion of the rats was 37.53±9.87(μEq HCl / 20min), which was not significantly different from the blank control group. The inhibition rate of gastric acid secretion was 11.6%±0.82, which was statistically different from the solvent control group. After 180min after administration, the gastric acid secretion of the rats was 10±5.32(μEq HCl / 20min), which was 73.68%±1.47, which was significantly different from the blank control group.

[0100] HW-N2001 (1.5mg / kg) group: After histamine was administered to the animals, the gastric acid secretion reached a steady state; 20min-80min after administration, the rat gastric acid secretion gradually decreased, and the pharmacodynamic effect reached a stable state; 20min after administration, the rat gastric acid secretion was 25.67±10.15 (μEq HCl / 20min), the gastric acid secretion inhibition rate was 33.28%±0, which was statistically different from the solvent control group; 80min after administration, the rat gastric acid secretion was 3.4±1.18 (μEq HCl / 20min), the gastric acid secretion inhibition rate was 89.64%±0, which was significantly different from the blank control group; 180min after administration, the rat gastric acid secretion was 2.2±0.56 (μEq HCl / 20min), the gastric acid secretion inhibition rate was 93.56%±0.09, which was significantly different from the blank control group.

[0101] (6) Conclusion The present invention continuously measures histamine-induced gastric acid secretion in SD rats and studies the pharmacodynamic effect of intravenous administration of vonoprazan pyroglutamate, Form I (HW-N2001), on gastric acid secretion in rats.

[0102] Vonoprazan fumarate was administered intraduodenally with no significant effect after 1 mg / kg administration, but with doses of 2 and 4 mg / kg, the pharmacodynamic effect appeared gradually at 60 and 140 minutes after administration, respectively, and inhibited gastric acid secretion in rats, with a certain dose-effect relationship. The drug's effect was gradual, with the strongest pharmacodynamic effect at 180 minutes, and the ID 50was 1.87 mg / kg.

[0103] Intravenous administration of HW-N2001 (0.375 mg / kg, 0.75 mg / kg, 1.5 mg / kg) significantly reduced gastric acid secretion in the treatment groups compared to the vehicle control group, with a certain dose-effect relationship. In the 0.75 and 1.5 mg / kg dose groups, the pharmacodynamic effect gradually appeared from 20 minutes after administration, the pharmacodynamic effect reached a plateau at 60-100 minutes after administration, and the ID 50 was 0.53 mg / kg.

[0104] In conclusion, intravenous administration of crystalline form I (HW-N2001) of vonoprazan pyroglutamate of the present invention can significantly inhibit histamine-induced gastric acid secretion in rats and exert its pharmacodynamic effect more quickly than intraduodenal administration of vonoprazan fumarate.

Claims

1. Crystalline form I of vonoprazan pyroglutamate, The X-ray powder diffraction patterns, expressed at 2θ diffraction angles, using Cu / Kα radiation, were 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, and 22.000±0.

200. Crystalline form I of vonoprazan pyroglutamate, characterized by having characteristic peaks at 0, 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, and 43.460±0.

200.

2. Crystalline form I of vonoprazan pyroglutamate, Crystalline form I of vonoprazan pyroglutamate is characterized in that the X-ray powder diffraction pattern displayed at 2θ diffraction angles using Cu / Kα radiation has 27 characteristic peaks, and the X-ray powder diffraction pattern is as shown in FIG. 1 or FIG.

2.

3. The infrared spectrum of the crystalline form I is 3255±5, 2738±5, 1690±5, 1657±5, 1573±5 cm －１ 3. The crystalline form I of vonoprazan pyroglutamate according to claim 1 or 2, characterized in that it has a characteristic absorption peak at

4. The crystalline form I of vonoprazan pyroglutamate according to any one of claims 1 to 3, characterized in that the crystalline form I is initially weightless at 297.9±1.0°C through thermogravimetric analysis, and has a weightlessness rate of 50.3±2.0%.

5. The crystalline form I of vonoprazan pyroglutamate according to any one of claims 1 to 4, characterized in that N-methyl-5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrole-3-methanamine pyroglutamate is dissolved in methanol, a ketone solvent is added dropwise, and the crystals are stirred, filtered, and dried to obtain crystalline form I.

6. Use of the crystalline form I of vonoprazan pyroglutamate or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 4 in the production of a potassium ion-competitive acid blocker.

7. 5. Use of the crystalline form I of vonoprazan pyroglutamate or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 4 in the manufacture of a gastric acid secretion inhibitor.

8. 8. The use according to claim 7, characterized in that the medicament is used for the treatment and / or prevention of diseases such as gastric ulcer, duodenal ulcer, reflux esophagitis, erosive esophagitis, gastroesophageal reflux disease, Helicobacter pylori infection and peptic ulcer.

9. The use of the pharmaceutical composition in the treatment and / or prevention of one or more diseases of gastric ulcer, duodenal ulcer, reflux esophagitis, erosive esophagitis, gastroesophageal reflux disease, Helicobacter pylori infection and peptic ulcer, The pharmaceutical composition comprises the crystalline form I of vonoprazan pyroglutamate or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 4 as an active ingredient.

10. The pharmaceutical composition according to claim 9, characterized in that it contains a drug comprising a potassium ion-competitive acid blocker or a gastric acid secretion inhibitor.

11. The pharmaceutical composition according to any one of claims 9 to 10, characterized in that the pharmaceutical composition comprises pharmaceutical excipients such as pharma- ceutically acceptable carriers, diluents or excipients including solubilizers, surfactants, film formers, antioxidants, stabilizers, adhesives and lubricants.

12. The pharmaceutical composition according to any one of claims 9 to 11, wherein the dosage form of the pharmaceutical composition may be a solid preparation, a liquid preparation or a semi-solid preparation, preferably a tablet, a capsule, an injection (including a powder injection), a microemulsion, a submicroemulsion, including a sustained release tablet, a sustained release capsule, and a sustained release injection.

Images