Crystalline form of vonerogivir glutamate and methods of making the same

Abstract

This invention relates to the crystal form of vonorazine pyroglutamate and its preparation method. The vonorazine pyroglutamate crystal form obtained by this invention not only has good solubility but also excellent storage stability; in vitro activity experiments show that crystal form I of this invention is effective against H+. + K + The inhibitory effect of ATPase is comparable to that of TAK-438; animal experiments show that the crystal form of the present invention, when administered intravenously, can significantly inhibit histamine-induced gastric acid secretion in rats, and can exert its pharmacological effect faster than that of TAK-438 administered duodenally.

Description

Crystal forms and preparation methods of vonoprazan pyroglutamate Technical Field

[0001] This invention belongs to the field of pharmaceutical crystal forms, specifically relating to the crystal form of vonoprazan pyroglutamate and its preparation method. Background Technology

[0002] Vonoprazan, chemically named 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine, has the following chemical structural formula:

[0003]

[0004] Vonoprazan fumarate (TAK-438) was developed by Takeda Pharmaceutical Company of Japan and approved for marketing in Japan on December 26, 2014. This drug belongs to a new class of inhibitors of potassium-competitive acid blockers (P-CABs). P-CABs are characterized by lipophilicity, weak basicity, high dissociation constant, and stability at low pH values. Therefore, vonoprazan fumarate exhibits rapid, potent, and long-lasting inhibition of gastric acid secretion. Simultaneously, it inhibits potassium ions in the final step of gastric acid secretion by parietal cells. + For H + -K + The compound binds to ATPase (proton pump) and prematurely terminates gastric acid secretion, but it has poor water solubility. This invention investigates the crystal form and in vitro / in vivo bioactivity of vonorazine pyroglutamate, which has good water solubility, and obtains a vonorazine pyroglutamate crystal form I with good solubility, stability, and in vivo bioactivity. Summary of the Invention

[0005] This invention provides a crystal form I of vonorazine pyroglutamate, characterized by: X-ray powder diffraction patterns expressed as 2θ diffraction angles using Cu / Kα radiation at 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± Characteristic peaks are observed at 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, and 43.460±0.200. Crystal form I may also exhibit the characteristics represented by the X-ray powder diffraction pattern shown in Figure 1.

[0006] Another embodiment of the present invention provides a crystal form I of vonorazan pyroglutamate, characterized in that: using Cu / Kα radiation, the crystal form I has 27 characteristic peaks, and its X-ray powder diffraction pattern is basically as shown in Figure 2.

[0007] Those skilled in the art will understand that when measurements are performed on substantially the same crystal form, the peak height and relative intensity of the 2θ characteristic peak in the XRPD spectrum may vary for various reasons.

[0008] The infrared spectra of vonoprazan pyroglutamate crystal form I described in this invention are at approximately 3255±5, 2738±5, 1690±5, 1657±5, and 1573±5 cm⁻¹. -1 The position has a characteristic absorption peak. Crystal form I can also have the basic characteristics represented by the infrared spectrum shown in Figure 3.

[0009] Thermogravimetric analysis (TGA) of the vonoprazan pyroglutamate crystal form I described in this invention showed that it experienced its first weight loss at 297.9 ± 1.0 °C, with a weight loss rate of 50.3 ± 2.0%, which was attributed to the decomposition of the substance. The TGA chart of crystal form I is shown in Figure 4.

[0010] Another embodiment of the present invention provides a method for preparing the above-mentioned vonorazan pyroglutamate crystal form I, characterized by comprising the following steps: dissolving 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine pyroglutamate in methanol, then adding a ketone solvent dropwise, stirring to precipitate crystals, filtering, and drying to obtain crystal form I.

[0011] Methanol dissolution can be carried out at room temperature or artificially heated to 20–35°C. Preferred ketone solvents include acetone and methyl ethyl ketone. Crystallization can be performed at room temperature or artificially cooled to 0–5°C. The amount of methanol and ketone solvent, temperature, and other parameters can be rationally selected by those skilled in the art based on the actual recrystallization conditions (such as experimental phenomena).

[0012] Another embodiment of the present invention provides the use of the above-described vonorazan pyroglutamate crystal form I or a pharmaceutically acceptable salt thereof in the preparation of a potassium-competitive acid blocker.

[0013] 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 medicament for inhibiting gastric acid secretion. The medicament is used to treat and / or prevent diseases such as gastric ulcers, duodenal ulcers, reflux esophagitis, erosive esophagitis, gastroesophageal reflux disease, Helicobacter pylori infection, and peptic ulcers.

[0014] Another embodiment of the present invention provides the use of a pharmaceutical composition in the treatment and / or prevention of diseases, including one or more of gastric ulcers, duodenal ulcers, reflux esophagitis, erosive esophagitis, gastroesophageal reflux disease, Helicobacter pylori infection, and peptic ulcers; the pharmaceutical composition uses vonoprazan pyroglutamate crystal form I or a pharmaceutically acceptable salt thereof as the active ingredient; the pharmaceutical composition may also include other potassium-competitive acid blockers or drugs that inhibit gastric acid secretion; the pharmaceutical composition may also include pharmaceutically acceptable excipients (e.g., pharmaceutically acceptable carriers, diluents, or excipients, including solubilizers, surfactants, film-forming agents, antioxidants, stabilizers, binders, lubricants, etc.). The dosage form of the pharmaceutical composition may be a solid dosage form, a liquid dosage form, or a semi-solid dosage form, preferably tablets, capsules, injections (including powder for injection), microemulsions, or submicroemulsions, including sustained-release tablets, sustained-release capsules, and sustained-release injections.

[0015] In this invention, the term "pharmaceutically acceptable salt" refers to the addition salt of a non-toxic inorganic or organic acid and / or base. See "Salt selection for basic drugs", Int. J. Pharm. (1986), 33, 201-217.

[0016] The various dosage forms involved in the pharmaceutical compositions of this invention can be prepared according to the technical specifications and requirements in the pharmaceutical field (such as the general requirements and specifications of the Chinese Pharmacopoeia (2015 edition), textbooks, or other existing technologies) to meet the clinical treatment and / or prevention of gastric acid secretion inhibition requirements, as well as their various specifications and dosage forms (including solid dosage forms, liquid dosage forms, and semi-solid dosage forms) such as sustained-release, controlled-release, and enteric-coated capsules, tablets, microemulsions, submicroemulsions, or various injections containing the same single or multi-component combinations.

[0017] The X-ray powder diffraction (XRPD) analysis of vonoprazan pyroglutamate crystal form I described in this invention was performed under ambient temperature and humidity conditions using a Rigaku MiniFlex 600 powder X-ray diffractometer (3-80°, step: 0.0200°, speed: 10° / min) and a Cu / Kα source (40kV, 40mA). The infrared spectroscopy analysis of this invention was performed under ambient temperature and humidity conditions using a Thermo Nicolet 6700 infrared spectrophotometer (USA) via the KBr pellet method. The thermogravimetric analysis (TGA) of this invention was performed under ambient temperature and humidity conditions using a Mettler TGA / DSC1 / 1100LF simultaneous thermal analyzer (Switzerland), with a scan rate of 10.00℃ / min, a maximum temperature of 800.00℃, and nitrogen as the protective gas. "Ambient temperature" is generally 0-40℃; "ambient humidity" is generally 30%-80% relative humidity.

[0018] The representative X-ray powder diffraction pattern, infrared spectrum, and thermogravimetric analysis diagram of vonoprazan pyroglutamate crystal form I described in this invention are listed in the accompanying drawings. "Representative X-ray powder diffraction pattern or infrared spectrum" means that the X-ray powder diffraction characteristics or infrared characteristics of this crystal form basically conform to the overall morphology shown in this spectrum. It is understood that during the testing process, due to the influence of various factors (such as the particle size of the test sample, the sample processing method, the instrument, the test parameters, the test operation, etc.), the peak positions or peak intensities of the X-ray powder diffraction pattern or infrared spectrum measured for the same crystal form may have certain differences. The 2θ value of the X-ray powder diffraction pattern may vary slightly between machines or between samples, and the experimental error of the 2θ value is generally ±0.2°; the experimental error of the absorption peak in the infrared spectrum is generally ±5 cm⁻¹. -1 The experimental error of weight loss temperature in thermogravimetric analysis is generally ±1℃, and the experimental error of weight loss percentage is generally ±2.0%.

[0019] The "vonorazine pyroglutamate" described in this invention refers to a molecule of vonorazine corresponding to a molecule of L-pyroglutamic acid, meaning that the molar ratio of 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine to L-pyroglutamic acid in vonorazine pyroglutamate is 1:1. It can be purchased commercially or prepared according to prior art (e.g., Chinese Patent Application No. 201410154778.8) using 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine and L-pyroglutamic acid. The "Vonoprazan fumarate" described in this invention is 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine fumarate (TAK-438), which was purchased commercially with a purity of 99.5%.

[0020] The chromatographic conditions for HPLC purity detection of the product in this invention are as follows:

[0021] Chromatographic column: Ultimate XB-C18, column length: 150 mm, inner diameter: 4.6 mm, particle size: 5 μm

[0022] Flow rate: 1.2 ml / min; Column temperature: 30℃; Wavelength: 210 nm; Injection volume: 20 μl; Solvent: Acetonitrile-water (v / v 5:95)

[0023] Mobile phase A: Acetonitrile - 0.02 mol / L potassium dihydrogen phosphate (adjust pH to 2.5 with phosphoric acid) (volume ratio 5:95)

[0024] 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)

[0025] The proportional relationship between mobile phases A and B over time is as follows:

[0026]

[0027] Compared with the prior art, the advantages of the present invention are: (1) The present invention obtains a new crystal form of vonoprazan pyroglutamate, namely crystal form I, which not only has good solubility, but also has excellent storage stability; (2) In vitro activity experiments show that crystal form I of the present invention is effective against H+. + K +- The inhibitory effect of ATPase is comparable to that of TAK-438, which is superior to that of vonorazine pyroglutamate (another crystal form, Figure 5) in the prior art (Chinese patent application number: 201410154778.8); (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 can exert its pharmacological effect faster than that of duodenal administration of TAK-438. Attached Figure Description

[0028] Figure 1 is an X-ray powder diffraction pattern of vonoprazan pyroglutamate crystal form I of the present invention;

[0029] Figure 2 shows the 27 characteristic peaks of X-ray powder diffraction for crystal form I, along with their diffraction angle 2θ and interplanar spacing d. Detailed parameter charts such as relative strength (%);

[0030] Figure 3 is the infrared spectrum of crystal form I;

[0031] Figure 4 is a thermogravimetric analysis diagram of crystal form I;

[0032] Figure 5 is the X-ray powder diffraction pattern of product A;

[0033] Figure 6 shows the effects of TAK438, Product A, and Crystal Form I on microvesicles H in porcine gastric mucosa. + K + - Inhibition rate curve of ATPase;

[0034] Figure 7 is a comparison of the body weight of rats in each group;

[0035] Figure 8 shows the effect of each group of test substances on histamine-induced gastric acid secretion.

[0036] Figure 9 shows the effect of each test substance on the inhibition rate of histamine-induced gastric acid secretion in rats. Detailed Implementation

[0037] To facilitate a further understanding of the present invention, the following embodiments are provided for more detailed description. However, these embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The implementation of the present invention is not limited to the following.

[0038] Example 1

[0039] Following the method described in Example 1 of Chinese Patent Application No. 201410154778.8, 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine pyroglutamate (10.0 g, HPLC purity 98.0%, i.e., vonorazan pyroglutamate, hereinafter referred to as Product A, the X-ray powder diffraction pattern of which is shown in Figure 5) was obtained.

[0040] 2.00 g of 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine pyroglutamate sample was weighed and added to a 100 mL flask. Methanol (3.0 mL) was added, and the mixture was stirred at 20–25 °C for 10 minutes to dissolve. Acetone (approximately 10.0 mL) was added dropwise at 20–25 °C. During the addition of acetone, a solid precipitated out. The mixture was stirred for 30 minutes, cooled to 0–5 °C, stirred for 1 hour, filtered, and dried under vacuum at 50 °C to obtain 1.54 g of white solid, which is vonorazan pyroglutamate crystal form I (hereinafter referred to as crystal form I). Its X-ray powder diffraction pattern is shown in Figure 1. The specific diffraction angle 2θ and interplanar spacing d are shown in Figure 1. The relative intensity (%) is shown in Figure 2, its infrared spectrum is shown in Figure 3, its thermogravimetric analysis is shown in Figure 4, and its HPLC purity is 99.8%.

[0041] Example 2 Stability Test

[0042] (1) Deliquescence test: Under the conditions of 40℃ and 75% relative humidity, product A, crystal form I and TAK-438 were stored and observed to see if deliquescence occurred. The test lasted for 30 days and the results were recorded every 10 days. See the table below for details.

[0043] Test Sample Day 10 Day 20 Day 30 Product A Slight Deliquescence Significant Deliquescence Significant Deliquescence Crystal Form I No Deliquescence Slight Deliquescence Significant Deliquescence TAK-438 No Deliquescence No Deliquescence No Deliquescence surface

[0044] No deliquescence means no moisture absorption was observed on the sample surface; slight deliquescence means moisture absorption was observed on the sample surface; significant deliquescence means the sample surface was observed to be slippery.

[0045] (2) Thermal stability test: Product A and crystal form I were stored in open containers at 60℃ for 30 days (day 31) and their storage stability was tested. The purity of the samples at the initial stage and after 30 days of storage was tested by HPLC. Three parallel groups were set up for each sample and the average value was taken. The results are shown in the table below.

[0046]

[0047] As can be seen from the deliquescence and thermal stability tests, the crystal form I of this invention has good stability and is easy to store for a long time.

[0048] Example 3: Solubility Test and Melting Point Test

[0049] Basic method for solubility test: Take an appropriate amount of the sample to be tested, grind it into a fine powder, place it in a suitable container, add an appropriate amount of water, and shake vigorously for 30 seconds every 5 minutes at 37℃±2℃. Observe the solubility within 30 minutes. If it does not dissolve, continue to add a measured amount of water, place it at 37℃±2℃, and shake vigorously for 30 seconds every 5 minutes. Observe the solubility and calculate the solubility at 37℃±2℃. Repeat the test 3 times and take the average value.

[0050] Basic method for melting point testing: Take an appropriate amount of the sample to be tested, grind it into a fine powder, and load it into a capillary tube with one end sealed, ensuring the powder is tightly packed at the sealed end of the capillary. The height of the sample loaded should be approximately 3 mm, and the heating rate of the melting point apparatus should be 1 °C / min. Repeat the test three times and take the average value. The results are shown in the table below.

[0051] Test sample solubility (mg / mL) Melting point (°C) Product A1018174-175 Crystal form I901167-168 surface

[0052] Example 4: Microsomal vesicles of porcine gastric mucosa H + K + - Inhibition of ATPase

[0053] Test method: H + K + -ATPase is an enzyme located in the parietal cells of the gastric mucosa that can be activated by K+. + Specifically activated ATPase. This experiment extracted H2 from microsomal vesicles of porcine gastric mucosa. + K + -ATPase, an enzyme that breaks down ATP to produce ADP and inorganic phosphate, the test substance binds to H + K + - ATPase activity was inhibited by measuring the amount of inorganic phosphorus, thus determining the level of ATPase activity and the inhibitory effect of the test substance. The specific method is as follows:

[0054] This experiment used porcine gastric mucosal microparticle vesicles H + K + ATPase was extracted by Beijing Huizhi Taikang.

[0055] (1) BCA protein quantification

[0056] 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 to 50 volumes of BCA reagent A (50:1) and mixing thoroughly. For example, add 100 μL of BCA reagent B to 5 mL of BCA reagent A, mix well, and prepare 5.1 mL of BCA working solution. The BCA working solution is stable at room temperature for 24 hours.

[0057] (2) Protein concentration detection

[0058] 1) Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard to the wells of a 96-well plate, and add standard diluent to bring the total to 20 μL. This corresponds 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.

[0059] 2) Dilute the sample to 2x, 5x, and 10x respectively, and add 20 μL of sample to the wells of a 96-well plate. Please record the sample volume and dilution factor.

[0060] 3) Add 200 μL of BCA working solution to each well and incubate at 37°C for 20-30 minutes.

[0061] Note: Alternatively, it can be incubated at room temperature for 2 hours or at 60°C for 30 minutes. When determining protein concentration using the BCA method, the color will deepen over time. Furthermore, the colorimetric reaction will accelerate with increasing temperature. If the concentration is low, it is suitable to incubate at a higher temperature or appropriately extend the incubation time.

[0062] 4) Use an ELISA reader to measure the absorbance at wavelengths between A562 and 540-595 nm.

[0063] 5) Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0064] (3) Configuration of the reaction system

[0065] 1) Components and configuration of the enzyme reaction system

[0066] 100mM TRIS-HCl (pH 6.8): Add 100ul of 1M TRIS-HCl to 900ul of ddH2O and mix well.

[0067] 37.5mM MgCl2: Weigh out 50mg of MgCl2. 2， Add 14 ml of ddH2O and mix well.

[0068] 200mM KCl: Weigh 500mg of KCl, add 33.5ml of ddH2O, and mix well.

[0069] 10mM Nigerian styramine and valacyclovir: Add 669ul and 890ul of DMSO respectively, mix well, dispense into 1ul tubes of Nigerian styramine and 5ul tubes of valacyclovir, and store at -20℃.

[0070] 0.1 mg / ml microsomal vesicles: After arrival, determine the protein concentration, dispense into aliquots for use, store at -80℃, and dilute with 5 mM TRIS-HCl before use.

[0071] 2) Preparation of the test sample

[0072] Control compound: TAK438 was prepared as a 10 mM stock solution with DMSO and dispensed in 1 μL tubes.

[0073] Test compounds: Product A and crystal form I were prepared into 10 mM stock solutions using ddH2O, and dispensed in 1 μL tubes. Using 20 μM (or 40 μM) as the highest concentration, the control compound and the test compound were gradually diluted 3-fold with DMSO and ddH2O, respectively, to obtain 6 (or 7 or 8) concentration gradients of test samples.

[0074] (4) The reaction proceeds

[0075] Enzymatic reaction

[0076] Test tube (µL) Control tube (µL) 100mM TRIS-HCl 60 60 37.5mM MgCl2 20 20 200mM KCl 15 / ddH2O / 15 Nigeriencin 1.5 1.5 Valinemycin 1.5 1.5 0.1mg / mL Microvoids 50 50 Compound 1.5 1.5 surface

[0077] Incubate at 37°C for 30 minutes, add 16.5 μL of ATP, mix well, and incubate at 37°C for 30 minutes.

[0078] (5) Color reaction

[0079] According to *H + K + Prepare the colorimetric reagent according to the requirements of the ATP reagent kit instructions (Nanjing Jiancheng).

[0080] (6) Reading the board

[0081] Add 45 μL of colorimetric reagent to every 150 μL of enzyme reaction volume, mix well, let stand for 2 min, and read the absorption light signal at 660 nm using an ELISA reader.

[0082] (7) Data Statistics

[0083] 1) Calculate the inhibition rate using the following formula: Inhibition rate (%) = (1 - (RLU) compound -RLU blank ) / (RLU DMSO –RLU blank ))×100%.

[0084] 2) Use Graphpad Prism5 to plot the drug efficacy inhibition rate curve and calculate the IC50. 50 The value is calculated using a 4-parameter model: [fit = (A + ((BA) / (1 + ((C / x)^D))))].

[0085] (8) Experimental Results

[0086] 1) BCA protein quantification: Based on the BSA standard curve, the average protein concentration was calculated to be 3 mg / mL.

[0087] 2) TAK438, Product A, and Crystal Form I on microvesicles of porcine gastric mucosa H + K + The inhibitory effect of ATPase is shown in the table below and Figure 6.

[0088] Test Sample IC 50 (nM)TAK43841.5 Product A53.4 Crystal form I40.4 surface

[0089] Example 5: Pharmacodynamic study of intravenous administration of histamine in a rat model of gastric acid secretion.

[0090] Surgical modeling: Rats were anesthetized by intraperitoneal injection of 1.5 g / kg of malol. After complete anesthesia, the hair on the neck and abdomen of the rats was shaved, and endotracheal intubation was performed to maintain airway patency during the operation. A cardia irrigation tube was inserted into the rat's stomach approximately 1 cm orally, and the other end of the cardia irrigation tube was connected to a constant flow pump. A small incision was made below the xiphoid cartilage to locate the duodenum. A transverse incision was made, and the end of the pyloric sample collection tube was inserted into the stomach approximately 1 cm into the duodenum through the incision. The duodenum and the pyloric sample collection tube were sutured together, and the other end of the duodenal incision was also ligated. The gastric contents were rapidly flushed with 150 mL of preheated 37°C saline solution through the esophageal-cardia cannula, and the contents were drained through the pyloric sample collection tube. The abdominal wall and skin were sutured, and the gastric contents were continued to be perfused with saline solution at a rate of 1 mL / min for 30 minutes. The other end of the pyloric sample collection tube was left outside the body, and gastric fluid samples were collected in conical flasks (one sample every 20 minutes). Two basal gastric fluid samples were collected, at which point the basal gastric acid secretion reached a stable state.

[0091] Histamine-induced gastric acid secretion: Histamine was continuously administered to rats via tail vein using a microinfusion pump at a dose of 20 μmol / kg / hr. Gastric acid was collected after 80 min. After collecting data at three points, histamine secretion in rats reached a steady state. Rats were grouped according to the amount of gastric acid secreted after histamine administration, and either the candidate drug or a solvent control was administered according to the protocol. Gastric acid collection continued, and data was collected at nine points after drug administration, at which point the drug efficacy reached a steady state.

[0092] Specifically as follows:

[0093] (1) Test drugs

[0094] Positive drug: Vonoprazan fumarate (TAK-438). Preparation method: The whole preparation process is carried out under light protection. Weigh the required amount of vonoprazan fumarate, add an appropriate amount of 0.5% CMC-Na, stir and sonicate to obtain a uniform suspension, and dilute it to the corresponding concentration successively according to the concentration required by the protocol.

[0095] Test substance: Polymorph I (represented by HW-N2001 in the experiment). Preparation method: The whole preparation process is carried out under light protection. Weigh the required amount of HW-N2001, add an appropriate amount of normal saline, mix well to obtain a clear solution, and adjust the pH value to the range of 3.2 - 3.8. Dilute it to the corresponding concentration successively according to the concentration required by the protocol.

[0096] (2) Animal feeding and instructions

[0097] 1) Animal information

[0098] Species and strain: SD rats. Grade: SPF grade. Gender: female and male. Source: Shanghai Jihui Laboratory Animal Breeding Co., Ltd. (License number: SCXK (Shanghai) 2017-0012). Certificate numbers: for females: 20170012019182, 20170012019726; for males: 20170012017891, 20170012018177, 20170012018680, 20170012018969, 20170012019726. Number of animals: 130 were ordered, and 118 healthy animals (51 females and 67 males) were selected for the experiment. Animal age at the start of the experiment: 6 - 9 weeks old. Animal weight at the start of the experiment: females 160 - 220 g, males 180 - 260 g. Adaptation time to the environment: 5 - 7 days.

[0099] 2) Feeding environment

[0100] SPF-class breeding room, meeting the national standard GB14925-China 2010. Temperature: 23 ± 2 °C. Humidity: 40 - 70%. Lighting: artificial lighting, with 12-hour light-dark alternation. Type of bedding: corncob bedding (Dezhou Gumei Agricultural Technology Co., Ltd., batch number: GMCC202102170-2). Type of feed: SPF low-protein maintenance feed for mice and rats (Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., 20210225(X)). Feeding method: free intake. Type of drinking water: high-pressure sterilized drinking water for experimental animals. Water supply method: contained in drinking water bottles, with free intake.

[0101] 3) Animal selection and fasting

[0102] Animals used in the experiment will maintain good health. Fast for 24 hours before the experiment without water restriction.

[0103] 4) Ethical approval for animal use

[0104] This experimental project has been approved by the Laboratory Animal Ethics Committee of the Suzhou Institute of Drug Innovation, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, with IACUC number 2021-06-WZC-10.

[0105] 5) Instructions for selecting laboratory animals

[0106] Common mammals, such as mice, rats, dogs, and monkeys, are used as laboratory animals in this experiment. These animals are typically used for studies of pharmacokinetic parameters, pharmacodynamics, and toxicology. The number of animals used is the minimum required to determine inter-individual variability. The animals used must be healthy and adapted to their environment, with free access to food and water. This experiment used bisexual SD rats.

[0107] (3) Experimental Design

[0108] 1) Experimental Grouping

[0109] Of the 130 SD rats, 118 were in good health. After the adaptation period, they were randomly divided into 8 groups according to their body weight and baseline values ​​after histamine administration. See the table below for details.

[0110]

[0111] 2) Animal model preparation and experimental process

[0112] Instrument and reagent preparation:

[0113] Place physiological saline in a beaker and put it in a water bath, maintaining the water temperature at 37°C.

[0114] Adjust the constant flow pump speed (1 mL / min) and set it aside.

[0115] Histamine dihydrochloride: Freshly prepared histamine dihydrochloride (0.6 mg / mL, dissolved in physiological saline).

[0116] Ulai sugar: Weigh an appropriate amount of utlai sugar, add physiological saline, sonicate to dissolve, and prepare a final concentration of 0.3g / mL for later use.

[0117] Phenolphthalein: Weigh an appropriate amount of phenolphthalein, add anhydrous ethanol, and prepare a final concentration of 0.5% for later use.

[0118] Basic titration solution: Take an appropriate amount of 0.1 mol / L NaOH solution, add fresh deionized water, and dilute to 0.01 mol / L for later use.

[0119] Heparin sodium: Weigh an appropriate amount of heparin sodium, add it to physiological saline, and dissolve it to a final concentration of 100 U / mL for later use.

[0120] Animal preparation:

[0121] After the acclimatization period, 118 SD rats (male and female) were fasted but allowed free access to water for 24 hours. They were then randomly divided into 8 groups (male and female).

[0122] Preparation and fabrication of intubation tubes:

[0123] Endotracheal intubation: Take 2-3cm of medical polyethylene plastic tube (outer diameter 2mm, inner diameter 1.5mm) for later use.

[0124] Gastric cardia irrigation tube: Take a medical polyethylene tube about 20cm long (outer diameter 2mm, inner diameter 1.5mm) and use it as a gastric tube for cardia irrigation.

[0125] Pyloric sample collection tube: Take a medical polyethylene tube (outer diameter 4mm, inner diameter 3mm) about 30cm long and use it as the pyloric insertion end.

[0126] Histamine perfusion tubing: Draw 10 mL of histamine dihydrochloride into a 10 mL syringe, connect it to the infusion extension tubing, wrap it with aluminum foil to protect it from light, and fix it inside the syringe pump for later use.

[0127] Animal anesthesia and care: Rats were anesthetized by intraperitoneal injection of 1.5 g / kg of ursotose. Hair was shaved off the neck and abdomen.

[0128] Endotracheal intubation:

[0129] To maintain an unobstructed airway during surgery, a 2cm longitudinal incision is made in the neck (at the epiglottis). The muscles are separated to expose the epiglottis and trachea. A suture is then passed under the trachea after the trachea is dissected. A T-shaped incision is made in the trachea, and the endotracheal tube is gently inserted approximately 1cm proximal to the heart through the incision and secured with sutures. The patency of the endotracheal tube is checked, the wound is sutured, and the other end of the endotracheal tube is exposed outside the skin incision.

[0130] Gastric cardia irrigation tube insertion:

[0131] Mark the distance from the inlet to the stomach (approximately 15cm) on the gastric cardia irrigation tube. Insert the tube through the esophagus to the marked point and stop, ensuring the tube passes through the esophageal sphincter to reach the stomach. Connect the gastric cardia irrigation tube to the silicone tubing on the constant flow pump.

[0132] Gastric pyloric sample collection tube insertion:

[0133] A small incision is made below the xiphoid cartilage along the linea alba in the abdomen to open the abdominal cavity and check the location of the gastric cardia irrigation tube. A transverse incision is made in the duodenum 1 cm below the pylorus. The end of the gastric pyloric sample collection tube is inserted into the stomach approximately 1 cm into the duodenum through the incision. The duodenum and the gastric pyloric sample collection tube are tied tightly with a suture, and the other end of the duodenal incision is also tied tightly. The stomach contents are rapidly flushed with 150 ml of preheated 37°C saline solution along the esophagus-cardia tube, and the contents flow out through the gastric pyloric sample collection tube. The abdominal wall and skin are sutured. Perfusion with saline solution continues at a rate of 1 ml / min for 30 minutes. The other end of the gastric pyloric sample collection tube is left outside the body. Gastric fluid samples are collected using conical flasks (one sample every 20 minutes), resulting in two basal gastric fluid samples. At this point, basal gastric acid secretion has reached a stable state.

[0134] Sample collection and processing:

[0135] Collected gastric fluid samples were titrated with 0.01N sodium hydroxide solution using an alkaline burette, with phenolphthalein indicator as the color indicator. The titration was completed to pH 7. The titration volume was measured, and the acid secretion was calculated.

[0136] Acid secretion (μEq) = NaOH volume (mL) × 10 -3 ×0.01mol / L×10 6

[0137] Tail vein indwelling needle: Disinfect the tail vein with alcohol, insert the indwelling needle into the tail vein, remove the needle core, push in 0.5ml of heparin sodium solution, remove the syringe and screw on the heparin sodium cap.

[0138] Two basal gastric fluid samples were collected. After the basal gastric acid level stabilized, the infusion extension tube containing pre-absorbed histamine was connected to the tail vein indwelling needle of the rat. The infusion pump was turned on, and the histamine was calculated at a rate of 20 μmol / kg / hr and the infusion pump was set to deliver the infusion rate to each animal intravenously. After continuously injecting the histamine solution for 80 minutes, gastric acid samples were collected after histamine administration. Gastric fluid was collected after three histamine administrations. When gastric acid secretion reached a stable state, the test substance was administered intravenously or the positive drug or solvent control was administered duodenally according to the group. Gastric fluid collection continued. Approximately 3 hours after administration, alkali consumption reached a stable state. A total of about 14 points were collected.

[0139] Weight: Weighed before fasting the day before the experiment and before anesthesia on the day of the experiment.

[0140] Total acidity of gastric juice: The total acidity of gastric juice was determined by acid-base titration, with 0.5% phenolphthalein as the color indicator, and the titration endpoint was pH 7.0.

[0141] (4) Data Statistics

[0142] Unless otherwise specified, experimental data are expressed as mean ± standard deviation (Mean ± SD). The Kolmogorov-Smirnov test was used to test for normality. When the data within a group conformed to a normal distribution, one-way ANOVA was used. When variances were homogeneous, multiple comparisons between multiple sample means were performed using the LSD test, with P < 0.05 considered statistically significant. When variances were unequal, multiple comparisons between multiple sample means were performed using the Games-Howell test. When the data within a group did not conform to a normal distribution, the independent samples test (a non-parametric test) was used, with P < 0.05 considered statistically significant. All statistical analyses were performed using SPSS 16.0 software.

[0143] (4) Experimental Results

[0144] 1) Comparison of rat body weight among groups

[0145] Throughout the experiment, no abnormalities were observed in the condition of the experimental animals in any group, and there was no significant difference in body weight before and after fasting. (Figure 7)

[0146] 2) Effect of the test substance on histamine-induced gastric acid secretion

[0147] There was no significant difference in baseline values ​​among the SD rat groups before histamine administration. After histamine administration, the Vehicle group showed a significant difference compared to the others. Significant differences were observed between the groups, and within each dose group of the positive control (vonoprazan fumarate) or the test substance (HW-N2001). After duodenal administration of the positive control or intravenous administration of the test substance, the gastric acid secretion and gastric acid secretion inhibition rate of rats in each group showed varying degrees of difference compared to the solvent control (Vehicle) group. Details are as follows (Figures 8 and 9).

[0148] Solvent control (Vehicle group): Gastric acid was collected from two points in rats before histamine administration. The baseline values ​​of gastric acid secretion were 6.43±2.37 and 8.83±5.1 (μEq HCl / 20min), respectively. There were no significant differences between the groups; after rats were given histamine for 80 minutes, gastric acid secretion was collected at three points, and the gastric acid secretion significantly increased and reached a steady state, at 42.3±14.58, 45.96±16.61, and 46.65±16.96 (μEq HCl / 20min), respectively. All group ratios showed significant differences; after intravenous solvent administration, the differences at each time point were statistically significant. The group ratios showed significant differences.

[0149] Vonorazan fumarate (1 mg / kg) group: After administration of histamine, the gastric acid secretion of animals reached a steady state. After administration, the gastric acid secretion of rats did not decrease significantly, and there was no statistical difference compared with the Vehicle group.

[0150] Vonorazan fumarate (2 mg / kg) group: After administration of histamine, the gastric acid secretion of animals reached a steady state. From 60 min to 180 min after administration, the gastric acid secretion of rats gradually decreased. At 60 min after administration, the gastric acid secretion of rats was 33.21 ± 15.63 (μEq HCl / 20 min), and the gastric acid secretion inhibition rate was 13.71% ± 2.12, which was statistically different from the solvent group. At 180 min after administration, the gastric acid secretion of rats was 16.21 ± 14.46 (μEq HCl / 20 min), and the gastric acid secretion inhibition rate was 57.48% ± 2.27, which was significantly different from the blank control group.

[0151] Vonorazan fumarate (4 mg / kg) group: After administration of histamine, the gastric acid secretion of animals reached a steady state. From 40 min to 180 min after administration, the gastric acid secretion of rats gradually decreased. At 40 min after administration, the gastric acid secretion of rats was 33.18 ± 11.15 (μEq HCl / 20 min), which was not statistically different from the solvent group. The gastric acid secretion inhibition rate was 12.47% ± 1.13, which was statistically different from the solvent control group. At 180 min after administration, the gastric acid secretion of rats was 4.41 ± 4.02 (μEq HCl / 20 min), and the gastric acid secretion inhibition rate was 87.03% ± 0.81, which was significantly different from the blank control group.

[0152] HW-N2001 (0.375 mg / kg) group: After administration of histamine, the gastric acid secretion of animals reached a steady state. From 60 min to 180 min after administration, the gastric acid secretion of rats gradually decreased. At 60 min after administration, the gastric acid secretion of rats was 39.15 ± 8.54 (μEq HCl / 20 min), and the gastric acid secretion inhibition rate was 4.34% ± 1.28, which was not statistically different from the solvent control group. At 180 min after administration, the gastric acid secretion of rats was 28.46 ± 5.71 (μEq HCl / 20 min), and the gastric acid secretion inhibition rate was 27.52% ± 1.43, which was significantly different from the blank control group.

[0153] HW-N2001 (0.75 mg / kg) group: After administration of histamine, the gastric acid secretion of animals reached a steady state. From 20 min to 180 min after administration, the gastric acid secretion of rats gradually decreased. At 20 min after administration, the gastric acid secretion of rats was 37.53 ± 9.87 (μEq HCl / 20 min), which was not significantly different from the blank control group. The gastric acid secretion inhibition rate was 11.6% ± 0.82, which was statistically different from the solvent control group. At 180 min after administration, the gastric acid secretion of rats was 10 ± 5.32 (μEq HCl / 20 min), and the gastric acid secretion inhibition rate was 73.68% ± 1.47, which was significantly different from the blank control group.

[0154] In the HW-N2001 (1.5 mg / kg) group: After administration of histamine, gastric acid secretion reached a steady state in the animals. From 20 to 80 minutes after administration, gastric acid secretion in rats gradually decreased, and the drug effect reached a stable state. At 20 minutes after administration, the gastric acid secretion in rats was 25.67 ± 10.15 (μEq HCl / 20 min), with an inhibition rate of 33.28% ± 0, which was statistically different from the control group. At 80 minutes after administration, the gastric acid secretion in rats was 3.4 ± 1.18 (μEq HCl / 20 min), with an inhibition rate of 89.64% ± 0, which was significantly different from the blank control group. At 180 minutes after administration, the gastric acid secretion in rats was 2.2 ± 0.56 (μEq HCl / 20 min), with an inhibition rate of 93.56% ± 0.09, which was significantly different from the blank control group.

[0155] (5) Conclusion

[0156] This invention employs a histamine-induced gastric acid secretion model in SD rats to investigate the pharmacodynamic effects of intravenous administration of vonoprazan pyroglutamate crystal form I (HW-N2001) on gastric acid secretion in rats.

[0157] When vonoprazan fumarate is administered duodenally, the effect is not significant after a dose of 1 mg / kg. At doses of 2 and 4 mg / kg, the drug gradually exerts its effects at 60 min and 140 min, respectively, inhibiting gastric acid secretion in rats, and exhibiting a dose-response relationship. The drug has a slow onset of action, reaching its peak efficacy at 180 min. 50 It is 1.87 mg / kg.

[0158] Intravenous administration of HW-N2001 (0.375 mg / kg, 0.75 mg / kg, and 1.5 mg / kg) significantly reduced gastric acid secretion in the treated groups compared to the control group, exhibiting a dose-response relationship. The 0.75 mg / kg and 1.5 mg / kg groups began to exert their effects gradually 20 minutes after administration, reaching a stable state 60-100 minutes after administration, with an effective dose of 180 minutes after administration. 50 It is 0.53 mg / kg.

[0159] In summary, 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 can exert its pharmacological effect faster than vonoprazan fumarate administered duodenally.

Claims

1. A crystal form I of vonorazine pyroglutamate, characterized in that: X-ray powder diffraction patterns expressed as 2θ diffraction angles using Cu / Kα radiation are shown at 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, and 20.820±0. Characteristic peaks are observed at 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, and 43.460±0.

200.

2. A crystal form I of vonorazine pyroglutamate, characterized in that: Using Cu / Kα radiation, the X-ray powder diffraction pattern expressed in 2θ diffraction angle has 27 characteristic peaks, and its basic X-ray powder diffraction pattern is shown in Figure 1 or Figure 2.

3. The vonorazine pyroglutamate crystal form I according to any one of claims 1-2, characterized in that... The infrared spectrum of crystal form I is at 3255±5, 2738±5, 1690±5, 1657±5, and 1573±5 cm⁻¹. -1 The position has a characteristic absorption peak.

4. The vonorazine pyroglutamate crystal form I according to any one of claims 1-2, characterized in that... Thermogravimetric analysis showed that crystal form I had a value of 297.9 ± 1.

0. o The first weightlessness occurred at time C, with a weightlessness rate of 50.3 ± 2.0%.

5. The method for preparing vonoprazan pyroglutamate crystal form I according to any one of claims 1-4, characterized in that... The process includes the following steps: dissolving 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine pyroglutamate in methanol, then adding a ketone solvent, stirring to induce crystallization, filtering, and drying to obtain crystal form I.

6. The use of vonorazan pyroglutamate crystal form I or a pharmaceutically acceptable salt thereof as described in any one of claims 1-4 in the preparation of a potassium-competitive acid blocker.

7. The use of vonoprazan pyroglutamate crystal form I or a pharmaceutically acceptable salt thereof as described in any one of claims 1-4 in the preparation of a medicament for inhibiting gastric acid secretion.

8. The application according to claim 7, characterized in that... The drug is used to treat and / or prevent the following conditions: gastric ulcer, duodenal ulcer, reflux esophagitis, erosive esophagitis, gastroesophageal reflux disease, Helicobacter pylori infection, and peptic ulcer.

9. A pharmaceutical composition for treating and / or preventing diseases, characterized in that... The disease is selected from one or more of gastric ulcer, duodenal ulcer, reflux esophagitis, erosive esophagitis, gastroesophageal reflux disease, Helicobacter pylori infection, and peptic ulcer; the pharmaceutical composition uses vonoprazan pyroglutamate crystal form I or a pharmaceutically acceptable salt thereof as the active ingredient.

10. The pharmaceutical composition according to claim 9, characterized in that... The pharmaceutical composition also includes other potassium-competitive acid blockers or drugs that inhibit gastric acid secretion.

11. The pharmaceutical composition according to any one of claims 9-10, characterized in that... The pharmaceutical composition also includes pharmaceutically acceptable excipients.

12. The pharmaceutical composition according to claim 11, characterized in that... The pharmaceutically acceptable pharmaceutical excipients are selected from pharmaceutically acceptable carriers, diluents, or excipients.

13. The pharmaceutical composition according to claim 11, characterized in that... The pharmaceutically acceptable pharmaceutical excipients are selected from solubilizers, surfactants, film-forming agents, antioxidants, stabilizers, adhesives, and lubricants.

14. The pharmaceutical composition according to claim 11, characterized in that... The dosage form of the pharmaceutical composition is a solid dosage form, a liquid dosage form, or a semi-solid dosage form.

15. The pharmaceutical composition according to claim 14, characterized in that... The dosage form of the pharmaceutical composition is tablet, capsule, injection, microemulsion, or submicroemulsion.

16. The pharmaceutical composition according to claim 14, characterized in that... The dosage form of the pharmaceutical composition is a sustained-release tablet, a sustained-release capsule, or a sustained-release injection.

17. The pharmaceutical composition according to claim 15, characterized in that... The dosage form of the pharmaceutical composition is a powder for injection.

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