Tablet, injection and compound formulation of vonoprazan pyroglutamate

Abstract

The present invention relates to a tablet, injection and compound formulation of vonoprazan pyroglutamate. The tablet comprises a core and a film coating layer, the core comprising a vonoprazan pyroglutamate crystal form I, a filler, a binder, a pH regulator, a disintegrating agent and a lubricant, and the film coating layer comprising an Opadry film coating premix. An oral solution comprises the vonoprazan pyroglutamate crystal form I, a preservative, a sweetening agent, a flavoring agent, a pH regulator and water. The compound formulation is a vonoprazan pyroglutamate-aspirin compound tablet and sequentially comprises, from inside to outside, an aspirin plain core, an enteric coating layer, an isolation coating layer, the vonoprazan pyroglutamate crystal form I loaded layer and a film coating layer.

Description

Tablets, injections, and compound preparations of Vonoprazan pyroglutamate Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and in particular relates to tablets, injections and compound preparations of vonoprazan pyroglutamate. Background Art

[0002] Vonoprazan, whose chemical name is 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine, has the following chemical structure:

[0003] Vonoprazan Fumarate (TAK-438) was developed by Takeda Pharmaceuticals of Japan and obtained marketing authorization in Japan on December 26, 2014. The drug belongs to a new class of inhibitors called potassium-competitive acid blockers (P-CABs). P-CABs are lipophilic, weakly alkaline, have high dissociation constants, and are stable at low pH values. Therefore, Vonoprazan Fumarate has a fast-acting, strong, and long-lasting inhibitory effect on gastric acid secretion. At the same time, in the last step of gastric acid secretion by gastric parietal cells, by inhibiting K + For H + -K + -ATPase (proton pump) binding effect, and also has an early termination effect on gastric acid secretion, but the water solubility of this compound is poor. The applicant previously described a crystalline form I of vonoprazan pyroglutamate in an international patent application (PCT / CN2022 / 131030). This crystalline form I not only has good solubility, but also has excellent storage stability; and it is resistant to H + K + The inhibitory effect of Form I on α-ATPase is comparable to that of TAK-438. Animal experiments have shown that intravenous administration of Form I can significantly inhibit histamine-induced gastric acid secretion in rats and can exert its pharmacological effect more quickly than intraduodenal administration of TAK-438. The present invention provides tablets, injections, compound preparations, and pharmaceutical compositions of Form I.

[0004] Summary of the Invention

[0005] The applicant described a crystalline form I of vonoprazan pyroglutamate in a previous international patent application (PCT / CN2022 / 131030, WO2023221419), characterized in that: using Cu / Kα radiation, the X-ray powder diffraction pattern expressed in 2θ diffraction angles is 9.000±0.200, 10.280±0.200, 11.340±0.200, 12.440±0.200, 13.480±0.200, 14.360±0.200, 15.640±0.200, 17.100±0.200, 18.000±0.200, 18.500±0.200 There are characteristic peaks at 19.240±0.200, 19.720±0.200, 20.820±0.200, 21.660±0.200, 22.500±0.200, 24.040±0.200, 24.860±0.200, 25.720±0.200, 26.400±0.200, 27.220±0.200, 28.600±0.200, 31.320±0.200, 33.200±0.200, 34.100±0.200, 34.600±0.200, 36.480±0.200, and 43.460±0.200. Form I may also have an X-ray powder diffraction pattern substantially as shown in FIG1 .

[0006] The above-mentioned crystalline form I of vonoprazan pyroglutamate is characterized in that: using Cu / Kα radiation, the crystalline form I has 27 characteristic peaks, and its X-ray powder diffraction pattern is basically as shown in Figure 2.

[0007] It will be understood by those skilled in the art that the relative intensities of the peak heights of characteristic 2θ peaks in an XRPD pattern may vary for various reasons when measured on substantially the same crystalline form.

[0008] The infrared spectrum of the above-mentioned vonoprazan pyroglutamate salt form I is at about 3255±5, 2738±5, 1690±5, 1657±5, 1573±5cm -1 The crystalline form I may also have the characteristics represented by the infrared spectrum shown in Figure 3.

[0009] Thermogravimetric analysis of the above-mentioned vonoprazan pyroglutamate salt Form I showed that it lost weight for the first time at 297.9±1.0°C, with a weight loss rate of 50.3±2.0%, which was caused by decomposition of the substance. The thermogravimetric analysis TGA graph of Form I is generally shown in Figure 4.

[0010] The preparation method of the above-mentioned vonoprazan pyroglutamate salt crystal form I is characterized by comprising the following steps: dissolving 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine pyroglutamate salt with methanol, then adding a ketone solvent dropwise, stirring to crystallize, filtering, and drying to obtain crystal form I. The methanol dissolution can be carried out at room temperature or artificially heated to 20-35°C. The ketone solvent is preferably acetone, methyl ethyl ketone, etc. The crystallization process can be carried out at room temperature or artificially cooled to 0-5°C. Parameters such as the amount of methanol and ketone solvents and the temperature can be reasonably selected by those skilled in the art according to the actual situation of recrystallization (such as experimental phenomena).

[0011] The present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition uses the above-mentioned vonoprazan pyroglutamate salt crystal form I or a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] Another embodiment of the present invention provides the above-mentioned pharmaceutical composition, characterized in that the pharmaceutical composition may further include a pharmaceutically acceptable pharmaceutical excipient. The dosage form of the pharmaceutical composition is selected from a solid preparation (preferably a tablet or capsule) or a liquid preparation (preferably an injection or oral solution).

[0013] Another embodiment of the present invention provides the above pharmaceutical composition, characterized in that the pharmaceutical composition may further comprise another or several marketed drugs (as active ingredients), preferably one or several of amoxicillin, aspirin or metronidazole.

[0014] Another embodiment of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition is a vonoprazan pyroglutamate tablet, which includes a tablet core and a film coating layer; the tablet core includes vonoprazan pyroglutamate crystal form I, a filler, a binder, a pH adjuster, a disintegrant, and a lubricant; the film coating layer includes Opadry film coating premix.

[0015] The filler is selected from one or a mixture of microcrystalline cellulose, lactose, mannitol or corn starch; preferably one or a mixture of two of microcrystalline cellulose and mannitol.

[0016] Wherein, the binder is selected from one or a mixture of hydroxypropyl cellulose, hydroxypropyl methylcellulose or povidone; preferably hydroxypropyl cellulose.

[0017] Wherein, the pH regulator is L-pyroglutamic acid.

[0018] The disintegrant is selected from one or a mixture of cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone or sodium carboxymethyl starch; preferably cross-linked sodium carboxymethyl cellulose.

[0019] Wherein, the lubricant is selected from stearic acid, magnesium stearate, calcium stearate or talc; preferably magnesium stearate.

[0020] In a preferred embodiment, the present invention provides a vonoprazan pyroglutamate tablet, characterized in that the tablet comprises a tablet core and a film coating layer, wherein the composition of the tablet core comprises, by weight percentage, 10-20% of vonoprazan pyroglutamate crystal form I, 40-60% of mannitol, 20-30% of microcrystalline cellulose, 0.1-0.3% of L-pyroglutamic acid, 1-3% of hydroxypropyl cellulose, 5-10% of cross-linked sodium carboxymethyl cellulose, and 0.5-2% of magnesium stearate; and the composition of the film coating layer comprises 3-5% of Opadry film coating premix.

[0021] In another preferred embodiment, the present invention provides a vonoprazan pyroglutamate tablet, characterized in that the tablet comprises a tablet core and a film coating layer, and the composition of the tablet core includes, by weight percentage, 12% of vonoprazan pyroglutamate crystal form I, 50% of mannitol, 25% of microcrystalline cellulose, 0.1% of L-pyroglutamic acid, 1.5% of hydroxypropyl cellulose, 7% of cross-linked sodium carboxymethyl cellulose, and 0.8% of magnesium stearate; the composition of the film coating layer includes 3.6% of Opadry film coating premix.

[0022] The present invention also provides a method for preparing the above-mentioned vonoprazan pyroglutamate tablets, which comprises the following steps:

[0023] dissolving vonoprazan pyroglutamate salt form I, a binder, and a pH adjuster in water to obtain a binding solution;

[0024] Mix Opadry film coating premix and water to obtain a film coating slurry;

[0025] The binding solution is added to the filler, granulated, dried, and sieved to obtain dry granules;

[0026] The dry granules are mixed evenly with a disintegrant and a lubricant, and tabletted to obtain vonoprazan pyroglutamate tablets;

[0027] The film coating slurry is used to perform film coating on vonoprazan pyroglutamate plain tablets to obtain the vonoprazan pyroglutamate tablets.

[0028] Preferably, the drying parameters include: setting the temperature to 50-80° C. and controlling the moisture content to be less than 5%.

[0029] Preferably, the granulation parameters include: the dry granules passing through a 20-40 mesh sieve.

[0030] Preferably, the tabletting parameters include: an average main pressure of 40±10 kN, and a controlled hardness of 40 to 60 N.

[0031] Preferably, the coating parameters include: main machine speed of 4-7 r / min, air inlet temperature of 50-70°C, exhaust temperature of 40-50°C, tablet bed temperature of 38-45°C, peristaltic pump speed of 15-35 r / min, and atomization pressure of 0.18-0.25 MPa.

[0032] Another embodiment of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition is an oral solution of vonoprazan pyroglutamate, characterized in that the oral solution comprises vonoprazan pyroglutamate crystal form I, a preservative, a sweetener, a flavoring agent, a pH adjuster and water.

[0033] The preservative can be selected from one or a mixture of methylparaben, propylparaben, potassium sorbate or sodium benzoate.

[0034] The sweetener can be selected from one or a mixture of sucrose, stevioside, sucralose, saccharin sodium or aspartame.

[0035] The flavoring agent can be selected from one or a mixture of strawberry flavor, orange flavor, apple flavor, cherry flavor or grape flavor.

[0036] The pH regulator is selected from one of acetic acid, hydrochloric acid, citric acid, L-pyroglutamic acid, anhydrous citric acid, sodium citrate, sodium dihydrogen phosphate or disodium hydrogen phosphate, or a mixture of several of them.

[0037] In a preferred embodiment, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition is a vonoprazan pyroglutamate oral solution, characterized in that the concentration of vonoprazan pyroglutamate crystal form I in the oral solution is 5 to 30 mg / ml; the concentration of the preservative is 0.5 to 2.0 mg / ml; the concentration of the sweetener is 1 to 10 mg / ml; and the concentration of the flavoring agent is 0.01 to 0.2 mg / ml.

[0038] In another preferred embodiment, the pH value of the vonoprazan pyroglutamate oral solution provided by the present invention is 3.0-5.0; more preferably, the pH value is 3.5-4.5.

[0039] The present invention also provides a method for preparing the above-mentioned vonoprazan pyroglutamate oral solution, which comprises the following steps:

[0040] (1) Dissolve one or more of sodium citrate, sodium dihydrogen phosphate, or disodium hydrogen phosphate in purified water to prepare a buffer solution of a certain concentration;

[0041] (2) Heat the buffer solution to a suitable temperature, add an appropriate amount of preservative, and stir until dissolved;

[0042] (3) Cooling the solution in step (2) to room temperature, adding vonoprazan pyroglutamate salt form I, sweetener and flavoring agent, and stirring until dissolved;

[0043] (4) Using a pH regulator, the pH of the solution in step (3) is adjusted to 3.0-5.0, preferably, the pH is adjusted to 3.5-4.5.

[0044] Preferably, the buffer solution is heated to a suitable temperature of 50-90°C, more preferably, the suitable temperature is 60-80°C, and more preferably, the suitable temperature is 80°C.

[0045] The present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition is a vonoprazan pyroglutamate-aspirin compound tablet, characterized in that the pharmaceutical composition comprises, from the inside to the outside, an aspirin tablet core, an enteric coating layer, an isolation coating layer, a vonoprazan pyroglutamate crystal form I upper drug layer and a film coating layer.

[0046] In a preferred embodiment, the present invention provides a vonoprazan pyroglutamate-aspirin compound tablet, characterized in that, in terms of mass percentage, the composition of the aspirin tablet core comprises: 40% to 50% aspirin, 2% to 7% microcrystalline cellulose, and 2% to 7% corn starch;

[0047] The composition of the enteric coating layer includes: 3% to 6% enteric coating powder, 0.28% to 1.5% triethyl citrate, and 1.23% to 4.5% sunscreen;

[0048] The isolation coating layer comprises: 0.4% to 8% of hypromellose;

[0049] The composition of the vonoprazan pyroglutamate salt crystal form I drug layer includes: vonoprazan pyroglutamate salt crystal form I 4% to 8%, binder 0.46% to 5%, filler 18% to 25%; pH regulator 0.1% to 1.0%;

[0050] The film coating layer comprises: 3% to 8% of Opadry film coating premix powder.

[0051] Preferably, the enteric coating powder comprises one or more of methacrylic acid copolymer type C, ethyl acrylate-methyl methacrylate copolymer dispersion, and methacrylic acid-ethyl acrylate copolymer aqueous dispersion.

[0052] Preferably, the sunscreen is one or more of talc, calcium carbonate and iron oxide.

[0053] Preferably, the filler is one or more of lactose and mannitol.

[0054] Preferably, the binder is selected from one or a mixture of hydroxypropyl cellulose, hypromellose or povidone; preferably hydroxypropyl cellulose.

[0055] Preferably, the pH adjuster is L-pyroglutamic acid.

[0056] Another embodiment of the present invention provides the use of the above-mentioned pharmaceutical composition in the treatment and / or prevention of diseases, wherein the diseases are selected from one or more of gastric ulcer, duodenal ulcer, reflux esophagitis, erosive esophagitis, gastroesophageal reflux disease, Helicobacter pylori infection, peptic ulcer, gastrointestinal diseases caused by long-term use of aspirin, etc.

[0057] The term "pharmaceutically acceptable salt" in the present invention refers to non-toxic inorganic or organic acid and / or base addition salts, as described in "Salt selection for basic drugs", Int. J. Pharm. (1986), 33, 201-217.

[0058] The various dosage forms of the pharmaceutical composition of the present invention can be prepared into preparations that meet the needs of clinical treatment and / or prevention of inhibiting gastric acid secretion and their appropriate various specifications and dosage forms (including solid preparations, liquid preparations and semi-solid preparations) such as sustained-release, controlled-release and enteric-coated capsules, tablets, microemulsions, submicroemulsions or various injections containing the same single and multiple components in combination according to the technical specifications and requirements of the pharmaceutical field (for example, the general requirements and specifications of the preparations in the Chinese Pharmacopoeia (2015 edition), textbooks or other methods in the prior art).

[0059] X-ray powder diffraction (XRPD) analysis of the vonoprazan pyroglutamate salt Form I described herein was performed at ambient temperature and humidity using a Rigaku MiniFlex 600 powder X-ray diffractometer (3-80°, step: 0.0200°, speed: 10° / min) with a Cu / Kα source (40 kV, 40 mA). Infrared spectroscopy analysis was performed at ambient temperature and humidity using a Thermo Nicolet 6700 infrared spectrophotometer (USA) using the KBr pellet method. Thermogravimetric analysis (TGA) was performed at ambient temperature and humidity using a Mettler TGA / DSC1 / 1100LF simultaneous thermal analyzer (Switzerland) with a scan rate of 10.00°C / min, a maximum temperature of 800.00°C, and nitrogen as the protective gas. "Ambient temperature" is generally 0-40°C; "ambient humidity" is generally 30%-80% relative humidity.

[0060] The representative X-ray powder diffraction patterns, infrared patterns and thermogravimetric analysis patterns of the vonoprazan pyroglutamate salt crystal form I described in the present invention are listed in the accompanying drawings of the specification. "Representative X-ray powder diffraction patterns or infrared patterns" means that the X-ray powder diffraction characteristics or infrared characteristics of the crystal form basically conform to the overall morphology shown in the patterns. It is understandable that during the test process, due to the influence of various factors (such as the particle size of the test sample, the processing method of the test sample, the instrument, the test parameters, the test operation, etc.), the peak position or peak intensity of the X-ray powder diffraction pattern or infrared pattern measured for the same crystal form will 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 its 2θ value is generally ±0.2 ° ; The experimental error of the absorption peak in the infrared spectrum is generally ±5cm -1 The experimental error of weight loss temperature in thermogravimetric analysis is generally ±1°C, and the experimental error of weight loss percentage is generally ±2.0%.

[0061] The "vonoprazan pyroglutamate salt" described herein refers to one molecule of vonoprazan corresponding to one molecule of L-pyroglutamic acid, i.e., the molar ratio of 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine to L-pyroglutamic acid in vonoprazan pyroglutamate salt is 1:1. It can be purchased commercially or prepared from 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine and L-pyroglutamic acid according to methods described in prior art (e.g., Chinese Patent Application No.: 201410154778.8). The "Vonoprazan Fumarate" described in the present invention is 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine fumarate (Vonoprazan Fumarate, abbreviated as TAK-438), which is purchased through commercial channels and has a purity of 99.5%.

[0062] The chromatographic conditions for HPLC purity testing of the product in the present invention are as follows:

[0063] Chromatographic column: Ultimate XB-C18, column length: 150 mm, inner diameter: 4.6 mm, particle size: 5 μm

[0064] Flow rate: 1.2 ml / min; column temperature: 30°C; wavelength: 210 nm; injection volume: 20 μl; solvent: acetonitrile-water (volume ratio 5:95)

[0065] Mobile phase A: acetonitrile-0.02 mol / L potassium dihydrogen phosphate (adjust pH to 2.5 with phosphoric acid) (volume ratio 5:95)

[0066] Mobile phase B: methanol-acetonitrile-0.02 mol / L potassium dihydrogen phosphate (adjust pH to 2.5 with phosphoric acid) (volume ratio 7:63:30)

[0067] The ratio of mobile phase A and B over time is as follows:

[0068] 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 the crystal form I of the present invention has good solubility and excellent storage stability; + K + -ATPase inhibition is comparable to that of TAK-438 and is superior to that of vonoprazan pyroglutamate (another crystal form, Figure 5) in the prior art (Chinese patent application number: 201410154778.8); (3) Animal experiments have shown that intravenous administration of the present invention's crystal form I can significantly inhibit histamine-induced gastric acid secretion in rats and can exert its pharmacological effect faster than that of TAK-438 administered intraduodenally; (4) The IC value of the present invention's crystal form I on Helicobacter pylori urease is 50 The inhibition rate of TAK-438 on urease activity was only 17% at 100 μM (its IC 50 (5) The results of the antibacterial experiment on Helicobacter pylori NCTC11637 showed that when the crystal form I of the present invention was used in combination with amoxicillin, the MIC of amoxicillin was 0.2 μg / ml, while when TAK-438 was used in combination with amoxicillin, the MIC of amoxicillin was 0.4 μg / ml, that is, when the crystal form I of the present invention was used in combination with amoxicillin, the antibacterial activity of amoxicillin was stronger than that of TAK-438 at the same concentration; (6) The crystal form I of the present invention can be prepared into stable clinical tablets, oral liquids, compound preparations, etc. The vonoprazan pyroglutamate-aspirin compound tablets of the present invention can be used to treat and / or prevent gastrointestinal diseases caused by long-term use of aspirin. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG1 is an X-ray powder diffraction pattern of vonoprazan pyroglutamate salt Form I of the present invention;

[0070] Figure 2 shows the 27 characteristic peaks of X-ray powder diffraction of Form I and their diffraction angles 2θ and interplanar spacings. Specific parameter diagrams such as relative intensity (%);

[0071] FIG3 is an infrared spectrum of Form I;

[0072] FIG4 is a thermogravimetric analysis diagram of Form I;

[0073] FIG5 is an X-ray powder diffraction pattern of product A;

[0074] Figure 6 shows the microsomal H-value of TAK-438, product A and crystal form I on porcine gastric mucosa. + K + - ATPase inhibition rate curve;

[0075] FIG7 is a comparison of the body weights of rats in each group;

[0076] FIG8 is a graph showing the effects of the test substances in each group on histamine-induced gastric acid secretion;

[0077] FIG9 is a graph showing the effects of various test substances on the inhibition rate of histamine-induced gastric acid secretion in rats;

[0078] FIG10 is a graph showing the inhibitory activity of Form I of the present invention and TAK-438 against Helicobacter pylori urease;

[0079] FIG11 is the IC of the crystal form I and TAK-438 against Helicobacter pylori urease of the present invention 50 picture;

[0080] Figure 12 is a dissolution curve of vonoprazan pyroglutamate tablets in Example 8;

[0081] Figure 13 is a dissolution curve of vonoprazan in a pH 1.2 medium in the compound tablet of Example 10;

[0082] FIG14 is a dissolution curve of aspirin from the compound tablet of Example 10 in a pH 6.8 medium. DETAILED DESCRIPTION

[0083] In order to facilitate further understanding of the present invention, the following examples are provided to illustrate it in more detail. However, these examples are only for better understanding of the invention and are not intended to limit the scope or implementation principles of the present invention. The implementation methods of the present invention are not limited to the following.

[0084] Example 1

[0085] According to 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., vonoprazan pyroglutamate, hereinafter referred to as Product A, whose X-ray powder diffraction pattern is shown in Figure 5) was obtained.

[0086] 2.00 g of a sample of 5-(2-fluorophenyl)-N-methyl-1-(3-pyridylsulfonyl)-1H-pyrrole-3-methylamine pyroglutamate was weighed and added to a 100 ml flask. Methanol (3.0 ml) was added and stirred at 20-25° C. for 10 minutes to dissolve the mixture. Acetone (about 10.0 ml) was added dropwise at 20-25° C. During the addition of acetone, solid precipitated. 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 a white solid, which is vonoprazan pyroglutamate Form I (hereinafter referred to as Form I). The X-ray powder diffraction pattern is shown in FIG1 , with the specific diffraction angle 2θ and the interplanar spacing being 0. The relative intensity (%) is shown in FIG2 , its infrared spectrum is shown in FIG3 , its thermogravimetric analysis is shown in FIG4 , and the HPLC purity is 99.8%.

[0087] Example 2 Stability Test

[0088] (1) Deliquescence test: Product A, Form I, and TAK-438 were stored at 40°C and 75% relative humidity to observe whether deliquescence occurred. The test lasted for 30 days, and the results were recorded every 10 days. See the table below for details.

[0089] No deliquescence means that no moisture absorption is observed on the sample surface, slight deliquescence means that moisture absorption is observed on the sample surface, and obvious deliquescence means that a smooth sample surface can be observed.

[0090] (2) Thermal Stability Test: Product A and Form I were stored in an open container at 60°C, and their storage stability was tested after 30 days (day 31). Purity tests were performed on the initial and 30-day samples by HPLC. Three parallel groups were set up for each sample, and the average values ​​were calculated. The results are shown in the following table.

[0091] It can be seen from the deliquescence and thermal stability tests that the crystal form I of the present invention has good stability and is convenient for long-term storage.

[0092] Example 3 Solubility test and melting point test

[0093] Solubility test basic method: Grind an appropriate amount of the sample to be tested into a fine powder, place in a suitable container, add an appropriate amount of water, and incubate at 37°C ± 2°C. Shake vigorously for 30 seconds every 5 minutes. Observe the dissolution within 30 minutes. If it does not dissolve, add a certain amount of water, incubate at 37°C ± 2°C, and shake vigorously for 30 seconds every 5 minutes. Observe the dissolution and calculate the solubility at 37°C ± 2°C. Repeat the test three times and take the average value.

[0094] Basic melting point testing method: Grind an appropriate amount of the sample to be tested into a fine powder and place it into a capillary tube sealed at one end, ensuring that the powder is tightly packed against the sealed end. The sample should be approximately 3 mm high and the melting point instrument should be heated at a rate of 1°C / min. Repeat the test three times and take the average value. The results are shown in the table below.

[0095] Example 4: Porcine gastric mucosal microsomes + K + -ATPase inhibition

[0096] Test method: H + K + -ATPase is a kind of enzyme located in the gastric mucosal parietal cells that can be + Specifically activates ATPase. In this study, microsomal H + K + -ATPase, ATPase can decompose ATP to generate ADP and inorganic phosphorus, and the test substance binds H + K + -ATPase, inhibit its activity, determine the level of ATPase activity by measuring the amount of inorganic phosphorus, and determine the inhibitory effect of the test substance. The specific method is as follows:

[0097] In this study, porcine gastric mucosal microsomes were used + K + ATPase was extracted by Beijing Huizhi Taikang.

[0098] (1) BCA protein quantification

[0099] Preparation of BCA working solution: Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA Reagent A to 1 volume of BCA Reagent B (50:1) and mix thoroughly. For example, add 5 mL of BCA Reagent A to 100 μL of BCA Reagent B and mix thoroughly to make 5.1 mL of BCA working solution. BCA working solution is stable at room temperature for 24 hours.

[0100] (2) Protein concentration detection

[0101] 1) Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard to the standard wells of a 96-well plate, and add standard diluent to make up to 20 μL. These concentrations correspond 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.

[0102] 2) Dilute the sample at 2x, 5x, and 10x dilutions, and add 20 μl of sample to each well of a 96-well plate. Be sure to record the sample volume and dilution factor.

[0103] 3) Add 200 μL of BCA working solution to each well and incubate at 37°C for 20-30 minutes.

[0104] Note: Alternatively, the sample can be placed at room temperature for 2 hours or at 60°C for 30 minutes. When the protein concentration is determined by the BCA method, the color will deepen over time.

[0105] The color development reaction is accelerated by increasing the temperature. If the concentration is low, it is appropriate to incubate at a higher temperature or extend the incubation time appropriately.

[0106] 4) Measure A562 or the absorbance at a wavelength between 540-595 nm using a microplate reader.

[0107] 5) Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0108] (3) Configuration of reaction system

[0109] 1) Composition and configuration of enzyme reaction system

[0110] 100mM TRIS-HCL (pH 6.8): Add 100µl of 1M TRIS-HCL to 900µl of ddH2O and mix well.

[0111] 37.5mM MgCl2: Weigh 50mg of MgCl 2， Add 14 ml of ddH2O and mix well.

[0112] 200mM KCl: Weigh 500mg of KCl, add 33.5ml of ddH2O, and mix.

[0113] 10mM nigericin and valinomycin: add 669ul and 890ul DMSO respectively, mix well, and aliquot into 1ul per tube of nigericin and 5ul per tube of valinomycin, and store at -20℃.

[0114] 0.1 mg / ml microsomal vesicles: Determine the protein concentration upon arrival and aliquot for use. Store at -80°C and dilute with 5 mM TRIS-HCl.

[0115] 2) Preparation of test samples

[0116] Control compound: TAK438 was prepared into a 10 mM stock solution using DMSO and aliquoted at 1 μl per tube.

[0117] Test compounds: Product A and Form I were prepared into 10 mM stock solutions using ddH2O and dispensed into tubes at 1 μl.

[0118] With 20 μM (or 40 μM) as the highest concentration, the control compound and the test compound were diluted stepwise 3-fold with DMSO and ddH2O, respectively, to obtain 6 (or 7 or 8) samples with a concentration gradient to be tested.

[0119] (4) Reaction progress

[0120] Enzymatic reaction

[0121] Incubate in a 37°C water bath for 30 minutes, add 16.5ul ATP and mix well, then incubate in a 37°C water bath for 30 minutes

[0122] (5) Color reaction: According to *H + K + Prepare the color developing reagent according to the instructions of the ATP kit (Nanjing Jiancheng).

[0123] (6) Plate reading

[0124] Add 45uL of color developer to every 150uL of enzymatic reaction volume, mix well, let it stand for 2 minutes, and read the absorbance signal at 660nm with a microplate reader.

[0125] (7) Data statistics

[0126] 1) Calculate the inhibition rate according to the following formula: Inhibition rate (%) = (1-(RLU compound -RLU blank ) / (RLU DMSO –RLU blank ))×100%.

[0127] 2) Use Graphpad Prism5 to draw the drug efficacy inhibition rate curve and calculate IC 50 The values ​​were obtained using a 4-parameter model [fit = (A + ((BA) / (1 + ((C / x)^D))))].

[0128] (8) Experimental results

[0129] 1) BCA protein quantification: Based on the BSA standard curve, the average protein concentration was calculated to be 3 mg / mL.

[0130] 2) Effects of TAK-438, Product A, and Form I on microsomal H in porcine gastric mucosa + K + The inhibitory effect of ATPase is shown in the table below and Figure 6.

[0131] Example 5: Study on the efficacy of intravenous administration on histamine-induced gastric acid secretion in rats

[0132] Surgical modeling: Rats were anesthetized with an intraperitoneal injection of 1.5 g / kg of ulanose. After complete anesthesia, the neck and abdomen of the rats were shaved and the rats were intubated to maintain airway patency during surgery. The cardiac perfusion tube was orally inserted into the rat's stomach approximately 1 cm, and the other end of the cardiac perfusion tube was connected to a constant flow pump. A small incision was made below the xiphoid cartilage to find the duodenum. A transverse incision was made, and the end of the gastric pyloric sample collection tube was passed through the incision along the duodenum into the stomach approximately 1 cm. The duodenum and gastric pyloric sample collection tube were tied tightly with sutures, and the other end of the duodenal incision was tied tightly. Use 150 mL of 37°C preheated normal saline to quickly flush out the gastric residue along the esophageal-cardia intubation, and let it flow out through the gastric pyloric sample collection tube. Suture the abdominal wall and skin, and continue to perfuse with normal saline at a rate of 1 mL / min for 30 minutes. Leave the other end of the gastric pyloric sample collection tube outside the body, and collect gastric juice samples in a conical flask (one sample is collected every 20 minutes). Collect two basal gastric juice samples, at which time the basal gastric acid secretion reaches a stable state.

[0133] Histamine-induced gastric acid secretion: Rats were continuously administered histamine via the tail vein using a microinjection pump at a dose of 20 μmol / kg / hr. Gastric acid was collected 80 minutes later. After three sampling points, histamine secretion reached a steady state. Rats were grouped according to their post-hitamine gastric acid secretion values ​​and administered the candidate drug or solvent control according to the protocol. Gastric acid collection was continued until nine sampling points were collected after drug administration, at which point the drug's efficacy reached a steady state.

[0134] The details are as follows:

[0135] (1) Test drugs

[0136] Positive drug: Vonoprazan fumarate (TAK-438). Preparation method: The entire preparation process is carried out under dark conditions. Weigh the required amount of Vonoprazan fumarate, add an appropriate amount of 0.5% CMC-Na, stir, and sonicate to obtain a uniform suspension. Dilute to the corresponding concentration according to the protocol.

[0137] Test substance: Form I (represented as HW-N2001 in the experiment). Preparation method: The entire preparation process was carried out in the dark. Weigh the required amount of HW-N2001, add an appropriate amount of physiological saline, mix thoroughly to obtain a clear solution, and adjust the pH to 3.2-3.8. Dilute to the desired concentration according to the protocol.

[0138] (2) Animal husbandry and description

[0139] 1) Animal information

[0140] Breed and strain: SD rats, grade: SPF grade, sex: female, male, source: Shanghai Jihui Experimental Animal Breeding Co., Ltd. (license number: SCXK (Shanghai) 2017-0012), certificate number: female: 20170012019182, 20170012019726; male: 20170012017891, 20170012018177, 20170012018680, 20170012018969, 20170012019726. Number of animals: 130 animals were ordered, and 118 animals (51 females and 67 males) in good health were selected for the experiment. The age of the animals at the beginning of the experiment was 6-9 weeks old. The weight of the animals at the beginning of the experiment was 160-220 g for females and 180-260 g for males. The adaptation time was 5-7 days.

[0141] 2) Rearing environment

[0142] The SPF-grade housing room complies with the national standard of the People's Republic of China, GB14925-2010: temperature: 23 ± 2°C, humidity: 40-70%, and lighting: artificial lighting with 12-hour light-dark cycles. Bedding: Corncob bedding (Dezhou Gumei Agricultural Technology Co., Ltd., batch number: GMCC202102170-2). Feed: SPF low-protein maintenance feed for mice and rats (Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., batch number: 20210225(X)). Feeding method: Ad libitum. Drinking water: Autoclaved drinking water for experimental animals, supplied in bottles, ad libitum.

[0143] 3) Animal selection and fasting

[0144] The animals used in the experiment were kept in good health and fasted for 24 hours before the experiment.

[0145] 4) Ethical approval for animal use

[0146] This experimental project has been approved by the Experimental Animal Ethics Committee of Suzhou Institute for Drug Innovation, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, with IACUC number 2021-06-WZC-10.

[0147] 5) Instructions for the selection of experimental animals

[0148] Common mammals, such as mice, rats, dogs, and monkeys, were used as experimental animals. These animals are typically used to study pharmacokinetic parameters, pharmacodynamics, and toxicology. The number of animals used was the minimum required to assess inter-individual variability. Animals must be healthy and acclimated to their environment, with free access to food and water. This experiment was conducted using Sprague-Dawley rats of both sexes.

[0149] (3) Experimental design

[0150] 1) Experimental groups

[0151] 118 healthy SD rats were selected from 130 SD rats. After the adaptation period, they were randomly divided into 8 groups according to their body weight and baseline values ​​after histamine administration. Details are shown in the table below.

[0152] 2) Animal model preparation and experimental process

[0153] Instrument and reagent preparation:

[0154] Place physiological saline in a beaker and place it in a water bath to maintain the water temperature at 37°C.

[0155] Adjust the flow rate of the constant flow pump (1 mL / min) for standby use.

[0156] Histamine dihydrochloride: Freshly prepare histamine dihydrochloride (0.6 mg / mL, dissolved in normal saline).

[0157] Ulticum saliva: Weigh an appropriate amount of ulicum saliva, add physiological saline, and sonicate to dissolve to a final concentration of 0.3 g / mL. Set aside.

[0158] Phenolphthalein: Weigh an appropriate amount of phenolphthalein and add anhydrous ethanol to a final concentration of 0.5% for later use.

[0159] Alkaline titrant: Take an appropriate amount of 0.1mol / L NaOH solution, add fresh deionized water, dilute to 0.01mol / L, and set aside.

[0160] Heparin sodium: Weigh an appropriate amount of heparin sodium, add normal saline, and dissolve to a final concentration of 100 U / mL for later use.

[0161] Animal preparation:

[0162] After the adaptation period, 118 SD rats (male and female) were fasted for 24 hours but not water and randomly divided into 8 groups (male and female).

[0163] Preparation and production of intubation:

[0164] Endotracheal intubation: Take a 2-3 cm medical polyethylene plastic tube (outer diameter 2 mm, inner diameter 1.5 mm) and keep it ready for use.

[0165] Gastric cardia perfusion tube: Take a medical polyethylene hose of about 20 cm (outer diameter 2 mm, inner diameter 1.5 mm) and use it as a gastric cardia tube.

[0166] Gastric pyloric sample collection tube: Take a medical polyethylene hose (outer diameter 4mm, inner diameter 3mm) about 30cm long and use it as the pyloric intubation end.

[0167] Histamine perfusion tube: Draw 10 mL of histamine dihydrochloride into a 10 mL syringe, connect the infusion extension tube, wrap it with tin foil to protect it from light, and fix it in the injection pump for later use.

[0168] Animal anesthesia and care: Rats were anesthetized by intraperitoneal injection of 1.5 g / kg of ulanose. The hair on the neck and abdomen was shaved.

[0169] Endotracheal intubation:

[0170] To maintain an open airway during surgery, a 2-cm vertical incision is made in the neck (at the epiglottis). The muscles are separated to expose the epiglottis and trachea. The trachea is separated and a suture is passed under the trachea. A T-shaped incision is made in the trachea. The endotracheal tube is gently inserted approximately 1 cm proximal to the incision and secured with suture. The patency of the tube is checked, the wound is sutured, and the other end of the tube is exposed through the skin incision.

[0171] Gastric cardia perfusion tube intubation:

[0172] Measure the distance from the mouth to the stomach (approximately 15 cm) and mark it on the gastric cardia perfusion tube. Insert the gastric cardia perfusion tube through the esophagus and stop at the mark, ensuring that the gastric cardia perfusion tube passes through the cardiac sphincter and reaches the stomach. Connect the gastric cardia perfusion tube to the silicone tube on the constant flow pump.

[0173] Intubation of the gastric pylorus sample collection tube:

[0174] A small incision is made along the linea alba below the xiphoid cartilage, and the abdominal cavity is opened to check the position of the gastric cardia perfusion tube. A transverse incision is made in the duodenum 1 cm below the pylorus. The end of the gastric cardia sample collection tube is passed through the incision and along the duodenum into the stomach approximately 1 cm. The duodenum and gastric cardia sample collection tube are tied tightly with a suture. The other end of the duodenal incision is tied tightly. 150 ml of 37°C preheated normal saline is used to rapidly flush gastric residues through the esophageal-cardia cannula. The residual fluid flows out through the gastric cardia sample collection tube. The abdominal wall and skin are sutured, and normal saline perfusion is continued at a rate of 1 ml / min for 30 minutes. The other end of the gastric cardia sample collection tube is left outside the body, and gastric juice samples are collected in a conical flask (one sample is collected every 20 minutes). Two basal gastric juice samples are collected, at which point basal gastric acid secretion reaches a stable state.

[0175] Sample collection and processing:

[0176] The collected gastric juice sample was titrated with 0.01N sodium hydroxide solution using a basic burette and phenolphthalein as a color indicator until the pH reached 7 as the end point. The titrated volume was measured and the acid secretion was calculated.

[0177] Acid secretion (μEq) = NaOH volume (mL) × 10 -3 ×0.01mol / L×106

[0178] Tail vein indwelling needle: Disinfect the tail vein with alcohol, insert the indwelling needle into the tail vein, pull out the needle core, push in 0.5 ml of sodium heparin solution, remove the syringe and screw on the sodium heparin cap.

[0179] Collect two basal gastric juice samples. After the basal gastric acid level stabilizes, connect the infusion extension tube that has been pre-inhaled with histamine to the rat's tail vein indwelling needle, turn on the injection pump, calculate the amount of histamine according to 20 μmol / kg / hr, and set the injection pump's intravenous drip rate for each animal. After continuous injection of histamine solution for 80 minutes, collect the gastric acid sample after histamine administration. Collect gastric juice after three histamine administrations until gastric acid secretion reaches a stable state. Administer the test substance intravenously or administer the positive drug or solvent control into the duodenum according to the grouping. Continue to collect gastric juice. Approximately 3 hours after administration, the alkali consumption reaches a stable state. A total of approximately 14 points are collected.

[0180] Body weight: The animals were weighed before fasting on the day before the experiment and before anesthesia on the day of the experiment.

[0181] Total acidity of gastric juice: The total acidity of gastric juice was determined by acid-base titration. The color indicator was 0.5% phenolphthalein and the titration endpoint was pH 7.0.

[0182] (4) Data statistics

[0183] Unless otherwise noted, experimental data are expressed as mean ± SD. Normal distribution was tested using the one-sample Kolmogorov-Smirnov test. When data within groups met normal distribution, one-way ANOVA was performed. When variances were equal, multiple comparisons between sample means were performed using the LSD test, and P < 0.05 was considered significant. When variances were unequal, multiple comparisons between sample means were performed using the Games-Howell test. When data within groups did not meet normal distribution, the nonparametric independent samples test was used, and P < 0.05 was considered significant. All statistical analyses were performed using SPSS 16.0 statistical software.

[0184] (4) Experimental results

[0185] 1) Comparison of rat body weight among groups

[0186] Throughout the experiment, no abnormalities were observed in the animals in each group, and there was no significant difference in body weight before and after fasting. (Figure 7)

[0187] 2) Effects of the test substance on histamine-induced gastric acid secretion

[0188] There was no significant difference in the baseline values ​​of SD rats in each group before histamine administration. After histamine administration, the values ​​of the Vehicle group and There were significant differences between the groups, and significant differences were observed within each dose group of the positive drug (vonoprazan fumarate) or the test substance (HW-N2001). Gastric acid secretion and gastric acid secretion inhibition rates in rats following intraduodenal administration of the positive drug or intravenous administration of the test substance were different to varying degrees compared with the vehicle control group. Details are as follows (Figures 8 and 9).

[0189] Solvent control (Vehicle) group: Gastric acid was collected at two points before histamine was administered to rats. The base values ​​of gastric acid secretion were 6.43±2.37 and 8.83±5.1 (μEq HCl / 20min), respectively. There was no significant difference between the two groups; 80 minutes after the rats were given histamine, gastric acid secretion was collected at three points, and the gastric acid secretion increased significantly and reached a steady state, which were 42.3±14.58, 45.96±16.61, and 46.65±16.96 (μEq HCl / 20min), respectively. There were significant differences between the two groups; after intravenous administration of solvent control at each time point There were significant differences among the groups.

[0190] Vonoprazan fumarate (1 mg / kg) group: After the animals were administered with histamine, gastric acid secretion reached a steady state. After administration, gastric acid secretion in rats did not decrease significantly, and there was no statistical difference compared with the Vehicle group.

[0191] Vonoprazan fumarate (2 mg / kg) group: After the animals were administered with histamine, gastric acid secretion reached a steady state. From 60 min to 180 min after administration, the gastric acid secretion of the rats gradually decreased. 60 min after administration, the gastric acid secretion of the 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 that of the solvent group. 180 min after administration, the gastric acid secretion of the 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 that of the blank control group.

[0192] Vonoprazan fumarate (4 mg / kg) group: After the animals were administered with histamine, gastric acid secretion reached a steady state. From 40 min to 180 min after administration, the gastric acid secretion of the rats gradually decreased. 40 min after administration, the gastric acid secretion of the 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. 180 min after administration, the gastric acid secretion of the 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.

[0193] HW-N2001 (0.375 mg / kg) group: After the animals were administered with histamine, gastric acid secretion reached a steady state. From 60 minutes to 180 minutes after administration, the gastric acid secretion of the rats gradually decreased. 60 minutes after administration, the gastric acid secretion of the rats was 39.15±8.54 (μEq HCl / 20 minutes), and the gastric acid secretion inhibition rate was 4.34%±1.28, which had no statistical difference compared with the solvent control group. 180 minutes after administration, the gastric acid secretion of the rats was 28.46±5.71 (μEq HCl / 20 minutes), and the gastric acid secretion inhibition rate was 27.52%±1.43, which was significantly different from the blank control group.

[0194] HW-N2001 (0.75 mg / kg) group: After the animals were administered with histamine, gastric acid secretion reached a steady state. From 20 minutes to 180 minutes after administration, the gastric acid secretion of the rats gradually decreased. 20 minutes after administration, the gastric acid secretion of the rats was 37.53±9.87 (μEq HCl / 20 minutes), which was not significantly different from that of the blank control group. The gastric acid secretion inhibition rate was 11.6%±0.82, which was statistically different from that of the solvent control group. 180 minutes after administration, the gastric acid secretion of the rats was 10±5.32 (μEq HCl / 20 minutes), and the gastric acid secretion inhibition rate was 73.68%±1.47, which was significantly different from that of the blank control group.

[0195] In the HW-N2001 (1.5 mg / kg) group, gastric acid secretion reached a steady state after administration of histamine. From 20 to 80 minutes after administration, gastric acid secretion in rats gradually decreased, and the drug efficacy reached a steady state. 20 minutes after administration, gastric acid secretion in rats was 25.67 ± 10.15 (μEq HCl / 20 minutes), and the gastric acid secretion inhibition rate was 33.28% ± 0, which was statistically different from the solvent control group. 80 minutes after administration, gastric acid secretion in rats was 3.4 ± 1.18 (μEq HCl / 20 minutes), and the gastric acid secretion inhibition rate was 89.64% ± 0, which was significantly different from the blank control group. 180 minutes after administration, gastric acid secretion in rats was 2.2 ± 0.56 (μEq HCl / 20 minutes), and the gastric acid secretion inhibition rate was 93.56% ± 0.09, which was significantly different from the blank control group.

[0196] (5) Conclusion

[0197] The present invention adopts a histamine-induced gastric acid secretion model in SD rats to study the pharmacodynamic effect of intravenous administration of vonoprazan pyroglutamate salt form I (HW-N2001) on gastric acid secretion in rats.

[0198] After intraduodenal administration of vonoprazan fumarate, the effect was not significant after 1 mg / kg administration. 2 and 4 mg / kg gradually exerted their pharmacological effects 60 minutes and 140 minutes after administration, respectively, inhibiting gastric acid secretion in rats, and there was a certain dose-effect relationship. The drug took effect slowly, reaching its maximum effect at 180 minutes, and ID 50 It is 1.87mg / kg.

[0199] HW-N2001 (0.375 mg / kg, 0.75 mg / kg, 1.5 mg / kg) was intravenously administered. The gastric acid secretion of the drug-treated groups was significantly reduced compared with the solvent control group, and there was a certain dose-effect relationship. The 0.75 and 1.5 mg / kg dose groups began to gradually exert their pharmacological effects 20 minutes after administration, and the pharmacological effects reached a stable state 60-100 minutes after administration. 180 minutes after administration, the ID 50 It is 0.53mg / kg.

[0200] In summary, intravenous administration of the vonoprazan pyroglutamate salt 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 intraduodenal administration of vonoprazan fumarate.

[0201] Example 6 Evaluation of the anti-urease activity of Form I of the present invention in vitro

[0202] The urease activity detection kit was purchased from Solebo, catalog number: BC4115, specification: 100T / 48S

[0203] Urease was purchased from Maclean, catalog number: U835597

[0204] Urease was diluted to 1.5 U / L and mixed with substrate and different concentrations of Form I of the present invention in a 96-well plate. The wells without test substance were used as solvent control groups. The reaction was incubated at 37°C for 1 hour. After the reaction was completed and thoroughly mixed, the OD value at 630 nm was measured, and the inhibition rate and IC of each concentration were calculated. 50 The results showed that the crystal form I of the present invention could significantly inhibit the urease activity of Helicobacter pylori, while the inhibitory effect of TAK-438 was weaker (Figure 10). 50 The inhibition rate of TAK-438 on urease activity was only 17% at 100 μM, so its IC 50 Much larger than 200 μM, we use 200 to represent its IC in Figure 11. 50 Example 7 Evaluation of the inhibitory activity of Helicobacter pylori proliferation by the combination of the crystal form I of the present invention and amoxicillin

[0205] The minimum inhibitory concentration (MIC) of TAK-438 + amoxicillin and the crystal form I + amoxicillin of the present invention was evaluated using the Helicobacter pylori NCTC11637 strain. According to the literature, we set the vonoprazan in crystal form I and TAK-438 to 128 μg / ml, and tested the MIC of amoxicillin. Crystal form I and TAK-438 were added to the Helicobacter pylori special culture medium to a final concentration of 128 μg / ml, and then different concentrations of amoxicillin were added. A small amount of Helicobacter pylori NCTC11637 was inoculated and cultured in a low-oxygen environment. The minimum inhibitory concentration (MIC) is the minimum inhibitory concentration (MIC). The results showed that the MIC of amoxicillin in the TAK-438 + amoxicillin combination group was 0.4 μg / ml, while the MIC of amoxicillin in the crystal form I + amoxicillin combination group of the present invention was 0.2 μg / ml.

[0206] Example 8 Preparation of Vonoprazan Pyroglutamate Tablets. The general preparation method of tablets is as follows:

[0207] dissolving vonoprazan pyroglutamate salt form I, a binder, and a pH adjuster in water to obtain a binding solution;

[0208] The binding solution is added to the filler, granulated, dried, and sieved to obtain dry granules;

[0209] The dry granules are mixed evenly with a disintegrant and a lubricant, and tabletted to obtain vonoprazan pyroglutamate tablets;

[0210] The Opadry film coating premix and water are mixed to obtain a film coating slurry, and the film coating slurry is used to film-coat the vonoprazan pyroglutamate tablets to obtain the vonoprazan pyroglutamate tablets.

[0211] Preferably, the drying parameters include: the temperature is set to 50-80°C, and the moisture content is controlled to be less than 5%. Preferably, the granulation parameters include: the dry granules pass through a 20-40 mesh sieve. Preferably, the tableting parameters include: the average main pressure is 40±10kN, and the hardness is controlled to be 40-60N. Preferably, the coating parameters include: the main machine speed is 4-7r / min, the inlet air temperature is 50-70°C, the exhaust air temperature is 40-50°C, the tablet bed temperature is 38-45°C, the peristaltic pump speed is 15-35r / min, and the atomization pressure is 0.18-0.25MPa.

[0212] Example 1

[0213] A vonoprazan pyroglutamate tablet, the prescription of which is as follows:

[0214] The preparation method of the above tablets is as follows:

[0215] (1) Solution preparation: Dissolve Form I, L-pyroglutamic acid, and hydroxypropyl cellulose in water (44 g) to obtain a binding solution for later use;

[0216] (2) Granulation: Mannitol and microcrystalline cellulose in the prescribed amount were weighed and placed in a wet mixing granulator (manufacturer: Shenzhen Xinyi Technology Co., Ltd., model: G6) and mixed for 15 minutes. The binding solution was evenly sprayed into the wet mixing granulator to prepare a soft material;

[0217] (3) Drying and granulation: The soft material was placed in an experimental fluidized bed (manufacturer: Shenzhen Xinyi Technology Co., Ltd., model: XYT-I), with the inlet air temperature set at 50°C and the fan frequency at 30 Hz. The material was dried for about 13 minutes and then discharged. A 20-mesh screen was used for dry granulation to obtain dry granules.

[0218] (4) Tableting: Dry granules, cross-linked sodium carboxymethyl cellulose, and magnesium stearate were placed in an experimental hopper mixer (manufacturer: Yichun Wanshen Pharmaceutical Machinery Co., Ltd., model: HLS-5) and mixed evenly. Tablets were compressed using a rotary tablet press (manufacturer: Beijing Xinlongli Technology Co., Ltd., model: ZP) to produce Devonoprazan pyroglutamate tablets.

[0219] (5) Coating: The vonoprazan pyroglutamate tablets were coated with a coating solution containing 10 wt% of Opadry film coating agent.

[0220] Tablets of Example 2 and Comparative Example can be prepared according to the general preparation method (or a similar method of Example 1)

[0221] Example 2

[0222] A vonoprazan pyroglutamate tablet, the prescription of which is as follows:

[0223] Comparative Example

[0224] A kind of vonoprazan fumarate tablets, its prescription is as follows:

[0225] Test experiment:

[0226] The contents and content uniformity of Examples 1 and 2 are shown in the following table.

[0227] The dissolution rate of the vonoprazan pyroglutamate tablets prepared in Examples 1 and 2, the comparative example TAK-438 tablets (homemade), and TAK-438 tablets (reference preparation, Takeda Pharmaceutical Company Limited, purchased) were tested.

[0228] It was determined according to the dissolution and release test method (Method 2, Part 4, General Chapter 0931, Chinese Pharmacopoeia 2020 Edition).

[0229] Dissolution conditions: 900 ml of acetate buffer (2.99 g of sodium acetate, adjusted to pH 4.5 by adding 14.0 ml of 2 mol / L acetic acid solution) was used as the dissolution medium. The rotation speed was set at 50 rpm. Samples were collected after 30 minutes. Dissolution experiments were conducted according to the procedure. The dissolution results are shown in the following table and Figure 12.

[0230] Comparison of dissolution test results of Examples 1-2 and the comparative example with the reference preparation

[0231] From the above experiments, it can be seen that the content and content uniformity of the crystal form I of the present invention meet the standard requirements, the content and content uniformity are good, its solubility meets the requirements, and the solubility is better than that of the TAK-438 homemade product and the TAK-438 tablet reference preparation.

[0232] Example 9: Vonoprazan pyroglutamate oral solution

[0233] The general preparation method of oral solution comprises the following steps:

[0234] (1) Dissolve one or more of sodium citrate, sodium dihydrogen phosphate, or disodium hydrogen phosphate in purified water to prepare a buffer solution of a certain concentration;

[0235] (2) Heat the buffer solution to a suitable temperature, add an appropriate amount of preservative, and stir until dissolved;

[0236] (3) Cooling the solution in step (2) to room temperature, adding vonoprazan pyroglutamate salt form I, sweetener and flavoring agent, and stirring until dissolved;

[0237] (4) Using a pH regulator, the pH of the solution in step (3) is adjusted to 3.0-5.0, preferably, the pH is adjusted to 3.5-4.5.

[0238] Preferably, the buffer solution is heated to a suitable temperature of 50-90°C, more preferably, the suitable temperature is 60-80°C, and more preferably, the suitable temperature is 80°C.

[0239] Example 1

[0240] A vonoprazan pyroglutamate oral solution, the prescription of which is as follows:

[0241] The preparation method of the above oral solution is as follows:

[0242] (1) Dissolve the prescribed amount of sodium dihydrogen phosphate and disodium hydrogen phosphate in purified water to prepare a buffer solution;

[0243] (2) Heat the buffer solution to 80°C, add the prescribed amount of methylparaben, and stir until dissolved;

[0244] (3) Cooling the solution in step (2) to room temperature, adding Form I, sucralose, and strawberry flavor, and stirring until dissolved;

[0245] (4) Using L-pyroglutamic acid, the pH of the solution in step (3) is adjusted to 4.0 to obtain the product.

[0246] According to the general method, the oral solution of Example 2 can be prepared

[0247] Example 2

[0248] A vonoprazan pyroglutamate oral solution, the prescription of which is as follows:

[0249] Test experiment:

[0250] The contents and related substances detected in Examples 1 and 2 are shown in the following table.

[0251] To investigate the stability of the samples under accelerated conditions, Example 1 was placed in a drug stability test chamber with the test conditions set at a temperature of 40°C and a relative humidity of 75% ± 5%. Samples were taken at 1 month, 3 months, and 6 months to test properties, pH value, content, and related substances.

[0252] Example 1 Accelerated test results

[0253] From the above experiments, it can be seen that the content and related substances of the oral solution of Form I of the present invention meet the standard requirements, the stability is good under accelerated test conditions, and all quality indicators do not change significantly, which meets the standard requirements.

[0254] Example 10 Vonoprazan pyroglutamate-aspirin compound tablets

[0255] Example 1

[0256] A vonoprazan pyroglutamate-aspirin compound tablet, the prescription of which is as follows:

[0257] The preparation method of the compound tablet is as follows:

[0258] Aspirin, corn starch and microcrystalline cellulose were mixed evenly and compressed using a Φ7mm punch with a pressure of 12kN to produce aspirin tablets.

[0259] Enteric coating: triethyl citrate is dissolved in purified water, talc is then added, and the mixture is sheared at 1000 rpm for 5 minutes. A methacrylic acid-ethyl acrylate copolymer aqueous dispersion is added under slow stirring (200 rpm), and the mixture is mixed uniformly to obtain an enteric layer coating solution; a coating pan is used to coat aspirin tablets with the enteric layer coating solution at a spray rate of 50 g / min, a coating pan speed of 3 rpm, and a material temperature of 26°C to obtain a weight gain of 17.5% to obtain enteric-coated aspirin tablets.

[0260] Isolation layer coating: spray rate of 50-110 g / min, coating pan speed of 3-7 r / min, material temperature of 26-36°C, tablet weight gain of 2%.

[0261] Drug application: Mannitol and hydroxypropyl cellulose are dissolved in water and added to the micronized Form I. The mixture is sheared at 1000 rpm for 5 minutes. The pH is adjusted with L-pyroglutamic acid to obtain a drug application solution. The coating process is performed at a spray rate of 50-240 g / min, a coating pan speed of 3-6 rpm, and a material temperature of 25-43°C. Enteric-coated aspirin tablets are coated with the drug application solution to a weight gain of 45.4%, thereby obtaining vonoprazan pyroglutamate-aspirin tablets.

[0262] Film coating: with a spray rate of 50-110 g / min, a coating pan speed of 3-7 r / min, and a material temperature of 26-36° C., the film coating slurry is used to coat the vonoprazan pyroglutamate-aspirin tablets containing the upper drug layer with a weight gain of 3.4% to obtain the compound tablets.

[0263] According to a similar method in Example 1, the compound tablets of Examples 2-4 can be prepared. The dissolution curves of the compound tablets are shown in Figures 13 and 14 (determined according to the dissolution and release determination method (Method 0931, Part 4, General Rules, 2020 Edition of the Chinese Pharmacopoeia)).

[0264] Example 2

[0265] A vonoprazan pyroglutamate-aspirin compound tablet, the prescription of which is as follows

[0266] Example 3

[0267] A vonoprazan pyroglutamate-aspirin compound tablet, the prescription of which is as follows

[0268] Example 4

[0269] A vonoprazan pyroglutamate-aspirin compound tablet, the prescription of which is as follows

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition uses vonoprazan pyroglutamate crystal form I or a pharmaceutically acceptable salt thereof as an effective ingredient; the pharmaceutical composition also includes pharmaceutically acceptable pharmaceutical excipients; the dosage form of the pharmaceutical composition is selected from solid preparations (preferably tablets, capsules) or liquid preparations (preferably injections, oral solutions); the crystal form I uses Cu / Kα radiation, and the X-ray powder diffraction pattern represented by the 2θ diffraction angle is 9.000±0.200, 10.280±0.200, 11.340±0.200, 12.440±0.200, 13.480±0.200, 14.360±0.200, 15.640±0.200, 17.100±0.200, 18.000±0.

200. There are characteristic peaks at .200, 18.500±0.200, 19.240±0.200, 19.720±0.200, 20.820±0.200, 21.660±0.200, 22.500±0.200, 24.040±0.200, 24.860±0.200, 25.720±0.200, 26.400±0.200, 27.220±0.200, 28.600±0.200, 31.320±0.200, 33.200±0.200, 34.100±0.200, 34.600±0.200, 36.480±0.200 and 43.460±0.

200.

2. The pharmaceutical composition according to claim 1, characterized in that The active ingredients of the pharmaceutical composition may also include one or more of amoxicillin, aspirin or metronidazole.

3. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition is a vonoprazan pyroglutamate tablet, which comprises a tablet core and a film coating layer; the tablet core comprises vonoprazan pyroglutamate crystal form I, a filler, a binder, a pH regulator, a disintegrant, and a lubricant; and the film coating layer comprises an Opadry film coating premix.

4. The pharmaceutical composition according to claim 3, characterized in that The filler is selected from one or a mixture of microcrystalline cellulose, lactose, mannitol or corn starch; preferably one or a mixture of microcrystalline cellulose and mannitol; the binder is selected from one or a mixture of hydroxypropyl cellulose, hydroxypropyl methylcellulose or povidone; preferably hydroxypropyl cellulose; the pH adjuster is L-pyroglutamic acid; the disintegrant is selected from one or a mixture of cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, cross-linked povidone or sodium carboxymethyl starch; preferably cross-linked sodium carboxymethyl cellulose; the lubricant is selected from stearic acid, magnesium stearate, calcium stearate or talc; preferably magnesium stearate.

5. The pharmaceutical composition according to claim 3, characterized in that The vonoprazan pyroglutamate tablet comprises a tablet core and a film coating layer. The composition of the tablet core comprises, by weight percentage, 10-20% of vonoprazan pyroglutamate crystal form I, 40-60% of mannitol, 20-30% of microcrystalline cellulose, 0.1-0.3% of L-pyroglutamic acid, 1-3% of hydroxypropyl cellulose, 5-10% of cross-linked sodium carboxymethyl cellulose, and 0.5-2% of magnesium stearate; the composition of the film coating layer comprises 3-5% of Opadry film coating premix.

6. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition is a vonoprazan pyroglutamate oral solution, which comprises vonoprazan pyroglutamate crystal form I, a preservative, a sweetener, a flavoring agent, a pH regulator and water.

7. The pharmaceutical composition according to claim 6, characterized in that The preservative can be selected from one or a mixture of methylparaben, propylparaben, potassium sorbate or sodium benzoate; the sweetener can be selected from one or a mixture of sucrose, stevioside, sucralose, sodium saccharin or aspartame; the flavoring agent can be selected from one or a mixture of strawberry flavor, orange flavor, apple flavor, cherry flavor or grape flavor; the pH adjuster can be selected from one or a mixture of acetic acid, hydrochloric acid, citric acid, L-pyroglutamic acid, anhydrous citric acid, sodium citrate, sodium dihydrogen phosphate or disodium hydrogen phosphate.

8. The pharmaceutical composition according to any one of claims 6 to 7, characterized in that The concentration of vonoprazan pyroglutamate salt form I in the oral solution is 5 to 30 mg / ml; the concentration of the preservative is 0.5 to 2.0 mg / ml; the concentration of the sweetener is 1 to 10 mg / ml; the concentration of the flavoring agent is 0.01 to 0.2 mg / ml; the pH value is 3.0 to 5.0; more preferably, the pH value is 3.5 to 4.

5.

9. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition is a vonoprazan pyroglutamate-aspirin compound tablet, which comprises, from the inside to the outside, an aspirin tablet core, an enteric coating layer, an isolation coating layer, a vonoprazan pyroglutamate crystal form I drug layer and a film coating layer.

10. The pharmaceutical composition according to claim 9, characterized in that The compound tablet comprises, by weight percentage, the composition of the aspirin tablet core comprising: 40% to 50% aspirin, 2% to 7% microcrystalline cellulose, and 2% to 7% corn starch; the composition of the enteric coating layer comprising: 3% to 6% enteric coating powder, 0.28% to 1.5% triethyl citrate, and 1.23% to 4.5% sunscreen; the composition of the isolation coating layer comprising: 0.4% to 8% hydroxypropyl methylcellulose; the composition of the vonoprazan pyroglutamate crystal form I drug layer comprising: 4% to 8% vonoprazan pyroglutamate crystal form I, 0.46% to 5% binder, 18% to 25% filler, and 0.1% to 1.0% pH adjuster; and the composition of the film coating layer comprising: 3% to 8% Opadry film coating premix powder.

11. The pharmaceutical composition according to claim 10, characterized in that The enteric coating powder comprises one or more of methacrylic acid copolymer type C, ethyl acrylate-methyl methacrylate copolymer dispersion, and methacrylic acid-ethyl acrylate copolymer aqueous dispersion; the sunscreen is one or more of talc, calcium carbonate, and iron oxide; the filler is one or more of lactose and mannitol; the binder is selected from one or a mixture of hydroxypropyl cellulose, hydroxypropyl methylcellulose, or povidone; preferably hydroxypropyl cellulose; and the pH regulator is L-pyroglutamic acid.

12. Use of the pharmaceutical composition according to any one of claims 1 to 11 in treating and / or preventing diseases, wherein the diseases are selected from one or more of gastric ulcer, duodenal ulcer, reflux esophagitis, erosive esophagitis, gastroesophageal reflux disease, Helicobacter pylori infection, peptic ulcer, gastrointestinal diseases caused by long-term use of aspirin, etc.

Images