Chemicals.

Tegoprazan, vonoprazan, omeprazole, and SCH28080 were synthesized by RaQualia Pharma Inc. (Kaminski et al., 1985; Gustavsson and Källstrӧm, 1997; Kajino et al., 2006; Hanazawa and Koike, 2007). All of the reagents and solvents used were analytical grade or higher.

Ethics Approvals and Animals.

All procedures were carried out with the approval of the Animal Ethics Committee at the institutes in which the study was conducted, RaQualia Pharma Inc. (Aichi, Japan) or Pfizer Nagoya Laboratories (Aichi, Japan), according to the Laboratory Animal Welfare guidelines. Porcine stomachs were purchased from a local abattoir. Beagle dogs were purchased from Oriental BioService Inc. (Kyoto, Japan), Marshall BioResources Japan (Tsukuba, Japan), and Beijing Marshall Biotechnology Co., Ltd (Beijing, China).

Preparation of Porcine and Canine Gastric H⁺/K⁺-ATPase Vehicles.

Porcine and canine H⁺/K⁺-ATPase vehicles were prepared as previously reported with some modifications (Mori et al., 2009). Briefly, a fresh porcine stomach was washed with saline on ice. The fundic mucosa was scraped off from the underlying muscular layer, minced and homogenized in a 250 mM sucrose solution. The homogenate was filtered with gauze, and then centrifuged at 20,000g for 30 minutes at 4°C. The supernatant was centrifuged at 115,000g for 30 minutes at 4°C. The pellet was suspended in a 250 mM sucrose solution and separated by a differential and zonal density gradient centrifugation, 250 mM sucrose (upper layer) and 7% (w/v) Ficoll in 250 mM sucrose (lower layer), at 130,000g for 60 minutes at 4°C with a vertical rotor. The fraction above the Ficoll interface was collected and diluted 10 times with water (ion-tight vehicle), permeabilized by freeze-dry processing (ion-leaky vesicle), and stored at −80°C. Before use, the freeze-dried vesicles were reconstituted with water up to the original volume. The protein concentration of reconstituted vesicles was determined using the BCA Protein Assay Kit protocol (Pierce Biotechnology, Rockford, IL).

Fresh canine stomachs were washed with saline on ice. The fundic mucosa was scraped, minced, and homogenized with a homogenizer in a solution containing 1 mM EGTA, 250 mM sucrose, and 5 mM Tris (pH 7.4 at 4°C). The homogenate was filtered with gauze, centrifuged at 20,000g for 30 minutes at 4°C, and then the supernatant was centrifuged at 115,000g for 30 minutes at 4°C. The pellet was suspended in a 250 mM sucrose solution and separated by density gradient centrifugation (the same construction as used for porcine preparation) at 132,000g for 90 minutes at 4°C with a swing rotor. The following procedures were the same as used for porcine stomach.

Preparation of Human Recombinant H⁺/K⁺-ATPase Vehicles.

Human recombinant H⁺/K⁺-ATPase was prepared as described previously with some modifications (Mori et al., 2009). Briefly, HEK293 cells stably expressing the human gastric H⁺/K⁺-ATPase were prepared by Pfizer Inc. (New York, NY). The cells were suspended in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum, 0.5 mg/ml G418, 0.1 mg/ml zeocin, 100 U/ml penicillin, and 100 µg/ml streptomycin, and were seeded in a T-225 cell culture flask. After 11-day culture in a humidified incubator at 37°C with 5% CO₂, the cells were harvested in 1 mM EDTA/phosphate-buffered saline without Mg²⁺/Ca²⁺. The cells were centrifuged at 1000 rpm for 5 minutes at 4°C. The packed cells were suspended in a buffer containing 0.5 mM MgSO₄, Roche Complete protease inhibitors (Mannheim, Germany), and 50 mM Tris-HCl (pH 7.4), and then homogenized with a Polytron homogenizer (Kinematica AG, Luzern, Switzerland) for 40 seconds. The homogenate was centrifuged at 1000 rpm for 5 minutes at 4°C. The supernatant was recentrifuged at 40,000g for 30 minutes at 4°C. The pellet was suspended in a 250 mM sucrose solution with the Polytron homogenizer and was aliquoted and stored at −80°C until use. The protein concentration of the membrane fraction was determined using the BCA Protein Assay Kit protocol (Pierce).

Measurement of H⁺/K⁺-ATPase Activities.

The H⁺/K⁺-ATPase activates were determined as described previously with some modifications (Keeling et al., 1988; Mori et al., 2009). The porcine and canine gastric H⁺/K⁺-ATPase activates were measured in a 60 µl reaction mixture containing 0.3 µg for porcine or 0.7 µg for canine freeze-dried vesicles (ion-leaky vesicles), test compounds, 5 mM KCl, 3 mM MgSO₄, 3 mM Na₂ATP, and 40 mM Bis-tris (pH 6.4) at 37°C in a 96-well polystyrene plate. For 0% and 100% inhibition controls, the enzyme reaction was performed in the presence of 1% dimethylsulfoxide and 100 µM SCH28080, respectively. The enzyme reactions using porcine ion-tight vesicles were assayed in a 60 µl reaction mixture containing 2 µg of vehicles, test compounds, 150 mM KCl, 3 mM MgSO₄, 3 mM Na₂ATP, 17 µM valinomycin (Sigma, St. Louis, MI), and 5 mM Tris (pH 7.4) at 37°C. The reaction was started by the addition of Na₂ATP and the mixture was incubated at 37°C for 30 minutes, and then 30 µl of 10% SDS containing antifoam A was added to stop the reaction. Then, 200 µl of the colorimetric reagent to analyze inorganic phosphate consisting of four parts of 10% L-ascorbic acid (pH 5) and one part of 35 mM ammonium molybdate in 15 mM zinc acetate (pH 5) was added into each well. Following incubation at 37°C, the optical density was measured at 750 nm with ARVOsx (PerkinElmer, Waltham, MA). The inorganic phosphate solution prepared from KH₂PO₄ and K₂HPO₄ solutions was used as a standard for the inorganic phosphate. Test compounds were dissolved in dimethylsulfoxide at 10 mM and then diluted in the reaction mixture to give appropriate final concentrations. The human recombinant H⁺/K⁺-ATPase activity was measured in a 60 µl enzyme reaction mixture containing 10 µg of H⁺/K⁺-ATPase vehicle, the test compound, 10 µM oligomycin, 100 nM bafilomycin A1, 10 µM thapsigargin, 10 µM ouabain, 5 mM KCl, 3 mM MgSO₄, 3 mM Na₂ATP, and 40 mM Bis-Tris (pH 6.4 at 37°C) in a 96-well plate. The reaction mixture was incubated for 50 minutes at 37°C after addition of Na₂ATP, and 30 µl of 10% SDS containing antifoam A was dispensed to stop the reaction. The colorimetric analysis was performed in the same procedure for the porcine assay.

Kinetic Analysis of H⁺/K⁺-ATPase Inhibition.

The enzyme kinetic study was performed based on the method of porcine ion-leaky assay described previously, except for the amount of vehicle (1 µg), and was tested with various concentrations of potassium (final concentrations of 2.0, 2.5, 3.5, 5.0, and 10 mM KCl) and tegoprazan (final concentrations of 0.15, 0.30, 0.45, and 0.60 µM).

Measurement of Canine Kidney Na⁺/K⁺-ATPase Activity.

Canine kidney Na⁺/K⁺-ATPase was purchased from Sigma (A-0142). The enzyme was diluted to 7 mg/ml with 250 mM sucrose solution. The canine kidney Na⁺/K⁺-ATPase activity was measured in a 60 µl enzyme reaction mixture containing 11 µg of protein, test compound, 100 mM NaCl, 2 mM KCl, 3 mM MgSO₄, 3 mM Na₂ATP, and 40 mM Tris (pH 7.4 at 37°C) in a 96-well polystyrene plate. For 0% and 100% inhibition controls, the enzyme reaction was performed in the presence of 1% dimethylsulfoxide and 100 µM ouabain, respectively. The reaction was started by the addition of Na₂ATP, the mixture was incubated for 30 minutes at 37°C, and then 30 µl of 10% SDS containing antifoam A was added to stop the reaction. The colorimetric analysis was performed in the same method for the porcine assay.

Data Analysis.

All of the H⁺/K⁺- ATPase and Na⁺/K⁺-ATPase assays were conducted in triplicate. The results of three independent experiments were used for analysis. The nonlinear curve fitting and IC₅₀ determination and the kinetic study using the Lineweaver-Burk plot were analyzed using GraphPad Prism version 5 (GraphPad Software Inc., San Diego, CA). The values are shown as mean ± S.E.M.

Measurement of Plasma and Gastric Juice Concentration of Tegoprazan in Dogs.

Male beagle dogs at 8–11 months old were fasted overnight prior to dosing and dosed tegoprazan with 0.3, 3, and 30 mg/kg by mouth (n = 2) and blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, and 24 hours post dosing. Male Heidenhain pouch (HP) dogs were orally dosed tegoprazan with 1 or 3 mg/kg, and blood and gastric juice were collected at 5 or 16 hours after the drug dose. Plasma and gastric concentrations of tegoprazan were quantified using liquid chromatography–tandem mass spectrometry (API4000 Triple Quadrupole mass spectrometer; Applied Biosystems, Waltham, MA) and calculated using Analyst Program version 1.4.1 (Applied Biosystems). Pharmacokinetic parameters were determined with noncompartmental analysis using WinNonlin version 5.2.1 (Pharsight Corporation, Sunnyvale, CA).

Inhibition of Binding/Function against Pharmacologically Relevant Molecules.

Inhibitions of binding or functional activity against pharmacologically relevant molecules, receptors, ion channels, transporters, and enzymes, were tested in the presence of 10 µM tegoprazan. Regarding the Na⁺/K⁺-ATPase, tegoprazan was tested at 30 µM. All of the assays were conducted by Eurofins Cerep (Celle-Lvescault France) under their validated assay methods in the presence of standard inhibitors as an assay control (http://www.cerep.fr/cerep/users/pages/catalog/assay/catalog.asp).

Measurement of Gastric Acid Secretion in the HP Dog Model.

A gastric pouch was constructed according to the method introduced by Heidenhain (1879). Briefly, male beagle dogs (7–12 kg body weight) were anesthetized with midazolam (0.2 mg/kg, i.m.) and medetomidine (0.05 mg/kg, i.m.) and isoflurane inhalation. The abdominal cavity was opened after the injection of atropine sulfate (0.5 mg/ml, 1 ml, i.m.) and infusion of Lactec D (100 ml/h). After exposing the stomach in the surgical field, a portion of the greater curvature opposite the splenic hilum was converted into a pouch with an adequate blood supply from the intact gastroepiploic artery. The main body of the stomach was reconstituted, while the pouch drained into an implanted metal cannula. After closing the pouch, the cannula was brought out of the abdominal cavity through the left lateral abdominal wall. Animals were allowed to recover from surgery for at least at last 3 weeks. The experiment was started after overnight fasting with free access to water. Acid secretion was stimulated by continuous intravenous infusion of histamine (80 μg/mg per hour; Sigma) and maintained throughout the experimental periods. Gastric juice samples were collected by gravity drainage every 15 minutes throughout the experiment. At 90 minutes after initiation of the histamine infusion, tegoprazan, omeprazole, or vehicle (0.5% methylcellulose; Wako, Osaka, Japan) was administrated orally. In the 5-day repeated dosing study, test drug or vehicle was administered orally for 5 days and its inhibitory effect on histamine-stimulated acid secretion was examined on treatment days 1 and 5. The collected gastric juice samples were centrifuged and acidity in the supernatant was determined using an automatic titrator (AUT-501; TOA/DKK, Tokyo, Japan). Gastric acid secretion at intervals of every 15 minutes (acidity × volume of the supernatant) was expressed as milliequivalents per 15 minutes. All results were calculated by MS EXCEL software (Microsoft, Redmond, WA).

Measurement of Gastric pH in Dogs.

Gastrostomy was conducted as follows. The dogs are anesthetized with midazolam (0.2 mg/kg, i.m.) and medetomidine (0.05 mg/kg, i.m.) and isoflurane inhalation. The abdominal cavity was opened after the injection of atropine sulfate (0.5 mg/ml, 1 ml, i.m.) and infusion of Lactec D (100 ml/h). A gastrostomy tube (D12-C, 6 cm; Natsume Seisakusho Co. Ltd., Tokyo, Japan) was connected to the center of the body of the stomach and closed. The gastrostomy tube was taken out from the abdominal cavity through the left lateral abdominal wall and closed. The experiments were started at least 1 month after the surgery.

After overnight fasting, the dogs were placed in suspended-type dog slings and attached to a dip-type reference microelectrode (MI-402; Microelectrode Inc.) in the back. An esophageal pH microelectrode (MI-508; Microelectrode Inc. Bedford, NH) was introduced into the gastrostomy tube and fixed at 10 mm above the gastric lumen. Both microelectrodes were connected to a pH meter (F52; Horiba, Kyoto, Japan) and measured the gastric pH values. During the pH measurement, pentagastrin (6 μg/kg per hour, s.c. infusion; Sigma) was injected continuously. Test drug or vehicle was administered orally or intravenously 1.5 hours after starting the pentagastrin injection and the pH was measured more than 5 hours after drug administration. In the 5-day consecutive administration experiment, drugs were administered before feeding on days 1–4 and the aforementioned procedures were done on day 5. The gastric pH values were gathered at 5- or 6-second intervals and the median of each 15 minutes was used for the analysis. Fig. 6 shows mean of the median from four dogs.

Measurement of Gastric Antral Motility in Conscious Dogs.

The gastric antral motility was determined as described previously (Mikami et al., 2008). Briefly, healthy male beagles weighing 9–15 kg were anesthetized with isoflurane, and the abdominal cavity was opened under aseptic conditions. Extraluminal force transducers (IS-12S; Star Medical, Tokyo, Japan) were sutured onto the seromuscular layer of the gastric antrum 3 cm proximal to the pyloric ring, according to the method from Itoh et al. (1977). The transducer’s lead wires were taken out of the abdominal cavity through a skin incision between the scapulae. After surgery, the dogs were placed in protective jackets and housed in individual cages. Gastric motility was recorded starting at least 2 weeks after surgery. After an overnight fast, the dogs were placed in a shielded room, and gastric motility was recorded in the fasted state. Gastric motility was measured with a telemetry system (GTS-800; Star Medical), and data were acquired on a personal computer with acquisition software (Eight Star; Star Medical). After confirming the incidence of an interdigestive migrating complex (a typical motility pattern of the upper gastrointestinal tract in the fasted state) at regular intervals, test drugs or vehicle (0.1% methylcellulose) were administered orally, and gastric motility was recorded for 8 hours. To measure the gastric motility quantitatively, motor indices that represent areas of contractions were calculated. The signals from the force transducer were acquired on a personal computer and analyzed by processing software (Analyze II; Star Medical).