The introduction of potassium-competitive acid blockers (P-CABs) has been a major innovation in gastric H,K-ATPase inhibition and many laboratories are actively engaged in the development of novel molecules within this class. This work investigates the interaction between H,K-ATPase and tegoprazan, a representative of the P-CABs group, in terms of K+ and H+ binding, through functional and structural analyses. First, by studying the H,K-ATPase activity, we found a model to describe the non-Michaelis-Menten kinetics through a "ping-pong" mechanism that explains a stoichiometry of 1 H+, 1 K+, and 1 ATP molecule, but also considering the influence of H+ on the ionization states of the protein. A kinetic evaluation of the inhibition of tegoprazan denotes the binding to two different intermediates states with apparent Kd (μM) 0.56 ± 0.04 and 2.70 ± 0.24 at pH 7.2. Molecular dynamics simulations revealed important changes in the interactions of tegoprazan with the transmembrane residues depending on whether the site contains K+ or not. This explains the decrease in affinity as a function of K+ concentration observed in the kinetic experiments. On the other hand, the structures predict that the protonation of tegoprazan is responsible for the change in its dihedral angle. The rotation of the benzimidazole ring allows the inhibitor to be positioned further into the luminal cavity, a situation compatible with the higher inhibition affinity of H,K-ATPase measured at low pH. Results presented herein will provide a basis for the rational design of novel P-CABs ligands.

Acid-related diseases (ARDs), such as peptic ulcers and gastroesophageal reflux disease, represent a major health-care concern. Some major milestones in our understanding of gastric acid secretion and ARD treatment reached during the last 50years include 1) discovery of histamine H₂-receptors and development of H₂-receptor antagonists, 2) identification of H⁺,K⁺-ATPase as the parietal cell proton pump and development of proton pump inhibitors (PPIs), and 3) identification of Helicobacter pylori (H. pylori) as the major cause of peptic ulcers and development of effective eradication regimens. Although PPI treatments have been effective and successful, there are limitations to their efficacy and usage, i.e. short half-life, insufficient acid suppression, slow onset of action, and large variation in efficacy among patients due to CYP2C19 metabolism. Potassium-competitive acid blockers (P-CABs) inhibit H⁺,K⁺-ATPase in a reversible and K⁺-competitive manner, and exhibit almost complete inhibition of gastric acid secretion from the first dose. Many pharmaceutical companies have tried to develop P-CABs, but most of their clinical development has been discontinued due to safety concerns or a similar efficacy to PPIs. Revaprazan was developed in Korea and was the first P-CAB approved for sale. Vonoprazan, approved in 2014 in Japan, has a completely different chemical structure and higher pKa value compared to other P-CABs, and exhibits rapid onset of action and prolonged control of intragastric acidity. Vonoprazan is an effective treatment for ARDs that is especially effective in healing reflux esophagitis and for H. pylori eradication. P-CABs, such as vonoprazan, promise to further improve the management of ARDs.

H(+),K(+)-ATPase is a key enzyme in the process of gastric acid secretion, and proton pump inhibitors (PPIs) have been accepted as one of the most effective treatments for peptic ulcer and gastroesophageal reflux disease. To discover a novel class of PPIs, the authors screened a low-molecular-weight compound library and identified two prospective acid blockers that were pyrrole derivatives. Both compounds inhibited H(+),K(+)-ATPase in a reversible and potassium-competitive manner. These compounds led to the development of TAK-438 (1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine monofumarate), which is currently undergoing clinical trials as a novel potassium-competitive acid blocker for the treatment of acid-related diseases.

Revaprazan is a novel acid pump antagonist. The aim of this study was to investigate the inhibitory effect of revaprazan on gastric acid secretion in healthy male subjects.

In a double-blind, three-way cross-over study, 30 healthy male volunteers were randomized to 100, 150 or 200 mg of oral revaprazan daily for 7 days. Serum gastrin concentration was measured, and 24-h intragastric pH was recorded at baseline and on days 1 and 7 of each administration period. Serial blood samples were processed for pharmacokinetics.

Median intragastric pH over 24 h and mean percentage time that pH was > 4 increased in a dose-dependent manner and were significantly higher on days 1 and 7 compared with baseline in all groups (P < 0.05). The antisecretory effect of revaprazan was rapid and nearly maximal on day 1 in all groups. Serum gastrin levels were rapidly normalized by 100 and 150 mg/day of revaprazan on days 1 and 7, but were significantly higher in the 200 mg/day revaprazan group. The pharmacokinetic effect was rapidly absorbed and eliminated on days 1 and 7 in all groups.

Revaprazan rapidly and effectively inhibits gastric acid secretion in healthy male subjects. Therefore, revaprazan can be used as an effective drug for acid-related disease.

In the present study, we evaluated the effect of the novel acid pump antagonist 7-(4-fluorobenzyloxy)-2,3-dimethyl-1-{[(1S,2S)-2-methylcyclopropyl]methyl}-1H-pyrrolo[2,3-d]pyridazine (CS-526) on the intragastric acidity of cynomolgus monkeys. The study was performed in a crossover manner with five male animals. CS-526 was administered orally or intravenously at doses of 3.0, 10 and 30 mg/kg, or 0.3, 1.0 and 3.0 mg/kg, respectively. The time period in which the intragastric pH was 4.0 or more (Time(pH > or = 4.0)) and the median pH were calculated for 24 h after the administration. The intragastric pH was elevated after CS-526 treatment. The Time(pH > or = 4.0) was increased in a dose-dependent manner (p = 0.0292) in the oral administration, and the median pH was also increased in a dose-dependent fashion (p = 0.0491) in the intravenous administration. The plasma concentration of CS-526 and its metabolite R-130185 was increased after oral and intravenous administration of CS-526, except for one animal which did not show any detectable amount of R-130185 after intravenous administration at the lowest dose. The area under the time-concentration curve of the active component was increased in the dose proportional manner after oral and intravenous administration. The absolute bioavailability of the active component was estimated to be approximately 1%. Correlation between the pharmacodynamic parameters and the pharmacokinetic parameters was observed in oral (p = 0.0029-0.0745), but not in intravenous administration (p = 0.0558-0.2789). In conclusion, oral and intravenous administration of CS-526 showed inhibition on gastric acidity in cynomolgus monkeys using intragastric pH-metry and some pharmacokinetic and pharmacodynamic parameters were well correlated.

In the present report, we evaluated the effect of the novel acid pump antagonist 7-(4-fluorobenzyloxy)-2,3-dimethyl-1-{[(1S,2S)-2-methylcyclopropyl]methyl}-1H-pyrrolo[2,3-d]pyridazine (CS-526) and 2-[3-methyl-4-(2,2,2-trifluoro-ethoxy)-pyridin-2-ylmethanesulfinyl]-1H-benzimidazole (lansoprazole) on rebound gastric acid secretion, using an intragastric dialysis membrane perfusion model and on the serum and antral gastrin level after a 14-day treatment in rats. The effect of CS-526 on gastric acid secretion was almost constant during the 14 days of treatment. After the 14-day treatment, gastric acid secretion had returned to pretreatment levels. However, CS-526 slightly increased and lansoprazole potently increased gastric acid secretion thereafter. In the posttreatment period, the influence on rebound gastric acid secretion by lansoprazole treatment was significant, but that by CS-526 was not. The serum gastrin concentration after the 14-day treatment with CS-526 did not increase significantly, even at 100 mg/kg/day. On the other hand, lansoprazole at 100 mg/kg/day significantly elevated the serum gastrin concentration. After the 14-day treatment with CS-526 at 100 mg/kg/day, the antral gastrin content significantly increased. Lansoprazole at the doses of 30 and 100 mg/kg/day also significantly increased the antral gastrin content after the 14-day treatment. The elevation of the serum gastrin level after the lansoprazole treatment was suppressed by the concomitant administration of CS-526. In conclusion, CS-526 has a potent antisecretory effect on gastric acid secretion without rebound gastric hypersecretion. Moreover, CS-526 had minimal effects on the serum and antral gastrin elevation. It is suggested that these effects on gastric acid secretion and serum gastrin after subchronic treatment with CS-526 would be beneficial in clinical use.

In the present study, a series of 179 quinoline and quinazoline heterocyclic analogues exhibiting inhibitory activity against Gastric (H+/K+)-ATPase were investigated using the comparative molecular field analysis (CoMFA) and comparative molecular similarity indices (CoMSIA) methods. Both the models exhibited good correlation between the calculated 3D-QSAR fields and the observed biological activity for the respective training set compounds. The most optimal CoMFA and CoMSIA models yielded significant leave-one-out cross-validation coefficient, q(2) of 0.777, 0.744 and conventional cross-validation coefficient, r(2) of 0.927, 0.914 respectively. The predictive ability of generated models was tested on a set of 52 compounds having broad range of activity. CoMFA and CoMSIA yielded predicted activities for test set compounds with r(pred)(2) of 0.893 and 0.917 respectively. These validation tests not only revealed the robustness of the models but also demonstrated that for our models r(pred)(2) based on the mean activity of test set compounds can accurately estimate external predictivity. The factors affecting activity were analyzed carefully according to standard coefficient contour maps of steric, electrostatic, hydrophobic, acceptor and donor fields derived from the CoMFA and CoMSIA. These contour plots identified several key features which explain the wide range of activities. The results obtained from models offer important structural insight into designing novel peptic-ulcer inhibitors prior to their synthesis.

AZD0865 belongs to a new class of acid-suppressing agents with rapid onset of action and potent acid inhibition. We evaluated its effectiveness for healing reflux esophagitis.

One thousand five hundred twenty-one patients with Los Angeles A-D esophagitis and heartburn of moderate or severe intensity for > or = 4 days/week were randomized to AZD0865 25, 50, or 75 mg or esomeprazole 40 mg once daily for 4-8 weeks. The primary end point was esophagitis healing by AZD0865 at 4 weeks. Healing and control of heartburn were also assessed at 2, 4, and 8 weeks for AZD0865 and esomeprazole.

After 4 weeks of treatment, healing rates were similar among AZD0865 doses (76.9%; confidence interval [CI], 72.4%-81.1%); 78.2% (CI, 73.7%-82.3%), and 81.1% (CI, 76.7%-84.9%) for 25, 50, and 75 mg, respectively). The healing rate with esomeprazole at 4 weeks was similar (81.9%; CI, 77.6%-88.7%), and healing rates also were comparable among all treatments at 2 and 8 weeks. There were no significant differences in heartburn control among treatments. AZD0865 and esomeprazole were well-tolerated, although reversible increases in transaminases occurred in a small number of patients receiving AZD0865, especially at the 75-mg dose.

AZD0865 25, 50, and 75 mg provided similar efficacy to esomeprazole 40 mg in terms of esophagitis healing and heartburn control. These findings suggest that increasing the degree of acid inhibition beyond that already achieved by esomeprazole 40 mg (or AZD0865 25 mg) does not translate into increased clinical efficacy in esophagitis patients.

AZD0865 is a member of a drug class that inhibits gastric H(+),K(+)-ATPase by K(+)-competitive binding. The objective of these experiments was to characterize the mechanism of action, selectivity and inhibitory potency of AZD0865 in vitro. In porcine ion-leaky vesicles at pH 7.4, AZD0865 concentration-dependently inhibited K(+)-stimulated H(+),K(+)-ATPase activity (IC(50) 1.0+/-0.2 microM) but was more potent at pH 6.4 (IC(50) 0.13+/-0.01 microM). The IC(50) values for a permanent cation analogue, AR-H070091, were 11+/-1.2 microM at pH 7.4 and 16+/-1.8 microM at pH 6.4. These results suggest that the protonated form of AZD0865 inhibits H(+),K(+)-ATPase. In ion-tight vesicles, AZD0865 inhibited H(+),K(+)-ATPase more potently (IC(50) 6.9+/-0.4 nM) than in ion-leaky vesicles, suggesting a luminal site of action. AZD0865 inhibited acid formation in histamine- or dibutyryl-cAMP-stimulated rabbit gastric glands (IC(50) 0.28+/-0.01 and 0.26+/-0.003 microM, respectively). In ion-leaky vesicles at pH 7.4, AZD0865 (3 microM) immediately inhibited H(+),K(+)-ATPase activity by 88+/-1%. Immediately after a 10-fold dilution H(+),K(+)-ATPase inhibition was 41%, indicating reversible binding of AZD0865 to gastric H(+),K(+)-ATPase. In contrast to omeprazole, AZD0865 inhibited H(+),K(+)-ATPase activity in a K(+)-competitive manner (K(i) 46+/-3 nM). AZD0865 inhibited the process of cation occlusion concentration-dependently (IC(50) 1.7+/-0.06 microM). At 100 microM, AZD0865 reduced porcine renal Na(+),K(+)-ATPase activity by 9+/-2%, demonstrating a high selectivity for H(+),K(+)-ATPase. Thus, AZD0865 potently, K(+)-competitively, and selectively inhibits gastric H(+),K(+)-ATPase activity and acid formation in vitro, with a fast onset of effect.

The gastric H+,K+-ATPase of the parietal cell is responsible for acid secretion in the stomach and is the main target in the pharmacological treatment of acid-related diseases. Omeprazole and other benzimidazole drugs, although having delayed efficacy if taken orally, have high success rates in the treatment of peptic ulcer disease. Potassium competitive acid blockers (P-CAB) compete with K+ for binding to the H+,K+-ATPase and thereby they inhibit acid secretion. In this study, the in vitro properties of AZD0865, a reversible H+,K+-ATPase inhibitor of gastric acid secretion, are described. We used a digital-imaging system and the pH sensitive dye BCECF to observe proton efflux from hand-dissected rat gastric glands. Glands were stimulated with histamine (100 microM) and exposed to a bicarbonate- and Na+-free perfusate to induce an acid load. H+,K+-ATPase inhibition was determined by calculating pHi recovery (dpH/dT) in the presence of omeprazole (10-200 microM) or AZD0865 (0.01-100 microM). The efficacies of both drugs were compared. Our data show that acid secretion is inhibited by both the proton pump inhibitor omeprazole and the P-CAB AZD0865. Complete inhibition of acid secretion by AZD0865 had a rapid onset of activation, was reversible, and occurred at a 100-fold lower dose than omeprazole (1 microM AZD0865 vs. 100 microM omeprazole). This study demonstrates that AZD0865 is a potent, fast-acting inhibitor of gastric acid secretion, effective at lower concentrations than drugs of the benzimidazole class. Therefore, these data strongly suggest that AZD0865 has great potential as a fast-acting, low-dose inhibitor of acid secretion.

In a program to identify new anti-ulcer compounds, a series of N-acyl derivatives of alpha-amino acids were screened for their in vitro H(+)/K(+) ATPase inhibitory activity, and in vivo efficacy in Pylorus ligation model. 3D-QSAR studies were carried out and a representative compound 13 was studied for the nature of its proton pump inhibition.

Vasorelaxant effects of H+/K+-ATPase were previously demonstrated in artery rings isolated from experimental animals. We examined the effects of clinically used H+/K+-ATPase inhibitors on isolated human internal mammary (n=19) and radial (n=5) arteries. Omeprazole and lansoprazole (30-300 microM) both induced concentration-dependent, reversible and reproducible relaxations of arteries which were precontracted with phenylephrine (5 microM), histamine (15 microM), high K+ (80 mM), ouabain (1 microM) and K+ free solution. Relaxant responses were similar in both arteries. Presence of Nomega-Nitro-L-arginine methyl ester (30 microM) had no effect on lansoprazole-induced responses, thus relaxations are independent from nitric oxide. Relaxation in the K+ free medium implies that this action could not be due to the inhibition of H+/K+-ATPase. Lansoprazole (300 microM) inhibited Ca2+-induced contractions in high K+-Ca2+ free medium. Omeprazole and lansoprazole may act on a common mechanism which plays a crucial role in regulating human vascular tone and that mechanism appeared to be involved in the regulation of intracellular Ca2+.

Gastric acid secretion is a complex process that requires hormonal, neuronal, or calcium-sensing receptor activation for insertion of pumps into the apical surface of the parietal cell. Activation of any or all these pathways causes the parietal cell to secrete concentrated acid with a pH at or close to 1. This acidic fluid combines with enzymes that are secreted from neighbouring chief cells and passes out of the gland up through a mucous gel layer covering the surface of the stomach producing a final intragastric pH of less than 4 during the active phase of acid secretion. Defects in either the mucosal barrier or in the regulatory mechanisms that modulate the secretory pathways will result in erosion of the barrier and ulcerations of the stomach or esophagus. The entire process of acid secretion relies on activation of the catalytic cycle of the gastric H+,K+-ATPase, resulting in the secretion of acid into the parietal cell canaliculus, with K+ being the important and rate-limiting ion in this activation process. In addition to K+ as a rate limiter for acid production, Cl- secretion via an apical channel must also occur. In this review we present a discussion of the mechanics of acid secretion and a discussion of recently identified transporter proteins and receptors. Included is a discussion of some of the recent candidates for the apical K' recycling channel, as well as two recently identified apical proteins (NHE-3, PAT-1), and the newly characterized calcium-sensing receptor (CaSR). We hope that this review will give additional insight into the complex process of acid secretion.

The effects of three specific H+/K+-ATPase inhibitors (omeprazole, lansoprazole and SCH 28080 (2-methyl-8-(phenylmethoxy)-imidazo[1,2-a] pyridine-3-acetonitrile)) were investigated on the mechanical and electrophysiological properties of rat atrium, in vitro. Omeprazole (100-300 microM), lansoprazole (100-300 microM) and SCH 28080 (10-100 microM) increased the amplitude of contractions and decreased the beating rate. These effects are reversible, reproducible and correlated with their order of potency as gastric H+/K+-ATPase inhibitors; SCH 28080 > omeprazole = lansoprazole. Cardiac effects of proton pump inhibitors were not inhibited with phentolamine (5 microM), propranolol (15 microM), atropine (1 microM), ouabain (2 microM), theophylline (300 microM) and milrinone (100 microM). Ouabain-induced increase in beating rate and contracture development were antagonized by H+/K+-ATPase inhibitors. Ouabain increased the positive inotropic effect of H+/K+-ATPase inhibitors. Lansoprazole (300 microM) significantly prolonged the duration of action potentials in rat atrial cells. H+/K+-ATPase may play a crucial role in the mechanical and electrophysiological properties of rat atrial myocardium.

Current therapies to treat gastroesophageal reflux disease (GERD), peptic ulcer disease (PUD), and other acid-related diseases either prevent stimulation of the parietal cell (H2 receptor antagonists, H2RAs) or inhibit gastric H+,K+-ATPase (e.g., proton pump inhibitors, PPIs). Of the 2 approaches, the inhibition of the final step in acid production by PPIs provides more effective relief of symptoms and healing. Despite the documented efficacy of the PPIs, therapeutic doses have a gradual onset of effect and do not provide complete symptom relief in all patients. There is scope for further improvements in acid suppressive therapy to maximize healing and offer more complete symptom relief. It is unlikely that cholecystokinin2 (CCK2, gastrin) receptor antagonists, a class in clinical trials, will be superior to H2RAs or PPIs. However, a new class of acid suppressant, the potassium-competitive acid blockers (P-CABs), is undergoing clinical trials in GERD and other acid-related diseases. These drugs block gastric H+,K+-ATPase by reversible and K+-competitive ionic binding. After oral doses, P-CABs rapidly achieve high plasma concentrations and have linear, dose-dependent pharmacokinetics. The pharmacodynamic properties reflect the pharmacokinetics of this group (i.e., the effect on acid secretion is correlated with plasma concentrations). These agents dose dependently inhibit gastric acid secretion with a fast onset of action and have similar effects after single and repeated doses (i.e., full effect from the first dose). Animal studies comparing P-CABs with PPIs suggest some important pharmacodynamic differences (e.g., faster and better control of 24-hr intragastric acidity). Studies in humans comparing PPIs with P-CABs will help to define the place of this new class in the management of acid-related diseases.

KR-60436 ([1-(4-methoxy-2-methylphenyl)-4-[(2-hydroxyethyl)amino]-6-trifluoromethoxy-2,3-dihydropyrrolo [3,2-c]quinoline]) is a new reversible H+/K+-ATPase inhibitor. The isoforms of human liver cytochrome P450 (CYP) responsible for the hepatic transformation of KR-60436 is identified. Dihydropyrrole oxidation and O-demethylation are major pathways for the metabolism of KR-60436 in human liver microsomes, whereas N-dehydroxyethylation and hydroxylation are minor pathways. The specific CYP isozymes responsible for KR-60436 oxidation to four major metabolites, pyrrole-KR-60436, O-demethylpyrrole-KR-60436, N-dehydroxyethyl-KR-60436 and an active metabolite, O-demethyl-KR-60436 were identified using the combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by expressed recombinant CYP enzymes. The inhibitory potency of KR-60436 on clinically major CYPs was investigated in human liver microsomes. The results show that CYP3A4 contributes to the oxidation of KR-60436 to pyrrole-KR-60436, O-demethylpyrrole-KR-60436 and N-dehydroxyethyl-KR-60436, and CYP2C9 and CYP2D6 play roles in demethylation of KR-60436 to form the active metabolite, O-demethyl-KR-60436. KR-60436 was found to inhibit potently the metabolism of CYP1A2 substrates.

The gastric H+, K+-ATPase is a proton pump that is responsible for gastric acid secretion and that actively transports protons and K+ ions in opposite directions to generate in excess of a million-fold gradient across the membrane under physiological conditions. This pump is also a target molecule of proton pump inhibitors which are used for the clinical treatment of hyperacidity. In this review, we wish to summarize the molecular regulation of this pump based on mutational studies, particularly those used for the identification of binding sites for cations and specific inhibitors. Recent reports by Toyoshima et al (2000, 2002) presented precise three-dimensional (3-D) structures of the sarcoplasmic reticulum (SR) Ca2+-ATPase, which belongs to the same family as the gastric H+, K+-ATPase. We have studied the structure-function relationships for the gastric H+, K+-ATPase using 3-D structures constructed by homology modeling of the related SR Ca2+-ATPase, which was used as a template molecule. We also discuss in this review, the regulation of cell surface expression and synthesis control of the gastric proton pump.

Dose-independent pharmacokinetic parameters of KR-60436, a new proton pump inhibitor, were evaluated after intravenous (i.v.; 5, 10, and 20 mg/kg) and oral (20, 50, and 100 mg/kg) administration to rats. The hepatic, gastric, and intestinal first-pass effects were also measured after iv, intraportal (i.p.), intragastric (i.g.), and intraduodenal (id) administrations to rats of a dose of 20 mg/kg. The areas under the plasma concentration-time curve from time to zero to time infinity (AUCs) were independent of iv and oral dose ranges studied; the dose-normalized AUCs were 83.0-104 microg. min/mL (based on 5 mg/kg) and 78.4-96.8 microg. min/mL (based on 20 mg/kg) for iv and oral administration, respectively. After an oral administration at a dose of 20 mg/kg, approximately 3% of the oral dose was not absorbed, and the extent of absolute oral bioavaliability (F) was estimated to be 18.8%. The AUCs of KR-60436 after i.g. and i.d. administration at a dose of 20 mg/kg were significantly smaller (82.4 and 57.5% decrease, respectively) than that after an i.p. administration at a dose of 20 mg/kg, suggesting that gastrointestinal first-pass effect of KR-60436 was approximately 80% of oral dose in rats (the gastric first-pass effect was approximately 25%). After an i.p. administration at a dose of 20 mg/kg, the AUC was 77.6% of an iv administration, suggesting that hepatic first-pass effect was approximately 22% of KR-60436 absorbed into the portal vein. Note that the value of 22% was equivalent to approximately 4% of the oral dose. Because only 17% of oral dose was absorbed into the portal vein, the low F of KR-60436 in rats was mainly due to considerable gastrointestinal first-pass effect, which was approximately 80% (the gastric first-pass effect was approximately 25%) of oral dose.

A high-performance liquid chromatographic (HPLC) method was developed for the determination of a new proton pump inhibitor, DBM-819, in human plasma and urine and rat tissue homogenates using KR-60461 as an internal standard. A 100-microl aliquot of acetonitrile (containing 0.5 microg/ml of the internal standard) and a 200-microl aliquot of 0.1 M Na(2)HPO(4) (adjusted pH 11 with 1 N NaOH) were added to a 100-microl aliquot of biological sample. After vortex-mixing, the mixture was extracted with 1 ml of ethylacetate. After centrifugation at 12000 x g for 3 min, the organic layer was collected and evaporated under nitrogen gas. The residue was then reconstituted with a 100-microl aliquot of mobile phase, and a 40-microl aliquot was injected onto the HPLC column. The mobile phase, 0.02 M phosphate buffer (pH 5): acetonitrile: methanol (46:44:10, v/v/v), was run at a flow rate of 0.5 ml/min and the column effluent was monitored by the fluorescence detector set at an excitation wavelength of 340 nm and an emission wavelength of 470 nm. The retention times for DBM-819 and the internal standard were approximately 10.5 and 12 min, respectively. The detection limits of DBM-819 in human plasma and urine, and rat tissue homogenates were 0.01, 0.02 and 0.02 (or 0.05) microg/ml. respectively. The coefficients of variation (CV) of the assay were below 11% for human plasma and urine, and rat tissue homogenates. No interferences from endogenous substances were found.

1. Cibenzoline, (+/-)-2-(2,2-diphenylcyclopropyl-2-imidazoline succinate, has been clinically used as one of the Class I type antiarrhythmic agents and also reported to block ATP-sensitive K(+) channels in excised membranes from heart and pancreatic beta cells. In the present study, we investigated if this drug inhibited gastric H(+),K(+)-ATPase activity in vitro. 2. Cibenzoline inhibited H(+),K(+)-ATPase activity of permeabilized leaky hog gastric vesicles in a concentration-dependent manner (IC(50): 201 microM), whereas no effect was shown on Na(+),K(+)-ATPase activity of dog kidney (IC(50): >1000 microM). Similarly, cibenzoline inhibited H(+),K(+)-ATPase activity of HEK-293 cells (human embryonic kidney cell line) co-transfected with rabbit gastric H(+),K(+)-ATPase alpha- and beta-subunit cDNAs (IC(50): 183 microM). 3. In leaky gastric vesicles, inhibition of H(+),K(+)-ATPase activity by cibenzoline was attenuated by the addition of K(+) (0.5 - 5 mM) in a concentration-dependent manner. The Lineweaver-Burk plot of the H(+),K(+)-ATPase activity shows that cibenzoline increases K(m) value for K(+) without affecting V(max), indicating that this drug inhibits H(+),K(+)-ATPase activity competitively with respect to K(+). 4. The inhibitory effect of H(+),K(+)-ATPase activity by cibenzoline with normal tight gastric vesicles did not significantly differ from that with permeabilized leaky gastric vesicles, indicating that this drug reacted to the ATPase from the cytoplasmic side of the membrane. 5. These findings suggest that cibenzoline is an inhibitor of gastric H(+),K(+)-ATPase with a novel inhibition mechanism, which inhibits gastric H(+),K(+)-ATPase by binding its K(+)-recognition site from the cytoplasmic side.

H 335/25, a 4-amino quinoline, belongs to a new class of reversible gastric acid pump inhibitors. A potential advantage of such drugs over the irreversible proton pump inhibitors (PPIs) is better control over the effect-time profile. Dose escalation studies were performed to characterize the effect on acid secretion in dogs (n=24) and healthy male subjects (n=12). The effect-time profile was delayed compared to the concentration-time profile. A model-based approach, using non-linear mixed effects modelling, was applied to quantify and elucidate the mechanism for the delayed effect. Three different models were investigated: (1) a slow equilibration preceding the formation of drug-enzyme complex, modelled by an effect-compartment, (2) a slow equilibration between free drug, free enzyme and drug-enzyme complex, described by a kinetic binding model, and (3) a delay between enzyme inhibition and the measured response, described by an indirect response model. Model 2 was shown to be superior to models 1 and 3, for both dog and human data. The dissociation rate constant, k(off), was estimated to be 0.85 and 0.88 h and the calculated equilibration constant, K(d), was 160 and 250 nM in dog and man, respectively. Simulations of the predicted time-course of the effect beyond the 4-5-h observation period was similar for the three models.

The dose-dependent pharmacokinetic parameters of DBM-819 were evaluated after intravenous (5, 10 and 20 mg/kg) and oral (10, 20 and 50 mg/kg) administrations of the drug to rats. The hepatic first-pass effect was also measured after intravenous and intraportal administrations of the drug, 10 mg/kg, to rats. After intravenous administration, the dose-normalized (based on 5 mg/kg) area under the plasma concentration-time curve from time zero to time infinity, AUC, at 20 mg/kg (27.0 and 45.8 microg min/ml) was significantly greater than that at 5 mg/kg due to saturable metabolism. After oral administration, the dose-normalized (based on 10 mg/kg) AUC(0-12 h) at 50 mg/kg (25.1, 18.3 and 49.2 microg min/ml) was significantly greater than those at 10 and 20 mg/kg again due to saturable metabolism. After oral administration of DBM-819, 10 mg/kg, 2.86% of oral dose was not absorbed and the extent of absolute oral bioavailability (F) was estimated to be 46.7%. After intraportal administration of DBM-819, 10 mg/kg, the AUC was 51.9% of intravenous administration, suggesting that approximately 48.1% was eliminated by liver (hepatic first-pass effect). The considerable hepatic first-pass effect of DBM-819 was also supported by significantly greater AUC of M3 (3.70 and 6.86 microg min/ml), a metabolite of DBM-819, after intraportal administration. The AUCs of DBM-819 were not significantly different (comparable) between intraportal and oral administrations of the drug, 10 mg/kg, suggesting that gastrointestinal first-pass effect of DBM-819 was almost negligible in rats. At 10 mg/kg oral dose of DBM-819, the hepatic first-pass effect was approximately 48.1%, F was approximately 46.7 and 2.86% was not absorbed from gastrointestinal tract in rats.

Prodigiosin, prodigiosin 25-C, and metacycloprodigiosin all strongly inhibited the acidification activity of (H(+)+K(+))-ATPase on membrane vesicles from hog gastric mucosa (IC(50) = 32 to 103 pmol/mg protein). But, the prodigiosins, unlike omeprazole, showed little inhibitory effect on K(+)-dependent ATPase (K(+)-ATPase) activity, although at higher concentrations they inhibited K(+)-ATPase activity with an IC(50) of 1.5 to 3.0 microM. Furthermore, the inhibitory effect of the prodigiosins was rapid and completely reversible unlike that of omeprazole, and the mode of inhibition was non-competitive with respect to ATP. Hog gastric (H(+)+K(+))-ATPase itself showed an absolute requirement of halide (effectively, chloride) for acidification activity. Prodigiosins also showed a chloride requirement for inhibition of vesicular acidification, and quickly reversed the acidification of vesicular pH to neutrality even in the presence of N, N'-dicyclohexylcarbodiimide (DCCD), showing their ionophoric nature of acidification inhibitory activity. In fact, tributyltin chloride (TBT, an OH(-)/Cl(-) exchange ionophore) also inhibited vesicular acidification, but it inhibited K(+)-ATPase activity too. Finally, the prodigiosins inhibited the acid secretion from parietal cells isolated from rabbit gastric mucosa. These results suggest that prodigiosins are potent reversible uncouplers of (H(+)+K(+))-ATPase that inhibit gastric acid secretion.

We purified a compound with strong inhibitory effect on H+, K(+)-ATPase from Paeoniae radix, which has been used in Japan for the treatment of gastritis and peptic ulcers. The compound was identified as 1,2,3,4,6,-penta-o-galloyl-beta-D-glucose by proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, and fast atomic bombardment mass spectrometry. The IC50 of the compound for H+, K(+)-ATPase was 166 nmol/l. Kinetic analyses indicated that the inhibition of the enzyme by pentagalloylglucose was noncompetitive with respect to K+. Pentagalloylglucose had relatively weak inhibitory effects for Mg(+)-ATPase (IC50: > 10 mumol/l) and Na+, K(+)-ATPase (IC50: 2.7 mumol/l). Pentagalloylglucose also inhibited the accumulation of [14C]aminopyrine in parietal cells that had been isolated from guinea pig stomach and stimulated by 10 mumol/l histamine (IC50: 7.8 mumol/l) and 1 mmol/l dbc-AMP (IC50: 10 mumol/l). These results suggest that pentagalloylglucose is a potent inhibitor of H+, K(+)-ATPase and may be responsible for inhibition of acid secretion by Paeoniae radix.

3-Amino-5-methyl-2(2-methyl-3-thienyl)- imidazo[1,2-a]thieno[3,2-c]pyridine, SPI-447, is a potent gastric H+,K(+)-ATPase inhibitor, but a detailed mechanism of the inhibition is unknown. This study was designed to investigate the mechanism by which SPI-447 inhibits gastric H+,K(+)-ATPase. For this purpose, the inhibitory action of SPI-447 on gastric H+,K(+)-ATPase from porcine gastric mucosa was compared with that of omeprazole (an irreversible inhibitor) and SCH28080 (a reversible inhibitor). All compounds produced dose-dependent inhibition of gastric H+,K(+)-ATPase, and the inhibitory intensities were increased under acidic conditions. The anti-H+,K(+)-ATPase actions of SPI-447 and SCH28080 were attenuated by dilution, but not influenced by glutathione pretreatment. In contrast, that of omeprazole was not influenced by dilution, but was suppressed by glutathione pretreatment. KCl addition reversed the inhibition of H+,K(+)-ATPase-mediated H(+)-transport by SPI-447 and SCH28080, but had no effect on that by omeprazole. The anti-gastric H+,K(+)-ATPase action of SPI-447 was additive with that of SCH28080. SPI-447 and SCH28080 had no effect on Na+,K(+)-ATPase activity. These findings indicated that the inhibitory mechanism of SPI-447 on gastric H+,K(+)-ATPase was similar to that of SCH28080, but different from that of omeprazole; i.e., 1) reversible, 2) SH-group independent, 3) K(+)-competitive, and 4) highly specific against gastric H+,K(+)-ATPase.

SK&F 97574 (3-butyryl-4-(2-methylamino)-8-(2-hydroxyethoxy)quinoline), is a potent inhibitor of the (H+/K+)-ATPase in membrane vesicles isolated from porcine gastric mucosa. It inhibits (H+/K+)-ATPase activity in lyophilised vesicles in a kinetically competitive manner with respect to the activating cation, K+, with an inhibition constant (Ki) of 0.46 +/- 0.003 microM. Inhibition of (H+/K+)-ATPase activity is freely reversible. Binding of SK&F 97574 was shown to be mutually exclusive and the previously reported reversible (H+/K+)-ATPase inhibitors, SCH 28080 and MDPQ. Therefore, despite its structural dissimilarity, SK&F 97574 appears to bind to the same lumenal region of the (H+/K+)-ATPase identified as the binding site for these compounds. SK&F 97574 is a weak base (pKa = 6.86), and would therefore be expected to accumulate in the acidic compartment at the lumenal face of the parietal cell. In intact gastric vesicles (which have the lumenal face of the ATPase on the interior), SK&F 97574 inhibited ATP-dependent H(+)-transport with a similar potency to ATPase activity. SK&F 97574 is therefore relatively membrane permeable, and would be predicted to gain access readily to its site of action in vivo. The effect of pH on inhibition of H+/K(+)-ATPase activity by SK&F 97574 is consistent with its being active only in its protonated form. The selectivity of SK&F 97574 for the gastric (H+/K+)-ATPase was tested by examining its ability to inhibit a closely related p-class pump, the (Na+/K+)-ATPase from dog kidney. SK&F 97574 was found to have a 60-fold greater sensitivity for the former enzyme. The (Na+/K+)-ATPase was not inhibited in a K(+)-competitive manner by SK&F 97574, indicating an entirely different, probably nonspecific, mechanism.

SK&F 97574 [3-Butryl-4-(2-methylamino)-8-(2-hydroxyethoxy) quinoline] is a potent, reversible inhibitor of the gastric (H+/K+)-ATPase. In an anaesthetised lumen-perfused rat preparation, it inhibited pentagastrin-stimulated gastric acid secretion with intravenous and intraduodenal inhibitory ED50 values of 2.40 mumol/kg and 4.43 mumol/kg, respectively. In the conscious fistula rat model, doses of 10 mumol/kg IV and 25 mumol/kg PO produced mean peak inhibitions of basal acid output of 91% and 97%, respectively. In these experiments, the duration of action of SK&F 97574 was much shorter than that of the covalent (H+/K+)-ATPase inhibitor, omeprazole. In the conscious Heidenhain pouch dog, SK&F 97574 inhibited histamine-stimulated gastric acid secretion after both intravenous and oral administration with ED50 values of 0.49 mumol/kg and 0.89 mumol/kg, respectively. In this model, duration of action studies showed that significant residual inhibition of acid secretion remained 8 hours after intravenous dosing with SK&F 97574 (producing peak inhibition of 92%). However, 24 hours after oral dosing of SK&F 97574 (10 mumol/kg), no significant inhibition remained. These data indicate that the duration of action of SK&F 97574 is longer than that of the histamine H2 receptor antagonists such as cimetidine, but shorter than that of covalent (H+/K+)-inhibitors such as omeprazole. Overall, the pharmacological properties of SK&F 97574 suggest that it could be a potentially useful clinical treatment for acid-related diseases.

Tobacco plants were transformed with a cDNA clone of chymotrypsin/trypsin-specific potato proteinase inhibitor II (PI2) under the control of a constitutive promoter. Although considerable levels of transgene expression could be demonstrated, the growth of Spodoptera exigua larvae fed with detached leaves of PI2-expressing plants was not affected. Analysis of the composition of tryptic gut activity demonstrated that only 18% of the proteinase activity of insects reared on these transgenic plants was sensitive to inhibition by PI2, whereas 78% was sensitive in insects reared on control plants. Larvae had compensated for this loss of tryptic activity by a 2.5-fold induction of new activity that was insensitive to inhibition by PI2. PI2-insensitive proteolytic activity was also induced in response to endogenous proteinase inhibitors of tobacco; therefore, induction of such proteinase activity may represent the mechanism by which insects that feed on plants overcome plant proteinase inhibitor defense.

The secretion of gastric acid is regulated both centrally and peripherally. The finding that H2-receptor antagonists are able to reduce or abolish acid secretion due to vagal, gastrinergic, and histaminergic stimulation shows that histamine plays a pivotal role in stimulation of the parietal cell. In the rat, the fundic histamine is released from the ECL cell, in response to gastrin, acetylcholine, or epinephrine, and histamine release is inhibited by somatostatin or by the H3-receptor ligand, R-alpha-methyl histamine. The parietal cell has a muscarinic, M3, receptor responsible for [Ca]i regulation. Blockade of muscarinic receptors by atropine can be as effective as H2-receptor blockade in controlling acid secretion. However, general effects on muscarinic receptors elsewhere produce significant side effects. The different receptor pathways converge to stimulate the gastric H+,K(+)-ATPase, the pump responsible for acid secretion by the stomach. This enzyme is an alpha,beta heterodimer, present in cytoplasmic membrane vesicles of the resting cell and in the canaliculus of the stimulated cell. It has been shown that acid secretion by the pump depends on provision of K+Cl- efflux pathway becoming associated with the pump. As secretion occurs only in the canaliculus, this K+Cl- pathway is activated only when the pump inserts into the canalicular membrane. Transport by the enzyme involves reciprocal conformational changes in the cytoplasmic and extracytoplasmic domain. These result in changes in sidedness and affinity for H3O+ and K+, enabling active H+ for K+ exchange. The acid pump inhibitors of the substituted benzimidazole class, such as omeprazole, are concentrated in the canaliculus of the secreting parietal cell and are activated there to form sulfenamides. The omeprazole sulfenamide, for example, reacts covalently with two cysteines in the extracytoplasmic loops between the fifth and sixth transmembrane and the seventh and eighth transmembrane segments of the alpha subunit of the H+,K(+)-ATPase, forming disulfide derivatives. This inhibits ATP hydrolysis and H+ transport, resulting in effective, long-lasting regulation of acid secretion. Therefore, this class of acid pump inhibitor is significantly more effective and faster acting than the H2 receptor antagonists. K+ competitive antagonists bind to the M1 and M2 transmembrane segments of the alpha subunit of the acid pump and also abolish ATPase activity. These drugs should also be able to reduce acid secretion more effectively than receptor antagonists and provide shorter acting but complete inhibition of acid secretion.

The parietal cells, which are responsible for the production of gastric HCl acid, are uniquely equipped for high-gradient ion transport. Adequate energy is supplied by oxidative metabolism in the mitochondria, which occupy an exceptionally high proportion of the cytoplasmic volume. Another characteristic feature is the secretory canaliculi. These are tortuous small channels lined by microvilli which penetrate all parts of the cytoplasm and which expand during stimulation of secretion. The activity of the parietal cell is controlled by receptors for acetylcholine, histamine and gastrin on the basolateral cell membrane. Stimulation of these receptors modulates the levels of protein kinases in the cell and brings about the changes from resting to stimulated structure. A key role in the production of acid is played by the gastric acid pump, also known as the H+, K(+)-ATPase, which exports hydrogen ions in 1:1 exchange for potassium ions. This protein is a member of the P-type ATP-driven ion pumps and appears to be uniquely located in the parietal cell. The gastric acid pump is found in the tubulovesicular membranes of the resting cell and moves to the membrane lining the secretory canaliculus when acid secretion is stimulated. Functional acid secretion also requires the presence of KCl pathways in the secretory membrane in order to supply the acid pump with a source of potassium ions. For each hydrogen ion secreted across the secretory membrane, one bicarbonate ion is generated in the cytoplasm and is transported across the basolateral membrane in exchange for chloride. The movement of ions across the apical membrane is followed osmotically by water, resulting in the secretion of 160 mM HCl from the parietal cell.