The Novel Acid Pump Antagonists for Anti-Secretory Actions with Their Peculiar Applications Beyond Acid Suppression

Review J. Clin. Biobem. Nutr., 38, 1–8, January 2006

The Novel Acid Pump Antagonists for Anti-secretory Actions with Their Peculiar Applications Beyond Acid Suppression

Marie Yeo,1 Dong Kyu Kim,1 In Sik Chung,2 Byoung Seok Moon,3 Keun Seog Song,3 and Ki-Baik Hahm1,*

1Genome Research Center for Gastroenterology and Department of Gastroenterology, Ajou University School of Medicine, Suwon 443-721, Korea 2Kangnam St. Mary Hospital, Catholic University School of Medicine, Seoul 137-701, Korea 3Yuhan Corp., Seoul 156-754, Korea

Received 28 October, 2005; Accepted 31 October, 2005

Summary The H+/K+-ATPase of gastric parietal cell exchanges luminal K+ for cytoplasmic + − H , of which outcome is gastric acidification with outflux of hydronium ion (H3O ). Secretion of gastric acid from the H+/K+-ATPase is stimulated by neuronal sensing and elaborately regulated various neuronal transmitters and hormones, consequently resulting in anchoring of the H+/K+-ATPase in canaliculi membrane of gastric parietal cell. Since hypersecretion of gastric acid or a defect of its barrier function is considered as a principal casual factor in the acid-related diseases such as duodenal and gastric ulcer, reflux esophagitis, and some types of gastritis, the development of anti-secretory agents including PPIs (proton pump inhibitors) and H2-RAs (histamine type 2 receptor antagonists) has revolutionized during the second millennium. Similar considerations applying to design of compounds substituting K+ led to the development of acid pump antagonists (APAs), of which advantages are independent of secretory status, no lag time required, reversible in actions allowing “on-demand dosage”. Our recent studies revealed that these inhibitors of H+/K+-ATPase could be extensively applied for the selective induction of cancer cell apoptosis, a significant anti-inflammatory and gastro- protective action beyond acid suppression. In the current review, we described the mechanistic regulation of gastric acid secretion with pump inhibitor, the difference and characteristics between PPI and APA based on the molecular mechanism, and their new applications beyond acid suppression.

Key Words: Gastric acid secretion, Acid pump, Helicobacter pylori, PPI, APA

They are fundamentally produced by specialized gastric Introduction proton pump, H+/K+-ATPase, presented in parietal cells of gastric glands. The enzyme exports one H+ ion and imports Gastric acids are necessary for sterilization and digestion one K+ ion with consuming one ATP molecule, by which of food, and specially required for maintenance of the H+/K+-ATPase is commonly annotated than simple nomina- activity of pepsin through the activation of pepsinogen. tion of proton pump [1, 2]. In the resting status, since H+/K+- ATPases exist in the tubulovescles of parietal cells of which membrane is impermeable to K+ ions, H+/K+-ATPase stays *To whom correspondence should be addressed. as inactive form in the vesicles. Tel: +82-31-219-4383 Fax: +82-31-219-4399 Hormones such as histamine, acetycholine, and gastrin E-mail: [email protected]

1 2 M. Yeo et al. promote the fusion of tubulovesicles with apical membrane including PPI and APA could regulate intracellular signaling of parietal cells, which results in elongation of microvilli and pathways of apoptosis as well as inflammatory propagation up to 10-fold expansion of the apical membrane area. H+/K+- [15, 16]. The studies also revealed that APA seems to be ATPase in the fused tubulovesicles are thus exposed to superior against Helicobacter pylori infection, based on the luminal fluid containing K+ ions and subsequently the findings that they exerted significant anti-inflammatory, enzyme is activated [3, 4]. Mis-regulation of acid secretion gastroprotective with overt molecular actions. Taken can cause several acid-related diseases of gastric, esopha- together, our novel findings shed lights on that gastric proton geal, and duodenal tissues, resulting in gastroduodenal ulcer, pump inhibitors including APAs could be extensively used erosive reflux gastritis, and Zollinger-Ellison syndrome in for a wide spectrum of gastric diseases comprising of extreme case. Besides of these hypersecretary conditions, therapeutic approach for gastric inflammatory disease as the acid related diseases of the upper gastrointestinal tract well as cancer. can be also caused by a defect in barrier function of the esophageal epithelium in the case of GERD or of the gastric The Gastric Acid Secretion and Its Suppression by mucosal or duodenal epithelium in the case of gastric or the H+/K+-ATPase Inhibitors duodenal ulcers [5, 6]. Treatment for acid-related disease has been revolution- The regulation of gastric acid secretion ized by histamin-2-receptor antagonists (H2RA) that signifi- The senses like as sight, smell, taste stimulate increase cantly suppress gastric-acid secretion and they have been vagal outflow from the dorsal motor nucleus of the hypo- used extensively for more than 25 years [7, 8]. The concept thalamus and the vagal impulses are transmitted to the that inhibition of the pump itself would be a more effective enteric neurons in the wall of the stomach [1, 2]. Secretory way of controlling acid secretion rather than the blockade of stimulation is induced by postganglionic fibers that release one of stimulatory signals led a development of proton pump PACAP (pituitary adenylate cyclase-activating polypeptide) inhibitor (PPI) [9–11]. As results, a benzimidazole ring was to the ECL (enterochromaffin like) cell and acetylcholine added to the pyridine-2-thoacetaminde of an antiviral agent and gastrin-releasing peptide (GRP) to the G cell and the proved to possess a strong antisecretory activity. The sulfide parietal cell [17]. The G cell is also stimulated to release of the compound was modified to a sulfoxide for stabiliza- gastrin by the aromatic amino acids, phenylalanine and tion, by which timoprazole, the word’s first proton pump tyrosine in the gastric lumen. Gastrin stimulates the ECL inhibitor, was born and the evolution into the invention of cell and the D cell. Release of histamine from the activated omeprazole was followed thereafter. ECL cell stimulates the parietal cell at the H2 receptor as As for the exact mechanism of acid inhibition by PPI, aceylcholine at M3 receptor does (Fig. 1). Receptors for the PPIs have a unique mode of action, being acid-activated acetylcholine, histamine, and gastrin are found in the prodrugs and covalently bind cysteine residues of the gastric basolateral membrane of parietal cells. These receptors are H+/K+-ATPase. The compound accumulated in the acid linked to two distinct intracellular pathways which ulti- space the secretory canaliculus of the stimulated parietal cell mately converge to stimulate secretion of acid [18]. Acetyl- and consequently converted to the active form, a cationic choline and gastrin bind to receptors that are coupled by a thiophilic reagent. These activated species binds covalently guanosine triphosphatatase-binding protein to phospholi- to one or more cysteines accessible from the luminal face pase C. Once activated, phospholipase C catalyzes the con- of the pump, interfering outflux of hydronium ion from version of membrane-bound phospholipids to diaglycerol cytoplasm. Similar considerations applying to design of and inositol tris-phosphate. Inositol trisphosphate then compounds substituting K+ on the outside surface led to the causes the release of free cytosolic calcium from intra- development of acid pump antagonists (APAs) [12–14]. cellular calcium stores. Histamine binds to histamine type 2 Whereas the PPIs have a unique targeting and covalent receptors which are linked by a stimulatory guanosine inhibitory action on the proton pump based on their triphosphatase-binding protein to adenylate cyclase. Acti- chemistry and the biology of the parietal cell, another class vated adenylate cyclase catalyzes the conversion of cyto- of compound, APA, have a structural specificity for their solic ATP to cAMP, which initiates a phosphorylation target region on the pump, close to the K+ binding region of cascade of cytoplasmic enzymes. The effect of increasing the H+/K+-ATPase and are, thus, K+ competitive and dis- cytoplasmic calcium and cAMP concentrations leads to acti- sociate from the pump when their blood concentration drops. vation of the proton pump [19, 20]. Within minutes of Therefore, this agent have great advantages in terms of stimulation of the parietal cell by either acetylcoline and independent of secretory status, no lag time required, histamine or gastrin, the cytoplasmic tubulovesicles hyper- reversible in actions, and could be therapeutic antacids, trophy and coalesce into canalicular folds which become the allowing “on-demand dosage”. secretory membrane of the parietal cells. The gastric proton Recently, we have found that the H+/K+-ATPase inhibitors pump, H+/K+-ATPase, translocates from the cytoplasmic

J. Clin. Biochem. Nutr. H+/K+-ATPase Inhibitors beyond Acid Suppression 3

for binding and interacting with ATP. The β-subunit com- poses a single transmembrane segment, a relatively short amino-terminal cytoplasmic segment and a longer extra- cellular domain including sites for glycosylation and three stabilizing disulfide bonds (Fig. 2). Interactions between the α- and β-subunits occur within the membrane and at the extracellular locus to stabilize functional activity and conformational status of the holoenzyme. Binding of K+ ion at its respective extracellular and intracellular sites is known of influence subunit interaction and protein conformation. The extensive glycosylation through seven N-linked oligo- saccarides may serve an important function in protecting the enzyme from the destructive extracellular environment. Kinetics of the H+/K+-ATPase have defined the reaction steps showing three major conformations, with ion-binding sites facing inward [the E1 conformation], ion sites occluded [the occ conformation], and ion-binding sites facing outward [the E2 conformation]. The transition between these confor- mations is driven by ATP binding, phosphorylation, and dephosphrylation. Transport-catalysis coupling in the gastric + + + H /K -ATPase, in which hydronium ions (H3O ) and MgATP bind to the cytoplasmic face of the enzyme [the E1 Fig. 1. A schematic model illustrating the regulation of gastric conformation], and then the enzyme is phosphorylated, acid secretion The parietal cells. moving the hydronium ion into the membrane domain and the out to the exoplasmic face [the E2 conformation]. After tubulovesicle to the membrane of the secretory canaliculus release of the hydronium ion, in the presence of K+ ion extra- where it is activated. cellularly enabled by the KCl channel in the canalicular membrane, K+ ion binds to the outward conformation of the The gastric acid pump, p-type H+/K+-ATPase phosphorlyated pump. The K+ ion is transported inwardly The gastric acid pump, p-type H+/K+-ATPase, on the during the dephosphorylation step. In the absence of K+ ion, parietal cell consists of two subunits, a 114 kDa α-subunit the pump stops in the E2P conformation [23, 24]. and a 35 kDa β-subunit [21, 22]. The α-subunit containing ATP and cation binding sites carries out the catalytic and Acid suppression through gastric pump inhibitors; PPI and transporting function of the proton pump. The heavily APA glycosylated β-subunit is required for endocytic retrieval of Gastric acid is pathogenic in many gastrointestinal dis- the H+/K+-ATPase from the canalicular membranes and is orders, such as gastroesphageal reflux disease and peptic also essential for protecting proton pump from environment ulcer disease. Treatment for acid-related disease has been of acid milieu. The α-subunit is shown with ten trans- revolutionized by drugs that significantly suppress gastric- membrane segments, a large cytoplasmic molecular mass acid secretion. Histamin-2-receptor antagonists (H2RA)

Fig. 2. Schematic representation of the H+/K+-ATPase heterodimer in the apical membrane of the parietal cell.

Vol. 38, No. 1, 2006 4 M. Yeo et al. have been used extensively for acid suppression for more nist (APA), is a reversible inhibitor of gastric H+/K+-ATPase, than 25 years. Subsequently introduced proton pump inhibi- which competitively binds with the luminal K+ ions to their tors (PPIs) provide more potent acid suppression and greater binding site of the proton pump and dissociates from the clinical efficacy than H2RA. PPIs are considered the drugs enzyme when their blood concentration falls. Since the of choice for acid-related diseases including gastro- inhibitor discriminates the K+ affinity site of H+/K+-ATPase esophageal reflux diseases (GERD) and peptic ulcer dis- from that of Na+/K+-ATPase, it has little effect on Na+/K+- eases. Regimens consisting of a PPI plus 2 or more anti- ATPase. In contrast to the PPIs, they are able to inhibit the microbials are also used for the eradication of Helicobacter pump without acid activation, but because of property of its pylori, a cause of PUD [25, 26]. Five PPIs are approved by week bases, they are accumulated in the canaliculus of the the US food and Drug Aministration (FDA)-osomeprazole, parietal cell. The K+-competitive acid pump antagonists lansoprazole, omeprazole, pantoprazole and rabeprazole such as the SCH28080, BY841, MeDAZIP+, and MDPQ are (Fig. 3A). bind noncovalently and their specific site of attachment is PPIs are substituted 2-pyridyl methyl-sulfiny benzimida- much harder to predict because the region of the protein zoles that share a similar core structure. These agents are that binds K+ or whose conformation prevents K+ binding is protonatable week bases with pKa values of ~4. Therefore not known (Fig. 3B). The binding can be investigated by PPIs accumulate selectively in acidic spaces with a pH <4, mutational analysis or by generating photoaffinity deriva- which are found primarily in the secretory cancliculus of tives. The photoaffinity derivative thereof, MeDAZIP+ was gastric parietal cell. In this acidic environment, protonation shown to be K-competitive and was covalently bound to the of pyridine and benzimidazole nitrogens results in formation TM1/TM2 domain of the H+/K+-ATPase. Substitution in the of a tetracyclic sulfonamide, which represents the active loop between TM1 and TM2 domain of the enzyme did not form of the drug. The sulfenamid binds to exposed cysteine affect the inhibitions, resulting in the conclusion that inhibi- residues in the α-subunit of H+/K+-ATPase to form covalent tion depends on interaction of the inhibitor with the TM1/ disulfide bonds, which inhibit the activity of the pump. TM2 membrane domain itself rather than with connecting Because PPIs bind covalently, the duration of action of these loop. Mutations have also shown that the loop between M5 drugs extends beyond their plasma half life of ~1 to 2 hours. and M6 is an important determinant of binding of Gastric acid secretion is restored by translocation of new SCH28080 and the binding sites for K+ and the inhibitor are H+/K+-ATPase to the secretory cancliculus membrane [27]. different and that competitive kinetics arises from mutual Because the PPIs have a covalent inhibitory action on the exclusion of binding. In contrast to PPIs, the action of APAs proton pump based on their chemistry and the biology of the is independent of the secretory status, and there is no lag parietal cell, there is lag time expected. time expected. Therefore, these are fast-acting compounds Recently another class of compound, acid pump antago- able to abolish acid secretion during their presence in the

Fig. 3. The structures of proton pump inhibitors (PPIs) and acid pump antagonists (APAs) The core structure of all the clinically approved PPIs is timoprazole and K+-competitive APAs showing the core SCH28080 with photoaffinity derivatives.

J. Clin. Biochem. Nutr. H+/K+-ATPase Inhibitors beyond Acid Suppression 5 blood. These drugs will be therapeutic antacids allowing without sustained hypergastrinemia. This inhibitor is com- on-demand dosage, but they will not have the extended petitive with the luminal K+ ions and shows a highly selec- inhibitory characteristics of PPIs. It remains to be seen tive affinity for H+/K+-ATPase, but displays low affinity to whether they will achieve the same healing rates of GERD Na+/K+-ATPase (Fig. 4C). Therefore it is expected that as PPIs although they are predicted to markedly improve the revaprazan would be a promising therapeutic agent for the onset of symptom relief. However, they are not introduced treatment of gastric acid-related disease. into clinical practice, yet. Revaprazan (Revanex®), 5,6-dimethyl-2-(4-fluorophenyl- APA beyond Acid Suppression; Another Potential amino)-4-(1-methyl-1,2,3,4,-tetrahydroisoquinoline-2-yl) Application for Gastric Inflammatory Disease as pyrimidine hydrochloride, is a novel APA that is currently well as Cancer approved by the Korea Food and Drug Administration (KFDA) as new drug for the treatment of gastric diseases As mentioned before, anti-secretory drugs had been including peptic ulcer disease (Fig. 4A and B) [28, 29]. This extensively applied for the treatment of acid-related dis- APA presents a reversible mode of action against H+/K+- eases. Also, regimens consisting of a PPI plus 2 or more ATPase, which is distinct from that of PPIs known for an antimicrobials are also used for the eradication of Helico- irreversible inhibition (Fig. 4D). The characteristics of the bacter pylori. Recently, we found that beside of the anti- drug have great advantages in aspect of inhibition of gastric secretory action, the H+/K+-ATPase inhibitors can regulate acid secretion rapidly as fast on-set action and effectively intracellular signaling pathways to apoptosis as well as

Fig. 4. New developed APA, revaprazan and its characteristics (A) Chemical structure of revaprazan 5,6-dimethyl-2-(4-fluorophenyl- amino)-4-(1-methyl-1,2,3,4,-tetrahydroisoquinoline-2-yl) pyrimidine hydrochloride (B) Inhibitory kinetics of revaprazan on acid pump compared to omeprazole. The advantage of superior action of acid suppression was noted in APA compared to PPI (C) Selectivity of revaprazan on the H+/K+-ATPase, and (D) Reversible mode of action. After washing out of drug, almost complete recovery of proton pump activity was noted in APA, whereas none in PPI treatment, suggesting the reversibility of pump inhibition only in APA, but renewal of parietal cells were required for that or the addition of glutathione.

Vol. 38, No. 1, 2006 6 M. Yeo et al. inflammatory response. Following treatment of panto- EtOH-induced acute gastritis animal model. Pretreatement prazole among PPIs, apoptotic cell death was seen selec- of the APA completely inhibited NSAID (indomethacin) or tively in gastric cancer cells, never seen in same cultured EtOH-induced gastric injury (Fig. 5). The effect of the APA non-cancerous cell lines, accompanied with MAPK ERK1/ might be induced by inhibiting the MAPK or NF-kappa B 2 deactivation. As for these selectivities of PPI’s apoptotic which is an executive critical enzyme in inflammatory activity, normal gastric mucosal cells showed the resistance response signaling. In vitro cell culture system, revaprazan to PPI-induced apoptosis through by the overexpression of has a strong inhibitory activity on Helicobacter pylori- anti-apoptotic regulators including HSP70 and HSP27. In a induced NF-κB transcriptional activation. The suppression xenograft model of nude mice, administration of the PPI of NF-κB by the agent also documented in different damage significantly inhibited tumorigenesis and induced large- factors such as H2O2 and EtOH. MAPK plays a critical role scale apoptosis of tumor cells. These data suggest that in inflammatory response as well as cell proliferation. proton pump inhibitors (PPIs) could be extensively used for Revaprazane like as pantoprazole shows a regulatory role selective anti-cancer effects. in MAPK ERK1/2 signaling provoked by Helicobacter In addition, conditioned media from H. pylori-infected pylori. In additional, this APA has been revealed to cyto- gastric epithelial cells directly induced a tubular formation protect gastric epithelium from cell injury provoked by of HUVEC cells and the increase of in vitro angiogenesis several damages such as alcohol or NSAIDs. Pretreatment of was suppressed by PPI treatment. Infection of H. pylori revaprazole prior to alcohol expose significantly rescued significantly up-regulated expression of HIF-1α and VEGF gastric epithelial cells from the ethanol-induced cell death in gastric epithelial cells and the expression of proangio- (Fig. 6A). The cytoprotective effect of this agent might be genic factors was mediated by MAPK activation and come from the induction of proteins of which function was partially responsible for NF-κB activation. PPI effectively well known as cytoprotection. Twenty five micromole of inhibited the phosphorylation of MAPK ERK1/2 that is a revaprazole stimulates expressions of the cytoprotective principal signal for H. pylori-induced angiogenesis. The fact heat shock proteins like as HO-1, HSP27, and HSP70 as well that PPI could down regulate H. pylori-induced angio- as cytoproliferative proteins like as IL-8, VEGF, and COX-2 genesis shed light on that anti-angiogenic treatment using (Fig. 6B and C). All together, our results shed bright light on PPI could be a promising protective therapeutic approach for speculation that gastric proton pump inhibitors including H. pylori-associated carcinogenesis. APA could be considered as wide spectrum of protective Interestingly, revaprazan (Revanex®), one of the first APA therapeutic approach for inflammatory disease as well as completed trials for clinical use and approved by Korea acid suppression. In addition, since APA could be effective FDA as new remedy for gastric diseases including duodenal so fast in acid suppression, it could be right drug for “on ulcer and gastritis, displayed a potent anti-inflammatory demand therapy” based on the action of therapeutic antacid. action on both the NSAID-induced acute gastritis and the

Fig. 5. Anti-inflammatory effect of revaprazan on both the NSAID-induced acute gastritis (A) and the EtOH-induced acute gastritis animal model (B). Six week-old Sprague-Dawley male rats are administrated with 10 mg/kg revaprazan via oro-gastric tube prior to exposure to either indomethacin (A, 40 mg/kg for 16 hrs) or absolute ethanol (B, 6 ml/kg for 1 hrs). Gross findings showing the gastric mucosal lesions in rat exposed to NSAID or EtOH significantly reduced in rat pre-administrated with revaprazan. Since the major principal actions of mucosal injuries were not related top gastric acidity, that is, direct toxicity, gastric inflammatory mediators, and oxidative stress related, APA might impose the anti-inflammatory and gastroprotective actions as well as acid inhibition.

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Fig. 6. Induction of cytoprotective proteins by revaprazan treatment. (A) Cell death, (B) RT-PCR for VEGF, IL-8, or COX-2 mRNA, and (C) Western blot for detection of induction of heat shock proteins. Rat gastric mucosa RGM-1 cells were incubated in the absence or presence of revaprazan for 16 h prior to expose to either ethanol (A) or NSAIDs (B & C). All of these molecular changes suggested that APA might be equal or superior with well-known gastroprotective drug, Mucosta (rebamipide), based on rescue of mucosal cells from direct toxicity of ethanol and through the inductions of cytoprotective proteins including HO-1, HSP70, HSP27, and VEGF.

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