Inhibitors of the H , K+-ATPase

Omeprazole is a racemate, from which the R- and S-isomers are isolated as reported by Kendall [155]. Both of these isomers convert to the same inhibitor of the H+/K+-ATPase and produce the same reduction in the gastric acid secretion. The S-isomer, esomeprazole, is metabolized more slowly and reproducibly than the R-isomer of omeprazole and therefore produces higher plasma concentrations for longer and, as a result, inhibits gastric acid production more effectively and for longer. Esomeprazole has the pharmacological properties of a more effective form of treatment for disorders related to gastric acid secretion. 

Omeprazole itself is not the active inhibitor of the H+, K+-ATPase, however. Rather, the transformation of omeprazole in acid is required to inhibit the H+, K+-ATPase in vitro and in vivo, whereas intact omeprazole is devoid of inhibitory action. Isolated H+, K+-ATPase is blocked by omeprazole only after the pretreatment of omeprazole with acid. Conversely, neutralization of the acid-secretory canaliculi of isolated gastric gland and parietal cell preparations by permeable buffers, which blocks the acid-catalyzed transformation of omeprazole, prevents inhibition of the enzyme. Furthermore, in-vivo blockade of acid secretion using an H2-receptor antagonist prior to omeprazole administration decreases the inhibitory potency of omeprazole, whereas the stimulation of acid secretion (e.g., by food intake) increases the potency. 

Another program to obtain a reversible proton-pump inhibitor came from workers at SmithKline French, who selected the substituted quinoline compound SK F 96067 as an early clinical candidate (164). SKF 96067 is a reversible inhibitor of the H /K -ATPase protein of the parietal cell (164). In clinical trials SKF 96067 was found to be a more potent inhibitor of gastric acid secretion than the Hz-receptor antagonist ranitidine. This compound reached Phase III clinical trials but has now been discontinued (165). The compound was followed by SKF 97574 (159) that, although of similar potency to that of SKF 96067, displayed a significantly longer duration of action in vivo. 

Rabon E, Sachs G, Bassilian S, et al. A K -competitive fluorescent inhibitor of the H,K-ATPase. J Biol Chem 1991 266 12395-12401. 

As for the Na pump, various cations such as Rb, Cs, NH/ and TV can act as surrogates. As will be seen later, design of competitive inhibitors of the H,K ATPase can take advantage of the surrogate properties of NH . 

The search for specific inhibitors of gastric H,K-ATPase has a dual purpose. First, with the help of suitable inhibitors it is possible to get insight into the molecular mechanisms of H,K-ATPase, and second, a specific inhibitor might be clinically useful for inhibition of gastric acid secretion in anti-ulcer therapy. 

As the H+, K+-ATPase inhibition is associated with the modification of mer-capto groups in the enzyme, the disulfide adduct (ESSR) can be considered as a model of the enzyme-inhibitor complex, and the sulfenamide, or possibly the sulfenic acid (C), formed from omeprazole can be considered to be the active inhibitor, which binds covalently to cysteine residues of the H+, K+-ATPase. 

There are currently four racemic PPIs available on the market omeprazole, lansoprazole, pantoprazole, and rabeprazole. (More recently, enantiomerically pure versions have also been studied and developed, e.g., S-omeprazole, marketed by AstraZeneca as esomeprazole see Chapter II-2.) Proton pump inhibitors share the same core structure, the substituted pyridylmethyl-sulfmyl-benzimidazole, but differ in terms of substituents on this core structure. The absolute requirements of the core structure for the activity of PPIs was not understood until it became clear that the active PPIs are derived from inactive prodrugs the prodrugs are transformed, in the acid-secreting parietal cells, by a unique cascade of chemical structural transformations leading to the active principle, a cyclic sulfenamide species. Inhibition of acid secretion in turn is then achieved by formation of covalent disulfide bonds with key cysteines of the (H+/K+)-ATPase. 

Systematic synthesis was carried out by Smith Khne French to identify freely reversible, noncovalent inhibitors of gastric H /K -ATPase with a mode of action comparable with SCH 28080. It was expected that gastric HVK -ATPase inhibitors with a shorter duration of action could be of therapeutic interest [155, 156]. This research resulted in the selection of compound SK F 96067 [157] Figure 4.10). 

Proton pump inhibitors inhibit the H/K-ATPase (proton pump) in oxyntic cells of the stomach, the final common pathway in the secretion of gastric acid in response to a variety of stimuli, such as gastrin and histamine. Their use in acid-related disorders has been extensively reviewed (1-3). 

Omeprazole belongs to a new class of antisecretory compounds, the snbstitnted benzimidazoles, that do not exhibit anticholinergic or H2-histamine-antagonistic properties bnt suppress gastric acid secretion by specific inhibition of the H+/K+ ATPase enzyme system at the secretory snrface of the gastric parietal cell. Because this enzyme system is the acid (proton) pump within the gastric mucosa, omeprazole has been characterized as a gastric acid pump inhibitor it blocks the final step of acid prodnction. This effect is dose-related, and inhibits both basal and stimnlated acid secretion irrespective of the stimnlns. 

FIGURE 36-2 Proton pump inhibitors. A. Inhibitors of gastric H , K -ATPase (proton pump). B. Conversion of omeprazole to a sulfenamide in the acidic secretory canaliculi of the parietal cell. The sulfenamide interacts covalently with sulfhydryl groups in the proton pump, thereby irreversibly inhibiting its activity. The other three proton pump inhibitors undergo analogous conversions. 

The concept that drove the development of alternatives to Hz receptor antagonists was the recognition that these would have limited efficacy given the multiplicity of secretory inhibitors. It seemed that inhibition of the pump itself would be a more effective way of controlling acid secretion. Work toward this end began at SK F in Philadelphia but was terminated with the launch of dmetidine. As will be discussed in the section on inhibition of the H,K ATPase, the synthesis of timoprazole heralded not only a novel therapeutic approach to... 

Analysis of their mechanism of action showed that the protonated form of an inhibitor such as SCH28080 was more effective, and that their reaction was with the external surface of the enzyme. This conclusion was reached because the quaternary form generated by methylation of the pyridine N of SCH28080 was ineffective in intact right-side-out vesicles of the H,K ATPase also, their K, decreased with decreasing pH. 

Biosynthesis of the two subunits of the ATPase takes place in the endoplasmic reticulum of the parietal cell. It appears that there is a need for coassembly of the two pump subunits for stabilization of the a subunit and for targeting of the dimer to the post-Golgi membrane compartment and the plasma membrane. The rate of synthesis of the H,K ATPase is directly relevant to the duration of action of proton pump inhibitors (PPIs). 

Figure 9. A schematic illustration of the regions of association between the a and P subunits of the H,K ATPase of gastric mucosa and the positions of the cysteines in the a sequence accessed by various proton pump inhibitors.

All these experiments clearly indicate that acid-catalyzed conversion of omeprazole to an active inhibitor is a necessary prerequisite for inhibition of the H K -ATPase. 

In this chapter we will review the recent investigations of the structure of both the a and P subunit, and the function of gastric H,K-ATPase. We will proceed from a brief overview of the tissue distribution to a successive discussion of structure, kinetics, transport properties, lipid dependency, solubilization and reconstitution, and inhibitors of H,K-ATPase that may label functionally important domains of the enzyme. 

ATPase also catalyzed a passive Rb -Rb exchange, the rate of which was comparable to the rate of active Rb efflux. This suggested that the K-transporting step of H,K-ATPase is not severely limited by a K -occluded enzyme form, as was observed for Na,K-ATPase. Skrabanja et al. [164] also described the reconstitution of choleate solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. With the use of a pH electrode to measure the rate of H transport they observed not only an active transport, which is dependent on intravesicular K, but also a passive H exchange. This passive transport process, which exhibited a maximal rate of 5% of the active transport process, could be inhibited by vanadate and the specific inhibitor omeprazole, giving evidence that it is a function of gastric H,K-ATPase. The same authors demonstrated, by separation of non-incorporated H,K-ATPase from reconstituted H,K-ATPase on a sucrose gradient, that H,K-ATPase transports two protons and two ions per hydrolyzed ATP [112]. 

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