The effects of varying acidity on Helicobacter pylori growth and the bactericidal efficacy of ampicillin.

BACKGROUND: Penicillins inhibit cell wall synthesis; therefore, Helicobacter pylori must be dividing for this class of antibiotics to be effective in eradication therapy. Identifying growth responses to varying medium pH may allow design of more effective treatment regimens. AIM: To determine the effects of acidity on bacterial growth and the bactericidal efficacy of ampicillin. METHODS: H. pylori were incubated in dialysis chambers suspended in 1.5-L of media at various pHs with 5 mM urea, with or without ampicillin, for 4, 8 or 16 h, thus mimicking unbuffered gastric juice. Changes in gene expression, viability and survival were determined. RESULTS: At pH 3.0, but not at pH 4.5 or 7.4, there was decreased expression of ~400 genes, including many cell envelope biosynthesis, cell division and penicillin-binding protein genes. Ampicillin was bactericidal at pH 4.5 and 7.4, but not at pH 3.0. CONCLUSIONS: Ampicillin is bactericidal at pH 4.5 and 7.4, but not at pH 3.0, due to decreased expression of cell envelope and division genes with loss of cell division at pH 3.0. Therefore, at pH 3.0, the likely pH at the gastric surface, the bacteria are nondividing and persist with ampicillin treatment. A more effective inhibitor of acid secretion that maintains gastric pH near neutrality for 24 h/day should enhance the efficacy of amoxicillin, improving triple therapy and likely even allowing dual amoxicillin-based therapy for H. pylori eradication.

Effects of TAK-637, a novel neurokinin-1 receptor antagonist, on colonic function in vivo.

Substance P (SP) is an important neurotransmitter that mediates various gut functions; however, its precise pathophysiological role remains unclear. In this study, we investigated the effect of SP on colonic function and the effect of TAK-637 [(aR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]naphthyridine-6,13-dione] a new neurokinin-1 (NK1) receptor antagonist, on colonic responses to SP or stress in Mongolian gerbils. SP and the selective NK1 agonist [pGlu6]SP6-11 significantly increased fecal pellet output. TAK-637 reduced [pGlu6]SP6-11-induced defecation, but did not significantly affect neurokinin A-, 5-hydroxytryptamine- or carbachol-stimulated defecation. Oral TAK-637 decreased restraint stress-stimulated fecal pellet output with an ID50 value of 0.33 mg/kg. Ondansetron and atropine, but not the peripheral kappa-receptor agonist trimebutine, also reduced restraint stress-stimulated defecation. TAK-637 inhibited the increase in fecal pellet output stimulated by intracerebroventricular injection of corticotropin-releasing factor, but did not affect the stress-induced increase in plasma adrenocorticotropic hormone levels. Denervation of the sensory neurons with capsaicin did not affect stress-stimulated defecation. These results suggest that NK1 receptors in the enteric plexus play an important role in stress-induced changes in colonic function, and that TAK-637 may be useful in the treatment of functional bowel diseases such as irritable bowel syndrome.

EM574, an erythromycin derivative, improves delayed gastric emptying of semi-solid meals in conscious dogs.

The gastroprokinetic effects of de(N-methyl)-N-isopropyl-8, 9-anhydroerythromycin A 6,9-hemiacetal (EM574), a non-peptide motilin receptor agonist, were investigated in conscious dogs in a normal state and with experimentally-induced gastroparesis. Gastric emptying of semi-solid meals was assessed indirectly from acetaminophen absorption with simultaneous recording of gastric antral motility. In the normal state, post-prandial intraduodenal administration of EM574 (0.03 mg/kg) [corrected] stimulated antral motility and significantly enhanced gastric emptying as potently as did intravenous porcine motilin (0.003 mg/kg/h). Intraduodenal cisapride at 1 mg/kg denal cisapride at 1 mg/kg elicited antral contractions and tended to accelerate gastric emptying but at 3 mg/kg, gastric emptying was not enhanced despite a further increase in the motor index. In dogs with gastroparesis induced by intraduodenal oleic acid or intravenous dopamine, EM574 (0.03 mg/kg) increased antral motility and reversed the delayed gastric emptying completely. Cisapride (1 mg/kg) partially ameliorated the impaired emptying under these conditions. In atropinized dogs, no acceleration of gastric emptying by EM574 was observed. These results indicate that EM574 potently accelerates gastric emptying of caloric meals in dogs in a normal state and with experimentally-induced gastroparesis, and also suggest that the effect is mediated through stimulation of a cholinergic neural pathway.

EM574, an erythromycin derivative, is a motilin receptor agonist in the rabbit.

This study was performed to examine whether an erythromycin derivative, de(N-methyl)-N-isopropyl-8,9-anhydroerythromycin A 6,9-hemiacetal (EM574) is a motilin receptor agonist in the rabbit gastrointestinal tract. EM574 and porcine motilin induced contractions in segments of isolated rabbit intestine with pEC50 values of 8.26 +/- 0.04 and 8.69 +/- 0.07, respectively, but not in rat or guinea pig preparations. The sensitivity and efficacy of the response to both compounds in rabbits decreased aborally and was insensitive to pretreatment with atropine or tetrodotoxin, but was markedly suppressed under Ca(2+)-free conditions. EM574 and porcine motilin specifically displaced [125I-Tyr23]canine motilin bound to gastric antral smooth muscle homogenates with plC50 values of 8.21 +/- 0.13 and 9.20 +/- 0.11, respectively. The pEC50 value for the contractile response and plC50 value for the receptor binding for motilin, EM574, erythromycin A and three other derivatives correlated well (r = 0.94, P < 0.01). Tissue section autoradiography in the antrum revealed that specific labeled motilin binding sites were localized in the circular muscle layer and myenteric plexus, and could be reduced in the presence of an excess of EM574. These results indicate that EM574 is a potent motilin receptor agonist in the rabbit gastrointestinal tract.

Role of endogenous basic fibroblast growth factor in the healing of gastric ulcers in rats.

Recently, it has been pointed out that growth factors play an important role in the healing of gastrointestinal ulcers. In the present study, we examined the role of endogenous basic fibroblast growth factor (bFGF) in the healing of gastric ulcers in the rat. In male SD rats, gastric ulcers were induced in the antrum by injection of acetic acid. Time-dependent changes in the area and bFGF content in the ulcerated area and distribution of bFGF in the ulcerated mucosa were examined. Effects of bFGF mutein CS23 (TGP-580) and a monoclonal antibody for bFGF (MAb 3H3) on the healing of the gastric ulcers and angiogenesis in the ulcer bed were also examined. The content of bFGF in the ulcerated area increased with time as the ulcer healed and reached a maximum 7 days after ulcer formation. In the gastric ulcer bed, many cells such as fibroblasts and macrophages were positively stained immunohistochemically by anti-bFGF antiserum. MAb 3H3 (0.1 mg/rat/day, i.v.) inhibited angiogenesis in the ulcer bed and significantly delayed ulcer healing, while TGP-580 (0.001-0.1 mg/kg x 2/day, p.o.) increased the number of microvessels in the ulcer bed and accelerated the healing. These results suggest that endogenous bFGF may play an important role in the healing of gastric ulcers in the rat and that the angiogenic properties of bFGF (TGP-580) may be involved in its effect on ulcer healing.

[Effects of intravenous lansoprazole on acute gastric mucosal lesions and acid secretion].

The effects of lansoprazole given intravenously on gastric mucosal lesions, gastric bleeding and acid secretion were investigated in rats in comparison with those of omeprazole, famotidine and ranitidine. Lansoprazole inhibited the formation of gastric mucosal lesions in rats induced by water-immersion stress or aspirin with ID50 values of 0.26 and 0.99 mg/kg, respectively, and also inhibited gastric bleeding induced by hemorrhagic shock or water-immersion stress with ID50 values of 0.46 and 1.22 mg/kg, respectively. Lansoprazole was more potent than omeprazole, famotidine and ranitidine in inhibiting gastric mucosal lesions and hemorrhagic shock- or stress-induced bleeding. Famotidine and ranitidine showed negligible inhibition of water-immersion stress-induced gastric bleeding. Lansoprazole strongly inhibited water-immersion stress-stimulated acid secretion in rats, whereas famotidine and ranitidine did not show a potent inhibitory effect. These results indicate that lansoprazole exerts prominent inhibitory actions against the formation of gastric mucosal lesions and gastric bleeding by inhibiting acid secretion, and they show that it is superior to histamine H2-receptor antagonists in inhibiting stress-induced gastric bleeding.

[Effects of lansoprazole on indomethacin-induced gastric bleeding and mucosal lesions in rats].

The effects of lansoprazole given intravenously on indomethacin-induced gastric bleeding and mucosal lesions were investigated in rats in comparison with those of omeprazole, famotidine and ranitidine. Lansoprazole inhibited gastric bleeding induced by indomethacin with an ID50 value of 0.29 mg/kg. Omeprazole and famotidine significantly inhibited gastric bleeding, but ranitidine provided negligible inhibition. A correlation was found between the inhibitory action of lansoprazole on gastric bleeding, and acid secretion, and its inhibitory action on gastric bleeding was almost completely abolished by adding 50 mM-HCl to the gastric perfusate, suggesting that lansoprazole's inhibitory action on gastric bleeding was mainly due to its antisecretory action. Lansoprazole inhibited the development of gastric lesions induced by indomethacin with an ID50 value of 0.10 mg/kg, whereas histamine H2-receptor antagonists did not display a potent inhibitory effect. ID50 values for omeprazole, famotidine and ranitidine were 0.69, 2.58 and 24.6 mg/kg, respectively. These results indicate that lansoprazole has a potent inhibitory action on indomethacin-induced gastric bleeding and mucosal lesions and that it is useful in the treatment of acute gastric mucosal lesions.

Bioactive metabolites of EM574 and EM523, erythromycin derivatives having strong gastrointestinal motor stimulating activity.

Two motilides, EM574 (N-demethyl-N-isopropyl-8,9-anhydroerythromycin A 6,9-hemiacetal) and EM523 (N-demethyl-N-ethyl-8,9-anhydroerythromycin A 6,9-hemiacetal) have strong gastrointestinal motor stimulating (GMS) activity. When administered orally to dogs, these agents showed strong GMS activity, but their plasma levels were very low and the metabolites which have been determined so far using the radio-labeled compounds show only weak GMS activity. The findings suggested that unknown bioactive metabolites might be responsible for the GMS activity. From the liver of dogs given EM574 intravenously, two bioactive metabolites, EM574 P1 and P2, were isolated by solvent extraction and chromatography as detected by contractile activity. They both showed the same UV spectra as EM574 and the molecular ion peaks at m/z 760 (MH+) and 602 (MH(+)-cladinose) in the FAB-MS. From 2D-NMR experiments, the structures of EM574 P1 and P2 were unveiled to be the 15- and 14-hydroxyl derivatives of EM574, respectively. EM523 P1 and P2 were also isolated in the same procedure. In order to prepare these bioactive metabolites, EM574 and EM523 were converted enzymatically with dog liver homogenates in the presence of co-enzymes to give the corresponding P1 and P2. The structures of the metabolites are shown in Fig 1. They exhibited stronger contractile activity in vitro and GMS activity in vivo than the parent compounds.

Microbial conversion of EM574 and EM523, gastrointestinal motor stimulating agents.

EM574 exerts gastrointestinal motor stimulating (GMS) activity even after being converted to its metabolites P1 and P2 in dogs. These metabolites were isolated from dog liver using a series of chromatographic procedures. Their structures were determined to be the 15- and 14-hydroxyl derivatives of EM574, respectively, by spectral analysis. Large scale preparation by microbial transformation was investigated for further evaluation of the metabolites, because the amounts obtained by oxidation with dog liver homogenate were limited. Three strains of actinomycetes, Amycolatopsis tolypophorus IFO 13151, Dactylosporangium variesporum IFO 14104 and Nocardia capreola IFO 12847, were found to have the aiming oxidative potency. HPLC analysis of the crude extracts from these three cultures showed that the bioactive metabolites, EM574 P1 and P2 were produced. They were isolated from the culture broth with the other bioactive products EM574 P3 and P4. These bioactive products were prepared by large scale cultivation. EM574 P3 and P4 showed GMS activity comparable to that of EM574 P1 and P2. The structures of EM574 P3 and P4 were elucidated by spectral analysis and found to be the 3"-O-demethyl derivatives of EM574 P2 and EM574, respectively. Moreover, the absolute configuration at the C14 position of P2 was determined to be R by spectral analysis of the 6-membered cyclic carbonate of EM574 P2.

Vagus-dependent and vagus-independent mechanisms of action of the erythromycin derivative EM574 and motilin in dogs.

The motor-stimulating action of de(N-methyl)-N-isopropyl-8,9-anhydroerythromycin A 6,9-hemiacetal (EM574) on the upper gastrointestinal tract was studied in fasted conscious dogs using chronically implanted force transducers and compared with those of porcine motilin and cisapride. EM574 induced gastric phase III-like migrating contractions and increased the plasma motilin levels slightly. The gastric motility induced by low doses of EM574 and motilin was abolished by a 5HT3-receptor antagonist ondansetron and acute vagal blockade, whereas under these conditions, high doses of both agents induced contractions, which were abolished by atropine. Cisapride-induced gastric motility was inhibited by atropine and acute vagal blockade, but not by ondansetron. EM574 did not stimulate gastric secretion in the basal state. These results indicate that EM574- and motilin-induced gastrointestinal motility is attributable mainly to motor-stimulating vagal cholinergic neurons, and 5HT3-receptors are probably involved in the process. At high doses, EM574 and motilin also appear to stimulate cholinergic neurons in a non-vagal pathway, probably the enteric nervous system.

Stimulatory mechanism of EM523-induced contractions in postprandial stomach of conscious dogs.

BACKGROUND & AIMS: EM523, a motilin agonist, is intended to be used as a gastroprokinetic during the postprandial period, but the mechanism(s) by which EM523 stimulates postprandial contractions in the stomach has not been studied before. The aim of this study was to examine the mechanism of contraction-stimulating activity by EM523 in fed dogs. METHODS: Contractile activity in the gastric antrum of 5 dogs was monitored using a long-term implanted force transducer and measured by integrating the area under the curve. Test materials were continuously infused or injected intravenously. RESULTS: EM523 (1-30 micrograms/kg) induced a dose-dependent increase in fed-type contractions. EM523-induced contractile activity was partially inhibited by atropine, hexamethonium, dopamine, 5-hydroxytryptamine 3 (5-HT3) receptor antagonist, and substance P antagonist. Atropine-resistant and EM523-induced contractions were further inhibited by 5-HT3 receptor antagonist and substance P antagonist, and the combined use of the two antagonists completely eliminated the atropine-resistant and EM523-induced contractions. CONCLUSIONS: EM523-induced contractions in the fed stomach are quite different from phase III contractions in the fasted state and are mediated partially through the cholinergic pathway. The noncholinergic pathway involves 5-HT3 and neurokinin 1 receptors.

Comparison of the motor-stimulating action of EM523, an erythromycin derivative, and prostaglandin F2 alpha in conscious dogs.

The effect of EM523 [de(N-methyl)-N-ethyl-8,9-anhydroerythromycin A 6,9-hemiacetal], an erythromycin derivative, on gastrointestinal motility was investigated using conscious dogs in the fasting state, and it was compared with those of motilin and prostaglandin F2 alpha (PGF2 alpha). EM523 and motilin given as i.v. infusions induced strong contractions in the stomach that migrated along the intestine. On the other hand, PGF2 alpha stimulated intestinal contractions, but its effect on gastric motility was weak. EM523 had 1/50 the potency of motilin and 6 times the potency of PGF2 alpha for stimulation of intestinal motility. Atropine at 0.1 mg/kg, i.v. strongly inhibited gastrointestinal contractions induced by EM52 EM523 or motilin and partly inhibited PGF2 alpha-induced intestinal motility. ICS-205-930, a 5HT3-receptor antagonist, at a dose of 1 mg/kg, i.v. strongly inhibited EM523 or motilin-induced gastric contractions but did not affect the action of PGF2 alpha. Infusion of EM523 at 100 micrograms/kg/hr induced strong migrating contractions even when motility was depressed by dopamine infusion or laparotomy. Infusion of PGF2 alpha at 300 micrograms/kg/hr stimulated intestinal but not gastric motility under these conditions. The results of this study indicate that the cholinergic pathway and 5HT3 receptors are involved in EM523 and motilin-induced migrating gastrointestinal contractions, whereas the cholinergic pathway seems to be only partly involved in PGF2 alpha-induced intestinal contractions.

Effects of the enantiomers of lansoprazole (AG-1749) on (H+ + K+)-ATPase activity in canine gastric microsomes and acid formation in isolated canine parietal cells.

The effects of the enantiomers of 2-[[[3-methyl-4-(2,2,2-trifluoroethoxy)-2-pyridyl]methyl]-sulfinyl ]- 1H-benzimidazole (lansoprazole, AG-1749) on acid formation in isolated canine parietal cells and (H+ + K+)-ATPase activity in canine gastric microsomes were investigated. Both the (+)-and the (-)-enantiomer of lansoprazole inhibited the acid formation stimulated by dibutyryl cyclic AMP (db-cAMP) in isolated canine parietal cells in a concentration-dependent manner with IC50 values of 59 and 82 nM, respectively. The enantiomers showed concentration-dependent inhibition of (H+ + K+)-ATPase with IC50 values of 4.2 and 5.2 microM, respectively. On the other hand, the IC50 values of lansoprazole for db-cAMP-stimulated acid formation and (H+ + K+)-ATPase were 59 nM and 2.1 microM, respectively. These results suggest that the two enantiomers of lansoprazole have antisecretory action due to inhibition of (H+ + K+)-ATPase.

Differences in the antisecretory actions of the proton pump inhibitor AG-1749 (lansoprazole) and the histamine H2-receptor antagonist famotidine in rats and dogs.

Antisecretory effects of a substituted benzimidazole, (+/-)-2-[[[3-methyl-4-(2,2,2-trifluoroethoxy)-2-pyridyl]methyl] sulfinyl]-1H-benzimidazole (AG-1749) were compared with those of a histamine H2-receptor antagonist, famotidine. AG-1749 inhibited acid formation regardless of the stimulant in isolated canine parietal cells, while famotidine inhibited the histamine-stimulated acid formation selectively. In pylorus-ligated rats, AG-1749 suppressed basal acid secretion, histamine-, bethanechol-, pentagastrin-, 2-deoxy-D-glucose- and stress (restraint and water-immersion)-induced acid secretion; ID50 values were 1.0-6.0 mg/kg. On the other hand, famotidine only partially inhibited the acid secretion induced by 2-deoxy-D-glucose or stress, although it suppressed the acid secretion stimulated by other secretagogues several times more potently than AG-1749. The antisecretory effect of AG-1749 lasted longer than that of famotidine, especially in the case of bethanechol-stimulated acid secretion. In Heidenhain pouch dogs, both AG-1749 and famotidine potently inhibited histamine-, bethanechol-, pentagastrin- and peptone meal-stimulated acid secretion, but the inhibitory effect of famotidine was short-lived in the case of bethanechol- and pentagastrin-stimulated acid secretion. These results suggest that AG-1749 persistently inhibits acid secretion induced by both peripheral and central stimuli and suggest that the antisecretory effect of famotidine depends on the nature of the stimuli.

Effects of a proton pump inhibitor, AG-1749 (lansoprazole), on reflux esophagitis and experimental ulcers in rats.

The effects of (+/-)-2-[[[3-methyl-4-(2,2,2-trifluoroethoxy-2- pyridyl]methyl]sulfinyl]-1H-benzimidazole (lansoprazole, AG-1749) and famotidine on various experimental ulcers in rats were compared. AG-1749 inhibited reflux esophagitis; gastric lesions induced by water-immersion stress, aspirin or ethanol; and duodenal ulcers induced by cysteamine or mepirizole in a dose-dependent manner: the ID50 values were 0.7, 2.4, 0.7, 8.5, 1.1 and 0.3 mg/kg, p.o. or i.d., respectively. Famotidine inhibited reflux esophagitis with an ID50 value of 12.9 mg/kg, but did not cause 50% inhibition of ethanol-induced gastric lesions even at 100 mg/kg, although it showed almost the same or a little stronger potency on other experimental ulcers: ID50 values were 0.3-1.4 mg/kg. Significant aggravation of ethanol- or water-immersion stress-induced lesions was observed in rats given famotidine at 30 mg/kg twice daily for 4 days, but not in rats given AG-1749 at 10 mg/kg twice daily. Administration of AG-1749 for 14 consecutive days markedly accelerated the healing of acetic acid-induced gastric and duodenal ulcers, and the healing effect was significant at 10 and 30 mg/kg/day, p.o. Famotidine also accelerated the healing of ulcers, but its potency was less than that of AG-1749. The results of this study indicate that although AG-1749 is slightly less potent than famotidine in inhibiting acutely induced gastroduodenal lesions, this agent is superior to famotidine in promoting the healing of ulcers and in inhibiting reflux esophagitis and ethanol-induced gastric lesions.

EM-523, an erythromycin derivative, and motilin show similar contractile activity in isolated rabbit intestine.

The effect of EM-523 [de(N-methyl)-N-ethyl-8,9-anhydroerythromycin A 6,9-hemiacetal], an erythromycin derivative, on preparations of isolated intestine of rabbits, rats and guinea pigs was investigated and compared to the effects of motilin and prostaglandin F2 alpha (PGF2 alpha). EM-523 and motilin induced contractions in the rabbit intestinal preparations but not in the rat or guinea pig preparations. In contrast, PGF2 alpha induced contractions in all three intestinal preparations. The rabbit duodenum and jejunum were more sensitive than the ileum to EM-523 and motilin, but no selectivity was observed with PGF2 alpha. The contractile responses induced by EM-523 and by motilin were not influenced by pretreatment with tetrodotoxin, atropine, naloxone or mepyramine but were greatly suppressed by pretreatment with verapamil or removal of calcium ions from the medium, suggesting that both agents induce intestinal contractions by acting directly on smooth muscle and that their actions depend largely on extracellular calcium. The contractile response to EM-523 or motilin was reduced markedly after treatment of the preparation with a high concentration of either agent, and cross-tachyphylaxis between EM-523 and motilin was also observed. These findings indicate that the contractile activity of EM-523 is very similar to that of motilin and that EM-523 and motilin may share the same site of action.

Ethanol oxidation by deermice mitochondria under physiologic conditions.

Mitochondria obtained from alcohol dehydrogenase-positive or - negative deermice do not oxidize significant amounts of ethanol at pH 7.4. A slight activity, equivalent to less than 0.3% of the elimination rate in alcohol dehydrogenase-negative deermice was observed at pH 10; it was strongly inhibited by cyanide and thiourea, and was not dependent on exogenous NAD. Whereas ethanol oxidation by the cytosol of alcohol dehydrogenase-positive deermice was time-dependent, that of mitochondria from alcohol dehydrogenase-negative deermice was not. These findings indicate that deermice mitochondria do not oxidize ethanol at physiological pH, and that the mitochondrial system is not likely to play a significant physiologic role.