Departments of 1 Gastroenterology and 4 Host Defense, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585; 2 Research Institute for Traditional Sino-Japanese Medicines, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-0194; 3 Department of Health and Epidemiology, Osaka City Institute of Public Health and Environmental Sciences, 8-34 Tohjo-cho, Tennoji-ku, Osaka 543-0026, Japan
Received 14 February 2002; returned 30 April 2002; revised 6 June 2002; accepted 21 June 2002
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Abstract |
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Keywords: H. pylori, alkyl methyl quinolones, eradication, respiration
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Introduction |
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Our previous studies demonstrated that, of numerous medical plants and spices, Gosyuyu, a crude extract from the fruit of Evodia rutaecarpa (E. rutaecarpa) and one component of Gosyuyu-to, a Chinese herbal medicine, had potent antibacterial activity against H. pylori in vitro, and that combination therapy including Gosyuyu-to, omeprazole and amoxicillin gave a better clinical eradication rate than dual therapy, without any significant adverse effects.9,10 In a previous study,11 we found that novel alkyl methyl quinolone alkaloids (AM quinolones): 1-methyl-2-[(Z)-8-tridecenyl]-4-(1H)-quinolone and 1-methyl-2-[(Z)-7-tridecenyl]-4-(1H)-quinolone (Figure 1) were the strongest and highly selective antibacterial components of Gosyuyu against H. pylori. Their in vitro antibacterial activity against H. pylori was better than or similar to those of amoxicillin and clarithromycin. AM quinolones as well as amoxicillin or clarithromycin may thus be useful for the treatment of H. pylori infection. However, not only the antibacterial mechanism(s) but also the in vivo efficacy of AM quinolones are still unknown.
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Materials and methods |
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We used the standard H. pylori strain (ATCC 43504). H. pylori was grown on Brucella agar (Becton Dickinson Microbiological Systems, Franklin Lakes, NJ, USA) supplemented with 5% final concentration defibrinated horse blood (blood agar), or with bovine serum albumin (BSA) fraction V (A-4503; Sigma Chemical Co., St Louis, MO, USA) sterilized using a 0.2 µm filter at 0.5 mg/mL (albumin agar), with 0.1% final concentration ß-cyclodextrin (Kishida Chemicals Co., Osaka, Japan).11 Cells were cultured for 2 or 3 days at 37°C in a glove box (Forma Scientific Anaerobic System Model 1024; Rankin Biomedical Co., Clarkston, MI, USA) under humidified microaerophilic conditions including 80% N2, 15% CO2 and 5% O2. The colonies of H. pylori were suspended in Brucella broth including BSA. At the time of harvest, the bacteria had grown to an OD550nm of 0.2, which corresponded to a viable cell concentration of 1 x 108 cfu/mL, and were in the early logarithmic growth phase, with good motility. The conditioned cells were used in the experiments described below.
Drug samples
Extraction and purification of the agents from the fruit of E. rutaecarpa purchased from Tsumura Company Ltd (Ibaraki, Japan) was carried out as described previously.11 In brief, the fruit of E. rutaecarpa was extracted twice with diethyl ether (300 mL x 2) under reflux for 2 h. The extracts were evaporated under reduced pressure. The diethyl ether extracts were separated into several fractions by preparative thin-layer chromatography (PTLC) with dichloromethane (first PTLC) as the solvent and then rechromatographed (second PTLC) with methanol:chloroform (2.5:97.5, v/v). Each fraction was detected by ultraviolet (253.7 nm) irradiation. Purification was repeated until a single spot was obtained on TLC. The compounds (AM quinolones) of 1-methyl-2-[(Z)-8-tridecenyl]-4-(1H)-quinolone and 1-methyl-2-[(Z)-7-tridecenyl]-4-(1H)-quinolone, which could not be separated from each other, were obtained; we used the compounds (AM quinolones) in the subsequent studies.
Effects of AM quinolones on respiration of H. pylori
AM quinolones were added to Brucella broth containing H. pylori (1 x 108 cfu) at a dose of 0, 0.01, 0.1 or 1 mg/L and incubated. During incubation, oxygen consumption by H. pylori was monitored polarographically using a Clark-type oxygen electrode fitted to a 2 mL water-jacketed closed chamber at 37°C.12 The same experiments were performed repeatedly to confirm reproducibility.
Testing for eradication of H. pylori by AM quinolones in the gastric tissues of H. pylori-inoculated Mongolian gerbils
Mongolian gerbils (6 weeks old, male, 4050 g) were purchased from Seac Yoshitomi Ltd (Fukuoka, Japan). Four weeks after H. pylori (1 x 108 cfu) inoculation, H. pylori-inoculated Mongolian gerbils were orally administered AM quinolones alone (2, 10 or 20 mg/kg), combined AM quinolones with omeprazole (30 mg/kg/day) or vehicle alone for 7 days. After 1 week of treatment, the stomach of each animal was excised and homogenized in Brucella broth containing BSA. Aliquots of dilutions of the homogenates were harvested on Brucella agar plates containing 5% horse blood at 37°C under microaerophilic conditions for 5 days. The number of colonies was counted and viable H. pylori were expressed as cfu/stomach. All experimental procedures were approved by the Animal Care Committee of Osaka City University Medical School.
Measurement of myeloperoxidase activity
MPO activity, a marker of neutrophil infiltration, of the gastric antral tissue was assayed by the method of Bradley et al.13 In brief, the gastric tissues were homogenized in 50 mM potassium phosphate buffer pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide (Sigma Chemical Co.). Suspensions were then centrifuged and MPO in the resulting supernatant was assayed with a spectrophotometer (Beckman Instruments, Fullerton, CA, USA). One unit of MPO activity was defined as that degrading 1 µmol of peroxidase per minute at 25°C. Proteins were measured with a modified bicinchonic acid method with a BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Results are expressed as units per mg protein.
Statistical analysis
All values are presented as mean ± S.E.M. Statistical significance was determined by two-way ANOVA, followed by the least-squares means test. Differences were considered significant at P < 0.05.
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Results |
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Effects of AM quinolones on respiration of H. pylori
AM quinolones inhibited the respiration of H. pylori in a dose-dependent manner: O2 consumption rates were 4.66 ± 0.21, 3.80 ± 0.09, 2.15 ± 0.05 and 2.12 ± 0.03 x 107 µmol/min/body for control, 0.01, 0.1 and 1 mg/L (20 x MIC noted above for H. pylori strain ATCC 43504) (Figure 2). However, AM quinolones had no effect on [3H]thymidine uptake into H. pylori (data not shown). High doses (0.1 and 1 mg/L) but not a low dose (0.01 mg/L (lower than the MIC noted above) of AM quinolones decreased the number of H. pylori over 2472 h. However, AM quinolones had no cytotoxic effect on gastric epithelial cells (MKN-45) (data not shown). The target of the agents in vitro was suggested to be only H. pylori and not gastric epithelial cells.
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After 1 week of treatment, the number of viable H. pylori in gastric tissues of inoculated animals decreased significantly (AM quinolones alone, at 2, 10 or 20 mg/kg/day: 46.0 ± 22.6 x 104, 17.3 ± 4.9 x 104 and 8.1 ± 6.6 x 104 cfu/stomach, respectively; combined AM quinolones at 2, 10 or 20 mg/kg/day with omeprazole at 30 mg/kg/day: 8.0 ± 5.6 x 104, 4.2 ± 2.5 x 104 and 5.5 ± 2.7 x 104 cfu/stomach, respectively) compared with the vehicle-treated group (control: 359.9 ± 180.3 x 104 cfu/stomach) (Table 1). During treatment, the agents caused no serious adverse effects such as diarrhoea, loss of appetite or body weight, and did not affect animal mortality. The presence of H. pylori in gastric tissues of treated Mongolian gerbils was histologically confirmed, and the scores of the presence of H. pylori by the updated Sydney System15 were consistent with the above-mentioned number of viable H. pylori in the gastric tissues.
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MPO activity of the H. pylori-infected gastric antral tissue with the vehicle treatment group (control) was 47.3 ± 10.8 units/mg protein (Figure 3). After 1 week of treatment with high-dose AM quinolones (20 mg/kg), MPO activity of the gastric tissue had decreased by 87.2%. In addition, combined AM quinolones (2, 10 or 20 mg/kg) and omeprazole treatment inhibited MPO activity by 83.1%, 69.0% or 80.3%, respectively. According to the updated Sydney System,15 histopathological grading for neutrophil infiltration was consistent with MPO activity in the gastric tissue of treated Mongolian gerbils.
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Discussion |
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Recent accumulating studies have yielded much knowledge of the basic metabolic pathways of H. pylori: the source of energy for H. pylori are metabolites of organic acids and amino acids obtained via the Krebs cycle; this energy is obtained by respiration; and H. pylori includes menaquinone-6 and an unidentified quinone as respiratory quinones.16,17 In addition, physiologically unfavourable conditions for H. pylori such as avoidance of its metabolic pathways and/or a lack of energy source result in transformation from the spiral to the coccoid form, which is dormant and not active.18,19 Thus, as one source of energy, respiration is a critical factor for H. pylori survival and/or growth in its life cycle. On the other hand, it has been reported that antimicrobial agents of the quinolone family competitively inhibit electron transport in the respiratory chain because of their structural similarity to microbial menaquinones.20,21 It was suggested that, given their characteristic chemical structure, AM quinolones inhibited respiration of H. pylori mediated by competitive inhibition of electron transport in the bacterial respiratory chain. Restriction of H. pylori growth associated with lack of energy caused by respiratory inhibition may be at least in part an antibacterial mechanism of AM quinolones.
The quinolone antimicrobial agents have been used clinically to prevent bacterial DNA synthesis by targeting two essential enzymes, DNA gyrase and DNA topoisomerase IV, that play important roles in DNA replication.22,23 However, in the present study, AM quinolones did not affect [3H]thymidine uptake into H. pylori. Urease activity is also a characteristic and essential property of H. pylori.24,25 In our other analysis using urea medium (Eiken Chemicals Co., Tokyo, Japan), AM quinolones did not affect urease activity. These findings suggest that the antibacterial activity of AM quinolones against H. pylori is mediated mainly by respiratory inhibition.
In in vivo studies, it is generally difficult to eradicate H. pylori, although many agents have potent antibacterial activity in vitro. Therefore, determination of both in vitro activity and in vivo efficacy is required for pharmacological evaluation of antimicrobial agents for treatment of H. pylori infection. In this study, AM quinolones decreased the number of viable H. pylori with reduction of neutrophil infiltration in the stomachs of H. pylori-inoculated animals. Although complete eradication was not achieved by the agents, their in vivo antibacterial activity was similar to or stronger than that of amoxicillin or clarithromycin alone.26,27 These findings indicate that AM quinolones exhibited in vivo efficacy as well as in vitro activity against H. pylori. Amoxicillin, clarithromycin and metronidazole are often used in therapeutic regimens for H. pylori eradication via their respective antimicrobial mechanism(s), such as inhibition of the organisms cell membrane and associated protein synthesis of H. pylori. Recently, however, increased occurrence of clarithromycin- or metronidazole-resistant strains of H. pylori is frequently encountered, thereby causing serious problems. Thus, AM quinolones possessing novel and different antimicrobial mechanism(s) from the above representative antibiotics might be useful for H. pylori eradication therapy, although not all antimicrobial agents in the quinolone family have strong antibacterial activity against the microorganism.2830 In addition, interestingly, combined administration of AM quinolones with omeprazole was more potent than AM quinolones alone. Omeprazole is usually used clinically for H. pylori eradication therapy. According to the increase in in vivo efficacy induced by combination with omeprazole, AM quinolones are probably applicable for the clinical situation. Notably, we observed previously that AM quinolones were highly selective for H. pylori but not for other intestinal bacterial species.11 Together with the finding that AM quinolones had no direct cytotoxic effects on gastric epithelial cells in vitro, oral administration of AM quinolones might thus cause no serious adverse effects in vivo, such as diarrhoea, loss of appetite or body weight, and animal mortality. These observations together suggest that AM quinolones exhibit strong antibacterial activity without harmful adverse effects and could be a candidate for use in eradication therapy for H. pylori.
In conclusion, the AM quinolone compounds 1-methyl-2-[(Z)-8-tridecenyl]-4-(1H)-quinolone and 1-methyl-2-[(Z)-7-tridecenyl]-4-(1H)-quinolone exhibited highly specific and strong antimicrobial activity against H. pylori mediated via respiratory inhibition without serious adverse effects. These agents, having a unique antimicrobial mechanism(s) different from that of other antibiotics such as amoxicillin, clarithromycin or metronidazole, may be beneficial in the treatment of H. pylori-associated gastroduodenal diseases, whether they are used alone or together with the above-mentioned antibiotics or proton pump inhibitor.
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Acknowledgements |
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Footnotes |
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References |
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