a Shionogi Research Laboratories, 3-1-1 Futaba-cho, Toyonaka, Osaka 561-0825; b Shionogi Research Laboratories, 5-12-4 Sagisu, Fukushima-ku, Osaka 553-0002; c Shionogi Institute For Medical Science, 2-5-1 Mishima, Settsu, Osaka 566-0022, Japan
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Abstract |
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Introduction |
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We recently developed a mouse helicobacter infection model, in which mice are infected with Helicobacter felis. The efficacies of amoxycillin, clarithromycin and metronidazole in H. felis eradication in the model were very similar to those reported clinically for H. pylori eradication.13 With this model, we noted that several 7-methoxy-cephems or 7
-methoxy-oxacephems showed higher rates of eradication in vivo than amoxycillin or clarithromycin even though they were less active in vitro. This suggested the importance of the 7
-methoxy substituent for in vivo activity. In the present study, we selected 7
-methoxy-cephems or 7
-methoxy-1-oxacephems and their demethoxy congeners with similar MICs for H. pylori and H. felis and compared their in vivo antibacterial activities in the mouse model.
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Materials and methods |
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Flomoxef, cefmetazole and amoxycillin were obtained from Shionogi & Co. Ltd (Osaka, Japan), Sankyo Co. Ltd (Tokyo, Japan) and Fujisawa Pharmaceutical Co. Ltd (Osaka, Japan), respectively. The demethoxy-flomoxef, 1-thia-flomoxef, demethoxy-1-thia-flomoxef, demethoxy-cefmetazole, compound M-1 and compound H-1 were synthesized at Shionogi Research Laboratories (Osaka, Japan). The chemical structures of these compounds are shown in Figure 1.
Bacteria
H. felis ATCC 49179 was used for in vitro and in vivo studies. Two clinical isolates and two standard strains of H. pylori were used for in vitro antimicrobial testing. Unless otherwise noted in the text, H. felis and H. pylori were grown in tissue culture flasks (Falcon 3109, Nippon Becton Dickinson Co., Ltd, Tokyo, Japan) containing Columbia broth (Difco Laboratories, Detroit, MI, USA) supplemented with 5% fetal bovine serum (FBS) under microaerophilic conditions at 37°C with constant rotation (45 rpm) for 48 h.
MIC determination
Because of poor growth of H. felis on the agar, the microbroth dilution method was adopted for MIC determination. No differences in amoxycillin or flomoxef MICs for H. pylori strains were seen between agar and broth dilution methods (data not shown). Bacterial cultures of H. felis and H. pylori were centrifuged and bacteria were suspended in Columbia broth supplemented with 5% FBS at an optical density of 0.9 at 660 nm. The bacterial suspensions were then diluted 50-fold, and 75 µL portions were inoculated into equal volumes of the broth containing serial two-fold dilutions of antimicrobial compounds. The final bacterial density in the assay was c. 106 cfu/mL. Assay plates were incubated for 3 days at 37°C under microaerophilic conditions before MICs were measured.
Timekill assay
For timekill studies, tissue culture flasks containing 10 mL of the prewarmed broth were inoculated with H. pylori at a final bacterial density of c. 106 cfu/mL and incubated at 37°C for 15 min. The antibiotics were added and incubation was continued at 37°C with constant rotation (45 rpm) under microaerophilic conditions. Viable bacterial counting was performed at 0, 3, 6, 9 and 24 h after antibiotic addition by plating serial 10-fold dilutions of 50 µL aliquots of each culture on to Columbia blood agar base supplemented with 5% horse blood. The plates were incubated for 5 days at 37°C under microaerophilic conditions before the colony numbers were counted. The lowest detectable number of organisms in this test was 20 cfu/mL.
Determination of in vivo antimicrobial activity
Seven-week-old female specific-pathogen-free BALB/c mice (Clea Japan, Inc., Tokyo, Japan) given free access to autoclaved laboratory chow and water were used for the determination of in vivo antimicrobial activity. H. felis ATCC 49179 was harvested from the broth culture by centrifugation at 3500g for 20 min, resuspended in Brucella broth containing 20% glycerol at an optical density of 0.9 at 660 nm and stored at 80°C until use for inoculation.
Mice (five mice in each group) were inoculated orally with 0.5 mL of the thawed bacterial suspension three times for 3 days (days 02). Clearance and eradication tests were performed 2 weeks after H. felis inoculation. For the clearance test, the mice were given oral antimicrobial agents dissolved in 0.3 mL of 0.5% methylcellulose twice on day 16 and were killed the next day (day 17) by cervical dislocation under ether anaesthesia. For the eradication test, the mice were given antimicrobial agents orally twice a day for 5 days (days 1620). Two weeks after completion of the therapy (day 34), mice were killed as above.
Presence of H. felis in the mouse stomach was detected by the urease test as described previously.13 The stomach was excised and cut along the greater curvature. The interior was rinsed with sterile saline to remove the remaining debris. The stomach was then cut along the lesser curvature into halves. One half of the gastric samples was homogenized in 1 mL of 10 mM sodium phosphate buffer (pH 6.5). The homogenate was serially diluted two-fold on a 96-well microtitre plate with the same buffer. The same volume of urease test solution (20 mg/mL urea, 0.05% phenol red and 0.2 mg/mL sodium azide in 10 mM sodium phosphate buffer, pH 6.5) was then added to each well. The plate was sealed and incubated for 16 h at 23°C. The gastric homogenate was judged to be urease negative when no significant colour change developed at any dilution.
The 50% eradication or clearance doses of antimicrobial agents were estimated by the logit method.14
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Results |
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The in vivo antibacterial activities of the compounds were evaluated in the mouse infection model (Table II). When the compounds were administered twice on the same day and H. felis was quantified in the stomach the next day (clearance test), all of the 7
-methoxy compounds in Figure 1 showed four-fold or more greater eradication activity than their demethoxy congeners. Compounds other than M-1 and H-1 were administered twice daily for 5 days and the mice were killed 14 days after administration (eradication tests). In these tests, the 7
-methoxy compounds also showed much higher activities.
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Discussion |
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Flomoxef and cefmetazole are parenteral antibiotics that are poorly absorbed after oral administration.15,16 Thus, the compounds used in this study are likely to have gained access to the bacteria on the gastric mucosa directly from the gastric cavity, not via the bloodstream. Indeed, flomoxef administered intravenously showed much lower in vivo activity (Table II). For many antimicrobial agents, there are no practical correlations between MICs and the clinical clearance of H. pylori5 and these disadvantages can probably be ascribed to difficulties for antimicrobial agents in accessing H. pylori in the stomach. The 7
-methoxy substituent may combat this difficulty by promoting permeability of the compounds through gastric mucus or by helping the compounds to remain in the stomach longer.
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Notes |
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References |
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2 . Marshall, B. J., Goodwin, C. S., Warren, J. R., Murray, R., Blincow, E. D., Blackbourn, S. J. et al. (1988). Prospective double-blind trial of duodenal ulcer relapse after eradication of Campylobacter pylori. Lancet ii, 143742.
3 . Graham, D. Y., Lew, G. M., Klein, P. D., Evans, D. G., Evans, D. J., Saeed, Z. A. et al. (1992). Effect of treatment of Helicobacter pylori infection on the long-term recurrence of gastric or duodenal ulcer. A randomized, controlled study. Annals of Internal Medicine 116, 7058.[ISI][Medline]
4 . Panel on Helicobacter pylori in Peptic Ulcer Disease. (1994). NIH Consensus Development Conference. Helicobacter pylori in peptic ulcer disease. Journal of the American Medical Association 272, 659.[ISI][Medline]
5 . Chiba, N., Rao, B. V., Rademaker, J. W. & Hunt, R. H. (1992). Meta-analysis of the efficacy of antibiotic therapy in eradicating Helicobacter pylori. American Journal of Gastroenterology 87, 171627.[ISI][Medline]
6 . Katelaris, P. H., Patchett, S. E., Zhang, Z. W., Domizio, P. & Farthing, M. J. (1995). A randomized prospective comparison of clarithromycin versus amoxycillin in combination with omeprazole for eradication of Helicobacter pylori. Alimentary Pharmacology and Therapeutics 9, 2058.[ISI][Medline]
7 . Labenz, J., Stolte, M., Peitz, U., Tillenburg, B., Becker, T. & Borsch, G. (1996). One-week triple therapy with omeprazole, amoxycillin and either clarithromycin or metronidazole for cure of Helicobacter pylori infection. Alimentary Pharmacology and Therapeutics 10, 20710.[ISI][Medline]
8 . Logan, R. P., Gummett, P. A., Hegarty, B. T., Walker, M. M., Baron, J. H. & Misiewicz, J. J. (1992). Clarithromycin and omeprazole for Helicobacter pylori. Lancet 340, 239.[ISI][Medline]
9 . Buckley, M., Xia, H. X., Hyde, D. M., Keane, C. T. & O'Morain, C. A. (1997). Metronidazole resistance reduces efficacy of triple therapy and leads to secondary clarithromycin resistance. Digestive Diseases and Sciences 42, 21115.[ISI][Medline]
10 . Lind, T., Mégraud, F., Unge, P., Bayerdörffer, E., O'Morain, C., Spiller, R. et al. (1999). The MACH2 study: role of omeprazole in eradication of Helicobacter pylori with 1-week triple therapies. Gastroenterology 116, 24853.[ISI][Medline]
11
.
Dore, M. P., Osato, M. S., Realdi, G., Mura, I., Graham, D. Y. & Sepulveda, A. R. (1999). Amoxycillin tolerance in Helicobacter pylori. Journal of Antimicrobial Chemotherapy 43, 4754.
12 . Realdi, G., Dore, M. P., Piana, A., Atzei, A., Carta, M., Cugia, L. et al. (1999). Pretreatment antibiotic resistance in Helicobacter pylori infection: results of three randomized controlled studies. Helicobacter 4, 10612.[ISI][Medline]
13 . Kobayashi, Y., Doi, M., Nagata, H., Sasaki, K., Maruyama, T. & Murakami, K. (1998). A model for anti Helicobacter pylori chemotherapy using an experimental murine infection with Helicobacter felis. Journal of Infection and Chemotherapy 4, 195200.
14 . Ashton, W. D. (1972). The logit transformation, with special reference to its use in bioassay. In Griffin's Statistical Monographs and Courses, (Stuart, A., Ed.), pp. 176. Charles Griffin, London.
15 . Kimura, Y., Nakashimizu, H., Nakano, M., Otubo, R., Matsubara, H. & Yoshida, T. (1987). Pharmacokinetic characterization of 6315-S (flomoxef) in experimental animals. Chemotherapy 35, S16175.
16 . Morita, E., Mizuno, N., Nishikawa, M. & Miyake, K. (1990). Comparison of the pharmacokinetics of five ß-lactam antibiotics between neonatal and adult rats. Developmental Pharmacology and Therapeutics 14, 22330.[ISI][Medline]
Received 9 August 1999; returned 9 November 1999; revised 20 December 1999; accepted 24 January 2000