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Antimicrobial Agents and Chemotherapy, June 2006, p. 2237-2239, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01118-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Idebenone Acts against Growth of Helicobacter pylori by Inhibiting Its Respiration

Sakiko Inatsu,¹ Ayumi Ohsaki,² and Kumiko Nagata^1*

Department of Microbiology, Hyogo College of Medicine, Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan,¹ Division of Medical Chemistry, Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, Kanda Chiyoda-ku, Tokyo 101-0062, Japan²

Received 7 September 2005/ Returned for modification 5 October 2005/ Accepted 23 March 2006

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Growth of Helicobacter pylori was inhibited by the quinones, idebenone, duroquinone, menadione, juglone, and coenzyme Q₁ at low concentrations of 0.8 to 3.2 µg/ml. Idebenone specifically inhibited H. pylori growth by inhibiting respiration and decreasing the cellular ATP level. The respiratory inhibition was accompanied by reduction of idebenone by the H. pylori cells.

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Helicobacter pylori is a major cause of chronic gastritis and peptic ulcers and has been implicated in the development of gastric cancer (1, 4, 16, 19). To eradicate H. pylori, triple therapy consisting of amoxicillin, clarithromycin, and proton pump inhibitors has been commonly used. However, relapse and reinfection often occur, and, more seriously, treatment failure can lead to increased antibiotic resistance (11). Therefore, new drugs are needed for effective chemotherapy.

One promising group of drugs was furanonaphthoquinone analogs, which we found could inhibit H. pylori growth with a low MIC of around 0.1 µg/ml. However, their inhibition was not specific to H. pylori (12). Continuing our search led us to idebenone, duroquinone, menadione, juglone and coenzyme Q₁, which could inhibit H. pylori growth at MICs of 0.8 to 3.2 µg/ml, as shown in Table 1.

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TABLE 1. Effect of quinone analogs on H. pylori ATCC 43504 growth

Among these quinone analogs, idebenone [6-(10-hydroxydecyl ubiquinone)] has no known adverse effects in humans (3, 15) and has been reported to be effective against mitochondrial diseases such as Friedreich's ataxia (10, 17). Further study of idebenone was conducted against the growth of various kinds of bacteria (Table 2). The strains of H. pylori and other species of bacteria and the media used to determine MIC were described in our previous report (12). Four strains of H. pylori showed similar MICs: 1.6 to 3.2 µg/ml. Idebenone did not inhibit the growth of other bacteria, including Campylobacter jejuni, with the exception of Streptococcus pyogenes and Clostridium perfringens, for which the MIC was 25 to 50 µg/ml.

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TABLE 2. Effect of idebenone on growth of various kinds of bacteria

The microaerophilic bacterium H. pylori does not catabolize saccharides (6) and does not use glucose as a preferred energy substrate (7, 8). Its potential respiratory substrates and energy sources are organic acids and amino acids. We reported previously that H. pylori cells could utilize amino acids such as L-serine, L-proline, and L-alanine as respiratory substrates and that these amino acids were predominantly found in human stomach juice (13).

In the present study, we examined the effect of idebenone on the respiratory activity of whole cells. Respiratory assay and measurement of the cellular ATP level in H. pylori with inhibitors were carried out as described previously (14). As shown in Table 3, idebenone inhibited cellular respiration with L-proline, L-serine, and L-alanine as well as pyruvate and succinate as respiratory substrates at 50% inhibitory concentration (IC₅₀) values of 2.0 to 3.5 µg/ml, which were close to the MIC against H. pylori growth.

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TABLE 3. IC50 of idebenone in respiration by various substrates in H. pylori ATCC 43504 cells

Since ATP production has been suggested to be coupled to respiration, as described previously for H. pylori (14), we examined the dose response of idebenone with respect to the cellular ATP level when succinate was used as a respiratory substrate. We found that idebenone dose-dependently decreased the cellular ATP level, suggesting that it can inhibit ATP production coupled to respiration (data not shown).

We next considered whether L-proline dehydrogenase, which is essential for respiratory activity, might be target of idebenone. An assay based on the 2,6-dichlorophenolindophenol-reducing activity of L-proline, described previously (13), showed that L-proline dehydrogenase was not inhibited by idebenone even at the concentration of 20 µg/ml.

Idebenone in the oxidized state has an adsorption peak at 275 nm, and the peak decreases in the reduced form (17). Assay of the reducing activity of idebenone by H. pylori cells showed that idebenone outside the cells was reduced time dependently when the cells were incubated with idebenone at 37°C (Fig. 1). After 2 min, almost maximum reduction of idebenone was observed and the mode of the reduction was similar to that of the respiratory inhibition (Fig. 1).

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FIG. 1. Inhibition of respiration and reduction of idebenone in H. pylori cells. (Open circles) Time course of preincubation of H. pylori cells with idebenone in inhibition of cellular respiration. Respiration of whole cells was monitored polarographically using a Clark-type oxygen electrode as described previously (14). Succinate was added after 0, 1, 2.5, and 5 min of preincubation of cells without or with idebenone (5 µg/ml), and respiratory activities were determined from polarographic traces after addition of succinate, as described previously (14). The percentage of control activity was determined using the following equation: % = [(respiratory activity with idebenone) x (respiratory activity without idebenone)–1] x 100. Data show means ± standard deviation obtained from triplicate experiments. (Solid circles) Time course of reduction of oxidized idebenone by H. pylori cells. H. pylori cells (2 x 108/ml) were added to the reaction mixture containing idebenone (5 µg/ml). After incubation of 1, 2.5, and 5 min at 37°C, cells were removed by centrifugation at 10,000 x g for 10 min at 4°C, and then the A275 of the supernatant was measured. The percentage of control was determined using the following equation: % = [(A275 with cells) x (A275 without cells) –1] x 100. Data show means ± standard deviation obtained from triplicate experiments.

Bacteria contain two main types of quinones, ubiquinone and menaquinone, which mediate electron transfer between dehydrogenase and reductase or oxidase components of respiratory chains (18). Menaquinone is the major quinone in H. pylori (5, 9). Wang et al., based on work using quinones such as coenzyme Q₁, menadione, and 1,4-naphthoquinone, reported that H. pylori has the activity of NADPH-specific quinone reductase (20). In the present study, the cell-free homogenate obtained from sonicated H. pylori showed NADPH-idebenone reductase activity (data not shown). We found that idebenone, which easily crosses the cell membrane (3, 15), is reduced time dependently by H. pylori cells (Fig. 1). The hydrogen derived from various respiratory substrates may reduce idebenone inside the cells, which may, in turn, affect the component(s) in the respiratory chain of H. pylori.

Support for our hypothesis comes from the finding that the cellular respiration of microaerophilic C. jejuni, which has a similar energy metabolic pathway to that of H. pylori, was not inhibited by idebenone (data not shown). C. jejuni cells did not reduce idebenone, and the cell-free homogenate did not show the activity of NADPH-idebenone reductase (data not shown).

Based on these observations, the action of idebenone against H. pylori growth is considered to be due to its inhibition of respiratory activity coupled to ATP production. Precise identification of the inhibitory target of idebenone in the respiratory chain of H. pylori will require further investigation using a cell-free system of H. pylori.

In contrast to the case for H. pylori observed in the present work, in human mitochondria, idebenone at 6.7 to 20 µg/ml was found to dose dependently stimulate respiration using succinate (17). Since idebenone seems to have no serious side effects in humans (3, 10, 15, 17), our findings should be useful for developing new quinone analogs to eradicate H. pylori.

 
We are indebted to Nobuhito Sone of the ATP System Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation and Junko Takashima of Mitsubishi Pharma Corporation for valuable discussions.

This research was financially supported by Grants-in-Aid for Researchers from the Hyogo College of Medicine Foundation.

 
* Corresponding author. Mailing address: Department of Microbiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. Phone: 81-0798-45-6548. Fax: 81-0798-40-9162. E-mail: kunagata{at}hyo-med.ac.jp.

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1
1. Blaser, M. J. 1990. Helicobacter pylori and the pathogenesis of gastroduodenal inflammation. J. Infect. Dis. 161:626-633.[Medline]
2
2. Briere, J. J., D. Schlemmer, D. Chretien, and P. Rustin. 2004. Quinone analogues regulate mitochondrial substrate competitive oxidation. Biochem. Biophys. Res. Commun. 316:1138-1142.[CrossRef][Medline]
3
3. Gillis, J. C., P. Benfield, and D. McTavish. 1994. Idebenone. Drugs Aging 5:133-152.[Medline]
4
4. Graham, D. Y., G. M. Lew, P. D. Klein, D. G. Evans, D. J. Evans, Jr., Z. A. Saeed, and H. M. Malaty. 1992. Effect of treatment of Helicobacter pylori infection on the long-term recurrence of gastric or duodenal ulcer. A randomized, controlled study. Ann. Intern. Med. 116:705-708.[Abstract/Free Full Text]
5
5. Marcelli, S. W., H. T. Chang, T. Chapman, P. A. Chalk, R. J. Miles, and R. K. Poole. 1996. The respiratory chain of Helicobacter pylori: identification of cytochromes and the effects of oxygen on cytochrome and menaquinone levels. FEMS Microbiol. Lett. 138:59-64.[CrossRef][Medline]
6
6. McNulty, C. A. M., and J. C. Dent. 1987. Rapid identification of Campylobacter pylori (C. pyloridis) by preformed enzymes. J. Clin. Microbiol. 25:1683-1686.[Abstract/Free Full Text]
7
7. Mendz, G. L., and S. L. Hazell. 1993. Glucose phosphorylation in Helicobacter pylori. Arch. Biochem. Biophys. 300:522-525.[CrossRef][Medline]
8
8. Mendz, G. L., S. L. Hazell, and B. P. Burns. 1993. Glucose utilization and lactate production by Helicobacter pylori. J. Gen. Microbiol. 139:3023-3028.[Abstract/Free Full Text]
9
9. Moss, C. W., M. A. Lambert-Fair, M. A. Nicholson, and G. O. Guerrant. 1990. Isoprenoid quinones of Campylobacter cryaerophila, C. cinaedi, C. fennelliae, C. hyointestinalis, C. pylori, and "C. upsaliensis." J. Clin. Microbiol. 28:395-397.[Abstract/Free Full Text]
10
10. Mowat, D., D. M. Kirdy, K. R. Kamath, A. Kan, D. R. Thorburn, and J. Christodoulou. 1999. Respiratory chain complex III (correction of complex) in deficiency with pruritus. J. Pediatr. 134:352-354.[CrossRef][Medline]
11
11. Murakami, K., R. Sato, T. Okimoto, M. Nasu, T. Fujioka, M. Kodama, J. Kagawa, S. Sato, H. Abe, and T. Akira. 2002. Eradication rates of clarithromycin-resistant Helicobacter pylori using either rabeprazole or lansoprazole plus amoxicillin and clarithromycin. Aliment. Pharmacol. Ther. 16:1933-1938.[CrossRef][Medline]
12
12. Nagata, K., K.-I. Hirai, J. Koyama, Y. Wada, and T. Tamura. 1998. Antimicrobial activity of novel furanonaphthoquinone analogs. Antimicrob. Agents Chemother. 42:700-702.[Abstract/Free Full Text]
13
13. Nagata, K., Y. Nagata, T. Sato, M. A. Fujino, K. Nakajima, and T. Tamura. 2003. L-Serine, D- and L-proline and alanine as respiratory substrates of Helicobacter pylori: correlation between in vitro and in vivo amino acid levels. Microbiology 149:2023-2030.[Abstract/Free Full Text]
14
14. Nagata, K., N. Sone, and T. Tamura. 2001. Inhibitory activities of lansoprazole against respiration in Helicobacter pylori. Antimicrob. Agents Chemother. 45:1522-1527.[Abstract/Free Full Text]
15
15. Nz-Nagy, I. 1990. Chemistry, toxicology, pharmacology and pharmacokinetics of idebenone: a review. Arch. Gerontol. Geriatr. 11:177-186.[CrossRef][Medline]
16
16. Parsonnet, J., G. D. Friedman, D. P. Vandersteen, Y. Chang, J. H. Vogelman, N. Orentreich, and R. K. Sibley. 1991. Helicobacter pylori infection and the risk of gastric carcinoma. N. Engl. J. Med. 325:1127-1131.[Abstract]
17
17. Rustin, P., J. C. von Kleist-Retzow, K. Chantrel-Groussard, D. Sidi, A. Munnich, and A. Rotig. 1999. Effect of idebonone on cardiomyopathy in Friedreich's ataxia. Lancet 354:477-479.[CrossRef][Medline]
18
18. Soballe, B., and R. K. Poole. 1999. Microbial ubiquinone: multiple roles in respiration, gene regulation and oxidative stress management. Microbiology 145:1817-1830.[Free Full Text]
19
19. Uemura, N., S. Okamoto, S. Yamamoto, N. Matsumura, S. Yamaguchi, M. Yamakido, K. Taniyama, N. Sasaki, and R. J. Schlemper. 2001. Helicobacter pylori infection and the development of gastric cancer. N. Engl. J. Med. 354:784-789.
20
20. Wang, G., and R. J. Maier. 2004. An NADPH quinone reductase of Helicobacter pylori plays an important role in oxidative stress resistance and host colonization. Infect. Immun. 72:1391-1396.[Abstract/Free Full Text]

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Antimicrobial Agents and Chemotherapy, June 2006, p. 2237-2239, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01118-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Inatsu, S. Articles by Nagata, K. Search for Related Content PubMed PubMed Citation Articles by Inatsu, S. Articles by Nagata, K.