In vitro anti-Helicobacter pylori activity of BAS-118, a new benzamide derivative

Intetsu Kobayashi1,*, Hiroe Muraoka1, Miyuki Hasegawa1, Takeshi Saika1, Minoru Nishida1, Makoto Kawamura2 and Ryoichi Ando2

1 Chemotherapy Division, Mitsubishi Kagaku Bio-Clinical Laboratories, 3-30-1 Shimura, Itabashi-ku, Tokyo 174-8555; 2 Pharmaceuticals Research Division, Mitsubishi Pharma Corporation, Kanagawa, Japan

Received 29 October 2001; returned 15 January 2002; revised 10 April 2002; accepted 2 May 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The antibacterial activity of BAS-118, a new benzamide derivative, against Helicobacter pylori and other species of bacteria was investigated, as was the in vitro ability of the compound to induce drug resistance in H. pylori. The MICs of BAS-118 for 155 isolates of H. pylori, including 30 clarithromycin (CAM)-resistant isolates (MIC >= 1.56 mg/L) and 25 metronidazole (MNDZ)-resistant isolates (MIC >= 6.25 mg/L), and 29 reference strains of other genera were determined by an agar dilution method. The MIC50, MIC90 and MIC range of BAS-118 for 100 randomly selected isolates of H. pylori were <=0.003, 0.013 and <=0.003–0.025 mg/L, respectively, with similar values obtained for CAM- and MNDZ-resistant isolates. Furthermore, MICs of BAS-118 for five H. pylori strains increased no more than two-fold after 10 serial passages in the presence of subinhibitory concentrations. BAS-118 exhibited a low antibacterial activity against the 29 non-H. pylori strains, with MICs of >=8 mg/L. In summary, BAS-118 is a novel anti-H. pylori agent with a potent and selective antibacterial activity, which includes CAM- and MNDZ-resistant isolates. Furthermore, BAS-118 does not appear to induce drug resistance readily in vitro.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Helicobacter pylori is well recognized as the major causative pathogen of peptic ulcers and active chronic gastritis. In patients with H. pylori infections in their gastric mucosa, eradication of the pathogen seems to cure both the infection and ulcers. H. pylori strains are generally susceptible to a wide range of antimicrobial agents in vitro, but clinical observations show that the successful treatment of H. pylori-infected patients is usually difficult using single antimicrobial therapy.1 Recent recommendations suggest that a combination of a proton pump inhibitor and two antimicrobial agents [mainly clarithromycin (CAM) and either amoxicillin or metronidazole (MNDZ)] is the most favourable treatment.2 Since there is great concern about the increasing resistance of clinical isolates of H. pylori to MNDZ and CAM,3 new antimicrobial agents active against H. pylori are needed. To generate new, selective anti-H. pylori agents, products synthesized in the research laboratories of Mitsubishi Pharma Corporation were screened and several new classes of anti-H. pylori compound were found. N-methylbenzamide derivatives were the most attractive of these compounds because of their simple structure, ease of synthesis, stability under acidic conditions and potent anti-H. pylori activity. Chemical modification of this series afforded many potent anti-H. pylori compounds, from which N-methyl-3-[2-(2-naphtyl)acetylamino]benzamide (BAS-118) (Figure 1) was selected as a candidate compound for further evaluation.4 The purposes of this study were to evaluate the anti-H. pylori activity of BAS-118 and the ability of H. pylori to acquire resistance to it in vitro.



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Figure 1. Chemical structure of BAS-118: N-methyl-3-[2-(2-naphtyl)acetylamino]benzamide.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

One hundred and fifty-five strains of H. pylori, including 30 CAM-resistant5 strains (MIC >= 1.56 mg/L) and 25 MNDZ-resistant6 strains (MIC >= 6.25 mg/L), were isolated from patients with peptic ulcers. The specimens were obtained between 1997 and 1998 at several hospitals in Japan and were cultured using selective and non-selective agar media, as described previously.7 Twenty-nine reference and clinically isolated strains of different species were also used: Staphylococcus aureus ATCC25923, Staphylococcus epidermidis ATCC12228, Micrococcus luteus ATCC9341, Streptococcus pyogenes S-23, Streptococcus pneumoniae ATCC33400, Enterococcus faecalis ATCC19433, Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC10031, Citrobacter freundii ATCC8090, Enterobacter cloacae ATCC13047, Proteus vulgaris ATCC13315, Proteus mirabilis ATCC21100, Pseudomonas aeruginosa ATCC27853, Acinetobacter calcoaceticus NCTC7844, Haemophilus influenzae ATCC9334, Bacillus subtilis ATCC6633, Neisseria gonorrhoeae WHO-A, Peptostreptococcus anaerobius ATCC27337, Peptostreptococcus magnus ATCC29328, Propionibacterium acnes ATCC11829, Bacteroides fragilis ATCC25285, Bacteroides vulgatus ATCC29327, Fusobacterium nucleatum ATCC25586, a Prevotella intermedia clinical isolate, Clostridium difficile ATCC9689, Clostridium perfringens NCTC4969, Veillonella parvula ATCC10790, a Campylobacter jejuni clinical isolate and a Campylobacter fetus clinical isolate. All strains were stored at –80°C until required.

Antimicrobials

Stock solutions of BAS-118 (Mitsubishi Pharma Co., Osaka, Japan), CAM (Taisho Pharmaceutical Co., Ltd, Tokyo, Japan), MNDZ (Sigma Chemical Co., St Louis, MO, USA) and amoxicillin (Sigma Chemical Co.) were prepared from powders of known potency in accordance with the manufacturers’ instructions.

Susceptibility testing

The MICs of BAS-118 for H. pylori and other species were determined by an agar dilution method according to the guidelines established by the Japanese Society of Chemotherapy.8 For the H. pylori strains, BAS-118 and the other drugs were tested at concentrations of 50–0.003 mg/L and 100–0.025 mg/L, respectively. For the other species, the drugs were tested at concentrations of 4–0.004 mg/L. The test strains (5 x 103 cfu/spot or 5 x 105 cfu/spot) were inoculated on to agar plates containing two-fold serial dilutions of the test agents with a multi-point inoculator (Sakuma Seisakusho, Tokyo, Japan). The agar plates inoculated with the test strains, including H. pylori, were cultured under the appropriate conditions for optimal growth: H. pylori and Campylobacter were inoculated on to Blood agar base No. 2 (Oxoid, Unipath Ltd, Basingstoke, UK) with 5% horse blood and incubated at 35°C for 72 h in a microaerophilic atmosphere; S. pneumoniae and H. influenzae were inoculated on to Mueller–Hinton agar (Difco Laboratories, Detroit, MI, USA) with 5% heat-lysed horse blood and incubated at 35°C for 18 h in 10% CO2; N. gonorrhoeae was inoculated on to GC agar (Becton Dickinson, Cockeysville, MD, USA) and incubated at 35°C for 24 h in 10% CO2; anaerobes were inoculated on to Brucella HK agar (Kyokuto Pharmaceutical Industrial Co., Ltd, Tokyo, Japan) supplemented with 5% defibrinated horse blood and incubated anaerobically at 35°C for 48 h; and all other aerobes were inoculated on to Mueller–Hinton agar and incubated in air at 35°C for 18 h. The MIC was defined as the lowest concentration of antimicrobial agent that completely inhibited visible bacterial growth.

Serial passage experiment

Five strains of H. pylori were subjected to a serial passage experiment for CAM and BAS-118 as described by Haas et al.9 The strains were each transferred with a swab on to the agars containing 0.5 x, 1 x and 2 x MIC of each drug. These agar plates were then incubated for 72 h under the above-mentioned conditions. This process was repeated serially until either no growth occurred or the tenth passage. The MICs of CAM and BAS-118 were determined at every passage.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MIC50, MIC90 and MIC range of BAS-118 for 100 randomly selected isolates of H. pylori were <=0.003, 0.013 and <=0.003–0.025 mg/L, respectively (Table 1). BAS-118 was the most active of the four anti-H. pylori agents tested against the 100 randomly selected clinical isolates. For the 30 CAM- and 25 MNDZ-resistant isolates, the MIC50, MIC90 and MIC range of BAS-118 were nearly the same, i.e. 0.006, 0.025 and <=0.003–0.025 mg/L, respectively. Furthermore, the MIC50s and MIC90s of BAS-118 for these resistant isolates were slightly higher than those for the 100 randomly selected isolates, but the MIC range of BAS-118 for the resistant isolates did not differ from that for the randomly selected isolates. BAS-118 exhibited a low antibacterial activity (>4 mg/L) against all ATCC and other reference strains of non-H. pylori species, with the exception of the single strain of C. jejuni tested (MIC <= 0.016 mg/L).


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Table 1.  In vitro activities of BAS-118 against clinical isolates of H. pylori
 
In serial passage experiments, no increases were noted in the MICs of BAS-118 for four of five H. pylori strains after the tenth serial passage on supplemented agar medium containing sub-MIC levels of BAS-118. The MIC for one isolate increased two-fold after the fifth serial passage, but no further increases were observed after the tenth passage. A parallel experiment was carried out using CAM; the MICs of CAM for four of the five strains increased four- and 16-fold after the fifth and tenth passages, respectively; the MIC of CAM for one isolate increased eight-fold after the tenth serial passage.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
H. pylori is highly susceptible in vitro to a wide range of antibacterial agents, but treatment of H. pylori infection using triple therapy (two antibiotics plus a proton pump inhibitor or bismuth salt) is recommended.2 Although H. pylori often disappears after treatment with antimicrobial agents, the infection usually relapses either because of incomplete eradication of H. pylori or the emergence of resistant strains.10 The increasing resistance of H. pylori to commonly used antimicrobial agents, especially CAM and MNDZ, is a great concern.3 Several mechanisms are thought to be involved in the acquisition of resistance in H. pylori, based on observations from passage experiments.6,9 The present results indicate that BAS-118 exhibits potent antimicrobial activity against clinical strains of H. pylori, including CAM- and MNDZ-resistant strains; no signs of cross-resistance between BAS-118 and commercially available anti-H. pylori agents were observed. Furthermore, BAS-118 was not active against other species of human intestinal flora and has potential to be useful for the eradication of H. pylori without affecting the normal intestinal bacterial flora. Serial passage experiments with BAS-118 did not result in the selection of resistance in five strains of H. pylori. Further studies are needed to investigate whether BAS-118 can be used to eradicate H. pylori in vivo or whether other alternatives for the treatment of H. pylori infections exist.

In conclusion, the potent and selective activity against H. pylori shown by BAS-118 may overcome the drawbacks of the present triple combination eradication regimen, including side effects and resistant strains. Moreover, the mechanism of its anti-H. pylori activity is expected to be novel because there have been no reports for structurally related compounds and because BAS-118 showed a very narrow antibacterial spectrum.


    Footnotes
 
* Corresponding author. Tel: +81-3-5994-2334; Fax: +81-3-5994-2939; E-mail: mbc-ka{at}sa2.so-net.ne.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . van der Hulst, R. W., Keller, J. J., Rauws, E. A. & Tytgat, G. N. (1996). Treatment of Helicobacter pylori infection: a review of the world literature. Helicobacter 1, 6–19.[Medline]

2 . Treiber, G., Ammon, S., Schneider, E. & Klotz, U. (1998). Amoxicillin/metronidazole/omeprazole/clarithromycin: a new, short quadruple therapy for Helicobacter pylori eradication. Helicobacter 3, 54–8.[ISI][Medline]

3 . Lind, T., Megraud, F., Unge, P., Bayerdorffer, 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, 248–53.[ISI][Medline]

4 . Ando, R., Kawamura, M. & Chiba, N. (2001). 3-(Arylacetylamino)-N-methylbenzamides: a novel class of anti-Helicobacter pylori agents. Journal of Medicinal Chemistry 44, 4468–74.[ISI][Medline]

5 . National Committee for Clinical Laboratory Standards. (2000). Performance Standards for Antimicrobial Susceptibility Testing—Tenth Informational Supplement (Aerobic Dilution): M100-S10. NCCLS, Wayne, PA.

6 . Jenks, P. J., Ferrero, R. L. & Labigne, A. (2001). The role of the rdxA gene in the evolution of metronidazole resistance in Helicobacter pylori. Journal of Antimicrobial Chemotherapy 43, 753–8.[Abstract/Free Full Text]

7 . Kobayashi, I., Hasegawa, M., Saika, T., Nishida, M., Fujioka, T. & Nasu, M. (1997). A new semi-solid agar dilution method for determining amoxycillin, clarithromycin and azithromycin MICs for Helicobacter pylori isolates. Journal of Antimicrobial Chemotherapy 40, 713–6.[Abstract]

8 . Japanese Society of Chemotherapy. (1981). Standard method of MIC determinations. Chemotherapy 29, 76–9.

9 . Haas, C. E., Nix, D. E. & Schentag, J. J. (1990). In vitro selection of resistant Helicobacter pylori. Antimicrobial Agents and Chemotherapy 34, 1637–41.[ISI][Medline]

10 . Adamek, R. J., Suerbaum, S., Pfaffenbach, B. & Opferkuch, W. (1998). Primary and acquired Helicobacter pylori resistance to clarithromycin, metronidazole, and amoxicillin—influence on treatment outcome. American Journal of Gastroenterology 93, 386–9.[ISI][Medline]





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