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JAC Advance Access originally published online on September 7, 2007
Journal of Antimicrobial Chemotherapy 2007 60(5):1060-1063; doi:10.1093/jac/dkm329
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Effect of plaunotol in combination with clarithromycin against clarithromycin-resistant Helicobacter pylori in vitro and in vivo

Makoto Sasaki1,*, Tsutomu Mizoshita1, Takashi Mizushima1, Harumi Inoue2, Takeshi Kamiya1, Hiromi Kataoka1, Naotaka Ogaswara1, Tsuneya Wada1, Eiji Kubota1, Yoshinori Mori1, Takaya Shimura1, Hirokazu Hirata1, Kenji Ando1, Yasuyuki Okamoto1, Hirotaka Ohara1, Haruhisa Nakao1 and Takashi Joh1

1 Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho, Nagoya 467-8601, Japan 2 Biological Research Laboratories IV, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo, Japan

* Corresponding author. Tel: +81-52-8538211; Fax: +81-52-8520952; E-mail: msasaki{at}med.nagoya-cu.ac.jp

Received 28 June 2007; returned 9 June 2007; revised 3 August 2007; accepted 4 August 2007


   Abstract
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 Abstract
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Objectives: Recently, there has been a decrease in the eradication rate of Helicobacter pylori due to the increase in antibiotic resistance of this bacterium. Plaunotol, a cytoprotective anti-ulcer agent, exhibits antibacterial activity against H. pylori. The purpose of the present study was to investigate the effect of plaunotol in combination with clarithromycin against clarithromycin-resistant H. pylori clinical isolates.

Methods and results: In the chequerboard titration method, the combination of plaunotol and clarithromycin showed a synergistic effect against 67% (10/15) clarithromycin-resistant strains and an additive effect against the other strains. No indifferent and antagonistic effects were observed against any of the strains tested. In a gastritis model of Mongolian gerbils infected with clarithromycin-resistant H. pylori, the plaunotol (40 mg/kg) and clarithromycin (66.6 mg/kg) combination exhibited synergistic effects; however, neither plaunotol nor clarithromycin alone showed bactericidal effects.

Conclusions: These results suggest that plaunotol may play a useful role in combination with anti-H. pylori drugs in the treatment of diseases associated with clarithromycin-resistant H. pylori.

Keywords: synergistic effect , cytoprotective anti-ulcer agent , eradication


   Introduction
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 Introduction
Materials and methods
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Helicobacter pylori is a Gram-negative bacterium associated with peptic ulcer disease, gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. The discovery of H. pylori has already changed the prognosis of peptic ulcer disease, with most patients being cured at their first presentation.1,2 Eradication of H. pylori is very important to prevent relapse and accelerate the healing of peptic ulcer disease,3 and only triple-drug therapy [proton pump inhibitor (PPI) + amoxicillin + clarithromycin] is currently permitted in Japan. Triple-drug therapy has a strong antibacterial effect against H. pylori and has a high eradication rate.4 However, the eradication rate of H. pylori using triple-drug therapy has decreased5,6 due to the increase in clarithromycin-resistant strains of H. pylori.7,8 Plaunotol, a cytoprotective anti-ulcer agent, enhances the antibacterial activity of clarithromycin;9,10 however, it is unclear whether plaunotol enhances the antibacterial activity of clarithromycin against clarithromycin-resistant H. pylori. Plaunotol shows antibacterial activity against H. pylori and plaunotol in combination with clarithromycin exhibits synergistic or additive activity against clarithromycin-susceptible H. pylori in vitro and in vivo. Triple-drug therapy is instituted before the detection of clarithromycin-resistant H. pylori. Therefore, it is useful to evaluate the combined effect of plaunotol with clarithromycin against clarithromycin-resistant H. pylori. In this study, we evaluated the antibacterial activity of plaunotol in combination with clarithromycin against clarithromycin-resistant H. pylori isolates by using the agar dilution chequerboard method. The in vivo activity of the plaunotol/clarithromycin combination was also examined in a gastritis model in Mongolian gerbils infected with a clinical isolate of clarithromycin-resistant H. pylori.


   Materials and methods
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 Abstract
 Introduction
 Materials and methods
Results and discussion
 Funding
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 References
 
Antibacterial agents

Plaunotol (Daiichi Sankyo Co., Ltd, Tokyo, Japan) and clarithromycin (Taisho Pharmaceutical Co., Ltd, Tokyo, Japan) were used.

Microorganisms

Twenty-one H. pylori clinical isolates used in this study were isolated from gastric biopsy samples obtained during a routine endoscopy. The gastric biopsy material was processed according to the standard methodology, and H. pylori was identified based on colony morphology, Gram staining and catalase-, oxidase- and urease-positive reactions. Twenty-one clarithromycin-resistant strains (MIC ≥ 1 mg/L) were maintained at –80°C in Brucella broth with 25% glycerol until usage. Stock culture was grown in Brucella broth (Nippon Becton Dickinson Co. Ltd) containing 3% heat-inactivated fetal bovine serum (FBS) and antibiotics (2.5 mg/L amphotericin B, 5 mg/L vancomycin, 2.5 mg/L trimethoprim and 125 IU/L polymyxin B) under microaerobic conditions with gas generator envelopes (AnaeroPack, Mitsubishi Gas Chemical Co., Inc.) in gas jars at 37°C for 4 days.

Animals

A total of 36 specific-pathogen-free, 5-week-old male Mongolian gerbils (MGS/Sea, Kyudo Co., Ltd) were housed in plastic cages on hardwood chip bedding in an air-conditioned biohazard room with a 12:12 h light:dark cycle. The experimental design was approved by the Animal Care Committee of the Nagoya City University Animal Research Institute and the animals were cared for in accordance with institutional guidelines, which comply with the instructions of the Health, Labour and Welfare Ministry concerning animal experiments.11

Stock clarithromycin-resistant H. pylori culture was grown in Brucella broth containing 3% FBS under microaerobic conditions. After 1 day of fasting, 6-week-old Mongolian gerbils were orally inoculated with 2 mL of bacterial suspension having a density of 1 x 108 cfu/mL. Uninfected gerbils were mock-inoculated with sterile Brucella broth containing 3% FBS.

Determination of MIC

Stock cultures of bacteria were grown on brain heart infusion agar (BHIA; Difco Laboratories) supplemented with 5% defibrinated horse blood (Nippon Bio-Supply) at 37°C for 3 days in gas jars with CampyPack. The MICs were measured on BHIA supplemented with 5% defibrinated horse blood by using the 2-fold dilution method. Plaunotol and clarithromycin were dissolved in DMSO and methanol, respectively. Inocula were prepared by suspending the bacterial growth on BHIA supplemented with 5% defibrinated horse blood plates in physiological saline to achieve a density equivalent to that of a 2.0 McFarland standard. A final inoculum of 104–105 cfu/spot was employed using an Auto-Inoculator (Dai Nippon Seiki Co., Ltd, Kyoto, Japan). All the plates were incubated for 3 days at 35°C under microaerobic conditions. The MIC was defined as the lowest concentration of the drug that prevented visible growth after 3 days of incubation.

Determination of in vitro interaction

Antimicrobial interactions between plaunotol and clarithromycin against 15 clinical isolates were evaluated by the chequerboard titration method. The inoculum, media and culture conditions for this evaluation were the same as those described for the MIC determination.

The fractional inhibitory concentrations (FICs) were calculated as follows:

FIC = (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone).

The FIC indices were interpreted as follows: ≤0.5, synergy; >0.5–1, additive; >1–4.0, indifference; >4, antagonism.

Determination of in vivo interaction

The experimental design is illustrated in Figure 1. Thirty-six gerbils were grouped into five groups. H. pylori was inoculated into four (Groups A, B, C and D) of five groups at experimental week 1. The remaining group (Group E) received Brucella broth containing 3% FBS. Plaunotol and clarithromycin were dissolved in 0.5% sodium carboxymethyl cellulose (CMC solution, Kanto Chemical Co., Inc.). Group A received plaunotol [40 mg/(kg·dose)] and clarithromycin [66.6 mg/(kg·dose)], Group B received clarithromycin [66.6 mg/(kg·dose)], Group C received plaunotol [40 mg/(kg·dose)] and Groups D and E received oral CMC. The gerbils were sacrificed humanely at 12 weeks. All gerbils were deeply anaesthetized with ether, laparotomized and exsanguinated from the inferior vena cava, followed by excision of their stomachs. Each glandular stomach was fixed in 10% formalin neutral buffer solution, deodorized (Wako Pure Chemical Industries, Ltd) and routinely processed for histopathological examination.


Figure 1
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  		Figure 1. Experimental design. Five-week-old male Mongolian gerbils were used. Intragastric inoculation of H. pylori (filled triangles) or Brucella broth (open triangle) occurred at week 1. Clarithromycin + plaunotol, clarithromycin, plaunotol, or CMC was administered to the respective groups as described in the Materials and methods section. {dagger} The gerbils were sacrificed humanely at 12 weeks.

 
Histopathological analyses

Tissue sections were immunohistochemically stained for examination of H. pylori (anti-H. pylori serum, Dako, Copenhagen). The degree of H. pylori density was graded according to the criteria modified from the Updated Sydney System12 by scoring the following parameters: mononuclear cell infiltration (0–3: 0, normal; 1, mild infiltration into lamina propria; 2, moderate infiltration into lamina propria; 3, marked infiltration into lamina propria and multiple lymphoid follicle formation); neutrophil infiltration (0–3: 0, none; 1, number of neutrophils in the pyloric mucosa in a line from the forestomach to the duodenum <50/mm; 2, 50–100/mm; 3, >100/mm); and H. pylori density (0–3: 0, none; 1, mild H. pylori density; 2, moderate; 3, marked).13 Two independent individuals (M. S. and T. Mizoshita) were blind with regard to information concerning H. pylori infection and treatment, and judged the histology.

Statistical analysis

The Mann–Whitney U-test was used to establish the significance of differences in microscopic H. pylori density. P values of <0.05 were considered to be statistically significant.


   Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
Funding
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 References
 
Regarding the genetic diversity of clarithromycin-resistant H. pylori, Masaoka et al.14 have demonstrated that dilution agar methods should be combined with PCR-restriction fragment length polymorphism (PCR-RFLP) analysis before second-line eradication to increase the accuracy of clarithromycin susceptibility testing and to improve eradication efficacy. In this study, we demonstrated the effect of plaunotol in combination with clarithromycin against clarithromycin-resistant H. pylori.

Table 1 shows the MIC50, MIC80 and MIC90 values and range of MICs of both clarithromycin and plaunotol for the 21 clarithromycin-resistant H. pylori strains tested. The MIC50/MIC80/MIC90 values (mg/L) of clarithromycin and plaunotol for the 21 clarithromycin-resistant H. pylori strains were 4/16/16 and 2/4/4, respectively. By using the chequerboard titration method, the antibacterial effect of plaunotol in combination with clarithromycin was examined against 15 of the 21 clarithromycin-resistant H. pylori strains. The clarithromycin/plaunotol combination demonstrated a synergistic effect against 67% of the strains and an additive effect against 33% of the strains. No indifferent or antagonistic effects were observed against any H. pylori strain (Table 2). Thus, by using the chequerboard titration method, we demonstrated that plaunotol synergistically enhanced the antibacterial activity of clarithromycin against clarithromycin-resistant H. pylori in vitro. This tendency is in accordance with a previous report of clarithromycin-susceptible H. pylori from Koga et al.10 As plaunotol is suggested to cause a change in fluidity with the associated increased membrane permeability,15 plaunotol might potentiate the anti-H. pylori activity of clarithromycin by increasing the permeability of the membranes of even clarithromycin-resistant H. pylori.


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  		Table 1. MIC50, MIC80 and MIC90 values and MIC range of clarithromycin and plaunotol for the 21 clarithromycin-resistant H. pylori strains

 


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  		Table 2. Number and percentage of strains in which additive or synergistic effects were observed and the FIC index range for each group

 
Table 3 shows the therapeutic effects of plaunotol in combination with clarithromycin. The H. pylori infection scores of Groups B and C were lower than that of Group D; however, the difference was not significant. In contrast, the H. pylori infection score of Group A receiving the combination therapy was significantly lower than that of Group D; this indicated the synergistic/additive effect of the plaunotol/clarithromycin combination. However, acute inflammation scores and chronic inflammation scores based upon the Updated Sydney System were not significantly different. Fukuda et al.16 have reported that the combination of plaunotol, clarithromycin and a PPI showed a 69% eradication rate as compared with a 38% eradication rate achieved with a combination of clarithromycin and a PPI. Thus far, there has been no study on the synergistic effect of plaunotol in combination with clarithromycin against clarithromycin-resistant H. pylori strains. Our study is the first to demonstrate that plaunotol can synergistically enhance the antibacterial activity of clarithromycin even against H. pylori strains that exhibit resistance to clarithromycin. The increased occurrence of not only clarithromycin-resistant strains but also metronidazole- and other antibiotic-resistant strains of H. pylori has resulted in a decrease in the eradication rate. Regarding the unsuccessful H. pylori eradication, a high resistance rate (47.9%) to gatifloxacin (8-methoxy fluoroquinolone) in H. pylori strains is also observed.17 Therefore, plaunotol might prevent the decrease in the eradication rate that occurred due to clarithromycin-resistant H. pylori.


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  		Table 3. Therapeutic effects of plaunotol in combination with clarithromycin

 
Conclusions

We demonstrated that plaunotol, a cytoprotective anti-ulcer agent, in combination with clarithromycin exhibits a synergistic effect on clarithromycin-resistant H. pylori strains in vitro and in vivo. These results suggest that plaunotol could play a useful role in combination with clarithromycin in the treatment of diseases associated with clarithromycin-resistant H. pylori.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Funding
Transparency declarations
 References
 
No funding was received.


   Transparency declarations
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Funding
 Transparency declarations
References
 
None to declare.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Funding
 Transparency declarations
 References
 
1 Suzuki H, Marshall BJ, Hibi T. Overview: Helicobacter pylori and extragastric disease. Int J Hematol (2006) 84:291–300.[CrossRef][Web of Science][Medline]

2 Suzuki H, Hibi T, Marshall BJ. Helicobacter pylori: present status and future prospects in Japan. J Gastroenterol (2007) 42:1–15.[Web of Science][Medline]

3 Rauws EA, Tytgat GN. Cure of duodenal ulcer associated with eradication of Helicobacter pylori. Lancet (1990) 335:1233–5.[CrossRef][Web of Science][Medline]

4 Asaka M, Sugiyama T, Kato M, et al. A multicenter, double-blind study on triple therapy with lansoprazole, amoxicillin and clarithromycin for eradication of Helicobacter pylori in Japanese peptic ulcer patients. Helicobacter (2001) 6:254–61.[CrossRef][Web of Science][Medline]

5 Hoshiya S, Watanabe K, Tokunaga K, et al. Relationship between eradication therapy and clarithromycin-resistant Helicobacter pylori in Japan. J Gastroenterol (2000) 35:10–4.[Web of Science][Medline]

6 Murakami K, Sato R, Okimoto T, et al. Eradication rates of clarithromycin-resistant Helicobacter pylori using either rabeprazole or lansoprazole plus amoxicillin and clarithromycin. Aliment Pharmacol Ther (2002) 16:1933–8.[CrossRef][Web of Science][Medline]

7 Perez Aldana L, Kato M, Nakagawa S, et al. The relationship between consumption of antimicrobial agents and the prevalence of primary Helicobacter pylori resistance. Helicobacter (2002) 7:306–9.[CrossRef][Web of Science][Medline]

8 Masuda H, Hiyama T, Yoshihara M, et al. Characteristics and trends of clarithromycin-resistant Helicobacter pylori isolates in Japan over a decade. Pathobiology (2004) 71:159–63.[CrossRef][Web of Science][Medline]

9 Koga T, Kawada H, Utsui Y, et al. In-vitro and in-vivo antibacterial activity of plaunotol, a cytoprotective antiulcer agent, against Helicobacter pylori. J Antimicrob Chemother (1996) 37:919–29.[Abstract/Free Full Text]

10 Koga T, Inoue H, Ishii C, et al. Effect of plaunotol in combination with clarithromycin or amoxicillin on Helicobacter pylori in vitro and in vivo. J Antimicrob Chemother (2002) 50:133–6.[Abstract/Free Full Text]

11 Instructions of the Health, Labour and Welfare Ministry concerning animal experiments. http://www.mhlw.go.jp/general/seido/kousei/i-kenkyu/doubutsu/0606sisin.html.

12 Dixon MF, Genta RM, Yardley JH, et al. Classification and grading of gastritis. The updated Sydney System. International Workshop on the Histopathology of Gastritis, Houston 1994. Am J Surg Pathol (1996) 20:1161–81.[CrossRef][Web of Science][Medline]

13 Otsuka T, Tsukamoto T, Tanaka H, et al. Suppressive effects of fruit-juice concentrate of Prunus mume Sieb. et Zucc. (Japanese apricot, Ume) on Helicobacter pylori-induced glandular stomach lesions in Mongolian gerbils. Asian Pac J Cancer Prev (2005) 6:337–41.[Medline]

14 Masaoka T, SuzukiKurabayashi HK, Kamiya AG, et al. Second-line treatment of Helicobacter pylori infection after dilution agar methods and PCR-RFLP analysis. Aliment Pharmacol Ther (2004) 20(Suppl 1):68–73.[Medline]

15 Koga T, Watanabe H, Kawada H, et al. Interactions of plaunotol with bacterial membranes. J Antimicrob Chemother (1998) 42:133–40.[Abstract/Free Full Text]

16 Fukuda Y, Yamamoto I, Okui M, et al. Combination therapy with mucosal protective agent for the eradication of Helicobacter pylori. Eur J Gastroenterol Hepatol (1995) 7(Suppl 1):S45–7.[Web of Science][Medline]

17 Nishizawa T, Suzuki H, Kurabayashi K, et al. Gatifloxacin resistance and mutations in gyrA after unsuccessful Helicobacter pylori eradication in Japan. Antimicrob Agents Chemother (2006) 50:1538–40.[Abstract/Free Full Text]


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