Effect of plaunotol in combination with clarithromycin or amoxicillin on Helicobacter pylori in vitro and in vivo
Tetsufumi Koga*,
Harumi Inoue,
Chika Ishii,
Yoko Okazaki,
Haruki Domon and
Yukio Utsui
Biological Research Laboratories, Sankyo Co., Ltd, 2-58 Hiromachi 1-chome, Shinagawa-ku, Tokyo 140-8710, Japan
Received 22 October 2001; returned 26 February 2002; revised 10 April 2002; accepted 24 April 2002
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Abstract
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Plaunotol, a cytoprotective anti-ulcer agent, has antibacterial activity against Helicobacter pylori. The purpose of the present study was to investigate the effect of plaunotol when combined with clarithromycin or amoxicillin against H. pylori. When combined with clarithromycin, plaunotol showed synergic activity against 11 of 14 strains, and additive activity against the other three strains, by chequerboard titration. When combined with amoxicillin, plaunotol showed additive activity against 10 of 14 strains. No antagonistic effects were seen against any of the strains tested. The interactions between plaunotol and either clarithromycin or amoxicillin were determined by timekill assay against the Sydney Strain (strain SS1) of H. pylori. The combination of plaunotol with clarithromycin showed synergic activity and with amoxicillin showed additive activity. In a C57BL/6 mouse gastritis model infected with H. pylori SS1, the plaunotolclarithromycin and plaunotolamoxicillin combinations both exhibited synergic effects, which allowed the effective dose of clarithromycin to be reduced when co-administered with plaunotol. These results suggest that plaunotol may have a useful role in combination with anti-H. pylori drugs in the treatment of H. pylori-associated diseases.
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Introduction
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Eradication of Helicobacter pylori is reported to be important to prevent relapse and accelerate the healing of duodenal and gastric ulcers,1 and there are a number of treatment regimens currently used to eradicate this organism. Plaunotol is often used as a cytoprotective anti-ulcer medication for gastritis and gastric ulcers in Japan. It has also been used in combination therapies against H. pylori-associated diseases because of its strong bactericidal effect against H. pylori. Furthermore, the combination of plaunotol, clarithromycin and a proton-pump inhibitor (PPI) is reported to eradicate H. pylori better than the combination of clarithromycin and PPI alone.2 In a previous study, we demonstrated that plaunotol enhances the antibacterial activities of clarithromycin and amoxicillin in a nude mouse model.3 However, no in vitro data on the interaction of plaunotol in combination with clarithromycin or amoxicillin have been available to date. In this study, we have evaluated the antibacterial activity of plaunotol when combined with clarithromycin or amoxicillin against H. pylori by chequerboard titration and timekill assay. The in vivo activities of the plaunotolclarithromycin and plaunotolamoxicillin combinations were also examined in a C57BL/6 mouse gastritis model infected with the Sydney Strain (strain SS1) of H. pylori.
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Materials and methods
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Agents
Plaunotol (Sankyo Co., Ltd, Tokyo, Japan), clarithromycin (Taisho Pharmaceutical Co., Ltd, Tokyo, Japan) and amoxicillin (Sigma Chemical Co., St Louis, MO, USA) were used.
Bacteria
One standard strain (NCTC 11637), 12 clinical isolates from Japan and the Sydney Strain (SS1) of H. pylori were used. These strains were grown on brainheart infusion agar (BHI; Difco Laboratories, Detroit, MI, USA) supplemented with 5% defibrinated horse blood (BHI blood agar) at 37°C for 3 days, in GasPak jars (Becton Dickinson Microbiology Systems, Sparks, MD, USA) with CampyPak inserts (Becton Dickinson).
Chequerboard method
Antimicrobial interactions were evaluated by agar chequerboard titration.4 Plaunotol, clarithromycin and amoxicillin were dissolved in dimethylsulphoxide, methanol and distilled water, respectively. Inocula were prepared by suspending growth from BHI blood agar plates in physiological saline equivalent to a 2.0 McFarland standard (108 cfu/mL). A final inoculum of 105 cfu/spot was applied using an Auto-Inoculator (Dai Nippon Seiki Co. Ltd, Kyoto, Japan). Experiments were performed in triplicate, and all plates were incubated for 3 days at 35°C under microaerophilic conditions. 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)
FIC indices were interpreted as follows:
0.5, synergy; >0.51, additive; >14.0, indifference; >4, antagonism.4
Timekill assay
H. pylori SS1 cultured overnight in 1 mL (c. 2 x 108 cfu/mL) of Brucella broth (Becton Dickinson and Company, Cockeysville, MD, USA) supplemented with 2% heat-inactivated fetal bovine serum (FBS broth) were inoculated into a 50 mL flask containing 24 mL of drug supplemented with the same medium. The concentrations used were the MIC of plaunotol and 0.5 x MIC of clarithromycin or 0.5 x MIC of amoxicillin. Viability counts were carried out after incubation at 37°C on a gyratory shaker rotating at 110 rpm. At 0, 6, 12, 24 and 36 h, the samples were removed and 10-fold serial dilutions were plated on to BHI blood agar plates. This experiment was carried out in triplicate. Data obtained were analysed by determining the viable count that yielded a
log10 cfu/mL reduction after 36 h of incubation. Synergy was defined as a
2 log10 cfu/mL decrease in the viable count with the drug combination, in comparison with the most active agent alone.5 Additive activity was defined as a viability count of 01.9 log10 cfu/mL below the starting inoculum after combination treatment.5
Animals
Six-week-old male C57BL/6 mice were obtained from Charles River Inc. (Kanagawa, Japan). The mice were fed a commercial diet (FR-2; Funabashi Farms, Chiba, Japan) and given water ad libitum. All animal experiments were carried out according to the guidelines provided by the Institutional Animal Care and Use Committee of Sankyo Co., Ltd.
Induction of infection with H. pylori
H. pylori SS1 was cultured in FBS broth on a gyratory shaker at 110 rpm for 24 h at 37°C. Mice were orally inoculated with 1.5 mL of bacterial suspension of 2 x 108 cfu/mL after 1 day of fasting. Therapy began 5 days after infection.
Combination therapy
Plaunotol was dissolved in distilled water supplemented with 0.4% Tween 80 (Tween solution; Kanto Chemical Co., Inc., Tokyo, Japan). Clarithromycin and amoxicillin were suspended in 0.5% sodium carboxymethyl cellulose (CMC solution, Kanto Chemical Co., Inc.). The mice received 30 or 100 mg/kg/dose of plaunotol, or 20 mg/kg/dose of clarithromycin or amoxicillin once daily for 5 days. Additional administrations included 30 or 100 mg/kg/dose of plaunotol with 20 mg/kg/dose of clarithromycin, and 30 or 100 mg/kg/dose of plaunotol with 20 mg/kg/dose of amoxicillin. Control mice received oral administrations of CMC and Tween solutions.
Clarithromycin-sparing effect of plaunotol in vivo
To evaluate the clarithromycin-sparing effect of plaunotol in vivo, mice were administered 30 mg/kg/dose of plaunotol plus 10 mg/kg/dose of clarithromycin, or 10, 20, 30 or 40 mg/kg/dose of clarithromycin alone once daily for 5 days.
Measurement of the viable count of H. pylori in the stomachs of mice
After the final administration, the mice were fasted for 1 day, killed, and samples were obtained by incising the mucosa of the stomach. After homogenizing the samples with 2 mL of FBS broth, 100 µL aliquots from 10-fold serial dilutions were inoculated onto modified Skirrows agar plates.3 The plates were then incubated at 37°C for 7 days in a microaerophilic environment, and the colonies of H. pylori were counted. Bacterial counts are expressed as cfu per stomach.
Statistical analysis
The statistical analysis was performed by two-way ANOVA to determine whether the antimicrobial combinations showed synergic activity, and by Dunnetts test to determine the difference between the therapeutic effects of clarithromycin alone and clarithromycin in combination with plaunotol. P values of <0.05 were considered statistically significant.
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Results and discussion
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MIC50/MIC90 values (mg/L) of plaunotol, clarithromycin and amoxicillin against 14 H. pylori strains were 3.13/12.5, 0.006/0.025 and 0.1/0.1, respectively. The plaunotolclarithromycin and plaunotolamoxicillin combinations demonstrated synergic effects against 79% and additive effects against 71%, respectively, of the strains by chequerboard titration; the other strains showing additive effects or indifference, respectively. FIC indices of H. pylori SS1 exposed to the plaunotolclarithromycin (0.63) and plaunotolamoxicillin (1.0) combinations were additive. Figure 1 shows the timekill curves of exposure to the combination of plaunotol (1.56 mg/L) and clarithromycin at 0.5 x MIC (0.006 mg/L) or amoxicillin at 0.5 x MIC (0.05 mg/L). The combination of plaunotol with clarithromycin was synergic, with a 3.6 log10 decrease in the viable count relative to the most active drug alone (Figure 1a), and that with amoxicillin was additive, with a 1.1 log10 decrease relative to the starting inoculum (Figure 1b). Studies by others have established that the timekill assay is more discriminatory than the chequerboard method for synergy testing of a wide variety of bacteria.6 In this study, synergic activity of plaunotol and clarithromycin for H. pylori SS1 was determined by timekill assay, whereas additive activity was determined by chequerboard titration. The mechanism by which plaunotol directly fluidizes the H. pylori membrane7 might explain these synergic and additive interactions. As plaunotol is hypothesized to cause a change in membrane fluidity, with an associated increased membrane permeability,7 plaunotol might potentiate the anti-H. pylori activity of clarithromycin and amoxicillin by increased permeability of the membranes. Since clarithromycin is a larger hydrophobic molecule than amoxicillin, clarithromycin may be more susceptible to permeabilization of the membrane by plaunotol than amoxicillin.

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Figure 1. Timekill curves of exposure to the combination of plaunotol and (a) clarithromycin at 0.5 x MIC or (b) amoxicillin at 0.5 x MIC on H. pylori SS1. The data are expressed as mean ± S.E.m. of three experiments. Open circles, control; filled circles, plaunotol at 1 x MIC; triangles, clarithromycin or amoxicillin at 0.5 x MIC; squares, plaunotol + clarithromycin or plaunotol + amoxicillin.
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Figure 2 shows the therapeutic effects of plaunotol in combination with clarithromycin and amoxicillin. The combination therapies with 30 mg/kg/dose and 100 mg/kg/dose of plaunotol plus clarithromycin (20 mg/kg/dose), or 100 mg/kg/dose of plaunotol plus amoxicillin (20 mg/kg/dose) resulted in synergic activity. It is reported that plaunotol concentration in gastric mucosa 30 min after oral administration of 30 mg/kg of [14C]plaunotol in rats showed 196.8 µg/g tissue.8 These synergic results are supported by a clinical study in which a combination of plaunotol, clarithromycin and PPI was reported to achieve a 69% eradication rate, versus a 38% eradication rate achieved with a combination of clarithromycin and PPI.2 Although there have been no clinical trials based on the synergic activity of plaunotol when combined with clarithromycin or the additive activity when combined with amoxicillin, our results suggest that the administration of a combination of PPI, a half dose of amoxicillin and clarithromycin plus plaunotol deserves further evaluation.

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Figure 2. Clarithromycin (CAM) or amoxicillin (AMX) in combination with plaunotol against H. pylori infection in C57BL/6 mice. Plaunotol was given at 30 or 100 mg/kg/dose, and clarithromycin or amoxicillin was given at 20 mg/kg/dose, for 5 days. Each vertical bar indicates the mean ± S.E.m. of seven mice. Asterisks indicate a statistically significant reduction (*P < 0.01, **P < 0.001 versus control group). White bar, control; black bar, 30 mg/kg/dose of plaunotol; light grey bar, 100 mg/kg/dose of plaunotol; hatched bars, 20 mg/kg/dose of clarithromycin or amoxicillin; dark grey bars, 30 mg/kg/dose of plaunotol + clarithromycin or plaunotol + amoxicillin; striped bars, 100 mg/kg/dose of plaunotol + clarithromycin or plaunotol + amoxicillin.
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The therapeutic effect of clarithromycin at the 10 mg/kg/dose in combination with plaunotol at the 30 mg/kg/dose was comparable to that of clarithromycin at the 30 mg/kg/dose alone (Figure 3). While the dose of clarithromycin has been reported to be important for the management of H. pylori infection,9 adverse effects, especially taste disturbances, glossitis or other symptoms related to the oral cavity, were significantly more frequent at higher doses of clarithromycin.10 Since co-administration with plaunotol may reduce the required dose of clarithromycin, this could improve patient compliance.

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Figure 3. Clarithromycin (CAM)-sparing effects of plaunotol against H. pylori infection in C57BL/6 mice. Plaunotol was given at a 30 mg/kg/dose, and clarithromycin was given at a 10, 20, 30 or 40 mg/kg/dose, for 5 days. Each vertical bar indicates the mean ± S.E.m. of seven mice. The asterisk indicates a statistically significant reduction (*P < 0.01, **P < 0.001 versus control group). White bar, control; black bar, 30 mg/kg/dose of plaunotol; light grey bar, 10 mg/kg/dose of clarithromycin; hatched bar, 20 mg/kg/dose of clarithromycin; dark grey bar, 30 mg/kg/dose of clarithromycin; diagonally striped bar, 40 mg/kg/dose of clarithromycin; vertically striped bar, 30 mg/kg/dose of plaunotol plus 10 mg/kg/dose of clarithromycin.
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In conclusion, plaunotol has been demonstrated to interact synergically with clarithromycin in vitro and in vivo, and additively in vitro and synergically in vivo with amoxicillin.
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Footnotes
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* Corresponding author. Tel: +81-3-3492-3131 ext. 3516; Fax: +81-3-5485-8565; E-mail: tekoga{at}shina.sankyo.co.jp 
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