Susceptibility of Helicobacter pylori to simethicone

Journal of Antimicrobial Chemotherapy (1996) 37, 45-52

Susceptibility of Helicobacter pylori to simethicone and other non-antibiotic drugs R. Ansorg, G. von Recklinghausen and £ . Heintschel von Heinegg

Institute of Medical Microbiology, University of Essen, 45147 Essen, Germany

Introduction

Helicobacter pylori is the causative agent of chronic type B gastritis and peptic ulcer disease (Buck, 1990; Lee, Fox & Hazell, 1993; NIH, 1994). It may be associated with some forms of non-ulcerative dyspepsia, and is probably related to gastric adenocarcinoma (Parsonett et al., 1991), MALT lymphoma (Wotherspoon et al., 1991), and Non-Hodgkin's lymphoma (Parsonett et al., 1994). H. pylori infection is present throughout the world. In industrialized countries, the incidence of infection is about 0.5%-1.0% per year; approximately 50% of 60-year-old adults are infected (Blaser, 1992). In non-industrialized countries, the prevalence rates are considerably higher, and even children are commonly infected (Talley & Noack, 1993). Thus, it is likely that one-half of the world's population is infected with H. pvlori (Blaser, 1992). Effective preventative measures are lacking, particularly because the mode of transmission from person-to-person is unknown (Mitchell, 1993), and because a vaccine will not be available in the near future (Goodwin, 1993). Likewise, eradication of H. pylori is hard to achieve. Eradication rates in the range of 80%-90% require triple regimens, e.g. bismuth subsalicylate, tetracycline, and metronidazole (NIH, 1994), or ranitidine, amoxicillin, and metronidazole (Hentschel et al., 1993), or a dual regimen consisting of omeprazole plus amoxycillin (Bayerdorffer et al., 1992). These regimens have substantial side effects, in particular emergence of resistance to antimicrobials (NIH, 1994). Moreover, they are quite expensive. It is recommended, therefore, to confine antimicrobial eradication treatment to symptomatic patients with duodenal and gastric ulcers (NIH, 1994). 0305-7453/96/010045 + 08 $12.00/0

45 % 1996 The British Society for Antimicrobial Chemotherapy

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The antifoaming agent simethicone (Lefax), the protease inhibitor gabexate mesilate (FOY), the antimycotic ketoconazole, and the hydroxyl scavangers dimethylsulphoxide (DMSO) and allopurinol were investigated for growth inhibition of Helicobacter pylon and representative strains of other bacterial species. H. pylori were selectively inhibited by 64-128 mg/L of simethicone, 64-128 mg/L gabexate mesilate, and 16-64 mg/L ketoconazole Dimethylsulphoxide and allopurinol showed no antibacterial effect at concentrations used therapeutically. It is concluded that gabexate mesilate, ketoconazole and, particularly, simethicone are candidates for treatment of H. pylon infection.

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Materials and methods Drugs, strains, and culture conditions Simethicone was used in its therapeutic formulation Lefax (Asche AG, Hamburg, Germany). One mL of the suspension contains 41.2 mg of dimeticon3000-silicium dioxide 97:3 (simethicone). Gabexate mesilate (FOYlm) was purchased from Ono Pharmaceutical Co., Osaka, Japan. Ketoconazole was supplied by Janssen AG, Neuss, Germany. Dimethylsulphoxide and allopurinol were obtained from Sigma Chemical Co., USA. Except for ketoconazole, the drugs were dissolved and diluted in physiological saline. Ketoconazole was dissolved in 98% acetic acid and diluted in physiological saline reaching a final acetic acid concentration of 0.5%. Eleven clinical H. pylori strains (IMMI) isolated from antral biopsy specimens, and the reference strain H. pylori ATCC 43504 were used. For comparison and control, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Bacteroides thetaiotaomicron ATCC 29148, and Clostridium perfringens ATCC 13124 were included. The strains were cultured on sheep blood agar composed of blood agar base (CM 55, Oxoid, Basingstoke, UK) and 5% sheep blood (FSR 1055, Oxoid). The H. pylori strains were cultured at 37"C for 5 days under microaerophilic conditions (Anaerocult C. E. Merck, Darmstadt, Germany); S1. aureus, E. coli, and P. aeruginosa were cultured aerobically at 37"C for 24 h. B. thetaiotaomicron and C. perfringens were cultured anaerobically at 37"C for 48 h. Cultures were harvested by rinsing with physiological saline. Inoculum suspensions were adjusted to a turbidity corresponding to McFarland turbiditv standard no. 2.


However, if it is accepted that H. pylori infection results in a sequence of changes in the gastric mucosa that ultimately lead to intestinal metaplasia, dysplasia and gastric cancer (Telford et al., 1994), the indication for treatment will extend to patients with non-ulcer dyspepsia or asymptomatic individuals (O'Morain & Buckley, 1994). The classic antibiotics are not suitable for such a widespread use. Drugs or drug combinations which would allow the extension of eradication treatment to every individual harbouring H. pylori should be: (i) free of the ecological impact of antibacterial antibiotics, (ii) nontoxic even in high dosages, and applicable to young children, (iii) as inexpensive as possible, so as not to be restricted to rich people or industrial countries. In order to develop alternative H. pylori eradication regimens, drugs were selected that are not classic antibiotics but which are being used in gastrointestinal disorders, at least experimentally. Such drugs are the defoaming agent simethicone, prescribed for meteorism (Auteri, 1991); the protease inhibitor gabexate mesilate used in acute pancreatitis (Yang, Chang-Chien & Liaw, 1987; Biichler et al., 1993); the antimycotic ketoconazole used to reduce yeast colonization of the gut (Kucers & Bennett, 1987; von Recklinghausen, Di Maio & Ansorg, 1993), and the hydroxyl scavangers dimethylsulphoxide (DMSO) and allopurinol, both used in the prevention of duodenal ulcer relapse (Salim, 1990). The susceptibility of H. pylori to these substances is investigated in the present study.

Susceptibility of H. pylori to non-antibiotics

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Agar diffusion assay Test plates of 4 mm agar depth were prepared by pouring CD agar consisting of blood agar base (CM 55, Oxoid) supplemented with 1 g/L 2,6-di-o-methyl-/?-cyclodextrin (Teijin Lim., Tokyo, Japan) in Petri dishes. The agar plates were seeded with 0.1 mL inoculum suspensions. Circular wells of 4 mm diameter were removed from the seeded agar by means of a metal cylinder and slight negative pressure. The agar wells were filled with 25 jih of serial dilutions of the test drugs. After incubation under the same culture conditions as used for the inoculum preparation, the zones of growth inhibition were read.

Microagar dilution assay

Combination test The microagar dilution assay technique was used. Serial dilutions of the first drug were prepared in physiological saline containing the second drug. After addition of agar, the final concentrations of the first drug were the same as those used for the MIC determinations, and the final concentration of the second drug was 1/4 of its MIC for the strain tested.

Results In the agar diffusion test used for screening of the antibacterial effect, the test strains H. pylori ATCC 43504 and H. pylori IMMI 676/89 showed inhibition zones at 2.6 mg/mL simethicone, at 2 mg/mL gabexate mesilate, and at 0.2 mg/mL ketoconazole. No zones of inhibition were produced by 10% (v/v) DMSO and 10 mg/mL allopurinol. The drugs showing an antibacterial effect were investigated further using the microagar dilution test (Table I). The MICs of simethicone against H. pylori was 64 mg/L in three strains, and 128 mg/L in nine strains. The control strains of S. aureus, E. coli, P. aeruginosa, B. thetaiotaomicron, and C. perfringens were not inhibited up to a concentration of 4120 mg/L. Gabexate mesilate showed MICs in the range of 64-128 mg/L against the 12 H. pylori strains. Of the control strains, S. aureus had a MIC of 256 mg/L. The MICs for the other control strains were 1024 mg/L and higher. Ketoconazole inhibited the H. pylori strains with a MIC range of 16-64 mg/mL; the MICs for the control organisms were greater than 1024 mg/L.


Serial dilutions (20 fiL) of the drugs were prepared in microtiter plates with flat-bottomed wells (Nunc, Wiesbaden, Germany). The dilutions were mixed with melted CD agar (180 /iL) at 60°C. After solidification, the plates were incubated at 37°C for 2 h in order to dry the agar surface. The assay plates had the following final drug concentrations: simethicone 4120-4 mg/L, gabexate mesilate 1024—1 mg/L, ketoconazole 1024—1 mg/L. Inoculum suspensions (10/iL) were spotted on the agar-filled wells. Incubation of the inoculated plates was performed under the same culture conditions as used for inoculum preparation. Minimum inhibitory concentration (MIC) was recorded by reading the lowest drug concentration that inhibited visible growth. Growth controls were performed on agar containing physiological saline and 0.5% acetic acid, respectively, instead of drug dilution.

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R. Ansorg tt al. Table I. Minimum inhibitory concentrations (MIC) of simethicone, gabexate mesilate, and ketoconazole against H. pylori and control strains simethicone

Strain

128 128 64 128 128 64 128 128 64 128 128 128 >4120 >4120 >4120 >4120 >4120

128 128 64 128 64 64 64 64 32 64 64 128 256 1024 >1024 > 1024 >1024

ketoconazole

> > > > >

16 16 64 16 16 32 16 16 16 32 64 32 1024 1024 1024 1024 1024

Combinations of simethicone, gabexate mesilate, and ketoconazole were tested against H. pylori ATCC 43504 and H. pylori IMMI 676/89 by determining the MIC of one drug in the presence of a fixed subinhibitory concentration of the second drug (Table II). A synergistic influence of the second drug can be assumed when the MIC of the first drug is diminished at least fourfold in comparison to the MIC determined in the absence of the second drug. The MIC of simethicone was not altered significantly in the presence of gabexate mesilate or ketoconazole and vice versa. The combination of gabexate mesilate and ketoconazole had a different effect on the two test strains. Against H. pylori ATCC 43504, the MICs of gabexate mesilate and ketoconazole

Table II. Effect of combinations of simethicone, gabexate mesilate, and ketoconazole on H. pylori

Test drug Simethicone Gabexate mesilate Ketoconazole

Combination with — Gabexate mesilate 32 mg/L Ketoconazole 4 mg/L Simethicone 32 mg/L Ketoconazole 4 mg/L Simethicone 32 mg/L Gabexate mesilate 32 mg/L

MIC (mg/L) of the test drug against H. pvlori H. pvlori ATCC'43504 IMMI 676/89 128 128 256 128 64 128 16 16 16

128 64 128 128 64 32 16 16 8


H. pvlon ATCC 43504 IMMI 676/89 IMMI 32/93 IMMI 327/92 IMMI 99/93 IMMI 363/93 IMMI 371/93 IMMI 375/93 IMMI 377/93 IMMI 399/93 IMMI 405/93 IMMI 1/94 S. aureus ATCC 25923 E. coli ATCC 25922 P. aeruginosa ATCC 27853 B. thetaiotaomicron ATCC 29148 C. perfringens ATCC 13124

MIC (mg/L) gabexate mesilate


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remained unchanged by the addition of the second drug. Against H. pylori IMMI 676/89, however, the addition of ketoconazole diminished the MIC of gabexate mesilate from 128 to 32 mg/mL. However, the addition of gabexate mesilate to ketoconazole had no significant effect on the MIC of ketoconazole. An antagonistic interaction of simethicone, gabexate mesilate, and ketoconazole was not observed in the combination tests. Discussion

The activity of ketoconazole against H. pylori corroborates previous studies showing susceptibility of the bacterium to nitroimidazole antimycotics (Rautelin et al., 1992; von Recklinghausen, Di Maio & Ansorg, 1992). Ketoconazole is therapeutically administered by the oral route; the dose recommended for adults is 200 mg (one tablet) daily (Kucers & Bennett, 1987). Transformation of the compound into an active and absorbable hydrochloride salt occurs in the stomach (Kucers & Bennett, 1987), so that local concentrations exceeding the MICs of 16 to 64 mg/L for H. pylori are probably achieved. Pharmacological and toxicological data of the drug are well known. Thus, clinical studies to prove its value in the elimination of H. pylori from the stomach are justified. The antibacterial spectrum of ketoconazole is obviously confined to H. pylori, and probably staphylococci (Heeres et al., 1979). In contrast to the antibiotics used at


In a placebo-controlled study, it has been shown that administration of DMSO and allopurinol reduces the recurrence of duodenal ulceration to a significant extent (Salim, 1990). The patients received 5 mL of 10% DMSO or 5 mL of 1% allopurinol every 6 h over 1 year. It has been suggested that the preventative effect of the two drugs is based on their ability to remove oxygen-derived free radicals (Salim, 1990). The effect on H. pylori, which is implicated as a major factor in the pathogenesis of peptic ulceration, was not investigated. The present results show that therapeutically administered concentrations of DMSO and allopurinol have no growth-inhibiting effect on H. pylori in vitro. Thus, the two drugs may not be suitable for H. pylori eradication treatment. However, it is not ruled out that both drugs could interfere with adherence factors of H. pylori, and in this way prevent recolonization of the gastro-duodenal epithelium and duodenal ulcer relapse. The protease inhibitor gabexate mesilate (MG 417.48), which also diminishes tryptic activation of pancreatic phospholipase A2 (Hesse, Lankisch & Kunze, 1984), has been used in the therapy of acute pancreatitis. Doses between 900 mg and 4,000 mg were applied iv (Yang et al., 1987; Buchler et al., 1993). Since the half-life of gabexate mesilate is less than 1 min, the concentration in the blood is calculated to be less than 10- 6 mol/L (Freise, Wittenberg & Magerstedt, 1989) corresponding to 0.417 mg/L. H. pylori produces proteolytic and lipolytic enzymes (Ottlecz et al., 1993), and growth inhibition by gabexate mesilate demonstrated in the present experiments is probably based on inhibition of the bacterial enzymes. However, the MICs ranging from 32 mg/L-128 mg/L are much higher than the drug levels in the blood, and it is improbable that concentrations inhibiting H. pylori are reached in the stomach and duodenum after intravenous administration. Local concentrations higher than the MICs may be attained by oral administration of gabexate mesilate but its pharmacological and toxicological characteristics after oral administration are not known. These data must be available before gabexate mesilate can be considered as a candidate for treatment of H. pylori infection.

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In conclusion, H. pylori shows susceptibility to simethicone, gabexate mesilate, and ketoconazole in concentration ranges that are achievable in the stomach under therapeutic conditions. The resistance of representatives of other bacterial species indicates specific interactions of the drugs with H. pylori, and offers the possibility of selective therapy without adverse effects on the normal bacterial flora. In view of the pharmacological data, simethicone appears to be the most promising agent for the treatment of all forms of H. pylori infection.

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present in the H. pylori regimens, it has the benefit of leaving the normal bacterial flora of the body unchanged and not selecting resistant bacteria. On the other hand, the possibility of side effects (Kucers & Bennett, 1987) would confine the treatment to symptomatic H. pylori infections. For some forty years, simethicone has been used in the symptomatic treatment of meteorism, and in premedication before X-ray, endoscopic, and sonographic examinations of the gastrointestinal tract (Auteri, 1991). The compound acts as a defoaming agent by enhancing the surface tension of fluids, thus destroying the fine gaseous bubbles in foam. Doses between 40 mg and 250 mg are administered three to four times per day (Niedner, 1982), and can be increased because the substance is chemically inert, non-absorbable, non-toxic, and free of adverse side effects (Heldwein et al., 1987; Wabnitz, R. W., personal communication 1994). The concentrations that can be attained in the stomach are higher than the demonstrated MICs of 64-128 mg/L for H. pylori. Therefore, the drug could be considered as an alternative to the more aggressive dual and triple regimens for H. pylori eradication therapy, and should be evaluated in clinical studies. Very recently, it has been reported that dimethylsiloxane (a compound related to simethicone), has a therapeutic effectiveness in treating gastric ulcers comparable to that of the H2-blocking agent ranitidine (Groitl, Schmidt & Upmeyer, 1994). H. pylori was eliminated more effectively in patients in the dimethylsiloxane group than in the ranitidine group. These clinical results and the present microbiological data, both showing for the first time the effect of siloxanes against H. pylori, prompt more extensive investigations of that drug family. There is a real chance that a therapeutic regimen for the eradication of H. pylori can be found that is not confined to symptomatic patients with duodenal and gastric ulcers but includes treatment of non-ulcer dyspepsia and asymptomatic H. pylori infections.


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