In vitro transfer of clarithromycin and amoxicillin across the epithelial
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Journal of Antimicrobial Chemotherapy (2002) 50, 865–872 DOI: 10.1093/jac/dkf219 In vitro transfer of clarithromycin and amoxicillin across the epithelial barrier: effect of Helicobacter pylori Tamara Matysiak-Budnik1*, Martine Heyman1, Céline Candalh1, Denis Lethuaire2 and Francis Mégraud3 1INSERM EMI-0212, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris; 2Sanofi-Synthélabo, 82 avenue Raspail, 94255 Gentilly Cedex; 3Laboratoire de Bactériologie, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France Received 6 December 2001; returned 26 May 2002; revised 15 August 2002; accepted 22 August 2002 Downloaded from http://jac.oxfordjournals.org/ by guest on March 15, 2015 The mode of absorption of amoxicillin and clarithromycin, two antibiotics used in the treatment of Helicobacter pylori infection, has not been completely elucidated. The aims of this study were to investigate the passage of these antibiotics across normal and infected epithelium and to measure their accumulation in HT29-19A or Caco2 epithelial cell monolayers. In non-infected cultures, basal-to-apical fluxes were significantly higher than apical-to-basal fluxes for both antibiotics, but this difference disappeared in monolayers infected with H. pylori. In 24 h studies, clarithromycin, but not amoxicillin, showed rapid intracellular accumulation. No difference was found between the transepithelial passage of amoxicillin across the HT29-19A and Caco2 mono- layers. Keywords: Helicobacter pylori, amoxicillin, clarithromycin, Ussing chamber, absorption Introduction detected in the gastric mucosa after oral administration.5 However, after intravenous (iv) administration, amoxicillin Amoxicillin and clarithromycin are two antibiotics widely was found in gastric juice7 and led to eradication of used as antibacterials that have been used to treat Helico- H. pylori,8,9 suggesting that eradication of the bacteria may be bacter pylori infection. Resistance in H. pylori to amoxicillin achieved by a systemic action of amoxicillin. has rarely been reported, whereas resistance to clarithromycin In contrast to amoxicillin, which is present mainly in the is increasing steadily.1 Treatments with each one of these extracellular space, it has been reported that clarithromycin drugs when used as a single antibiotic therapy led to eradica- may accumulate in the cells, suggesting a mainly systemic tion of H. pylori in only 10–40% of infected patients, and effect.10 After iv injection, the plasma concentration of clari- when associated with a proton pump inhibitor, in 20–80% of thromycin decreases immediately, and after that it increases patients.2,3 The relatively low efficacy of amoxicillin and slowly with time.11 Based on this observation, it has been clarithromycin in vivo may be due to insufficient local drug hypothesized that the drug accumulates in the cells and is sub- concentrations at the locus of infection after oral adminis- sequently released gradually into the extracellular fluid.12 tration.4–6 Although the absorption of amoxicillin and clarithromycin H. pylori is located at the surface of the gastric epithelium, has been studied in vitro13,14 and in vivo,15 there is no direct in the mucus layer, and thus is not easily accessible to drugs. evidence supporting this hypothesis. For this reason, we have The question of whether bacterial eradication is achieved by undertaken studies to measure the transepithelial passage of the topical or systemic effect of amoxicillin in the gastric both drugs. mucosa remains the subject of discussion. For a long time, the Employing an epithelial barrier model, the absorption of main mode of action of amoxicillin has been considered to amoxicillin and clarithromycin was investigated as a function be topical since, in healthy volunteers, amoxicillin has been of the mode (mucosal or serosal) of their administration, as .................................................................................................................................................................................................................................................................. *Corresponding author. Tel: +33-1-40-61-56-34; Fax: +33-1-40-61-56-38; E-mail: matysiak@necker.fr ................................................................................................................................................................................................................................................................... 865 © 2002 The British Society for Antimicrobial Chemotherapy
T. Matysiak-Budnik et al. well as their intracellular absorption. The effects of the attached) bacteria, and the filter-grown monolayers (with presence of H. pylori on the absorption of the antibiotics by attached bacteria) were placed between two half chambers of HT29-19A epithelial cells grown as monolayers on micro- an Ussing chamber to study the fluxes of the antibiotics. porous filters and presenting tight junctions were measured using radiotracer analysis. Antibiotics Radiolabelled [14C]clarithromycin (specific activity: 56.6 mCi/mM; radiochemical purity: >99%, labelled with 14C in Materials and methods 6-O-methyl position) was provided by Abbott Laboratories Culture of the intestinal cell lines HT29-19A and Caco2 (Abbott Park, IL, USA) and [3H]amoxicillin (specific activ- ity: 43.7 mCi/mM; radiochemical purity: 96.4%, labelled The intestinal cell line HT29-19A is a clone derived from the with 3H in position 3 of the phenol moiety) was provided by human colonic adenocarcinoma cell line HT29, which forms SmithKline Beecham (King of Prussia, PA, USA). tight monolayers of differentiated cells, widely used in func- tional studies of the epithelial barrier.16 These cells were Short-term absorption of the antibiotics in Ussing cultured at 37°C in Dulbecco’s modified Eagle’s medium chamber (Figure 1a) (DMEM; GIBCO, Life Technologies) containing 10% heat- inactivated fetal calf serum (Boehringer Mannheim, Grenoble, The Ussing chamber system has already been used to study Downloaded from http://jac.oxfordjournals.org/ by guest on March 15, 2015 France), 4 mM glutamine and 50 µg/mL gentamicin, in a antibiotic transfer across the epithelium in vitro.13,19 We used humidified atmosphere of 5% CO2/95% air. In addition, the this system to study short-term absorption of the antibiotics Caco2 intestinal cell line was used for amoxicillin study across the HT29-19A epithelial monolayer. The study in the because these cells are known to express a specific transport Ussing chamber lasted 1 h 40 min, a period of time during carrier for β-lactam antibiotics (PepT1),17 which is not con- which the viability of the tissue is preserved, allowing the stitutively expressed in HT29-19A cells.18 These cells were study of the early absorption of the antibiotics. Each side of cultured in DMEM supplemented with 20% heat-inactivated the exposed monolayer (surface area 0.150 cm2) was bathed fetal calf serum, 4 mmol/L glutamine and 1% non-essential with 1.5 mL of Ringer solution, and oxygenated at 37°C for amino acids. the whole duration of the experiment. The electrical para- For experimental purposes, HT29-19A cells were seeded meters, potential difference (PD) and electrical resistance at a density of 0.6 × 106 per cm2 on polyethylene terephthalate (R), attesting the integrity of the epithelium, were checked for filters with a pore diameter of 0.4 µm and a surface area of 2 h at 30 min intervals. Administration of [3H]amoxicillin 0.9 cm2 (Falcon; Becton Dickinson Labware, NJ, USA). and [14C]clarithromycin started after a 30 min period of Caco2 cells were seeded at a density of 0.6 × 106 per cm2 on equilibration of the system. Both antibiotics were introduced Transwell polycarbonate insert filters with a pore diameter of together into the apical (mucosal) or basal (serosal) compart- 0.4 µm and a surface area of 1 cm2 (Costar, Corning, NY, ment of the Ussing chamber at a final concentration of 10 mg/L USA). each. Transport of the antibiotics was evaluated by sampling Upon reaching confluence, both HT29-19A and Caco2 the opposite compartment at 30 min intervals. At each time cells progressively formed confluent monolayers with apical point, a 500 µL sample was obtained from a receiver compart- and basolateral domains separated by tight junctions. They ment and was replaced with an equivalent volume of Ringer were studied after 21 days of culture, when the electrical solution. resistance, checked on the filter-grown monolayers mounted The concentration of radiolabelled amoxicillin and clari- in an Ussing chamber, reached stable values of ∼150 Ω/cm2. thromycin was measured on 500 µL samples by β scintillation counting. Unidirectional fluxes of amoxicillin (Jamox) and Bacterial strain clarithromycin (Jclari), apical-to-basal or basal-to-apical, were An H. pylori strain isolated from a patient with duodenal ulcer calculated using the following equation: and subcultured minimally (less than five times) was used in Jamox (or Jclari) = ∆Q/dt·1/A this work. Bacteria were grown on Vitox chocolate agar (Oxoid, Dardilly, France) under micro-aerobic conditions where ∆Q represents the amount of amoxicillin or clarithro- (CampyGen in jar) for 24–36 h at 37°C. A bacterial suspen- mycin accumulated in the opposite compartment during the sion, containing from 6 × 107 to 9 × 107 cfu/mL, was prepared time interval dt, and A the exposed area of tissue. The fluxes of and used to infect the epithelial cells on the apical side. After both antibiotics are expressed in ng/h·cm2. 24 h incubation, the cell monolayers were washed with The same experiment was carried out with non-infected phosphate-buffered saline in order to remove planktonic (not (n = 20) and H. pylori-infected (n = 10) cell monolayers. 866
Absorption of clarithromycin and amoxicillin Figure 1. (a) Ussing chamber. Epithelial cells grown as monolayers on filters are placed between two half chambers of the Ussing chamber separat- Downloaded from http://jac.oxfordjournals.org/ by guest on March 15, 2015 ing two compartments, basal and apical. Antibiotics are added to one of the compartments and their presence on the opposite side is measured as a function of time. (b) Cell culture insert. Inserts with filters bearing the epithelial cells, separating the basal and apical compartments, are bathed in cell culture medium. Antibiotics are added to the apical or basal compartment and their presence on the opposite side is measured by sampling this compartment at different time points. Long-term absorption of the antibiotics across described as bearing the specific oligopeptide transporter filter-grown intestinal monolayers (Figure 1b) PepT1, which can be used by this antibiotic to cross the epithelium. This part of the study was carried out in order to evaluate the long-term (up to 24 h) absorption of antibiotics across the HPLC chromatographic analysis of [14C]clarithromycin epithelium, and particularly their intracellular accumulation. Since the cell viability in Ussing chambers cannot be main- or its metabolites during transport across the HT29-19A tained for more than 2–3 h, these experiments were carried cell monolayers out using the cell monolayers grown on Falcon inserts. The While amoxicillin is excreted mainly in an intact form,20 inserts bearing the cells were bathed in the cell culture clarithromycin is known to undergo substantial metabolism.11 medium and kept at 37°C under an air 95%/5% CO2 atmo- Therefore, this part of the study was carried out in order to sphere, which maintained the viability of the tissue for the determine whether clarithromycin is metabolized during its whole duration of the experiment. Antibiotic, [14C]clarithro- mycin or [3H]amoxicillin, was added separately, at final transepithelial passage across the HT29-19A intestinal cell concentrations of 10 mg/L, to the basal (serosal) or apical monolayer. HPLC chromatographic methods have been used (mucosal) compartment of the insert (surface 0.9 cm2) bearing widely for the determination of macrolide antibiotics in the cell monolayers grown on the microporous filters. The different biological samples.21,22 We used a gel exclusion apical and basal compartments were sampled at 10, 30, 60 and HPLC method with in-line detection of radioactivity to 360 min and at 10 and 24 h, starting from introduction of the analyse clarithromycin and its metabolites. To this end, antibiotic. For each time point, six separate filter-grown [14C]clarithromycin was added at a concentration of 10 mg/L monolayers were studied. At the same time points, the filter- to the basal or apical compartment of filter-grown HT29-19A grown cells (one filter bearing ≈106 cells) were cut out of the monolayers. After 8 h incubation, samples from the media inserts, washed four times with Ringer solution in order to in the apical and basal compartments were collected and remove extracellular antibiotic, and placed into 1 M nitric assayed. The 500 µL samples were concentrated using the acid in order to obtain the cell lysis and liberation of intra- Speed-vac system by evaporating the excess of water during cellular drug. After a 24 h incubation, the lysed cells were centrifugation at low temperature in the Speed Vac Plus centrifuged for 3 min at 3000 rpm, and 500 µL of supernatant apparatus (Savant Instruments, Farmingdale, NY, USA). The was used to measure the radioactivity. resulting volume of 200 µL was injected into a steric exclu- Additionally, the same 24 h study was carried out using sion HPLC column (Superdex Peptide PE 7.5/300). The Caco2 cells with only amoxicillin. This cell line has been column was eluted at 30°C for 45 min with 0.1% trifluoro- 867
T. Matysiak-Budnik et al. acetic acid in 30% acetonitrile at a flow of 0.3 mL/min with centration of 10 mg/L each did not affect the integrity of the detection of eluting compounds using a radiochemical epithelial barrier as assessed by its electrical resistance, which detector (Berthold HPLC radioactivity monitor LB 506 6-1). did not differ between the amoxicillin + clarithromycin exposed cells (n = 8) and control cells (n = 8), whose resist- Analysis of antimicrobial activity of clarithromycin ances were 150 ± 20 and 135 ± 15 Ω/cm2, respectively. after its passage across the HT29-19A intestinal cell monolayers Short-term absorption of amoxicillin and clarithromycin This experiment was carried out in order to verify whether clarithromycin antimicrobial activity is preserved after across the control and H. pylori-infected HT29-19A cell passage across the epithelial monolayer. Antimicrobial monolayers activity in the culture media containing [14C]clarithromycin Time course of antibiotic absorption across control mono- was measured before and after its apical–basal passage across layers. Clarithromycin and amoxicillin fluxes (means ± S.D.) the HT29-19A cell monolayer according to a standard as a function of time are shown in Figure 2. The apical-to- procedure.23 Briefly, Micrococcus luteus, inoculated on basal fluxes of amoxicillin rapidly achieved a steady state, Mueller–Hinton agar (Bio-Rad, Marne la Coquette, France), and were 50 ± 40, 57 ± 35, 63 ± 27 and 86 ± 49 ng/h·cm2 at 10, was used as the test organism. The culture media were tested 40, 70 and 100 min, respectively. The clarithromycin fluxes directly and after dilution and the results were compared with increased throughout the duration of the experiment and were a standard curve made with concentrations of clarithromycin 14 ± 24, 33 ± 50, 56 ± 65 and 127 ± 110 ng/h·cm2 at 10, 40, 70 Downloaded from http://jac.oxfordjournals.org/ by guest on March 15, 2015 (0.015–2 mg/L). Readings were made after 24 h incubation at and 100 min, respectively. 30°C in air. Similar profiles were observed for the basal-to-apical Statistical analysis fluxes, with a steady state rapidly achieved by amoxicillin, and a progressive increase in the flux of clarithromycin. Data were analysed using the SAS package (SAS Institute, Although clarithromycin seemed to display a different profile Cary, NC, USA). The results are expressed as means ± S.D. from amoxicillin, there was no significant difference between Comparison of means was carried out by analysis of variance the amoxicillin and clarithromycin fluxes at any time point. and by non-parametric tests (Wilcoxon two-sample test). Differences were considered significant for P < 0.05. Dependence of end-point antibiotic fluxes on the side of drug application and effects of the presence or absence of Results H. pylori. This analysis was carried out by comparing the Effect of amoxicillin and clarithromycin on the epithelial mean fluxes at 70 and 100 min. Values of 72 ± 35 and 115 ± 84 ng/h·cm2 were measured for apical-to-basal and basal-to- barrier integrity apical amoxicillin fluxes, respectively. They were not signi- Amoxicillin and clarithromycin applied together on the apical ficantly different from those determined for clarithromycin or basal side of the epithelial monolayer at a final con- (88 ± 83 and 160 ± 107 ng/h·cm2, respectively). For both anti- Figure 2. Short-term (100 min) amoxicillin and clarithromycin apical-to-basal and basal-to-apical fluxes measured in an Ussing chamber (mean ± S.D.). 868
Absorption of clarithromycin and amoxicillin Long-term basal-to-apical absorption of clarithromycin and amoxicillin across HT29-19A cell monolayers Clarithromycin. The basal-to-apical transport of clarithro- mycin across the epithelial layer as a function of time is shown in Figure 4. At each time point, the results are presented as means ± S.D. of six independent measurements. Up to 60 min, very little clarithromycin was found in the apical compart- ment; its concentration was 64 ± 5 ng/mL at 120 min and increased gradually to a value of 686 ± 29 ng/mL after 24 h. In contrast, 10 min after the basal application of clarithromycin, the drug was detected inside the cells at a concentration of 70 ± 10 ng/106 cells. The intracellular concentration of clarithromycin increased rapidly to values of 141 ± 7, 237 ± 22 Figure 3. End-point (70 and 100 min) apical-to-basal and basal-to- and 408 ± 35 ng/106 cells after 30, 60 and 120 min, respect- apical fluxes of amoxicillin and clarithromycin across normal and ively, and reached a concentration of 571 ± 55 ng/106 cells H. pylori-infected HT29-19A monolayers measured in an Ussing after 24 h. The data showed that basally applied clarithro- chamber (mean ± S.D.). *Significantly different from apical-to-basal mycin was first captured by the epithelial cells and subse- fluxes, P < 0.02. quently released to the apical compartment. Downloaded from http://jac.oxfordjournals.org/ by guest on March 15, 2015 Amoxicillin. Figure 4 shows that after basal application biotics, the basal-to-apical fluxes were higher than the apical- of 10 mg/L amoxicillin, only a small quantity of antibiotic to-basal ones (P < 0.04 for amoxicillin and P < 0.02 for (13 ± 1 ng/mL) was found in the apical compartment after clarithromycin) (Figure 3). 60 min. The concentration of antibiotic in this compartment The presence of H. pylori did not modify the apical-to- increased gradually, reaching 56 ± 4 ng/mL at 6 h, 88 ± basal fluxes of both antibiotics. The values measured for 9 ng/mL at 10 h and 224 ± 7 ng/mL at 24 h. Virtually no H. pylori-infected cells were 93 ± 76 ng/h·cm2 for amoxi- amoxicillin (
T. Matysiak-Budnik et al. Figure 5. Comparison of the apical-to-basal and basal-to-apical Figure 6. HPLC chromatograms of [14C]clarithromycin before and amoxicillin fluxes across Caco2 monolayers in cell culture inserts after transepithelial transport. Different elution times (26.62 min for (mean ± S.D.). No significant differences were found between apical and native clarithromycin and 28.13 min for its metabolite) indicate that dur- basal fluxes of the antibiotic. ing the transport clarithromycin underwent a modification of its structure. Downloaded from http://jac.oxfordjournals.org/ by guest on March 15, 2015 Long-term absorption of amoxicillin across Caco2 cell 0.25 mg/L pure clarithromycin (reference) activity, whereas monolayers the antimicrobial activity of the medium in the basal compart- ment (where the antibiotic was recovered), was equivalent to The time course of the basal-to-apical and apical-to-basal 0.5 mg/L reference activity. These results indicate that there amoxicillin fluxes across the Caco2 cell monolayers was was no loss of antimicrobial activity during the transepithelial similar to that obtained with HT29-19A cell monolayers. No passage of clarithromycin. intracellular accumulation of antibiotic was observed, and the concentrations of amoxicillin at different time points in either compartment after its application on the opposite side were Discussion not significantly different from those observed with HT29- 19A cell monolayers. In addition, the transport of amoxicillin Amoxicillin and clarithromycin are antibiotics that are used across the Caco2 monolayers showed no significant differ- widely in the treatment of H. pylori infection. In the present ences between apical or basal drug application (Figure 5). study, using an in vitro epithelial barrier model, it has been demonstrated that these two antibiotics had different modes Analysis of [14C]clarithromycin metabolites of transport across the epithelium. Clarithromycin was initially taken up by the epithelial cells, and then gradually The HPLC chromatogram of clarithromycin after trans- released into the opposite compartment of the epithelium as epithelial passage across an HT29-19A monolayer was its 14-hydroxy metabolite. In contrast, amoxicillin did not slightly different from that of the parent clarithromycin, with accumulate in the cells and crossed the epithelium either via a elution times of 26.62 and 28.13 min for the parent clarithro- paracellular pathway, across the intercellular tight junctions mycin and its metabolite after passage across the monolayer, or via a transcellular pathway. This study also showed that the respectively (Figure 6). No evidence of other degradation basal-to-apical passage of amoxicillin and clarithromycin is products was found after transepithelial transport. This small larger than the apical-to-basal passage across the epithelium. difference in the elution times between the two molecules The presence of bacteria seemed to decrease the basal-to- suggests that both compounds have a similar molecular apical fluxes of both antibiotics but had no significant influ- weight. This result, together with the result of the micro- ence on their apical-to-basal fluxes. There was no significant biological assay (see below), strongly suggests that the com- difference between short-term (100 min) transepithelial pound found in the receiver compartment could correspond to fluxes of both antibiotics, but clarithromycin presented much 14-hydroxy-clarithromycin, the principal biologically active better long-term (24 h) transepithelial absorption than metabolite of clarithromycin. amoxicillin. Antimicrobial activity of clarithromycin after its passage The fact that the cells concentrated clarithromycin but not amoxicillin was already known,10,12 but the mode of absorp- across HT29-19A cell monolayers tion of these two antibiotics across the epithelial barrier had The antimicrobial activity of the medium in the apical com- not been established. Amoxicillin, like all β-lactam anti- partment (where clarithromycin was added) was equivalent to biotics, uses the apically located, energy-dependent dipeptide 870
Absorption of clarithromycin and amoxicillin carrier system to cross the intestinal epithelium along the follow serum concentrations. The improved eradication transcellular pathway. This system is present in human results obtained by administering a continuous infusion of enterocytes and has been found in some intestinal cell lines, amoxicillin would further support such an explanation. It has such as Caco2 and also HT29 cells,17,24 but is reported to be been hypothesized that eradication failure could also be due to absent in the HT29-19A cells used in this study.18,25 For this the persistence of a few intracellular H. pylori.30 The intra- reason, transport of amoxicillin across the latter cells was cellular penetration of clarithromycin, in contrast to amoxi- compared with that of Caco2 cells, known to express the cillin, may allow it to kill the few intracellular bacteria transporter. The results with the HT29-19A monolayer sug- present. gested that an amoxicillin transporter may be absent in this In conclusion, in this study, we have shown that amoxi- cell line since no antibiotic was found inside the cells. On the cillin and clarithromycin have different modes of transepi- other hand, the possibility cannot be excluded that the drug thelial absorption. The higher passage rate of clarithromycin crosses the epithelium, in part at least, through the tight and its capacity to accumulate inside the epithelial cells may intercellular junctions, as the lower molecular weight of explain, in part, its better efficacy when used alone, in the amoxicillin (mol. wt 365) relative to that of clarithromycin eradication of H. pylori. (mol. wt 747) may favour a paracellular diffusion of the former across the epithelium. Moreover, as a zwitterion, it is Acknowledgements easier for amoxicillin, compared with negatively charged clarithromycin, to cross negatively charged tight junctions. Excellent technical assistance from Clémence Puzin and Downloaded from http://jac.oxfordjournals.org/ by guest on March 15, 2015 Similar absorption profiles were observed with the Caco2 Cecile Miot-Sertier is acknowledged. The authors also thank cells to those seen with HT29-19A cells, despite the well- Dr Nathalie Dhoyen from SmithKline Beecham and Dr documented presence of a specific PepT1 transporter in this Robert Flamm from Abbott for providing the radiolabelled cell line.17,26,27 No intracellular antibiotic was found and there antibiotics, and acknowledge the kind financial support of was no significant difference between the apical-to-basal and Sanofi-Synthélabo, France, in the realization of this study. basal-to-apical amoxicillin fluxes across the Caco2 mono- layer. These observations suggested that either the specific References PepT1 transporter was not active, or that its presence did not modify significantly the kinetics of transepithelial transport 1. Megraud, F. (1998). Epidemiology and mechanism of antibiotic of amoxicillin. resistance in Helicobacter pylori. Gastroenterology 115, 1278–82. It was assumed that amoxicillin passed through the epi- 2. Labenz, J., Gyenes, E., Ruhl, G. H. & Borsch, G. (1993). thelium in a non-metabolized form as it has been demonstrated Omeprazole plus amoxicillin: efficacy of various treatment regimens in humans that >90% of amoxicillin is excreted intact.28 In to eradicate Helicobacter pylori. American Journal of Gastroentero- contrast, clarithromycin is known to undergo a rapid biotrans- logy 88, 491–5. formation leading to the production of the microbiologically 3. Axon, A. T. & Moayyedi, P. (1996). Eradication of Helicobacter active metabolite 14-hydroxy-clarithromycin.11 We have pylori: omeprazole in combination with antibiotics. Scandinavian Journal of Gastroenterology 215, Suppl. 21, 582–9. shown that transepithelial passage of clarithromycin slightly modified its HPLC profile, suggesting the metabolism of the 4. al Assi, M. T., Genta, R. M., Karttunen, T. J. & Graham, D. Y. parent compound. In all probability, the metabolite corre- (1994). Clarithromycin–amoxycillin therapy for Helicobacter pylori infection. Alimentary Pharmacology and Therapeutics 8, 453–6. sponded to its hydroxy-metabolite because it was confirmed that it preserved the antimicrobial activity of the parent 5. Cooreman, M. P., Krausgrill, P. & Hengels, K. J. (1993). Local compound. gastric and serum amoxicillin concentrations after different oral application forms. 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