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Brazilian Journal of Medical and Biological Research (2007) 40: 383-389ISSN 0100-879X

Effect of Helicobacter pylori infectionand acid blockade by lansoprazole onclarithromycin bioavailability

Unidade Integrada de Farmacologia e Gastroenterologia,Universidade São Francisco, Bragança Paulista, SP, Brasil

R.A.M. Ortiz, S.A. Calafatti,L.A. Moraes, M. Deguer,

C.C. Ecclissato,M.A.M. Marchioretto,

M.L. Ribeiro, G. Bernasconiand J. Pedrazzoli Jr.

Abstract

The effect of proton pump inhibitors and Helicobacter pylori infectionon the bioavailability of antibiotics is poorly understood. We deter-mined the effects of 5-day oral administration of 60 mg lansoprazoleon the bioavailability of clarithromycin in individuals with and with-out H. pylori infection. Thirteen H. pylori-infected and 10 non-infected healthy volunteers were enrolled in a study with an open-randomized two-period crossover design and a 21-day washout periodbetween phases. Plasma concentrations of clarithromycin in subjectswith and without lansoprazole pre-treatment were measured by liquidchromatography coupled to a tandem mass spectrometer. Clarithro-mycin Cmax and AUC0-10 h were significantly reduced after lansoprazoleadministration. In addition, lansoprazole treatment of the H. pylori-positive group resulted in a statistically significant greater reduction inCmax (40 vs 15%) and AUC0-10 h (30 vs 10%) compared to lansoprazole-treated H. pylori-negative subjects. Thus, treatment with lansoprazolefor 5 days reduced bioavailability of clarithromycin, irrespective of H.pylori status. This reduction, however, was even more pronounced inH. pylori-infected individuals.

CorrespondenceJ. Pedrazzoli Jr.

UNIFAG, Universidade São Francisco

Av. São Francisco de Assis, 218

12916-900 Bragança Paulista, SP

Brasil

Fax: +55-11-4034-1825

E-mail:pedrazzoli@saofrancisco.edu.br

R.A.M. Ortiz and S.A. Calafatti

were supported by FAPESP.

Received December 14, 2005

Accepted December 6, 2006

Key words• Clarithromycin• Helicobacter pylori• Lansoprazole• Peptic ulcer• Pharmacokinetics

Introduction

More than 50% of the world populationis believed to be infected by Helicobacterpylori, the most common chronic bacterialinfection in humans. This infection is a ma-jor pathogenic factor in gastroduodenal dis-eases, including chronic active gastritis, pep-tic ulcers and gastric neoplasm (1). Eradica-tion is recommended for patients with symp-tomatic H. pylori infection and can be

achieved by the combination of two antibiot-ics plus a proton pump inhibitor (2). H.pylori is capable of altering acid secretionand proton pump inhibitors have a morepronounced effect in H. pylori-infected thanin non-infected individuals (3-5). Anti-se-cretory drugs such as proton pump inhibitorscan potentially alter the pharmacokinetics ofantibiotics by reducing the gastric juice vis-cosity and/or volume (6), affecting the chem-ical stability of drugs (7), slowing gastric

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emptying (8), and possibly reducing the dis-solution of solid drug formulations.

Clarithromycin is commonly prescribedin most anti-H. pylori treatments (9). It is anadvanced-generation macrolide with a broadin vitro antimicrobial spectrum. It also ex-hibits a broad spectrum of in vitro activitycomparable with that of erythromycin and isactive against most Gram-positive aerobiccocci and Gram-positive bacilli includingmycobacteria, Gram-negative bacteria, someanaerobes, and chlamydiae (10). The activ-ity of clarithromycin is enhanced by its ex-tensive tissue distribution and by formationof the 14-(R)-hydroxyclarithromycin meta-bolite. Clarithromycin and 14-(R)-hydroxy-clarithromycin have a minimum inhibitoryconcentration (MIC90) of 0.03 and 0.06 µg/mL for H. pylori, respectively (11). The drugis well absorbed from the gastrointestinaltract (12), but undergoes rapid degradationas a result of the low pH of the stomach (13).Therefore, elevation of the gastric pH mightalter the bioavailability and distribution ofclarithromycin.

The aim of the present study was to de-termine the effect of both lansoprazole ad-ministration and H. pylori infection on thepharmacokinetics of orally administered cla-rithromycin.

Subjects and Methods

Subjects

Twenty-three healthy volunteers (aged 19to 35 years, height 165 to 180 cm, weight 55 to100 kg) were selected after clinical examina-tion and laboratory screening. The laboratoryscreening consisted of blood glucose, urea,creatinine, aspartate aminotransferase, alanineaminotransferase, gamma-glutamyl transpep-tidase, alkaline phosphatase, total bilirubin,total protein, albumin, hemoglobin, hemato-crit, total and differential white cell counts,routine urinalysis and serology for humanimmunodeficiency virus, hepatitis C virus,

hepatitis B virus, and H. pylori. The serologi-cal test had a sensitivity of 92% and a specific-ity of 93.1% for Helicobacter (ImmunoCombII Helicobacter pylori IgG, Orgenics, Yavne,Israel). Thirteen of the individuals were H.pylori positive and 10 were H. pylori negative.The exclusion criteria included abnormalitiesin laboratory screening, previous gastric sur-gery or antibiotic use for eradication of H.pylori, use of antibiotics or proton pump in-hibitors four weeks prior to the study, and ahistory of gastric ulcer or neoplasm. Writteninformed consent was obtained from eachvolunteer prior to the study, and the protocolwas approved by the Universidade São Fran-cisco Ethics Committee in accordance withthe Declaration of Helsinki and Brazilian law.

Lansoprazole administration

The study had an open-randomized two-period crossover design, with a 21-day wash-out period between phases. In the LANS(-)phase (no lansoprazole pre-treatment), thevolunteers were hospitalized at 9:00 pm af-ter having had a regular evening meal. Fol-lowing an overnight fast, they received asingle tablet of clarithromycin (500 mgKlaricid®; Abbott, São Paulo, SP, Brazil)with 200 mL water at 7:00 am. During theLANS(+) phase (lansoprazole pre-treatment),the volunteers came to the clinical pharma-cology and gastroenterology unit daily for 5days (Monday to Friday), where they re-ceived one capsule of lansoprazole (30 mgOgastro™; Abbott) at 9:00 am and 20:00pm. On Friday evening, they were admittedto the Clinical Pharmacology and Gastroen-terology Unit, and received another capsuleof lansoprazole at 9:00 pm. After an over-night fast, clarithromycin was administeredas described above for the LANS(-) group.

During both study periods, blood samples(10 mL) with EDTA for plasma clarithromy-cin quantification were taken from a conve-nient forearm vein before and 0.5, 1, 1.5,2.0, 2.5, 3, 3.5, 4, 6, 8, and 10 h following

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clarithromycin administration. The sampleswere centrifuged at 2,000 g for 10 min, andplasma was separated and stored at -70ºCuntil assayed for clarithromycin content byhigh-performance liquid chromatography(HPLC)-mass spectrometry.

Sample extraction procedure

Two hundred microliters of plasma wasextracted after adding 50 µL internal stand-ard (erythromycin, 10 µg/mL) and 50 µL 0.1M NaOH and 1 mL ethyl acetate. The tubeswere vortex-mixed briefly and allowed tostand at room temperature for 5 min. Thetubes were then centrifuged at 10,000 rpmfor 10 min at room temperature. The upperorganic layer was carefully removed and200 µL acetonitrile was added to the tubesfollowed by vortex-mixing for 15 s to recon-stitute the residue. The solutions were trans-ferred to microvials, capped and placed in anautosampler rack. Aliquots of 10 µL wereinjected automatically into the HPLC.

Preparation of standard solutions andreagents

Clarithromycin standards were preparedby dilution from a stock solution of 100 µg/mL. The internal standard (erythromycin)was prepared by dilution from a stock solu-tion (100 µg/mL) in acetonitrile to a finalconcentration of 10 µg/mL.

Preparation of calibration and quality controlsamples

The limit of quantification of clarithro-mycin was 10 ng/mL and the lower limit ofdetection was 2 ng/mL. Calibration stan-dards were prepared by spiking control hu-man plasma with standard solutions contain-ing clarithromycin to give standards of 100,500, 1000, 1500, 3000, and 5000 ng/mL. Afixed amount of erythromycin was added toall assay tubes as an internal standard. The

calibration standards and blanks were freshlyprepared for each assay and were extractedat the same time as the plasma samples andquality controls. Quality control sampleswere prepared by spiking control humanplasma with 300, 2000, and 4000 ng/mLclarithromycin. One quality control samplefor each of the three concentrations in everyassay was thawed and extracted with theplasma samples.

Under these conditions, the calibrationcurves for clarithromycin in plasma (100-5000 ng/mL) presented a straight line throughthe origin with a correlation of 0.9997. Intra-and interassay coefficients of variation were3 and 9%, respectively.

Mass spectrometry and chromatographyconditions

An HPLC model PRO STAR 410 system(Varian, Walnut Creek, CA, USA) equippedwith a Polaris C18 analytical column (3 µm,30 mm x 2.0 mm, ID, C18 Genesis guardcolumn, 4 µm, 10 mm x 4 mm, ID; JonesChromatography, Hengoed, UK) was used.The mobile phase consisted of 10 mM ammo-nium acetate in water (20%) and acetonitrile(80%). The column was eluted isocratically at0.1 mL/min at room temperature. Total runtime was 3.0 min. The temperature of theautosampler was maintained at 20ºC and theinjection volume was 10 µL. A wash bottlecontaining freshly prepared 50% acetonitrilein water was used to wash the autosamplerneedle to prevent a carry over effect.

Mass spectrometry was performed usingan LC 1200 triple-stage quadrupole massspectrometer, equipped with ESI electro-spray source operating in the positive-ionmode using a cross flow as the counter elec-trode (Varian). The mass spectrometric con-ditions (tuning and collision-induced disso-ciation) for all compounds studied were op-timized with standard solutions in the mo-bile phase (5 µg/mL each at a flow rate of 10µL/min) using an infusion pump connected

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directly to the electrospray probe.The full-scan single mass spectrum was

obtained after the infusion of pooled plasmafollowing the intravenous administration ofclarithromycin. The retention times for eryth-romycin and clarithromycin in the mass spec-trometer were 1.1 and 1.4 min, respectively.The mass spectrometer was operated in thepositive mode (ES+) and set for multiplereactions monitoring the following ions (m/z); 734.0 >576.0 for erythromycin and 748.5>590.0 for clarithromycin. The dwell timewas set at 0.8 s, the cone voltage was 30 Vand the collision energy and gas pressure(Argon) were 20 eV and 2.02 m Torr, re-spectively.

Pharmacokinetic and statistical analysis

Maximum plasma concentration (Cmax)and the time taken to reach it (Tmax) were

obtained directly from the individual con-centration vs time curves. A first-order ter-minal elimination rate constant (ke) for clari-thromycin was derived by log-linear regres-sion of selected data points from the concen-tration vs time curves describing a terminallog-linear decaying phase. The half-life (T½)was estimated from this rate constant (T½ =ln(2)/ke). The respective area under the time-concentration curves from 0 to 10 h forclarithromycin (AUC0-10 h CLA) were calcu-lated by the linear trapezoidal method usinga non-compartmental pharmacokinetic mo-del and performed by the addition of theC2 h/ke value, where C10 h = plasma CLAconcentration 10 h following clarithromycinadministration. The area under the time-con-centration curves was also extrapolated toinfinite (AUC0-∞ CLA) (14). All variables wereanalyzed by parametric (one-way ANOVA)and non-parametric tests (15). Parametric

Table 1. Clarithromycin pharmacokinetic parameters in plasma after a single oral (500 mg) dose of clarithro-mycin in Helicobacter pylori-positive and H. pylori-negative volunteers with (LANS(+)) and without (LANS(-))lansoprazole pre-treatment.

Variables H. pylori-positive (N = 13) H. pylori-negative (N = 10)

LANS(-) LANS(+) LANS(-) LANS(+)

AUC0-10 h (ng h-1 mL-1) 12598.44 8805.98* 14370.75 12932.55

Geometric mean (90% CI) 11068.38 to 7384.54 to 11989.11 to 11023.93 to

16709.09 12761.25 18216.40 16429.58

AUC0-∞ (ng h-1 mL-1) 17634.12 22501.10* 19308.07 19379.95

Geometric mean (90% CI) 14582.96 to 14053.89 to 16009.16 to 16485.56 to

25973.05 42227.06 24563.26 23728.02

Cmax (ng/mL) 2258.45 1366.87* 2352.80 2006.35

Geometric mean (90% CI) 2010.41 to 1139.93 to 1974.37 to 1703.67 to

2817.51 2003.54 3006.48 2614.27

T½ (h) 4.82 8.39* 4.59 4.68

Geometric mean (90% CI) 3.75 to 6.88 0.28 to 31.68 4.18 to 5.12 4.25 to 5.23

Tmax (h) 1.80 2.83 1.96 2.30

Geometric mean (90% CI) 1.58 to 2.27 0.81 to 8.02 1.70 to 2.40 1.41 to 4.49

*P < 0.05 compared to LANS(-) patients (one-way ANOVA). Note: In contrast to H. pylori-positive patients,LANS had no statistically significant effect on the pharmacokinetics of clarithromycin in H. pylori-negativesubjects. 90% CI = confidence interval at 90%; AUC0-10 h and AUC0-∞ = area under the time-concentrationcurves from 0 to 10 h for clarithromycin and extrapolated to infinite, respectively; Cmax = maximum plasmaconcentration; T½ = half-life estimated as T½ = ln(2)/ke, where ke is the rate constant; Tmax = time taken toreach the Cmax.

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and non-parametric tests were also used tocompare AUC0-10 h CLA and Cmax data. Indi-vidual AUC0-10 h, Cmax, T½, ke ratios, and Tmax

differences between the LANS(+) (test) andLANS(-) (reference) phases were calculated.

We used the assessment of bioequiva-lence to evaluate whether a pharmacokineticinteraction had taken place. To prove bio-equivalence there must be no difference be-tween the bioavailability of clarithromycinbefore (“reference”) and after lansoprazoleadministration (“test”). A 90% confidenceinterval approach, the bioequivalence inter-val, was used to compare the bioavailabilityof clarithromycin before and after lansopra-zole administration and a 90% confidencelimit was estimated for the sample means.For considering bioequivalence, there shouldalso be no statistical differences between themean AUC0-10 h AUC0-∞ and Cmax. A P valueof 0.05 was taken as the significance levelfor all statistical tests performed. ANOVAand the Mann-Whitney test were used.

Results

No side effects were reported by any ofthe volunteers after the administration ofeither lansoprazole or clarithromycin. Theclinical and biochemical data of patients andvolunteers were not affected by drug admin-istration and remained within the referencevalues of the laboratories.

The pharmacokinetic parameters for cla-rithromycin were similar in H. pylori-posi-tive or -negative volunteers before acid sup-pression (Table 1). A 5-day treatment withlansoprazole adversely affected the relativebioavailability of the antibiotic, as indicatedby a reduction in the peak plasma concentra-tion (Cmax) of clarithromycin, by the lack ofinclusion within the 90% confidence inter-val for AUC0-10 h CLA, Cmax and the individualAUC0-∞ CLA values in the range of 80-125%,as well as the LANS(-)/LANS(+) ratio forCmax and AUC0-10 h CLA. Lansoprazole admin-istration reduced the bioavailability of clari-

thromycin in both groups of individuals;however, this reduction was even more pro-nounced in H. pylori-infected individuals(Table 2).

In addition, lansoprazole treatment inthe H. pylori-positive group resulted in asignificant (P < 0.05) reduction in Cmax andAUC0-10 h CLA compared to lansoprazole-treated H. pylori-negative subjects (Table 1).

Discussion

Until now, two studies have evaluatedthe pharmacokinetic interaction between aproton pump inhibitor and clarithromycin.Gustavson et al. (11) evaluated the pharma-cokinetics of clarithromycin and its mainactive metabolite, 14-(R)-hydroxyclarithro-mycin, at steady state, with and withoutomeprazole administration in H. pylori-nega-tive healthy male volunteers, concluding thatomeprazole increased their bioavailability.More recently, Mainz et al. (16) investigatedthe pharmacokinetics of lansoprazole, amox-icillin, clarithromycin, and 14-(R)-hydroxy-clarithromycin after 5 days of administra-tion to uninfected volunteers, when givensimultaneously (16) and observed an increasein 14-(R)-hydroxyclarithromycin and lanso-prazole bioavailability. We did not measure14-(R)-hydroxyclarithromycin concentra-tions. It is well known that this metabolitehas in vitro activity against H. pylori; theMIC for this metabolite, however, is higher

Table 2. Bioequivalence analysis of clarithromycin (CLA) in Helicobacter pylori-posi-tive and H. pylori-negative volunteers with (LANS(+)) and without (LANS(-))lansoprazole pre-treatment.

LANS(+)/LANS(-) H. pylori-positive (N = 13) H. pylori-negative (N = 10)

Geometric 90% CI Geometric 90% CImean mean

Cmax (ng/mL) 62.74 49.69-79.21 85.27 61.74-117.79AUC0-10 h (ng h-1 mL-1) 71.37 52.08-97.75 51.66 68.74-117.81AUC0-∞ (ng h-1 mL-1) 114.00 78.22-166.10 101.73 86.37-119.78

For abbreviations, see legend to Table 1.

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than that observed for its parent compound(0.06 vs 0.03 mg/L) (10). Since H. pylori isnot supposed to interfere with the hepaticmetabolism of drugs and the bioavailabilityof clarithromycin was even lower in infectedindividuals than in non-infected ones afterlansoprazole administration, a reduction ofplasma 14-(R)-hydroxyclarithromycin con-centrations is also to be expected. The ob-served reduction in clarithromycin bioavail-ability and the increase in AUC0-∞ and Tmax

after lansoprazole administration suggest areduction, and the delay of its absorptionstrongly suggests that the reduced bioavail-ability of clarithromycin after lansoprazoleadministration was induced during the bio-pharmaceutical phase, i.e., before absorption.

The discrepancies between these studiesand ours may be partially explained by theanalytical methods employed (liquid chro-matography coupled to tandem mass spec-trometry vs liquid chromatography) with dif-ferent quantification limits (10 ng/mL in ourstudy, 500 ng/mL in the study by Mainz et al.(16)), different clarithromycin formulations,study design (multiple doses/steady-statestudy vs single dose), differences in druginteraction (omeprazole/lansoprazole andclarithromycin), the concomitant use ofamoxicillin in one of the studies (16), and bydemographic characteristics of volunteers,such as age, which are directly correlated toplasma concentrations of clarithromycin andH. pylori status (17). It should also be stressedthat H. pylori-infected individuals presenteda more pronounced effect of lansoprazole onclarithromycin pharmacokinetics and ourswas the only study evaluating these indi-

viduals.The efficacy of clarithromycin is sug-

gested to be related to a time above MIC forat least 40 to 50% of the dosing interval inotitis media (18,19). According to our re-sults, this was not achieved after lansoprazolepre-treatment if one considers the MIC forclarithromycin to be >2 µg/mL in our pa-tients (20,21), if the same relationship ap-plies to H. pylori infection. Despite present-ing a reduced bioavailability when associ-ated with lansoprazole, eradication thera-pies containing these two drugs plus amoxi-cillin have eradication rates similar to thoseachieved when other proton pump inhibitorsare used (22). Therefore, the reduction ofclarithromycin bioavailability after lanso-prazole administration appears to be clini-cally irrelevant for H. pylori eradication regi-mens. On the other hand, if the observedreduction of the bioavailability of clarithro-mycin during lansoprazole administration isrelevant for the therapy of other infectionsremains to be determined.

Our results suggest that other mechan-isms of action are also important for clari-thromycin activity when used in associationwith proton pump inhibitors against H. pylo-ri. Other possibilities may include a directmechanism of action, the postulated bacteri-cidal activity (23,24) or even the importanceof clarithromycin transfer from blood to gas-tric lumen, as described by our group andothers (25). Studies performed in a clinicalsetting may be needed for a better under-standing of the possible interactions thatmay occur among drugs ordinarily used inmulti-drug schedules.

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