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MATERIALS AND METHODS

The study took place at the Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria. The study protocol was approved by the local Ethics Committee, and the study was performed in accordance with the Declaration of Helsinki 1964 (including current revisions), the Austrian Drug Law, and the Good Clinical Practice Guidelines.

Healthy volunteers. Seven healthy male volunteers between the ages of 25 and 37 years were enrolled into the study. Written informed consent was obtained from each volunteer prior to any study-related investigation or intervention. Each volunteer underwent a screening examination consisting of the following: medical history, physical examination, routine laboratory tests, heart rate, blood pressure, and a 12-lead electrocardiography. These assessments were performed prior to inclusion and after completion of the study. All volunteers were initially drug free and received standardized meals on study days and were instructed to avoid caffeine and grapefruit juice during the entire study period.

Study protocol. (i) Study day 1 (250 mg clarithromycin single dose). The volunteers were admitted to the clinical research ward in the morning of study day 1. A plastic cannula was inserted into an antecubital vein to monitor blood concentrations of clarithromycin at defined time points. Concentrations in interstitial-space fluid of skeletal muscle and subcutaneous adipose tissue were determined by microdialysis. The principle of microdialysis has been described previously in detail ( 15 ). In brief, a microdialysis probe with a molecular weight cutoff of 20,000 (CMA12; CMA/Microdialysis AB, Solna, Sweden) was inserted into one thigh muscle and into the subcutaneous adipose tissue at the ventrolateral side of the thigh under aseptical conditions by use of a guidance cannula. The probe was constantly perfused with Ringer's solution at a flow rate of 1.5 µl/min by means of a precision pump (Precidor; Infors-AG, Basel, Switzerland). After a 60-min equilibration period, 250 mg of clarithromycin (Klacid 250 mg tablet; Abbott, Abbott Park, IL) was administered orally to the fasting volunteer. Sampling of microdialysates and venous blood was performed at 20-min intervals from h 0 to 4 and at 30-min intervals from h 4 to 8. After completion of the 8-h sampling period, the individual recovery values of clarithromycin were determined by use of the "retrodialysis" method ( 3 ). For that reason, clarithromycin was added at a concentration of 5 mg/liter to the perfusion fluid and its rate of disappearance through the microdialysis membrane was determined. The individual recovery was calculated by using the mean of two measurements by the following equation: recovery (%) = 100 – (100 x C dialysate / C perfusate ), where C represents the concentration. Blood was collected in tubes containing the lithium salt of heparin, kept on ice for a maximum of 30 min, and centrifuged at 1,600 x g for 5 min at 4°C. Plasma and microdialysates were stored at minus 80°C until analysis.

At 12 h after the initial single dose of 250 mg, each volunteer continued oral intake of clarithromycin at a dosage of 500 mg twice a day (b.i.d.) for 3 to 5 days until the morning of study day 2.

(ii) Study day 2 (steady state; 500 mg clarithromycin b.i.d.). After clarithromycin intake b.i.d. over a period of 3 to 5 days, the last dose of 500 mg of clarithromycin was administered in the morning of study day 2 under the supervision of the study staff. Before the last dosage of clarithromycin was taken, a baseline blood sample was drawn and a microdialysate for the determination of the through concentrations in tissue was collected over 2 h. The setting of study day 2 was identical to that of study day 1.

Chemical analysis. Clarithromycin concentrations in plasma and microdialysates were analyzed by use of a validated high-performance liquid chromatography method ( 23 ), applying moderate modifications. Pure clarithromycin was a gift from Abbott (Abbott Laboratories, Abbott Park, IL). Pure roxithromycin and all other chemicals were purchased from Sigma-Aldrich (Steinheim, Germany). In brief, 150 µl plasma containing the internal standard roxithromycin (1 mg/liter) and 10 µl of 1 M sodium hydroxide were extracted with 2 ml of tert -butyl methyl ether. The organic layer was evaporated to dryness, and the residue was dissolved with 50 µl of the mobile phase. The mobile phase consisted of 0.05 M citrate buffer (pH 6.5) and acetonitrile (71:29 ). The flow rate was 0.450 ml/min. Separation was performed isocratically on a reverse-phase column (Synergi max RP; Phenomenex, Torrance, CA) (150 by 2 mm; particle size, 4 µm) at ambient temperature. Microdialysates were spiked with the internal standard at a final concentration of 0.05 mg/liter and analyzed without further preparation. Clarithromycin and roxithromycin in the eluent were detected with an amperometric detector (BAS; West Lafayette, IN) at +950 mV oxidation potential. The lower limits of quantification were 0.04 mg/liter and 0.012 mg/liter in plasma and microdialysates, respectively. Intraday and interday inaccuracy were Intraday and interday imprecision were

Protein binding studies. Protein binding of clarithromycin was determined individually for each volunteer. Aliquots of 300 µl plasma from samples drawn 80 and 180 min after administration of the drug (for both single doses and steady state) were ultrafiltrated by use of centrifugal filter units with a low-binding regenerated-cellulose membrane (Ultrafree-MC; Millipore Corp., Bedford, MA) (nominal relative molecular weight cutoff, 5,000) at 5,000 x g for 30 min at ambient temperature. Ultrafiltrates were analyzed as described above for plasma. For determination of the binding of clarithromycin to the ultrafiltration membrane during the filtration process, standards in Ringer's solution were ultrafiltrated and analyzed in the same way. The ultrafiltrate concentrations were subsequently corrected (corr) by the mean membrane binding of 5% ( C ultrafiltrate corr ). The protein binding was calculated using the following equation: protein binding (%) = 100 – (100 x C ultrafiltrate corr / C plasma total ).

Pharmacokinetic calculations and statistical analysis. The individual protein binding values were used for the determination of free clarithromycin concentrations in plasma. The absolute interstitial concentrations were calculated by use of the following formula: interstitial concentration = 100 x ( C dialysate /recovery).

Pharmacokinetic calculations were carried out by use of commercially available computer software (Kinetica, version 3.0; Innaphase, Philadelphia, PA). Concentrations at 12 h and 24 h were calculated by the following equation: C = C 8 x e – k el x t, where C is the concentration at 12 h or 24 h, C 8 is the last concentration measured in vivo (at 8 h), k el is the elimination rate constant, and t is the time difference between C 8 and C. The areas under the concentration-time curves from 0 to 8 h (AUC 0-8 ), 0 to 12 h (AUC 0-12 ), and 0 to 24 h (AUC 0-24 ) in plasma and interstitial fluid were calculated by use of the linear trapezoidal rule. For calculation of the total drug clearance (CL) and the apparent volume of drug distribution during the terminal phase after a single dose ( V z ) and at the steady state ( V ss ), the oral dose of clarithromycin was corrected for a bioavailability value ( F ) of 55% ( 6 ). CL, V ss, and V z of clarithromycin were calculated for plasma as follows: CL = dose x ( F )/AUC 0-, where AUC 0- represents the AUC from 0 to infinity, V ss = CL x MRT (mean residence time), and V z = dose x ( F )/(AUC 0- x k el ). The AUC 0-24(b.i.d.) for 500 mg at the steady state was corrected for dosing twice daily by the equation AUC 0-24(500 mg b.i.d.) = AUC 0-12 x 2. Wilcoxon's paired test was used for comparison of AUC values in plasma and interstitial fluids within individuals. A two-sided P value of

RESULTS

The present study set out to test the ability of clarithromycin to penetrate the interstitial-space fluid of subcutaneous adipose tissue and skeletal muscle in healthy volunteers. The results for one volunteer had to be excluded because plasma concentrations were almost 0, indicating noncompliance of the subject with respect to the study protocol. Thus, results of six volunteers were eligible for pharmacokinetic analysis.

The mean plasma protein binding of clarithromycin was 71.3 ± 7.4% for a 250 mg single dose and 76.9 ± 8.1% for 500 mg b.i.d. at the steady state (drug intake for 3 to 5 days). The mean individual in vivo recovery values for clarithromycin in microdialysis were 57.7 ± 17.2% and 54.3 ± 19.0% for adipose and muscle tissue, respectively. In separate in vitro experiments (data not shown), we demonstrated that recovery was not dependent on concentration and time. Variability between probes was minimal.

Pharmacokinetic data for a single 250 mg dose of clarithromycin. Main pharmacokinetic data are summarized in Table 1.

TABLE 1. Main pharmacokinetic indices of clarithromycin following administration of a single oral dose of 250 mg ( n = 6) a

The concentration-time profiles for free clarithromycin in the interstitial-space fluid of adipose tissue resembled closely the concentration-time profiles for skeletal muscle. Detectable interstitial concentrations were observed about 1 h after drug administration (Fig. 1 ). The ratios of the f AUC 0-24 values in tissues to the f AUC 0-24 values in plasma were 0.29 ± 0.17 (range, 0.14 to 0.61) and 0.42 ± 0.18 (range, 0.17 to 0.60) for subcutaneous adipose and skeletal muscle tissue, respectively, after intake of a single oral dose of 250 mg clarithromycin. The differences between f AUC 0-24 values for plasma and tissues were significant ( P

FIG. 1. Clarithromycin concentrations in plasma and interstitial-space fluid of soft tissues from six male healthy volunteers after a single dose of 250 mg (mean ± standard deviation).

Pharmacokinetic data of 500 mg clarithromycin at the steady state. Main pharmacokinetic data are summarized in Table 2.

TABLE 2. Main pharmacokinetic indices of clarithromycin at the steady state after administration of 500 mg b.i.d. ( n = 6) a

Interstitial-space fluid concentrations of free clarithromycin in subcutaneous adipose tissue and skeletal muscle were descriptively identical (Fig. 2 ). The ratios of the f AUC 0-24(b.i.d.) values in tissues to the f AUC 0-24(b.i.d.) values in plasma were 0.39 ± 0.04 (range, 0.32 to 0.44) and 0.41 ± 0.19 (range, 0.19 to 0.75) for subcutaneous adipose tissue and skeletal muscle, respectively. The differences between f AUC 0-24(b.i.d.) values for plasma and tissues were significant ( P

FIG. 2. Clarithromycin concentrations in plasma and interstitial-space fluid of soft tissues from six male healthy volunteers after multiple doses of 500 mg b.i.d. (mean ± standard deviation).

The clearance values at steady state differed from the single dose data because dose and AUC 0- did not increase proportionally (nonlinear pharmacokinetics).

Safety and tolerability. The study drug was well tolerated by all subjects. Metal-like taste sensation and mild gastrointestinal disturbance were observed in one volunteer. Both adverse events subsided within the study period without therapeutic measures. No adverse events related to the microdialysis procedure were observed.

DISCUSSION