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The aim of the present study was to compare the pharmacokinetics and bioavailability of two commercial brands of clarithromycin (CAS 81103-11-9) suspensions in healthy male Iranian volunteers. In an open label, single-dose, randomized study with a cross over design an equivalent 500-mg clarithromycin suspensions were given orally to each of 24 subjects as a single dose on two treatment days. The treatment periods were separated by a one-week washout period. Blood samples were drawn at different time points and the separated plasma was kept frozen at -20 °C for subsequent analysis. The plasma concentrations of drug were analyzed by a rapid and sensitive HPLC method with UV detection.
Mean maximum serum concentration of 2256.5±590.1 ng/ml and 2840.2±717.5 ng/ml were obtained for the test and reference formulation, respectively. The AUC0-infinity of clarithromycin was on average 45008.7 ± 10989.9 ng h/ml for the test and 45221.3 ± 2155.7 ng h/ml for the reference formulation. The calculated 90% confidence intervals for the ratio of Cmax (81.98-94.26 %), (91.6-109.15 %) and (93.08-110.85 %) values for the test and reference products were all within the 85-120% interval proposed by FDA and EMEA. Therefore the clarithromycin suspension of Test and Reference formulations are bioequivalent in terms of rate and extent of absorption.
Key words: AUC, Bioavailability, Bioequivalence, Pharmacokinetics, Cmax , Clarithromycin.
Clarithromycin (CAS 81103-11-9) is a semisynthetic macrolide that exhibits broad-spectrum activity against gram-positive and gram-negative aerobes, atypical bacterial pathogens of the respiratory tract, mycobacterium species, and helicobacter pylori . Therefore it is indicated for the treatment of wide range of infections . However there are some bacterial species that are resistant to macrolides; Mycobacterium tuberculosis and Mycobacterium smegmatis are both intrinsically resistant to the macrolides, and resistance can be induced in both species [2, 3]. Although Clarithromycin has relatively poor in vitro activity against Mycobacterium tuberculosis, it has better activity against Mycobacterium avium (MIC90 = 8 mg/ml), and Mycobacterium kansasii (MIC90 = 0.5 mg/ml)  and is even 8 -32 fold more active than erythromycin against Mycobacterium avium . High intracellular drug concentration of Clarithromycin in macrophages was also observed . Clarithromycin is rapidly absorbed from the gastrointestinal tract, and undergoes first-pass metabolism through the liver to form the active 14-hydroxylated metabolite and this is a unique feature of Clarithromycin among macrolides. The bioavailability of the parent drug is about 55% and the extent of absorption is relatively unaffected by the presence of food. Clarithromycin may therefore be given without regard to meals. Peak concentrations of clarithromycin and its main active metabolite, 14-hydroxyclarithromycin, are reported to be about 900 and 600 ng/mL respectively following a single 250-mg dose by mouth. At steady state the same dose given every 12 hours as tablets produces peak concentrations of clarithromycin of about 1 μg/mL. However administration of the same dose as a suspension to fasting subjects produces a steady-state plasma concentration of about 2 μg/mL [7, 8]. Therefore it seems that the extent of absorption of clarithromycin from the suspension formulation is not significantly different from that of the reference tablet formulation, whereas the extent of formation of the active metabolite is significantly lower with the suspension formulation . Moreover a single/multiple dose study conducted in adults revealed a delay in the time to reach peak plasma concentration after suspension administered as compared with data obtained after tablet administration . On the other hand, many drugs are made and marketed by more than one pharmaceutical manufacture. For instance, in recent years, as a way of reducing healthcare costs, several countries including Iran have taken steps to increase the use of generic drugs. To establish that a generic drug product is therapeutically equivalent to and can be used interchangeably with the original product, scientific comparison of the pharmacokinetic properties is necessary. Therefore in the present study the rate and extent of absorption of two commercial brands of clarithromycin suspensions were determined and compared following oral administration in healthy Iranian volunteers.
2.1 Study design
Two brands of clarithromycin suspensions were used. The test product (Klarexir®, Batch no.T001) was manufactured by Exir (Boroujerd, Iran) and obtained from the respective manufacturer. The reference product (Batch no.38398TF01) was also obtained from the respective manufacturer. Both test and reference products contained 125 mg clarithromycin per 5 ml. The study was an open label, single-dose, randomized study with a cross over design carried out in accordance with the guidelines of the Declaration of Helsinki (World Medical Assembly 1964) as revised in Edinburgh (2000). Twenty four male healthy volunteers were enrolled in this study. They were all Iranians, aged between 21 and 37 years (24.4 ± 4.1 years) and weight from 56 to 90 kg (69.9 ± 9.3 kg). None of them had a history of hypersensitivity to medications and no one was smoker. The patients were informed of the nature of the study and written consent was obtained prior to enrollment in the study. The test product and the reference were randomized and given to the volunteers. Two-week washout period was included between two treatments. An equivalent 500-mg clarithromycin suspension was given orally to each subject as a single dose with 200 ml of water. Five milliliters of blood were drawn at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12 and 24 hours after each administration. The blood samples were taken from subject's forearm veins. All samples were centrifuged in heparinated tube. The plasma samples were separated and kept frozen at the temperature below -20 °C for subsequent analysis.
2.2 Analysis of plasma samples
The analytical procedure for determination of clarithromycin in plasma was adopted from the method of Amini and Ahmadiani . The method used was validated for specificity, accuracy, precision and sensitivity.
Fifty micro liters of the internal standard (1 µg/m1 of diltiazem HCl, Sigma, St. Louis, MO, USA) and 20 micro liters of NaOH 1 N (Merck, Darmstadt, Germany) were added on 1 ml of plasma, then the mixture were extracted with 2.5 ml hexane/isopropyl alcohol (98/2 %v/v) (Merck Darmstadt, Germany) by vortexing for 5 min. After centrifugation for 5 min at 1000 g, the upper organic phase was transferred to a 5-ml glass tube and 50 micro liters of 0.2% acetic acid (Merck Darmstadt, Germany) was added. The mixture was vortex-mixed for 2 min and then, some of the upper organic phase was discarded and the remaining mixture (about 1 ml) was transferred into a 1.5 ml microcentrifuge tube. After centrifugation for 2 min, the upper organic phase was discarded completely. Finally 50 micro liters of aqueous phase was injected onto the HPLC column. The mobile phase consisted of acetonitril (Merck Darmstadt, Germany) and 50 mM aqueous sodium dihydrogen phosphate (32:68 %v/v) (Merck Darmstadt, Germany), pH=4.5 (adjusted with concentrated phosphoric acid and 4M sodium hydroxide); Analytical column used for chromatographic separations was Shimpack CLC-CN 5 µm (250 - 4.6 mm) (Shimadzu, Cloumbia, MD) with a Shimpack CLC-CN 5 µm 4.6 - 20 mm guard column (Shimadzu, Cloumbia, MD) ; the flow rate was 1 ml/min at 40 °C and the detector wavelength was set at 205 nm. Under these conditions the retention times for clarithromycin and the internal standard (diltiazem) were 8.6 and 7.5 min respectively. Liquid chromatographic system (Knauer, Berlin, Germany) comprising of Knauer K1000 solvent delivery module equipped with a Rheodyne (Rheodyne, Cotati, CA) injector and a variable wavelength ultraviolet spectrophotometric detector (Knauer smartline 2500, Berlin, Germany). Eurochrom 2000 version 2.05 was used for data acquisition, data reporting and analysis. All plasma samples of a given volunteer collected in the two treatment periods were analyzed in the same chromatographic run (analytical own control). Each run had a separate daily calibration. Quality control samples (QC) at two concentrations levels were used in each run. Calibration curves were obtained by plotting the clarithromycin to diltiazem peak area ratio against the concentrations of the standard solutions.
2.3 Pharmacokinetic calculations
The plasma concentration-time profile of each individual treatment was constructed. Pharmacokinetic analysis consisted of visual identification of the maximum plasma concentration (Cmax) and the time at which this occurred (Tmax) from the individual subject plasma concentration-time profiles. The area under the plasma concentration - time curve from time zero to t () was calculated using the linear trapezoidal rule. The terminal first order constant (kel) was determined by a least squares fit of the terminal plasma concentrations (using Excel® for Windows®). The constant kel was used to extrapolate. was calculated acording to the following equation: = +Ct /kel . Finally the elimination half-life ( t1/2) was calculated by the quotient of 0.693/kel.
3. Results and Discussion:
The method of analysis was linear in the range of 62.5 to 4000 ng/ml, with a coefficient of correlation (r) of greater than 0.999. Limit of quantitation was 62.5 ng/ml, with a within-day reproducibility (intra assay accuracy) of 96.8-100.3% and a day-to-day reproducibility (inter assay accuracy) of 97.2-100.5%. All subjects completed the trial. No adverse events were considered to be related to the study medication, and no abnormalities in vital signs were observed. The average concentration - time plot after administration of reference and test products to 24 healthy volunteers was illustrated in Fig.1. It is seen that the mean clarithromycin concentration-versus-time plots are almost superimposable between two treatments. In Table 1 the individual as well as mean pharmacokinetic parameters after administration of both products to 24 healthy volunteers are reported. As it is seen, mean maximum serum concentration of 2256.5 ± 590.1 ng/ml (90% CI: 2258.4 - 2654.6) and 2840.2 ± 717.5 ng/ml (90% CI: 2599.3 - 3081.1) were obtained for the test and reference formulation, respectively. The respected value for Tmax, the time required to reach the maximum serum concentration, was 2.25 ± 0.59 h (90% CI: 2.05 - 2.45) and 1.83 ± 0.46h (90% CI: 1.68 - 1.99). The parameters used as measures of the extent of absorption are AUC0-t and AUC 0-∞. The AUC0-t and AUC 0-∞ for the test formulation were 16278.5 ± 4639.6 ng.h/ml (90% CI: 14720 - 17836.3) and 17102.9 ± 4761.6 ng.h/ml (90% C.: 15504.2 - 18701.6). The calculated values for the reference formulation were 16732.4 ± 4971.3 ng.h/ml (90% CI: 15063.3 - 18401.6) and 17358.4 ± 5282.6 ng.h/ml (90% CI: 15584.8 - 19132.1) respectively. The results of the analysis of variance (ANOVA) for the assessment of product, group and period effects and the 90% confidence intervals (90% CI) for the ratio of, and Cmax values for the test and reference products, are shown in Table 2. According to FDA and EMEA for guidance on the investigation of bioavailability and bioequivalence , two pharmaceutical products are considered bioequivalent if their rate and extent of absorption after administration of the same molar dose are similar to such degree that their effects (both efficacy and safety) will be essentially the same. This is fulfilled if the 90% C.I. of the AUC ratio and Cmax ratio are within acceptance range (80% - 125%) in which the calculated 90% confidence intervals for the ratio of Cmax , and for the test and reference products were (81.98-94.26%), (91.6-109.15%) and (93.08-110.85%) respectively. The findings reveal that no statistical differences were found between pharmacokinetic parameters for the test and reference products, thus no relevant differences were found for tmax, kel and T1/2.
From the obtained results bioequivalence between the investigated clarithromycin formulations was demonstrated and interchangeability of the products can be stated.