A bioequivalence study of two formulations of cefaclor

CURRENT THERAPEUTIC RESEARCH ® VOL. 58, NO. 6, JUNE 1997

A BIOEQUIVALENCE STUDY OF TWO FORMULATIONS OF CEFACLOR RUTH KITZES-COHEN, 1'2 DINA FARIN, 1 J U D I T H MARCKOVICH, 1 AMIT WASSERMAN, I IGAL GOZLAN, 1 AND ARIE IAOR 1

1Clinical Pharmacology and Infectious Diseases Unit, Carmel Medical Center, and 2Rappaport Faculty of Medicine Technion, Israel Institute of Technology, Haifa, Israel

ABSTRACT

A randomized, two-way, crossover,

bioequivalence s t u d y in 16 healthy male volunteers was conducted to compare two formulations of 250-mg capsules of cefaclor. The drug was given in a single d o s e o f t w o c a p s u l e s ( t o t a l , 500 mg), and b l o o d s a m p l e s w e r e w i t h d r a w n during the 12 hours after drug administration. C e f a c l o r plasma levels were determined by high-pressure liquid chromatogr a p h y a s s a y . T h e f o l l o w i n g p h a r m a c o k i n e t i c v a r i a b l e s w e r e computed for t h e t w o f o r m u l a t i o n s : a r e a u n d e r t h e plasma concentra-

tion-time curve, maximum concentration, time to maximum concentration, half-life of elimination, and mean residence time. The results of this study suggest that the two formulations are bioequivalent. Key words: cefaclor, bioequivalence, high-pressure liquid chromatography, volunteers. INTRODUCTION

Cefaclor, 3-chloro-7-D-(2-phenylglycinamido)-3-cephem-4-carboxylicacid monohydrate, is a second-generation semisynthetic cephalosporin antibiotic. Cefaclor is well absorbed from the gastrointestinal tract after oral administration. Peak serum concentrations are usually reached within 60 minutes and are dose dependent. Several pharmacokinetic studies of oral cefaclor1-1° have been conducted; however, to the best of our knowledge, no bioequivalence studies of two formulations of cefaclor have been reported in pharmacologic literature. This study compared the bioequivalence of two formulations ofcefaclor 250-rag capsules--Cefalor®* (test drug) and Ceclor®t (reference drug). SUBJECTS AND METHODS

Subjects Sixteen healthy male volunteers, aged 18 to 31 years, participated in Address correspondence to: R. Kitzes-Cohen, MD, Clinical Pharmacology and Infectious Diseases Unit, Carmel Medical Center, 7 Michal Street, 34362 Haifa, Israel. Received for publication on February 10, 1997. Printed in the U.S.A. Reproduction in whole or part is not permitted. * Trademark: Cefalor ® (Vitamed Pharmaceutical Industries Ltd., Binyamina, Israel). t Trademark: Ceclor~ (Lilly, Basingstake, England). 352

0011-393X/97/$3.50

R.KITZES-COHENETAL.

the study. None of the subjects smoked tobacco or abused drugs (ie, they denied use of illicit drugs), and all subjects were within 10% of ideal body weight for height. Each subject underwent physical examination, electrocardiography, and laboratory testing (complete blood count and biochemistry tests, urinalysis, and antibody testing for the human immunodeficiency virus, hepatitis B surface antigen, and hepatitis C virus).

Study Design The study was a randomized, two-way, single-dose, crossover design. Each subject initially received a single dose consisting of two 250-mg capsules of Cefalor (Batch Number 1192R13) or two 250-mg capsules of Ceclor (Batch Number 69923PE), expiration date: February 1995, with a 1-week washout period between drug administrations. Each subject received one randomly chosen formulation of cefaclor the first period and the other formulation the second period. No beverages containing alcohol and no food or beverages containing xanthines (eg, caffeine) were allowed for 24 hours before or during the study, and no medications were taken within 1 week of the study. The subjects fasted for 10 hours before each study period. The study was approved by the Helsinki Committee of the Carmel Medical Center and by the Helsinki Committee of the Israeli Ministry of Health. All volunteers provided informed written consent before participating in the study. Thirteen venous blood samples (10 mL each) were collected during each study period, as follows: before drug administration; at 20, 40, 60, and 100 minutes; and at 2, 2.5, 3, 4, 6, 8, 10, and 12 hours after drug administration. At each blood collection, 1.5 mL of blood was withdrawn and discarded before the blood sample was collected. After collection of the blood, the intravenous catheter was washed with 1 mL of saline with heparin (10 IU/mL). Immediately after collection, each blood sample was gently inverted a few times for complete mixing. The blood samples were collected with 2-minute intervals between subjects. Within 1 hour after collection, the blood samples were centrifuged at 3000 rpm for 10 minutes. The plasma samples were then transferred into three Eppendorf tubes (Sarstedt, Inc., Niimbrecht, Germany) and were frozen (-36 °C) until assayed. Analysts were not aware of which samples represented the test or the reference drug. The administration scheme was disclosed only after termination of the analytic phase of the study. Cefaclor plasma levels were determined by high-pressure liquid chromatography (HPLC). The chromatographic system consisted of an HP1050 HPLC (Hewlett-Packard Co.), with a UV/VIS variable wavelength detector (at 260 nm) and a reverse phase column (Cs, Spherisorb, 25 cm × 4 mm, 5-~m particles [Knauer, Bad Homburg, Germany]). The mobile phase consisted of buffer (12.5 mmol/L monopotassium phosphate, pH = 353

BIOEQUIVALENCE OF CEFACLOR

2.7) and acetonitrile. The following gradient method was used at a flow rate of 1.5 mL/min: acetonitrile, 3% at 5 minutes, 20% at 24 minutes, 20% at 30 minutes, 3% at 31 minutes, and posttime 14 minutes. Total run time was 45 minutes. A modification of the procedure was performed for the extraction of cefaclor from plasma samples. 11 After precipitation of proteins with a solution of 6% trichloroacetic acid, a 50-~LL sample of the upper layer was injected into the HPLC system. The internal standard (IS) was 40 ~g/mL cephalexin (Sigma Israel Chemicals Ltd., Rehovot, Israel) in water. The overall extraction recovery was 94% for cefaclor (at three concentration levels) and 95% for the IS, at the working concentration. All samples were assayed against freshly prepared calibration curves (concentration range: 0.2, 0.5, 1, 2, 4, 6, 8, 10, 20, and 30 ~Lg/mL). Three sets of quality controls (QCs), at concentrations of 1, 10, and 20 }xg/mL, were placed within each run. The analytic method was fully validated before the study. Linearity (over 10 calibration curves) and stability (on machine for 24 hours, longterm stability up to 14 weeks, stability to three freeze-thaw cycles, and inter- and intraday stability) were established. All data obtained were within 20% precision and accuracy. The limit of quantification (LOQ), which is the lowest concentration determined within 20% precision and accuracy, was 0.2 ~g/mL. (The same value was observed by Rotschafer et al ~ for the limit of sensitivity of their method.) Pharmacokinetic and Statistical Analyses For each subject at each trial phase, the following pharmacokinetic variables were computed: area under the plasma concentration-time curve (AUC), maximum concentration (Cm~), time to maximum concentration (Tm~x), elimination half-life (t1/2), and mean residence time (MRT). Residual plots against predicted value plots were determined for all models computed. The distributions of the residuals were computed and plotted. The normal plot and the Shapiro-Wilk statistics were used to test the hypothesis that the residuals are a random sample from a normal distribution. 12 If the assumptions for linear regression were not met by the residuals, the models were fitted by logarithmic transformation in cases where a multiplicative model could be assumed; otherwise, nonparametric analysis was performed. A decision rule accepted by the regulatory authorities uses the 90% shortest confidence interval (CI) for ratios of the expected characteristic (AUC-test/AUC-reference, Cmax-test/C~ax-reference, MRT-test/MRTreference). 13'1t In the case of AUC, a deviation of 20% of the reference is generally accepted. Using the preceding linear models, standard 90% CIs were computed. Nonparametric CIs for the median difference were computed by the method proposed by Hauschke et al, 15 which does not require 354

R.KITZES-COHENETAL.

the restrictive assumption of equal period effect. Tables for the MannWhitney test statistic by Conover ze were used. Statistical computations were performed using the SAS 6.08 software (SAS Institute, Inc., Cary, North Carolina). Cm~ and Tm~ were taken from the individual time-concentration curves. The tl/2 values were calculated by log(2)]Lz, where Lz is the negative, unweighted, least-squares estimate of the slope of a line describing the time versus log (concentration) pairs of values for the last three measurable concentration points. The AUCo_~ up to the last measured concentration above LOQ was calculated using the linear trapezoid rule. Extrapolation to infinity was performed by adding Chst/Lz to the trapezoidal estimate up to the last concentration value (Clast). The MRT was calculated as follows: MRT = AUMCo_JAUCo_~, where AUMC is the area under the moment curve. The AUMC up to the last measurable time was calculated using the linear trapezoid rule. Extrapolation to infinity was performed by adding Cl~t/Lz (Tlast + 1/az) to the trapezoidal estimate up to the last concentration value at time Tl~st. RESULTS

Cefaclor a p p e a r e d in the p l a s m a of all subjects within 20 minutes, achieved Cm~ between 40 minutes and 3 hours, and had no measurable concentrations (ie, all concentrations were below LOQ) after 8 hours. Therefore, these data were not used in the computations. The precision and accuracy of the analytic phase were evaluated from the QCs included in the analytic run. The values are summarized in Table I. The individual pharmacokinetic variables and the mean values for the 16 subjects are shown in Table II. The figure shows the mean concentration-time curve for both formulations of cefaclor. No adverse drug reactions with cefaclor were detected in any of the study periods. P e a k plasma levels were obtained after 1 hour and 0.83 hour and reached mean values of 11.3 + 3.2 ~g/mL and 11.8 + 3.5 tLg/mL for the test and reference formulations, respectively. The Cma~ ratio was 0.96, with a 90% CI of 0.82 to 1.11. This ratio was computed as the ratio of the arith-

Table I. Precision and accuracy of the analytic phase. Quality Control (pg/mL) 1 10 20

n

Measured Concentration (pg/mL)

Accuracy

Precision

(%)

(%)

48 48 48

1.01 ± 0.07 9.72 ± 0.70 19.37 ± 1.45

5.61 6.17 6.41

7.36 7.18 7.47

355

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Time ~ Figure. Plasmalevels (mean ± SD) after oral administration of 500 mg of each of two different formulations of cefaclor (Ceclor~ [Lilly, Basingstake, England]; Cefalor~ [Vitamed Pharmaceutical Industries Ltd., Binyamina,Israel]). metic m e a n of the test preparation parameter to the arithmetic mean of the reference p a r a m e t e r (mean Cma~-test/mean Cm~-reference); no transformation was needed because of good distributional properties of the residuals from the linear models investigated. The AUC values were 16.6 + 3.6 ~g- h/mL and 17.8 ± 2.8 ~g" ldmL for the test and reference formulations, respectively. The geometric mean of the ratios between the test and reference parameter was 0.93, with a 90% CI of 0.86 to 1.00. For pharmacokinetic reasons proposed by Steinijans and Hauschke, 17 the 90% analysis of variance CI was presented afLer logarithmic transformation and is included inside the 0.80 to 1.25 range. MRT values were also in agreement (1.65 ± 0.30 and 1.68 ± 0.45 hours) for the test and reference formulations, with a ratio of 1.05. The 90% nonparametric CI for the ratio was 0.97 to 1.08. The median difference, used in the computations ofTma~ and tl/2, with its corresponding median estimate, was computed by the method proposed by Hauschke et al, 15 which does not require the restrictive assumption of equal period effect required in previous methods. The rate of absorption of cefaclor, reflected by Tm~, h a d a difference of 0 hour, with a 90% CI of -0.17 to 0.33 for the two formulations. The m e a n t1/2 values were 0.71 hour (range, 0.54 to 0.86) and 0.73 hour (range, 0.41 to 1.80) for the test and reference formulations, respectively. The tl/2 difference was -0.04 hour, with a 90% CI o f - 0 . 0 9 to 0.01 hour. 357

BIOEQUIVALENCEOF CEFACLOR

D I S C U S S I O N AND CONCLUSIONS

In this study, two 250-mg tablets were given as a single oral dose in an effort to obtain higher plasma concentrations of cefaclor, and, therefore, to achieve better sensitivity in the analytical method. 6 We found that cefaclor is rapidly absorbed after oral administration, and peak plasma levels are reached not later than 1 hour after drug administration. No detectable concentration of cefaclor was observed at 8 hours, whereas other investigators reported no detectable concentration by 4 or 6 hours after dosing. 2'4 In this study, we used the actual values derived from the timeconcentration curves for Cm= and Tin= according to the recommendations of the US Food and Drug Administration and other regulatory agencies. is'x9 As mentioned, a deviation of 20% in the test formulation is generally accepted because two different drug preparations will not be equivalent, even when prepared by the same manufacturer. Increasing the sample size or decreasing the variance (mean square of error) decreases the CI. This is a general rule, and CIs that do not include unity are seen more often as the sample size is larger and the variability is smaller. We found that the AUC ratio between the test and reference preparations was 0.93. The coefficient of variation for the linear model was 11%. Therefore, using the table given by Diletti et al, 2° the power of this study is more than 90% to detect a 20% difference between the preparations. Because we used samples instead of populations and probability models instead of deterministic models, a 20% difference cannot be excluded. The only possibility that statistics can provide is to decrease the probability for such an event. Our conclusions support the findings of several studies 2'16 of the pharmacokinetics of orally administered cefaclor 500 mg. Barbhaiya et al6 investigated the absorption of 500 mg of cefaclor,* and reported the following values: AUC = 17.5 _+2.1 l~g" h/mL, tl/2 = 0.6 _+0.1 hour, and MRT = 1.2 _+0.2 hours', all of which are similar to our findings. In addition, they found Cn~, = 17.3 txg/mL at Tma~ = 0.7 -+ 0.2 hour, which is slightly higher than the C ~ x found in our study (11.3 ~g/mL). Meyers et al 2 reported a value similar to ours (12.8 ~g/mL) 1 hour after dosing. The results of this study show that, after a single oral dose of two 250mg capsules of cefaclor, the two formulations are similar in terms of the following pharmacokinetic variables: the extent of absorption, reflected by AUCo_~; the rate of absorption, reflected by C ~ = and Tma~; the rate of terminal elimination, tl/2; and the MRT, which characterizes the overall absorption and elimination process. We conclude, therefore, that the two cefaclor formulations are bioequivalent.

* Trademark: Distaclor~ (Dista Products and Eli Lilly and Co., Indianapolis, Indiana).

358

R. KITZES-COHENETAL.

Acknowledgment This study was supported by Vitamed Pharmaceutical Industries Ltd., Binyamina, Israel. References: 1. Lode H, Stahlmann R, Dzwillo G, Koeppe P. Comparative pharmaeokinetics of oral cephalosporins: Cephalexine, cefaclor and cefadroxil. Arzneim-forsch Drug Res. 1980;30:505--509. 2. Meyers BR, Hirschman SZ, Wormser G, et al. Pharmacologic studies with cefaclor, a new oral cephalosporin. J Clin Pharmacol. 1978;18:174-179. 3. Welling PG, Dean S, Selen A, et al. The pharmacokinetics of the oral cephalosporins cefaclor, cephradine and cephalexin, lnt J Clin Pharmacol Biopharm. 1979;17:397-400. 4. Oksana M, Korzeniowski W, Michael S, Sande MA. Comparative pharmacology of cefaclor and cephalexin. Antimicrob Agents Chemother. 1977;12:157-162. 5. Rotschafer JC, Crossley KB, Lesar TS, et al. Cefaclor pharmacokinetic parameters: Serum concentrations determined by a new HPLC technique. Antimicrob Agents Chemother. 1982;21:170-172. 6. Barbhaiya RH, Shukla UA, Gleason CR, et al. Comparison of cefprozil and cefaclor pharmacokinetics and tissue penetration. Antimicrob Agents Chemother. 1990;34:1204-1209. 7. Barbhaiya RH, Shukla UA, Gleason CR, et al. Comparison of the effects of food on the pharmacokinetics of cefprozil and cefaclor. Antimicrob Agents Chemother. 1990;34:1210-1213. 8. Oguma T, Yamada H, Sawaki M, Narita N. Pharmacokinetic analysis of the effects of different foods on absorption of cefaclor. Antimicrob Agents Chemother. 1991;35:17291735. 9. Yamaguchi M, Satoh T, Kikuchi F, et al. Comparative study on human blood concentration of oral cephem antibiotics cefuroxime axetil vs cefaclor--effect of food on bioavailability. Oral Ther Pharmacol. 1992;11:99-104. 10. James NC, Donn KH, Collins JJ, et al. Pharmacokinetics of cefuroxime axetil and cefaclor: Relationship of concentrations in serum to MICs for common respiratory pathogens. Antimicrob Agents Chemother. 1991;35:1860-1863. 11. Barbhaiya RH, Shukla UA, Gleason CR, et al. Phase I study of multiple-dose cefprozil and comparison with cefaclor. Antimicrob Agents Chemother. 1990;34:1198-1203. 12. Shapiro SS, Wilk MB. An analysis of variance test for normality. Biometrika. 1965;52: 591-611. 13. Studies on Bioavailability and Bioequivalence---APV Guideline, International Views of Politics and Science with Contributions on Development, Manufacture, Marketing, Distribution and Therapeutic Use of Drugs. 1987;30:131-136. 14. Bioavailability Protocol Guidelines for ANDA and NDA Submission. Rockville, Md: US Food and Drug Administration, Division of Biopharmaceutics; 1977. 15. Hauschke D, Steinijans VW, Diletti EA. Distribution free procedure for the statistical analysis of bioequivalence studies. Int J Clin Pharmacol Ther Toxicol. 1990;28:72-78. 359

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16. Conover WJ, ed. Practical Nonparametric Statistics. New York: Wiley; 1971:384-389. 17. Steinijans VW, Hauschke D. Update on the statistical analysis of bioequivalence studies. Int J Clin Pharmacol Ther Toxicol. 1990;28:105-110. 18. Guidance for Statistical Procedures for B~oequivalence Studies Using a Standard Two Treatment Crossover Design. Rockville, Md: US Food and Drug Administration, Office of Generic Drugs; July 1992. 19. Shein CC, Lin JP. Recent statistical developments in bioequivalence trials---A review of the FDA guidance. Drug lnfJ. 1994;28:51-64. 20. Diletti E, Hauschke D, Steinijans VW. Sample size determination for bioequivalence assessment by means of confidence intervals. Int J Clin Pharmacol. 1991;29:1-8.

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