Bioequivalence study of two formulations of doxycycline

CURRENT THERAPEUTIC RESEARCH” VOL. 59, NO. 5, MAY 1998

BIOEQUIVALENCE STUDY OF TWO FORMULATIONS OF DOXYCYCLINE RUTH RITZES-COHEN, DINA FARIN, ARIE LAOR, GUILLERhIO PIVA, RLARA HAZAN, AMIT WASSERMAN, AND IGAL GOZLAN Clinical Pharmacology and Znfectious Diseases Unit, Cannel Medical Center, The Rappaport Faculty of Medicine, Technion, Haifa, Israel

ABSTRACT A randomized, two-way, crossover bioequivalence study in 22 healthy male volunteers was conducted to compare 100~mg tablets of a monohydrate formulation with a hydrochloride formulation of doxycycllne. The drug was given in a single dose of 100 mg, and blood samples were collected duriug the 72-hour period after drug administration. Doxycycliue levels in plasma were determined by high-performance liquid chromatography assay. The pharmacolcinetic variables of area under the plasma concentration-time curve, maximum concentration, time to maximum concentration, elimination half-life, and mean residence time were computed. The results of this study suggest that the two formulations are bioequivalent. Keg words: doxycyeline, bioequivalence, high-performance liquid chromatography, healthy volunteers. INTRODUCTION

Doxycycline is a once-daily tetracycline antibiotic with a wide spectrum of activity. The drug is almost completely absorbed from the gastrointestinal tract after oral administration, and serum concentrations are usually reached within 2 hours. Doxycycline is 80% to 93% protein bound. It is metabolized to bacteriologically inactive derivatives in the liver, and about one half of an administered dose is transformed to inactive compounds. In healthy adults approximately 35% to 45% of doxycycline is excreted unchanged in the urine; biliary excretion of the drug also occurs. The half-life of doxycycline in patients with normal renal function ranges from 15 to 24 hours. Penetration of doxycycline into different body tissues allows a wide spectrum of clinical applicati0ns.l Several studies2-l2 have been performed comparing different forms of doxycycline made by different manufacturers. These studies have confirmed that differences are not significant in the bioavailability of various doxycycline preparations. Address correspondence k: Dr. R. Kitzes-Cohen, Clinical Pharmacology Carmel Medical Center, 7 Michal St., 34362 Haifa, Israel. Received for publication on January 21, 1998.Printed in the USA. Reproduction in whole or part is not permitted.

315

and Infectious

Diseases Unit,

0011-393x/934$19.00

BIOEQUNALENCEOF TWO DOXYCYCLINE FORMULATIONS

Several derivatives and preparations of doxycycline are presently in use. The commonly used preparations are the hydrochloride and the hyelate salts, as tablets or capsules, and the monohydrate free-base formulation. These forms have different physicochemical and solubility characteristics. Doxycycline monohydrate dissolves more slowly in the esophagus than the hydrochloride derivative; a solution containing doxycycline monohydrate has a higher pH (5.0 to 6.5) than a solution containing doxycycline hydrochloride (2.5 to 3.0); therefore, it is considered less likely to cause esophageal ulcers.10,13P14 The aim of this study was to compare the lOO-mg tablet of doxycycline monohydrate* (the test drug) with the lOO-mg capsule of doxycycline hydrochloride? (the reference drug). SUBJECTS

AND METHODS

Subjects

Twenty-two healthy male volunteers aged 18 to 32 years participated in the study. AI1 subjects were nonsmokers and denied use of illicit drugs; they were within 10% of ideal body weight for height. Subjects underwent physical examination, electrocardiography, and laboratory testing (complete blood count; biochemistry; urinalysis; and antibody testing for the human immunodeficiency virus, hepatitis B surface antigen, and hepatitis C virus). Study Design

In this randomized, two-way, single-dose, crossover study, each subject initially received one 100-mg tablet of doxycycline monohydrate (B.N. E1456/95; expiration date, August 1997) or one lOO-mg capsule of doxycycline hydrochloride (B.N. 301-58603; expiration date, August 1998), with a 2-week washout period before administration of the other formulation. No beverages containing alcohol and no food or beverages containing xanthines (eg, caffeine) were allowed for 24 hours before the study or during the study. No medications were to be taken for 1 week before the study. Subjects fasted for 10 hours before and 4 hours after drug administration. The study was approved by the Helsinki committees of the Carmel Medical Center, Haifa, Israel, and the Israeli Ministry of Health, Jerusalem. All subjects provided written informed consent before entering the study. Seventeen venous blood samples (10 mL each) were collected during both study periods as follows: 0 (before drug administration), 0.5, 1,1.5,2, * Trademark: Doxytrim@ (Trima Pharmaceutical Industries Ltd, Kibbutz Ma’abarot, Israel). t Trademark: Vibramycin” (Wzer Inc, New York, New York).

316

R. KI’lZES-COHEN

El’ AL

2.5,3,4,6,8,10,12,15,24,36,48, and 72 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, the intravenous catheter was washed with 1 mL saline with heparin (10 IU/mL). Immediately after collection, each blood sample was gently inverted several times for complete mixing. The blood samples were collected with 2-minute intervals between subjects. Within 1 hour of collection, the blood samples were centrifuged at 3000 rpm for 10 minutes. The plasma samples were then transferred into three Eppendorf tubes (Srastdet, Niimbrecht, Germany) and were frozen (-36 “C) until assayed. Analysts were masked as to whether the samples contained the test or the reference drug. The administration scheme was disclosed only after termination of the analytical phase of the study. Doxycycline plasma levels were determined by high-performance liquid chromatography (HPLC). The chromatographic equipment consisted of an HP-1050 HPLC system with a UVMS variable wavelength detector (at 357 nm), and a reverse-phase column (C, BDS, 10 cm x 4.6 mm, 3-pm particles, Hypersil, Shandon HPLC, Cheshire, United Kingdom). The mobile phase consisted of buffer (lo-mM oxalic acid, pH 2.37 [70%1, acetonitrile [18%1, and methanol [12%1), at a flow rate of 1.5 mumin. Total run time was 10 minutes. The extraction of doxycycline from plasma samples was performed as follows: O.l-mL internal standard solution (chlortetracycline, 5 Fg/rnL in water) and 2 mL phosphate sulfite buffer were added to 0.5 mL of plasma. The mixture was shaken and 6 mL of an ethyl acetate-isopropanol mixture (88:12 v/v) was added. After centrifugation the organic layer was isolated and evaporated to dryness at 33 “C using nitrogen. The residue was reconstituted with 0.3 mL of the mobile-phase solvent and 1 mL hexane. After mixing and centrifugation (10,000 rpm, 5 minutes), 100 ~.LLof the lower layer was injected into the HPLC system. The overall extraction recovery was 75% for doxycycline (at concentrations of 0.05, 0.50, and 2.50 Fg/mL) and 69% for the internal standard solution at the working concentration (1 Fg/mL). All samples were assayed against freshly prepared calibration curves (concentration range, 0.02, 0.03, 0.05,0.1, 0.2, 0.5, 1.0, 2.5, 4.0, and 5.0 kg/mL). Three sets of quality controls, at concentrations of 0.2,1.0, and 2.5 pg/mL, were included in 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 22 weeks, stability to three freeze-thaw cycles, and inter- and intraday stability) were established. Interday coefficients of variation were 10.9%, 6.4%, 7.7%, 6.0%, 4.8%, and 16.3% at concentrations of 0.03, 0.05, 0.2, 0.5, 2.5, and 5.0 bg/mL, respectively. All data obtained were within 20% precision and accuracy. The limit of 317

BIOEQUIVALENCE OF TWO DOXYCYCLINE FORMUIATIONS

quantification, which is the lowest concentration determined within ~15% precision and accuracy, was 0.02 pg/mL, and the limit of detection was 0.008 pg/mL. Pharmacokinetic and Statistical Analysis For each subject at each trial phase the following pharmacokinetic variables were determined: area under the plasma concentration-time curve (AUC), maximum concentration (C,,), time to maximum concentration (t,,), elimination half-life (tl,,), and mean residence time (MRT). Residual plots against predicted value plots were calculated for all models. The distributions of the residuals were computed and plotted. The normal plot and the Shapiro-Wilk statistic8 were used to test the hypothesis that the residuals were a random sample from a normal distribution. For the cases in which the assumptions for linear regression were not met by the residuals, the models were fitted by logarithmic transformation when a multiplicative model could be assumed; otherwise, nonparametric analysis was performed. A decision rule accepted by the regulatory authorities16,17 uses the 90% confidence interval (CI) for ratios of the expected characteristic of the test and reference drugs (AUC test/AUC reference, C,,, test/C,,, reference, MRT test.MRT reference). In case of AUC, a deviation of 20% of the reference is generally accepted. Using these linear models, standard 90% CIs were computed. Nonparametric CIs for the median difference were computed using the method proposed by Hauschke et al,ls which does not require the restrictive assumption of the equal period effect. Tables for the MannWhitney U test by Conoverlg were used. Statistical computations were performed using the SAS 6.12 software @AS Institute, Inc, Cary, North Carolina).20 were taken from the individual concentration-time Cmax and t,, curves. T, was calculated as log(2YLz, where Lz is the negative unweighted least-squares estimate of the slope of a line describing the timeversus-log (concentration) pairs of values for the last three measurable concentration points. AUC,, to the last measured concentration above the limit of quantification was calculated using the linear trapezoid rule. Extrapolation to infinity was performed by adding Ci,,&z to the trapezoidal estimate to the last concentration value (Ci,,,). MRT was calculated as where AUMC is the area under the moment curve. The AUMC,JAUC,, AUMC to the last measurable time (T,,,,) was calculated by the linear trapezoid rule. Extrapolation to infinity was performed by adding Ci,,&z (Ti,, + l/Lz) to the trapezoidal estimate to the last concentration value at Tlast* 318

R. KJTZES-COHEN

ET AL.

RESULTS

Doxycycline appeared in the plasma of all volunteers within 0.5 hour after administration and achieved maximal concentrations from 0.5 to 3 hours. The precision and accuracy of the analytical phase were evaluated from the quality controls included in the analytical run (Table I). The individual and mean values of the pharmacokinetic variables for the 22 subjects are presented in Table II. The mean concentration-time curves for both formulations of doxycycline are shown in the figure. No adverse effects of doxycycline were detected in any of the study periods. Mean peak plasma levels were obtained after 1.5 and 1.7 hours and reached values of 1.7 * 0.4 kg/mL and 2.0 * 0.6 &mL for the test and reference formulations, respectively. The C,, ratio was 0.9 (90% CI, 0.8 to 0.9). This ratio was computed as the ratio of the arithmetic mean of the test preparation variable to the arithmetic mean of the reference variable (mean C,, test/mean C,, reference). No transformation was needed because of good distribution of the residuals from the linear models investigated. The mean AUC values were 32.0 f 8.6 l&h per mL and 37.2 f 9.0 ~_~g/hper mL for the test and reference formulations, respectively. The geometric mean of the ratios between the test and reference variables was 0.9 (90% CI, 0.8 to 0.9). For pharmacokinetic reasons discussed by Steinijans and Hauschke, ” the 90% analysis of variance CI was presented after logarithmic transformation and was included within the 0.8 to 1.3 range. Mean MRT values were also similar (27.0 f 4.1 and 26.1* 3.9 hours) for the test and reference formulations, with a ratio of 1.0. The median difference used in the computations oft,, and t,, and the corresponding median estimate were computed by the method proposed by Hauschke et al,‘* which does not require the restrictive assumption of the equal period effect, as did previous methods. The rate of absorption of doxycycline reflected by t,, had a difference of -0.2 hour (range, -2 to 1 hours), with a 90% CI of -0.5 to 0 for the two formulations. The mean t,, values were 20.4 hours (range, 13.8 to 27.8 hours) and 19.6 hours (range, 14.4 to 25.6 hours) for the test and reference formulations, respectively. The tl,z difference was 0.8 hour (90% CI, -0.3 to 1.6 hours).

Table I. Precision and accuracy of the analytical phase based on quality control samples at concentrations of 0.2, 1.0, and 2.5 pg/mL.

Nominal

Ei 2.50

No. of Samples

2: 50

Observed Concentration

(PohL)

0.21 f 0.03 1.02 f 0.10 2.38 * 0.21

319

Accuracy

WI

Precision

WI

13.31 9.92 8.70

E 14:9

Z

E 30.8 35.8 39.4

45.5 17.0

34.7 30.2 23.4

E 31.6 26.i 43.0

E"

E

F J :

N"

F Q

s"

,

E 4618

34:7 36.2 38.0

Ki

40.1 24.4 19.9 48.3

%

DH

47.9 27.1 26.9 33.7 29.2 32.5 34.8 53.0 37.22 9.0 19.9-53.0

0.9 0.8-O.9 o.E.9

t: 2.0 1.3-3.5 f 0.6

1.4 1.9

{I$

1.5 1.2 1.6 1.7 2.1 'lE;l4

2.0 3.1 2.6 3.5

Z:! 1.7 1.7 2.3

1:: 2.3 i1

2.2 1.6 1.4 2.4

1.; 1:s 1.5 2.5

1.8

1.7 1.3 1.6 1.6 1.0 2.2

DH

DM

C,, (WmL)

1.0 1.0-1.1

;t:

E

:3":: 24.8 E3 26.1; 3.9 20.5-33.0

:::: 27.0+ 4.1 18.5-36.7

;:::

z:: 29.5 28.8 27.2 24.7 28.9 34.1 30.1

E:! 29.8 20.6

;;:; 25.0

DH

23:6

DM

MRT (h)

;:: 2.0 3.0 2.0 1.0 1.0 1.0 1.0 1.5 ._ 2.0 1.5

1.5 1.0 1.5

7::

::: 1.5 1.5 1.7kO.6 -.1.0-3.0

-0.5-0.0

-0.2f 0.7 ($J)

1:: 1.5 1.5 1.5* 0.6 0.5-3.0

:i

11:: .-

1:: 0.5 1.0

1:5 2.0 1.0 1.5 2.0 2.0 1.0

2.0

1.0

E

DH

DM

t,,,(b)

(-2h&7.2) -0.3-1.6

0.8+ 2.4

;::: 18.2 23.9 19.6+ 3.2 14.4-25.6 E 18.0 27.8 20.4f 3.2 13.8-27.8

:;:: 17.5 18.0 20.2 :::: 17.3 17.8 22.6

Z

1::; 19.9

1:::

15.6

OH

:A:: 22.6 19.8 17.5 21.4 22.8 21.6 22.4 18.7 21.1

:;:; 17.0 20.0 20.4

13.8 19.2

DM

t,, (b)

MRT = mean residencetime;t,, = time to maximum concentration; AIJC = area under the plasma concentration-time curve;C,, = maximum concentration; t,,= elimination half-life; Cl = confidence interval.

M$~~gee..timate 90% nonparametric Cl

90% Cl Difference median

;;;y

:! Mean

y&

;:::

A :

7

DM

Subject

AUC (wlh * mL)

Table II. Individual and mean values of pharmacokinetic parameters in 22 subjects after oral administration of one lOO-mg dose of doxycycline monohydrate (DM) or doxycycline hydrochloride (DH).

R. KITZES-COHEN ET AL.

2.5-

- - = - - Doxycycline monohydrate I - - e - - Doxycycline hydrochloride I

I

2.0'

|.J=

1.5"

~

1.0' 0.5,

1 0.0 I

I

0

20

I

40

I

60

Time (h)

Figure. Mean plasma levels after oral administration of 100 mg doxycyclinemonohydrateor doxycyclinehydrochloride. DISCUSSION AND CONCLUSIONS In the present study, two 100-rag formulations of doxycycline were given orally. It was found t h a t doxycycline was rapidly absorbed, and peak plasma levels were reached within 1.5 hours. Seventy-two hours after dosing, plasma levels reached values below the limit of quantification or lower t h a n 10% of Cm~, indicating t h a t most of the drug had been eliminated. We used the actual values derived from the concentration-time curves for Cma~ and tma~, according to the recommendations of the US Food and Drug Administration and other regulatory agencies. 22'23 As mentioned previously, a deviation of 20% in the test formulation is generally accepted. This is because two different drug preparations are rarely exactly the same, even when prepared by the same manufacturer. We found an AUC ratio of 0.9 between the test and reference preparations. The coefficient of variation for the linear model was 16%. Therefore, using the table given by Diletti et al, 24 the power of this study was more t h a n 90% to detect a 20% difference between the preparations. Because we use samples r a t h e r t h a n populations and probability models r a t h e r t h a n deterministic models, a 20% difference cannot be excluded. Statistical tools can only help to assess the probability of such an event. Only MRT parametric estimators and parametric CIs based on multiplicative model are brought. For pharmacokinetic reasons, 21 we preferred to use CIs based on a multiplicative model; however, the linear model is 321

BIOEQUNALENCE

OF TWO DOXYCYCLINE

FOBMULATIONS

adequate statistically and the CIs based on that model differ by less than 2% from the Cls derived from the multiplicative model. Several bioequivalence studies of orally administered doxycycline 100 mg7,10-12or 200 mg81ghave been rep orted in the literature. Our pharmacokinetic findings support the findings of other studies of doxycycline 100 mg administered orally. In a comparison of doxycycline monohydrate and doxycycline hydrochloride tablets, Malmborg” reported a C,, of 1.7 pg/ mL for both formulations and an AUC of 40.1 and 37.4 &h per mL for the two formulations, respectively. Lode et al” compared the lOO-mg preparation of Sigadoxine (Sigfried GmbH, Bad Sackingen, Germany) with a reference preparation and found C,, to be 2.9 and 3.2 Fg/mL, t,, 3.6 and 3.2 hours, and AUC 44.9 and 50.3 p&h per mL for the two formulations, respectively. These values are somewhat higher than the values found for the formulations we studied. The findings of Lode et al” were similar to those obtained by Balogh et al l2 for Doxycycline Kapselne (Jenapharm, Jena, Germany) (B.N. 050786) and for a reference preparation (B.N. 88 320650) (C,,, 3.1 and 3.2 pg/mL, t,,, 2.3 and 2.1 hours, and AUC 56.5 and 56.2 &h per mL, respectively). Saket et al7 compared lOO-mg Doxydare capsules (Dar Al Dawa Development Investment Co., Na’ur, Jordan) with Vibramycine (Pfizer, Brussels, Belgium). Their findings for C,, (2.0 and 1.8 Fg/mL) were similar to ours; however, they reported values for AUCo-is, which could not be compared with our findings. Our study indicates that the two formulations of doxycycline, both given as a single lOO-mg dose, are comparable in terms of the following pharmacokinetic variables: the extent of absorption, reflected by AUC,; the rate of absorption, reflected by C,, and t,,; the elimination half-life, represented by t,; and MRT, which characterizes the overall absorption and elimination process. The results suggest that the monohydrate and hydrochloride formulations of doxycycline used in this study are bioequivalent. Acknowledgment This study was supported by Trima Pharmaceutical Industries Ltd, Kibbutz Ma’abarot, Israel. References: 1. Bennett WM, Aronoff GR, Golper TA. Drug Prescribing in Renal Failure: Dosing Guidelines for Adults. 3rd ed. Philadelphia, PA: American College of Physicians; 1994. 2. Petrick RJ, Love SJ, Noris E. The bioavailability and gastrointestinal cycline hyclate tablets. Postgrad Med Comm. 1981;20. 3. Cohen RK, Grauer F, Weisenberg Isr J Med Sci. 1980;16:545-546.

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322

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