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Introduction

Candesartan is a long-acting and selective nonpep-tide AT

1 subtype angiotensin II receptor antagonist1.

Candesartan blocks the vasoconstrictor and aldosterone-secreting effects of angiotensin II by selectively blocking the binding of angiotensin II to the AT1 receptor in many tissues, such as vascular smooth muscle and the adre-nal gland. Candesartan is indicated for the treatment of hypertension or heart failure2.

Candesartan is administered orally as a prodrug, candesartan cilexetil, which is completely hydrolyzed to the active compound during absorption from the

gastrointestinal tract. The absolute bioavailability of candesartan was estimated to be 15%. After oral administration, the time to peak candesartan concentrations was 3–4 h in the adult. There are no clinically significant effects of food on the bioavailability of candesartan after candesartan cilexetil administration2,3. Candesartan is highly bound to plasma proteins (>99%) and the volume of distribution of candesartan is 0.13 L/kg3. Candesartan is mainly excreted unchanged in urine and feces. It undergoes minor hepatic metabolism by O-deethylation and glucuronidation to an inactive metabolite. Total plasma clearance of candesartan is

ReseaRch aRtIcle

Pharmacokinetic Properties and Bioequivalence of Candesartan Cilexetil in Korean Healthy Volunteers

Ji-Young Jeon1,2, Yong-jin Im1,2, Yunjeong Kim1,2, Su-mi Han1,2, Myeong-jin Jo1,2, Dae-Hee Shin3, Ji-Seok Yoo3, Byung-Kwan Moon3, Byoung-Ki Kim3, Byung-Hoon Lee3, Yoon-Ho Choi4, Beom-Sik Cho4, Han-Young Jang4, Soo-Wan Chae1,5, and Min-Gul Kim1,2

1Clinical Trial Center, Chonbuk National University Hospital, Jeonju-si, Jeollabuk-do, Korea, 2Biomedical Research Institute, Chonbuk National University Hospital, Jeonju-si, Jeollabuk-do, Korea, 3Yungjin Pharm. Co., Ltd., Gandong-gu, Seoul, Korea, 4BioInfra Co. Ltd., Suwon-city, Gyunggi-do, Korea, and 5Department of Pharmacology, Medical School, Chonbuk National University, Jeonju-si, Jeollabuk-do, Korea

abstractCandesartan is a long-acting and selective nonpeptide AT1 subtype angiotensin II receptor antagonist. The aim of this study was to compare the pharmacokinetics and to evaluate the bioequivalence of two candesartan cilexetil 16 mg formulations. Forty healthy volunteers were randomly assigned into two groups. After a single dose of 16 mg candesartan cilexetil oral administration, blood samples were collected at specific time intervals from 0–36 h. The plasma concentrations of candesartan cilexetil were determined by LC-MS/MS. The pharmacokinetic parameters such as AUClast, AUCinf and Cmax were calculated and the 90% confidence intervals of the ratio (test/reference) pharmacokinetic parameters were obtained by analysis of variance on logarithmically transformed data. The mean for AUClast in the reference and the test drug were 1530.1 ± 434.6 and 1315.7 ± 368.6 ng·h/mL. The mean for AUCinf in the reference and the test drug were 1670.0 ± 454.5 and 1441.2 ± 397.8 ng·h/mL. The mean value for Cmax in the reference and the test drug was 142.6 ± 41.0 and 134.9 ± 41.4 ng/mL. The 90% confidence intervals for the AUClast, AUCinf and Cmax were in the range of log 0.81–log 0.91, log 0.81–log 0.91 and log 0.88–log1.01, respectively. No adverse events were reported by subjects or found on analysis of vital signs or laboratory tests. This single dose study found that the test and reference products met the regulatory criteria for bioequivalence in these health volunteers. Both formulations were safe and well tolerated in 16 mg of candesartan cilexetil hydrochloride.Keywords: Candesartan cilexetil, pharmacokinetics, bioequivalence, Korean healthy volunteers

Address for Correspondence: Min-Gul Kim, Clinical Trial Center, Chonbuk National University Hospital, 20, Geonji-ro, Deokjin-Gu, Jeonju-si, Jeollabuk-do, 561–712, Korea. Tel: +82-63-250-2532. Fax: +82-63-274-5546. E-mail: mgkim@jbnu.ac.kr

(Received 07 March 2012; revised 22 August 2012; accepted 26 August 2012)

Drug Development and Industrial Pharmacy, 2013; 39(9): 1296–1299© 2013 Informa Healthcare USA, Inc.ISSN 0363-9045 print/ISSN 1520-5762 onlineDOI: 10.3109/03639045.2012.725732

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Bioequivalence of Candesartan Cilexetil

J.-Y. Jeon et al.

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0.37 mL/min/kg, with a renal clearance of 0.19 mL/min/kg. The elimination half-life of candesartan is approximately 9 h2.

The pharmacokinetics of candesartan has been stud-ied in the elderly (65–78 years)4. The plasma concentra-tion of candesartan was higher in the elderly (C

max and

AUC of candesartan were approximately 50% higher) compared to younger subjects (19–40 years) admin-istered the same dose. The half-life of candesartan in the elderly (9–12 h) was somewhat longer than in the younger healthy adult volunteers4. However, in children aged 6–17 years, the pharmacokinetic profile of candes-artan is similar to adults5.

In hypertensive patients with renal insufficiency, serum concentrations of candesartan were elevated. After repeated dosing, the AUC and C

max were approxi-

mately doubled in patients with severe renal impairment (creatinine clearance <30 mL/min/1.73 m2) compared to patients with normal kidney function6. The pharmaco-kinetics of candesartan in hypertensive patients under-going hemodialysis is similar to those in hypertensive patients with severe renal impairment7.

The aim of this study was to compare the pharma-cokinetic properties and to evaluate the bioequiva-lence of two Candesartan Cilexetil formulations, Test drug (Cansartan Tab 16 mg, Yungjin Pharm. Co., Ltd.), Reference drug (Atacand Tab 16 mg, Yuhan Corporation), in healthy Korean subjects.

Methods

SubjectsA total of forty healthy Korean male adults, aged between 19 and 55 years, were enrolled in the study. Subjects were excluded if they had clinically or biologically significant abnormalities.

The protocol was approved by the institutional review board of Chonbuk National University Hospital, and all subjects provided written informed consent prior to par-ticipation. This study was performed in accordance with the revised Declaration of Helsinki8 and the guidance for bioequivalence study9.

ProtocolThis study was an open-labeled, randomized, single dose, crossover design. Subjects were randomized to group A or B. After a wash out period of 7 days, each sub-ject received the other formulation drug. All volunteers avoided taking other drugs for at least 10 days prior to the study. Alcohol and intense physical activity were not allowed during the study period.

Subjects were hospitalized to the clinical trial cen-ter on the evening before drug administration. After a 10 h overnight fast, each subject received 16 mg of Candesartan Cilexetil with 240 mL of water and fasted for 4 hours post-dose. All subjects received standardized meals at 4-h post-dose.

Subjects were continuously monitored by investiga-tors throughout the study period. Adverse events were

collected based on observation and direct question-ing. Vital signs were measured before and after drug administration.

Analytic proceduresFrozen human plasma samples were thawed at ambient temperature. 10 µL of IS working solution (candesartan-d

5 1.50 µg/mL in 70% methanol) was added to 50 µL

aliquot of plasma sample, vortexed for 60 s and then centrifuged at 13,000 rpm for 1 min. The mixture sample was deproteinized by 150 µL volume of pure acetonitrile, vortexed for 60 s and then centrifuged at 13,000 rpm for 3 min. Each 0.100 mL of the supernatant was diluted with 200 µL of 0.1% formic acid and then 10 µL aliquot of the sample was injected into the LC/MS/MS system.

LC/MS/MS analysis was performed on Micromass Quattro Premier XE Mass spectrometer (Waters, USA). The mobile phase was delivered using an Acquity UPLC system (Waters, USA). Chromatographic separations were performed on Acquity UPLC BEH C18, 1.7 µm col-umn. The mobile phase composition was a mixture of 0.1%(v/v) formic acid in Distilled water and 0.1%(v/v) formic acid in acetonitrile (60: 40, v/v). The flow rate was 0.4 mL/min. The peak area was measured for calculation of the peak area ratio of the analytes to their correspond-ing IS, and the plasma concentrations were estimated.

A calibration curve was constructed using eight non-zero calibrators covering the total range (5–1000 ng/mL). Linearity was assessed by a weighted (1/x2) least squares regression analysis. The calibration curves were linear over the concentration range 5–1000 ng/mL for all analytes (r2 > 0.995). The intra-day accuracy was 104.7–107.5% with precision of 2.7–9.8%. The inter-day accuracy was 96.6–107.1% with precision of 5.2–10.4%. These results indicated that the bioanalytical method has a satisfactory accuracy and precision.

Pharmacokinetic parameters and statistical analysesBlood samples (5 mL) for the determination of plasma candesartan cilexetil concentrations were collected prior to dosing and 0.5, 1, 2, 3, 4, 5, 6, 8, 12, 24 and 36 h after drug administration. Blood was immediately centrifuged at 3000 rpm for 10 min and stored at −70°C until analysis.

Pharmacokinetic analysis was performed using WinNonlin 5.3 software (Pharsight Corporation, CA, USA) Pharmacokinetic parameters were analyzed by using a noncompartmental method. The linear up/log down trapezoidal rule was used to calculation the AUC

last.

AUCinf

was calculated as AUClast

+ Clast

/ke, where C

last was

the last measured concentration, and ke was the slope of

the linear regression of the log-transformed plasma con-centration-time in the terminal phase. C

max and T

max were

directly obtained from measured values. The plasma elimination half-life (T

1/2) was calculated as ln 2/k

e.

The statistical analysis was performed by using SAS 9.2 (SAS Institute Inc., Cary, NC, USA). The general linear model used for statistical analysis includes sequence, formulation, period, and subject (subject is nested within

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1298 J.-Y. Jeon et al.

Drug Development and Industrial Pharmacy

sequence random effect) as factors. The 90% confidence intervals for the ratio of AUC

last, AUC

inf and C

max values for

the test and reference drug were used the logarithmic transformed data. Bioequivalence could be concluded if the 90% confidence intervals for AUC and C

max were

within log 0.80–log 1.25.

Results

SubjectsA total of forty volunteers were enrolled in the study and were randomized to treatment. The demograph-ics of enrolled subjects are shown in Table 1. Mean age of the 40 subjects was 22.9 ± 2.5 years, mean height was 176.2 ± 5.3 cm, and mean weight was 69.7 ± 7.5 kg. Since both groups did not show significant differences in age, height or weight, the randomized assignments were judged to have no problem. A total of 40 subjects com-pleted the study. Both formulations were well tolerated and no clinically significant changes or adverse events were observed.

PharmacokineticsFigure 1 shows the mean plasma concentration-time curves of candesartan following oral administration, and Table 2 represents the pharmacokinetic parameters for two formulations. The mean for AUC

last in the reference

and the test drug were 1530.1 ± 434.6 and 1315.7 ± 368.6 ng·h/mL. The mean for AUC

inf in the reference and the

test drug were 1670.0 ± 454.5 and 1441.2 ± 397.8 ng·h/mL, respectively. The mean value for C

max in the reference

and the test drug was 142.6 ± 41.0 and 134.9 ± 41.4 ng/mL, respectively. The median value for T

max was 5.0 h in

two formulations, and t1/2

was 8.3 ± 2.4 h for the reference drug and 8.1 ± 2.6 h for the test drug.

Log transformation of AUClast

, AUCinf

and Cmax

were performed prior to the statistical analysis, and Table 3 illustrates the statistical analysis. The parametric 90% confidence intervals for the AUC

last, AUC

inf and C

max were

in the range of log 0.81–log 0.91, log 0.81–log 0.91, and log 0.88–log 1.01, respectively. The intra-individual coef-ficient of variation of parameters are shown in Table 3.

Discussion

We studied the pharmacokinetics of candesartan in Korean healthy male subjects. AUC

last, AUC

inf and C

max

were similar for the reference drug and the test drug. Ratio estimates for the parameters AUC

last and C

max

were log 0.86 and 0.94 and the 90% confidence intervals were satisfied with the acceptance range of log 0.80–log 1.25. There was no carryover effect for any pharmacoki-netic parameters on analysis of variance. In this study the intra-individual coefficient of variation of AUC

last,

AUCinf

and Cmax

was around 14.6–18.5%. In other phar-macokinetic study, the intra-individual variability of AUC

last, AUC

inf and C

max was around 30.7–40.8%10. The

Table 1. Demography characteristics of study subjects. Group A (n = 20)

Group B (n = 20)

Total (n = 40) p valuea

Age (year) 22.8 ± 2.4 23.0 ± 2.7 22.9 ± 2.5 0.805Height (cm) 175.3 ± 5.6 177.1 ± 4.9 176.2 ± 5.3 0.285Weight (kg) 69.6 ± 7.9 69.8 ± 7.4 69.7 ± 7.5 0.951Values are presented as mean ± standard deviation.aIndependent t-test.

Figure 1. Mean plasma concentration-time profiles of candesartan cilexetil after reference and test 16 mg tablet formulation of candesartan cilexetil in 40 healthy Korean subjects. Each point represents the mean ± standard deviation.

Table 2. Pharmacokinetic parameters of candesartan cilexetil after a single oral administration of two formulations.

ParametersReference drug

(n = 40) Test drug (n = 40)AUC

last (ng·h/mL) 1530.1 ± 434.6 1315.7 ± 368.6

AUCinf

(ng·h/mL) 1670.0 ± 454.5 1441.2 ± 397.8C

max (ng/mL) 142.6 ± 41.0 134.9 ± 41.4

Tmax

(h) 5.0 [2.0–8.0] 5.0 [2.0–8.0]t

1/2 (h) 8.3 ± 2.4 8.1 ± 2.6

Clearance (L/h) 10.3 ± 2.7 12.0 ± 3.4Volume of distribution (L)

120.7 ± 42.2 135.5 ± 41.1

Values are presented as mean ± standard deviation.AUC

last, area under the concentration-time curve from time zero

to the time of the last quantifiable concentration; AUCinf

, area under the concentration-time curve with the last concentration extrapolated based on the elimination rate constant; C

max,

maximum observed concentration; Tmax

, time of maximum observed concentration; t

1/2, terminal half-life.

Table 3. Bioequivalence assessment for pharmacokinetic parameters of two candesartan cilexetil formulations.

ParametersGeometric Mean

Ratio (90% CI)Intra-individual

Variability (CVintra

%)

AUClast

(ng h/mL) 0.86 (0.81–0.91) 15.58AUC

inf (ng h/mL) 0.86 (0.81–0.91) 14.65

Cmax

(ng/mL) 0.94 (0.88–1.01) 18.35

AUClast

, area under the concentration-time curve from time zero to the time of the last quantifiable concentration; AUC

inf, area

under the concentration-time curve with the last concentration extrapolated based on the elimination rate constant; C

max,

maximum observed concentration.

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low intra-individual variability with sufficiently high number of volunteers in our study means very narrow confidence interval. The single dose, open label, a fast-ing study was conducted only in healthy male subjects. Therefore further studies in a patient population might be useful to show pharmacokinetic and to evaluate clini-cal efficacy of candesartan cilexetil.

In this single dose study, the two candesartan cilexetil 16 mg tablet met the KFDA regulatory criteria for bio-equivalence in healthy volunteers. All two formulations were well tolerated.

Declaration of interest

This research was supported by Yungjin Pharm. Co., Ltd. (Seoul, Korea).

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