Pharmacokinetics of the combination hydrochlorothiazide and ...

Ke X et al. Asian Journal of Pharmacodynamics and Pharmacikinetics 2008; 8(2):131-137
Asian Journal of Pharmacodynamics and Pharmacokinetics Paper ID 1608-2281-2008-08020131-07
Copyright by Hong Kong Medical Publisher Received March 15, 2008
ISSN 1608-2281 2008; 8(2):131-137 Accepted May 15, 2008
Pharmacokinetics of the combination hydrochlorothiazide and bisoprolol
tablet in Chinese healthy young male and female volunteers
Ke Xu, Chen Hui*, Xu Rong, Wang Ping,Gu Shifen, Ru Lin, Zeng Fandian
Tongji Medical College of Huazhong University of Science & Technology, Wuhan 430030, People’s Republic of China
Abstract The pharmacokinetics of bisoprolol/hydrochlorothiazide compound formulation (A: 2.5/6.25, B: 5/6.25, or C:
10/6.25 mg in each tablet) was studied after oral administration in Chinese healthy volunteers that was
divided into 3 groups of 8 males and 8 females per group. Each subject was randomized to receive a single
oral dose of the compound either A, B or C in a one-way crossover. The pharmacokinetic parameters such as
C max, T max, AUC (0-t), AUC (0-∞), T 1/2 were calculated and compared between the two genders and three groups.
The results of the single-dose study indicated the linear relationship between the mean values for AUC 0-t,
AUC 0-∞, C max and dose (r = 0.999,r =0.995 and r =0.999, respectively) for bisoprolol. It is founds that T max
and T 1/2 are dose-independent for bisoprolol, and all parameters are of no significant difference between the
three groups for hydrochlorothiazide. The results from this study demonstrated the gender factor gave little
impact to the pharmacokinetic property. Moreover, the testing tablets were well tolerated in Chinese healthy
young volunteers.
Key words hydrochlorothiazide; bisoprolol ; pharmacokinetics
Introduction
Hydrochlorothiazide is a diuretic and
antihypertensive agent within the class of
benzothiadiazine drug that reduces plasma volume by
increasing the excretion of sodium, chloride and
water and, to a lesser extend, that of potassium ion as
well [1-3] . Bisoprolol, which is indicated for the
treatment of hypertension and angina pectoris, is a
highly β1-selective adrenoceptor blocking agent
without membrane-stabilizing activity or intrinsic
sympathomimetic activity [4,5] . Diuretics and
β-adrenergic blocking drugs are the only two classes
of antihypertensive drugs that have been tested and
shown to reduce morbidity and mortality. They have
been recommended as preferred initial therapy in
patients with systemic hypertension [6] . They are often
combined to maximize blood pressure control either
as separate agents or in single fixed-dose
formulations [7–11] . Recently, the combination of
low-dose bisoprolol (2.5, 5, or 10 mg) combined with
low-dose hydrochlorothiazide (6.25 mg) has been
approved as first-line therapy for hypertension.
Though pharmacokinetics of bisoprolol and
131
hydrochlorothiazide have been studied separately
[12-16], a review of data in literature shows that the
pharmacokinetics of the combination of low-dose
bisoprolol combined with low-dose
hydrochlorothiazide in human has not been
extensively studied. Therefore, the study was
designed to explore the pharmacokinetics of the
combination products after single oral administration
in Chinese healthy young volunteers
Material and Methods
Chemicals and reagents
Compound recipe of bisoprolol and
hydrochlorothiazide were synthesized by MERCK
KGaA in and packaged by Merck Santé SA in France
into three batchs (A: bisoprolol 2.5mg
/hydrochlorothiazide 6.25mg with batch NO. 1058, B:
bisoprolol 5mg /hydrochlorothiazide 6.25mg with
batch NO. 2066, C: bisoprolol 10mg
/hydrochlorothiazide 6.25mg with batch NO. 3055).
Bisoprolol hemifumarate reference standard (99.6%
purity), hydrochlorothiazide reference standard
(99.5% purity), EDM 34800 reference standard (IS,

Ke X et al. Asian Journal of Pharmacodynamics and Pharmacikinetics 2008; 8(2):131-137
98.5% purity)and hydroflumethiazide reference
standard (IS, 97% purity) were supplied by MERCK
KGaA (German). Purified water from a Milli-Q
system (Millipore, Bedford, MA, USA) was used
throughout. The control human plasma was
purchased from Blood Bank (Tongji Hospital, Wuhan,
China). HPLC grade acetonitrile, methanol was
purchased from Merk (German). All other chemicals
were of analytical grade.
Study design
In accordance with the Declamtion of Helsinki,
all volunteers who had been told the aim and risk of
the study gave their written consent to their
participation in the study, and the protocol was
approved by an Institutional Review Board of Tongji
Medical College of Huazhong University of Science
& Technology.
The study was conducted as a single-dose,
open-label, randomized study in 48 Chinese healthy
volunteers (M 24, F 24, age 19–27 years, body mass
index between 18.6 and 23.3) that was divided into 3
groups of 8 male and 8 female per group. All the
mean demographic data of age, height and weight of
volunteers are summarized in Table 1. All subjects
passed complete physical examination, revealing no
clinically relevant hepatic, renal, cardiac,
hematologic, or other diseases, and all had no other
drugs two weeks before and during the study. A
standardized meal was served. Cigarettes and alcohol
were restrained. Volunteers were provided with
standard diet 3 times per day. Each group received
single oral drug A, B, or C a tablet together with 200
ml water respectively. During the whole study course,
subjects were instructed by the investigator to report
immediately the occurrence of any adverse event.
After completing the study, all subjects received
complete physical examination.
Sample collection
Blood samples were drawn predose and 20min,
40min and 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24, 36 and 48 h
postdose (6 ml each time). After centrifugation, the
plasma was isolated and stored at -20 ℃ until analysis.
Vital sign examination was taken predose and 1, 2, 4,
8, 24 and 48 h postdose.
Chromatographic analysis
132
After adding the internal standard, bisoprolol
plasma samples were adjusted to proper PH with
saturated natronite and were precipitated using
pentane-dichloromethane, 70:30(v/v), and then were
freezing at -60°C~-70°C. The water layer separated
from the clear supernatant was melting to room
temperature and injected into a integrated HPLC
system (LC1100 Agilent, Hewlett-Packard, USA)
equipped with a quaternary pump, a degasser, an
auto-sampler, an injector with a 50-μL loop, a column
oven , a UV detector (set as 271nm) and a data
system (Agilent ChemStation). As to
hydrochlorothiazide plasma samples,
hydrochlorothiazide and IS were extract by methyl
tert-butyl ether (MTBE). The extract was dried under
nitrogen and analyzed by the same HPLC system
with a fluorescence detector (setting as EX:225nm
and EM : 300nm). The data were acquired and
processed with the 3P97 software package (The
Mathematics Pharmacology Committee, Chinese
Pharmacological Society) and SAS 8.02 (Carey,
North Carolina, USA).
The assay method was validated with respect to
accuracy, precision, linearity, sensitivity, and
specificity. The standard calibration curves were
linear over the concentration range 0.5–50 ng·mL -1
for bisoprolol and 2–75 ng·mL -1 for
hydrochlorothiazide. The accuracy and precision of
this method was examined using human plasma
freshly prepared at hydrochlorothiazide
concentrations of 3, 35, 70 ng·mL -1 and at bisoprolol
concentrations of 0.7, 20, 45 ng·mL -1 , which had
coefficients of variation less than or equal to 7.2%
and 8.35%, respectively.
Pharmacokinetic analysis
Pharmacokinetic parameters were calculated by
employing standard non-compartmental analysis
(Gibaldi and Perrier, 1982) and using Practical
Pharmacokinetic Program 3P97 (Math-pharmacology
Committee, Chinese Academy of Pharmacology,
Beijing, China). The peak plasma concentration (Cmax)
and the corresponding time (Tmax) were directly
obtained from the raw data. The area under the
plasma concentration vs time curve up to the last
quantifiable time point(AUC(0−t)) was obtained by
the linear and log–linear trapezoidal summation. The
AUC(0−t) extrapolated to infinity (i.e. AUC(0−∞)) by

Ke X et al. Asian Journal of Pharmacodynamics and Pharmacikinetics 2008; 8(2):131-137
adding the quotient of Clast/Kel, where Clast represents
the last measurable time concentration and Kel
represents the apparent terminal rate constant. Kel
was calculated by the linear regression of the
log-transformed concentrations of the drug in the
133
terminal phase. The half-life (t1/2) of the terminal
elimination phase was obtained using the relationship
t1/2 = 0.693/Kel.
Table 1. Mean demographic data for subjects (n =48)
A B C
Male (8) Female (8) Male (8) Female (8) Male (8) Female (8)
Year (y) 23.0±2.3 21.4±1.7 22.1±1.0 21.1±1.7 23.6±1.7 22.9±2.1
Weight (kg) 59.6±6.5 52.7±3.5 55.8±5.3 50.0±4.8 59.0±5.4 52.2±5.6
Height (cm) 168.7±6.7 159.1±4.6 167.1±6.5 155.4±4.0 169.6±7.0 157.8±5.9
BMI 20.9±1.2 20.8±0.6 20.0±1.3 20.7±1.5 20.5±1.6 20.9±1.2
Table 2. Pharmacokinetic parameters of 48 health Chinese volunteers after oral administration
of compound hydrochlorothiazide and bisoprolol tablets (n = 48) (n=8, mean±SD)
PK parameters
bisoprolol hydrochlorothiazide
A B C A B C
AUC 0-tlast(ng·h·ml -1 ) 200.2±35.4 414.3±102.7 914.8±190.9 264.4±97.8 254.3±58.8 257.4±52.6
AUC 0-inf(ng·h·ml -1 ) 213.8±38.4 431.7±112.2 946.6±216.6 296.0±110.6 275.7±60.2 280.1±55.3
C max(ng·ml -1 ) 14.4±2.2 30.0±5.3 65.8±13.8 47.3±10.0 48.5±8.8 46.4±10.0
Tmax (h) 2.4±0.9 2.4±1.0 2.4±0.9 2.1±0.6 2.1±0.6 1.9±0.4
T 1/2(h) 10.5±2.7 9.2±1.4 9.2±1.7 5.9±1.7 5.1±1.7 5.0±1.6
Vd (L·kg -1 ) 3.2±0.8 3.1±0.7 2.6±0.4 2.6±0.5 2.4±0.7 2.8±1.0
Table 3. Pharmacokinetic parameters of bisoprolol in healthy male and female volunteers
Parameter A B C
Male Female Male Female Male Female
C max(ng·ml -1 ) 13.5±2.7 15.2±1.3 29.0±6.6 31.0±4.0 66.0±14.0 65.6±14.5
T max(h) 2.2±0.7 2.6±1.0 2.4±0.9 2.4±1.2 2.5±0.9 2.3±0.9
AUC (o-t)( ng·h·ml -1 ) 178.1±24.6 222.3±31.1 370.2±80.3 458.4±108.1 902.6±177.7 927.0±214.9
AUC (0-∞)(ng·h·ml -1 ) 191.2±28.1 236.4±34.7 385.8±88.8 477.5±119.4 932.9±206.6 960.3±239.5
T1/2(h) 10.8±2.6 10.1±3.0 9.7±1.6 8.7±0.9 9.0±1.9 9.4±1.5
Table 4. Pharmacokinetic parameters of hydrochlorothiazide in healthy male and female volunteers( n=8, mean±SD)
Parameter A B C
Male Female Male Female Male Female
Cmax(ng·ml -1 ) 41.5±8.6 53.1±8.8 43.2±5.0 53.9±8.8 40.4±8.1 52.5±8.2
T max(h) 2.0±0.7 2.3±0.7 2.3±0.6 1.9±0.6 2.1±0.4 1.8±0.4
AUC (o-t)( ng·h·ml -1 )) 215.1±52.7 313.8±116.2 225.0±46.5 283.6±57.4 238.1±56.7 276.6±43.2
AUC (0-∞)( ng·h·ml -1 )) 242.6±52.9 349.3±136.6 248.5±51.7 302.9±58.3 264.7±62.1 295.5±46.6
T1/2(h) 5.7±0.9 6.0±2.4 5.9±1.7 4.2±1.2 5.7±1.7 4.3±1.1

Ke X et al. Asian Journal of Pharmacodynamics and Pharmacikinetics 2008; 8(2):131-137
Fig 1. Individual plasma concentration-time profiles of bisoprolol (1) and hydrochlorothiazide (2) in healthy male and female
Chinese young volunteers after oral administration one tablet of bisoprolol/hydrochlorothiazide combination
(A: 2.5/6.25, B: 5/6.25, or C: 10/6.25 mg/tablet).
Fig 2. Mean plasma concentration-time profiles of bisoprolol and hydrochlorothiazide in healthy male and female Chinese
young volunteers after single oral administration one tablet of bisoprolol/hydrochlorothiazide combination
(A: 2.5/6.25, B: 5/6.25, or C: 10/6.25 mg/tablet).
Fig 3. Mean AUC 0-48, AUC 0-∞ or C max of bisoprolol versus dose after after single oral administration one tablet of
bisoprolol/hydrochlorothiazide combination (A: 2.5/6.25, B: 5/6.25, or C: 10/6.25 mg/tablet).
134

Ke X et al. Asian Journal of Pharmacodynamics and Pharmacikinetics 2008; 8(2):131-137
Fig 4. Mean plasma concentration-time profiles of bisoprolol (1) and hydrochlorothiazide (2) in healthy male and female
Chinese young volunteers after oral administration one tablet of bisoprolol/hydrochlorothiazide combination
(A: 2.5/6.25, B: 5/6.25, or C: 10/6.25 mg/tablet).
Statistical evaluation
Data are expressed as mean±SD. An unpaired
t-test with significance set at α=0.05 was used for all
parameters to compare the pharmacokinetic
parameters of bisoprolol and hydrochlorothiazide in
males and females. And an analysis of variance
(ANOVA) was used to examine a different in Tmax,
T1/2 and dose-normalized AUC0-t, AUC0-∞ and Cmax
for bisoprolol and hydrochlorothiazide across the
three doses. The p value

Ke X et al. Asian Journal of Pharmacodynamics and Pharmacikinetics 2008; 8(2):131-137
was not possible to determine definitively if the
difference was real or due to the limited number of
subjects. There was little inter-variability between
subjects when compound hydrochlorothiazide and
bisoprolol tablet was administered orally.
Mean plasma concentration-time curves in both
sexes are shown in Figure 4 and the related
pharmacokinetic variables are summarized in Table 3
and 4. There was no significant difference in all
calculated pharmacokinetic parameters between the
male and female subjects (p>0.05).
Tolerability
In the whole study, We had only found that
several adverse reactions, including headache,
dizziness, chillsz hands and sweats, were observed on
four female volunteers,half after treatment with A
and half after treatment with C.
Discussion
According the former study after oral
administration in humans, bisoprolol is rapidly
absorbed (tmax:2~3h)with an absorptivity of more
than 90% and has an absolute bioavailability of
about 88% .The first pass metabolism of bisoprolol
fumarate is about 20%. And the rate of plasma
protein binding is about 30%. Hydrochlorothiazide is
well absorbed (65%-75%) following oral
administration and Cmax are observed within 1-5
hours of dosing, ranging from 70-490 ng·ml -1
following oral doses of 12.5-100 mg. Plasma
concentrations are linearly related to the administered
dose and 35-38% of the whole blood concentration.
Hydrochlorothiazide is eliminated primarily by renal
pathways and T1/2 has been reported to be 6-15
hours [17] . The combination of bisoprolol and
hydrochlorothiazide was well tolerated, and overall
quality-of-life questionnaire scores indicated an
improvement after bisoprolol/hydrochlorothiazide
therapy. The binary mixture of bisoprolol and
hydrochlorothiazide is widely used and studied in the
treatment of hypertension but still isn’t studied and
used on Chinese. To the best of our knowledge, few
documents have been described for the
pharmacokinetics of the combination product of two
drugs on Chinese. Therefore, it is necessary to
136
conduct the pharmacokinetic study for the drug using
in Chinese. This technique can add general
knowledge on pharmacokinetics with
co-administration of compound hydrochlorothi- azide
and bisoprolol tablets in 3 dosage groups on Chinese.
Twenty-four female and twenty-four male
adolescents enrolled in the study, complied with the
protocol and completed the required follow-up visits.
All participants enrolled in the study completed the
protocol.
Through use of the bridging study, the
international harmonization for new drug
development makes extrapolation of foreign clinical
trial data to a new region possible for new drug
application. As a bridging study, this study can
provide evidence for the investigational drug using in
China. On the basis of results of pharmacokinetic
study, all the pharmacokinetic parameters were
similar to the result of clinical trial for the respective
pharmacokinetics study [16] . Therefore, the study
gives clue to the clinical use of the combination in
Chinese patiens.
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*Correspondence to:Hangkong Road 13, Department of
clinical pharmacology, Tongji Medical College of Huazhong
University of Science & Technology at Wuhan, Hubei, China.
E-mail:chenhuitjmu@163.com,Tele: 02783657947