CNS-active Drugs · Hypnotics · Psychotropics · Sedatives

Comparative Bioavailability of
Two Estazolam Tablet Formulations
in Indonesian Healthy Volunteers
Yahdiana Harahap1, Lucy Sasongko2, Budi Prasaja3, Ega Indriati3, Windy Lusthom3, Lipin3
1 Department of Pharmacy, Faculty of Mathematic and Science, University of Indonesia, Depok (Indonesia)

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2 School of Pharmacy, Bandung Institute of Technology, Bandung (Indonesia)
3 PT. Clinisindo Laboratories, Jakarta (Indonesia)

Corresponding author: Budi Prasaja, S. Si, Apt, MM, PT Clinisindo Laboratories, Jl. Ulujami Raya 12,
Jakarta 12250 (Indonesia); email: budi.prasaja@clinisindo.com

Abstract
Key words
Aim: To compare the bioavailability of 95.90 % (92.60–99.31 %) respectively, sat-
two estazolam (CAS 29975-16-4) tablet isfying the bioequivalence criteria of the j Benzodiazepines
formulations (Estalin® 2 mg tablets as European Committee for Proprietary j CAS 29975-16-4,
test formulation and 2 mg tablets of the ­Medicinal Products and the US Food and j Estalin®
originator product as reference formula- Drug Administration guidelines. j Estazolam, bioequivalence,
tion). Conclusion: These results indicate
pharmacokinetics
Methods: The study was conducted that two formulations of estazolam are
j Hypnotics
­according to an open label, randomized bioequivalent and, thus, may be pre-
j Sedatives
two-way cross-over design with a two- scribed interchangeably.
week washout period. Twenty-four sub-
jects received each of the two estazolam
Arzneimittel-Forschung
formulations. Blood samples for pharma- (Drug Research)
cokinetic profiling were taken up to 72 h 2008;58(10):501–504
after drug administration in fasting con-
dition. Plasma concentrations of estazo-
lam were determined with a validated
HPLC method with ultraviolet detection.
Pharmacokinetic parameters were calcu-
lated from observed plasma concentra-
tion-time profiles.
Results: The mean AUC0-t, AUC0-∞ and
Cmax were 2581.38 ng ∙ h/mL, 2934.37 ng ∙
h/mL and 95.25 ng/mL, respectively for
the test formulation and 2835.75 ng ∙ h/
mL, 3207.73 ng ∙ h/mL and 99.32 ng/mL,
respectively, for the reference formula-
tion. The median Tmax for both formula-
tions was 1 h. The point estimates and
90 % confidence intervals for AUC0-t,
AUC0-∞ and Cmax were 91.03 % (87.48–
94.72 %), 91.48 % (86.67–96.55 %) and

Arzneimittel-Forschung (Drug Research) 2008;58(10):501–504

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Harahap et al. – Estazolam
CNS-active Drugs · Hypnotics · Psychotropics · Sedatives
1. Introduction
Estazolam (8-chloro-6-phenyl-4H-1,2,4-triazolo [4.3-α]-
1,4-benzodiazepine, CAS 29975-16-4) is a triabenzo-
lobenzodiazepine derivative. The spectrum of pharma-
cological actions of benzodiazepines, including seda-
tive/hypnotic, anxiolytic, anticonvulsant and muscle
relaxant effects, is elicited by binding to the supramo-
lecular benzodiazepine GABA receptor complex. They
have less risk of overdose and abuse potential than bar-
biturates. Benzodiazepines increase sleep time and im-
prove sleep quality by reducing sleep-onset latency and
wakefulness after sleep onset and by increasing sleep ef-
ficiency. Benzodiazepines that have been approved by
the FDA for treating chronic insomnia include estazo-
lam, flurazepam, temazepam, quazepam and triazolam.
Rapidly acting drugs with shorter half-lives (i.e., estazo-
lam, triazolam and temazepam) are preferred. Temaze-
pam has a slower onset of action and is less effective for
initiating sleep [1, 2].
Because tolerance and dependence occur with pro-
longed use, benzodiazepines are most useful for short-
term treatment of insomnia. Using benzodiazepines for
more than four weeks increases the likelihood of de-
pendence and withdrawal phenomena. Benzodiaz-
epines withdrawal may cause anxiety, depression, nau-
sea, perceptual changes, rebound insomnia, intense
dreams, nightmares and poor memory consolidation.
Withdrawal symptoms may develop within a few hours
of discontinuing a short-acting benzodiazepine, or up
to three weeks after discontinuing a long-acting benzo-
diazepine [2].
Estazolam is classified as a short-acting benzodiaz-
epine with general properties similar to diazepam. It is
given by mouth as a hypnotic in the short-term man-
agement of insomnia usually in a dose of 1 to 2 mg at
night. Estazolam has shown that this drug is rapidly ab-
sorbed following oral administration and is rapidly dif-
fused into brain and other tissues. Peak plasma concen-
trations of estazolam are reached on average within 2 h
(0.5–6 h) of oral administration. Estazolam is extensive-
ly metabolized, mainly to 1-oxo-estazolam and 4-hy-
droxy-estazolam. These metabolites are excreted either
free or conjugated in the urine with small quantities
­detected in the feces. Only a small proportion of a dose
is excreted as unchanged drug. The mean elimination
half-life has generally been reported to be in the range
of 10 to 24 h [2–5].
This study was designed to investigate the pharma-
cokinetics and bioavailability of two estazolam tablet
formulations in order to prove bioequivalence between
both formulations.
2. Subjects, materials and methods
2.1 Subjects and study design
Twenty-four healthy adult volunteers (17 males and 7 females)
were selected among Indonesian residents and participated in
this study. The ages of the subjects were between 18 and 37
502  Harahap et al. – Estazolam
years (25.8 ± 5.2 years), the body weights were between 46.5
and 73 kg (56.2 ± 7.5 kg) and the heights were between 148 and
173 cm (162.9 ± 6.5 cm). The sample size n = 24 subjects was
based on a previous report [6]. The subjects were selected after
passing a clinical screening procedure including a physical ex-
amination, ECG and clinical laboratory tests (blood urea nitro-
gen, sGPT, sGOT, alkaline phosphatase, total bilirubin, total
protein, cholesterol, fasting glucose, albumin, hemoglobin, he-
matocrit, WBC and urine analysis). The volunteers were exclud-
ed if they had a history of any illness of the hepatic, renal and
cardiovascular system, had taken alcohol or other medications
for a prolonged period of time or had a history of benzodiaz-
epine derivative intake.
The test preparation was Estalin® (2 mg estazolam tablets,
manufacturer: PT Novell Pharmaceutical Laboratories, Jakarta,
Indonesia). The reference formulation was purchased from the
representative manufacturer.
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This study was performed at PT Clinisindo Laboratories (Ja-
karta, Indonesia) according to the Declaration of Helsinki for
biomedical research involving human subjects and the rules of
Good Clinical Practice. The protocol of this study was reviewed
by the Committee of The Medical Research Ethics of the Faculty
of Medicine University of Indonesia and was approved by the
National Agency of Drug and Food Control, Indonesia. All par-
ticipants signed a written informed consent after they had been
informed of the nature and details of the study in accordance
with Indonesian Guidelines for Bioequivalence Studies.
All subjects avoided using other drugs for at least two weeks
prior to the study and until after its completion. They also re-
frained from ingesting alcohol, caffeine, chocolate, tea or coke-
containing beverages at least 48 h before each dosing and until
the collection of the last blood sample. Each volunteer received
an oral dose of 2 mg of estazolam according to a standard two-
way crossover randomized design. The dose was taken with
240 ml of tap water. There was a two-week washout period be-
tween the doses. The subjects were asked to fast from 10 h be-
fore until 4 h after drug administration.
Serial venous blood samples (10 mL) were collected pre-
dose and at 1, 1.5, 2, 2.5, 3, 5, 8, 12, 24, 36, 48 and 72 h after the
administration in heparinized tubes. After blood separation,
plasma was frozen at –20 °C until drug analysis.
2.2. Analytical method
The concentrations of estazolam in plasma were analyzed by a
validated HPLC method with ultraviolet detector following the
GLP rules. The mobile phase was acetonitrile:disodium hydro-
gen phosphate 5 mmol/L, pH 6.0 (35:65) pumped isocratically
at 1 mL/min through a Purospher® STAR RP-18, 5 μm, 250 mm
x 4.6 mm i.d column (Merck, Darmstadt, Germany) maintained
to 40 °C. The wavelength was set at 254 nm. Briefly, a 20 µL ali-
quot of internal standard (alprazolam in methanol) was added
to a 1.5 mL aliquot of plasma sample. After mixing, to the mix-
ture was added 2 mL of water, 2 mL of NaOH 0.1 N and 8 mL of
diethyleter. The mixture was vortexed for 5 min and centrifuged
for 15 min at 3500 rpm. After centrifugation, the organic phase
was removed and evaporated to dryness under vacuum at 60 °C.
The residue was reconstituted with 150 µL of the mobile phase
and removed to the HPLC vial. The assay was linear over the
concentration range of 1–250 ng/mL.
2.3 Pharmacokinetic and statistical analysis
The bioequivalence was studied using the primary parameters,
AUC0-t, AUC0-∞ and Cmax. The maximum plasma concentrations
(Cmax) and time to reach maximum plasma concentrations
Arzneimittel-Forschung (Drug Research) 2008;58(10):501–504
 CNS-active Drugs · Hypnotics · Psychotropics · Sedatives

120.00

100.00

Concentration (ng/mL)
80.00
Test

Reference
60.00

40.00

20.00

0.00
0 10 20 30 40 50 60 70 80
Time (h)

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Fig. 1: Plasma concentration-time curves of estazolam after oral administration of two different formulations containing 2 mg
of estazolam. Data are shown as mean ± SD from 24 subjects. The vertical bars represent the standard deviation of the mean.

(tmax) were obtained directly by inspection of the individual marized in Table 1 which shows the geometric mean
drug plasma concentration time data, and were used as mea- values and the range for the AUC0-t, AUC0-∞, Cmax, and
sures of the absorption rate. The area under the plasma con- t½ values obtained for each formulation. The pharma-
centration-time curve up to the last time (t) showing a measur- cokinetic characteristic tmax is presented as mean (±
able concentration (Ct) of the analyte (AUC0-30h) was calculated
SD).
using the trapezoidal rule. The elimination rate constant (Kel)
The results of the bioequivalence analysis are given
was calculated by the technique of least-squares regression
in Table 2. The parametric 90 % confidence intervals for
from the data of the last 5 to 8 points of each plasma concentra-
tion-time curve. The AUC0-∞ value was computed using the the T/R ratio ranged from 87.48–94.72 (point estimate
equation 91.03) for AUC0-t, 86.67–96.55 (point estimate 91.48) for
AUC0-∞, 92.60-99.31 (point estimate 95.90) for Cmax and
AUC0-∞ = AUC0-t + *Ct/Kel
93.05–107.06 (point estimate 99.81) for t1/2, respective-
where *Ct is the estimated last plasma concentration. The ap-
ly.
parent elimination half-life (t½) of estazolam in plasma was de-
The intra-individual variations of AUC0-t, AUC0-∞,
termined by using the following equation
Cmax and t1/2 estimated from the coefficients of varia-
t1/2 = (ln2)/Kel
tion as determined by ANOVA were 8.0 %, 10.86 %, 7.0 %
For the parameters of AUC0-t, AUC0-∞, and Cmax, a multiplicative and 14.14 %, respectively.
model was assumed, and analysis of variance (ANOVA) was ap-
plied using the respective ln-transformed data. For estimation
of bioequivalence the 90 % confidence of intervals of the geo-
Table 1: Mean pharmacokinetic characteristics for estazolam
metric mean ratio test/reference for AUC0-t, AUC0-∞, and Cmax
after administration of the two formulations to 24 subjects.
were calculated assuming a multiplicative model. The accepted
bioequivalence range for these parameters was 80 % to 125 %. Test Reference
Parameter
formulation formulation
All statistical analyses were performed using EquivTest 2.0 sta-
tistical program (Statistical Solutions, Cork, Ireland). AUC0-t (ng ∙ h/mL)
Geometric mean 2581.38 2835.75
Range 1830.08–3759.74 1988.32–4179.97
AUC0-∞ (ng ∙ h/mL)
3. Results Geometric mean 2934.37 3207.73
Range 1958.09–4299.59 2181.25–4999.83
All 24 volunteers successfully completed the trial ac-
cording to the protocol. Both estazolam formulations Cmax (ng/mL)
Geometric mean 95.25 99.32
were well tolerated at the administered dose and no seri- Range 68.06–130.92 67.78–141.63
ous adverse clinical events were observed. The mean es-
t½ (h)
tazolam concentrations versus time profiles for both for- Geometric mean 22 22
mulations are shown in Fig. 1. Range 15–32 15–37
The pharmacokinetic parameters used to assess the tmax (h)
bioequivalence of the test formulation versus the refer- Mean 1.73 1.94
ence were AUC0-t, AUC0-∞ for the extent of the absorp- + SD 1.34 1.86
tion and Cmax and tmax for the rate of absorption. De- AUCt-∞ /AUC0-∞
scriptive statistics of the pharmacokinetic parameter Mean 11.9 11.5
+ SD   5.1   5.1
for estazolam test and reference preparations are sum-

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Harahap et al. – Estazolam   503
CNS-active Drugs · Hypnotics · Psychotropics · Sedatives

Table 2: Statistical evaluation of the comparison of AUC0-t, suggest a sample size of 12 subjects as a minimal size
AUC0-∞, Cmax and t½ of two formulations administered to 24 that would be necessary in order to conclude bioequiv-
subjects.
alence with a power of 80 % at the 5 % nominal level
Parameter AUC0-t AUC0-∞ Cmax t½
[8].
T/R point estimate 91.03 91.48 95.90   99.81 In conclusion, the two estazolam formulations are
90 % CI equivalent with respect to the rate and extent of absorp-
Lower limit 87.48 86.67 92.60   93.05
tion and they can be assumed to be therapeutically
Upper limit 94.72 96.55 99.31 107.06
CV ANOVA (%)   8.0 10.86   7.0   14.14 equivalent and exchangeable in clinical practice.

4. Discussion
The aim of this study was to demonstrate the compara-
ble bioavailability of two estazolam tablet formulations.
We found that the 90 % CI for AUC0-t, AUC0-∞ and Cmax
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504  Harahap et al. – Estazolam