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Clinical Therapeutics/Volume 29, Number 6, 2007

Brief Report

Bioavailability of Two Oral Suspension and Two Oral

Tablet Formulations of Acyclovir 400 mg: Two Single-Dose,
Open-Label, Randomized, Two-Period Crossover
Comparisons in Healthy Mexican Adult Subjects
Jose Antonio Palma-Aguirre, PhD1; Jose Antonio Absaln-Reyes, MS1;
Germn Novoa-Heckel, MD1; Alberto de Lago, MD2; Ivn Oliva, MS2;
Zulema Rodrguez, BS2; Mario Gonzlez-de la Parra, PhD2; Victoria Burke-Fraga, BS2;
and Salvador Namur, MS2
1Centro

de Estudios Cientficos y Clnicos Pharma, S.A. de C.V., Mexico City, Mexico; and 2Fundacin
Liomont A.C., Mexico City, Mexico
ABSTRACT
Background: Acyclovir is an important antiviral
drug, used extensively for treatment of herpes simplex
and varicella zoster. Six oral generic formulations of
acyclovir are available in Mexico; however, a literature search failed to identify data information concerning the bioavailability of these formulations in the
Mexican population.
Objective: The aim of these 2 studies was to compare the bioavailability of 4 oral formulations of acyclovir 400 mg2 tablet formulations and 2 suspension
formulationswith their corresponding listed drug
references in Mexico (a list issued by Mexican Health
Authorities).
Methods: Two separate, single-dose, open-label, randomized, 2-period crossover studies were conducted
at the Centro de Estudios Cientficos y Clnicos
Pharma, S.A. de C.V. (clinical unit), Mexico City,
Mexico. For each study, a different set of eligible subjects were selected. They included healthy Mexican
volunteers of either sex. For each study, subjects were
randomly assigned to receive 1 test formulation of
acyclovir 400 mg followed by the reference formulation, or vice versa, with a 1-week washout period between doses. After a 12-hour (overnight) fast, subjects
received a single 400-mg dose (tablet or 10-mL suspension) of the corresponding formulation. For the
analysis of pharmacokinetic properties, including
Cmax, AUC from time 0 (baseline) to time t (AUC0t ),
and AUC from baseline to infinity (AUC0 ), blood
1146

samples were drawn at baseline, 0.25, 0.5, 0.75, 1.0,

1.25, 1.5, 1.75, 2, 3, 4, 6, 8, 12, and 24 hours after
dosing. The formulations were considered bioequivalent if the natural logarithm (ln)-transformed ratios
of Cmax and AUC were within the predetermined
equivalence range of 80% to 125% and if P 0.05 for
the 90% CIs. Tolerability was assessed by monitoring vital signs, laboratory analysis results, and subject
interviews.
Results: Twenty-six subjects were enrolled in the
study for the suspension dosage form and 25 completed it (13 men, 12 women; mean age, 22.2 years).
Subjects in the suspension-dosage form study had the
following characteristics: age range, 18 to 48 years;
weight range, 46 to 86 kg; and height range, 145 to
179 cm. Thirteen subjects received the suspension-test
formulation first. Twenty-four subjects were enrolled
in the study for the tablet dosage form; all of them
concluded the study (13 men, 11 women; mean age,
24.7 years). Subjects had the following characteristics for
the tablet-dosage form study: age range, 18 to 49 years;
weight range, 46 to 84 kg; and height range, 147 to
185 cm. Twelve subjects received the tablet-test formulation first. No period or sequence effect was observed in

Accepted for publication March 29, 2007.

doi:10.1016/j.clinthera.2007.06.007
0149-2918/$32.00
Printed in the USA. Reproduction in whole or part is not permitted.
Copyright 2007 Excerpta Medica, Inc.

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either study. For the suspension dosage form, the 90%
CIs for the corresponding ratios of ln Cmax, ln AUC0t,
and ln AUC0 were 106.55 to 116.11, 99.29 to
113.19, and 98.37 to 112.04, respectively (all, P <
0.05). For the tablet dosage form, the 90% CIs for the
corresponding ratios of ln Cmax, ln AUC0t, and ln
AUC0 were 83.46 to 103.64, 86.16 to 111.05, and
87.77 to 109.99, respectively (all, P < 0.05).
Conclusions: In these 2 studies in healthy subjects,
single, 400-mg doses of the test brand of acyclovir administered either in tablet or suspension form, appeared to be bioequivalent to the reference brand
based on the rate and extent of absorption in accordance with the definition of the US Food and Drug
Administration. All formulations were well tolerated.
(Clin Ther. 2007;29:11461152) Copyright 2007
Excerpta Medica, Inc.
Key words: acyclovir, bioequivalence, bioavailability, pharmacokinetic, human, HPLC, fluorescence, suspension, tablets.

Zovirax tablet (GlaxoSmithKline, Research Triangle

Park, North Carolina; reference formulation). Cicloferon suspension (Laboratorios Liomont S.A. de C.V.,
Mexico City, Mexico; test formulation) versus Zovirax
suspension (GlaxoSmithKline Mexico, S.A. de C.V.,
Mexico City, Mexico; reference formulation).

INTRODUCTION

Study Design

Acyclovir (9-[2-hydroxyethoxy]-methylguanine) is a
nucleoside analog with antiviral activity for the treatment of herpes simplex viruses, varicella zoster virus,
Epstein-Barr virus, cytomegalovirus, and human herpes virus 6 (HHV-6).1,2 Acyclovir exerts its antiviral activity by acting as a substrate that inhibits viral DNA
polymerase.14 Because of the efficacy and tolerability
of this drug, it is widely used in Mexico.
The bioavailability of oral acyclovir is from 10% to
30% and decreases with increasing administration
frequency. The Tmax and t1/2 for a single, 400-mg oral
dose of acyclovir are 2.4 hours and 1.5 to 2 hours,
respectively.2
Although several oral generic formulations of acyclovir are available in Mexico, a MEDLINE search
(19912006; key terms: bioequivalence, bioavailability, Mexico, and acyclovir) failed to identify data concerning the bioavailability of each formulation in the
Mexican population. Thus, the aim of these studies
was to compare the bioavailability of 4 oral formulations of acyclovir 400 mg (2 tablet formulations and
2 suspension formulations) with their corresponding
listed drug references in Mexico. In each study, 2 formulations were compared to determine bioavailability: Cicloferon tablet (Laboratorios Liomont S.A. de
C.V., Mexico City, Mexico; test formulation) versus

The 2 protocols were approved by the ethics and research committees of the Centro de Estudios Cientficos
y Clnicos Pharma, S.A. de C.V. The studies were carried out in accordance with the principles of the
Declaration of Helsinki and its amendments5 and the
International Conference on Harmonisation Guideline
for Good Clinical Practice.6 A 2 2 crossover, openlabel, randomized design was used in both studies.
The subjects for each study arrived at the clinical
unit the day before the beginning of the study and
were randomized in a 1:1 ratio using a table of random numbers to receive Cicloferon followed by
Zovirax, or vice versa, with a 1-week washout period
between doses. After subjects were fasted for 12 hours
overnight, blood was drawn for baseline plasma measurements in the following manner: a 20-G catheter
(Jelco Plus, Medex Medical Ltd., Ascot, United
Kingdom) was placed in a suitable forearm vein and a
6-mL blood sample was drawn into a heparinized vacuum tube (Vacutainer , Becton, Dickinson and
Company, Franklin Lakes, New Jersey). Subjects received a single 400-mg tablet or 10 mL of suspension
(whichever was applicable in the corresponding study)
of the study medication, given with 250 mL of water
(either tablet or suspension formulation), and additional blood samples were drawn at 0.25, 0.5, 0.75, 1.0,

June 2007

SUBJECTS AND METHODS

Subjects
For each study, a different set of eligible healthy
subjects of either sex were selected, based on information from a database of the Centro de Estudios
Cientficos y Clnicos Pharma, S.A. de C.V. (clinical
unit), Mexico City, Mexico.
Healthy subjects aged 18 to 55 years were included
based on their medical history, clinical examination
results, and routine laboratory tests results. Subjects
with HIV or hepatitis B virus were excluded. All eligible subjects provided written informed consent for
participation in the corresponding study. Subjects
were compensated for participation.

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Clinical Therapeutics
1.25, 1.5, 1.75, 2, 3, 4, 6, 8, 12, and 24 hours after
study drug administration. Plasma was obtained by
centrifugation (2000g for 10 minutes at room temperature) and stored frozen at 70C until analyzed using
high-performance liquid chromatography (HPLC). After
the washout period, subjects returned to the clinical
unit where the alternative formulation was administered and samples were drawn and analyzed as before.

Determination of Plasma Acyclovir

Concentrations
In both studies, acyclovir plasma levels were determined using HPLC as adapted by personnel of
Fundacin Liomont A.C., Mexico City, Mexico, in
which acetaminophen was used as internal standard,
based on the methods of Testereci et al.7 Briefly,
0.2 mL of plasma, 0.1 mL of internal standard (acetaminophen, 200 g/mL), and 0.1 mL of 7% perchloric
acid were mixed by shaking in a test tube for
1 minute. The tube was then centrifuged at 1440g for
10 minutes at room temperature. The supernatant
was filtered through a regenerated cellulose syringe
filter (pore size, 0.45 micron) and injected into the
chromatographic system. The separation of compounds
was performed in a 15-cm 4.6-mm inside diameter
column of 5 m particle size, Zorbax SB-C8 (Agilent,
Palo Alto, California), and eluted with a mobile phase
consisting of 0.02 M perchloric acid (pH 2.0 0.1).
The column temperature was 25C. Flow rate was
maintained constant at 1.5 mL/min, and detection
was carried out using a fluorescence detector at 260
and 375 nm of excitation and emission wavelength,
respectively. The analytical equipment used included
an HPLC device with a fluorescence detector (model
1100, Agilent).
Under these conditions, the method was linear in
the range of 35 to 1500 ng/mL, accuracy was between
90% and 109%, and the relative standard deviation
of the method was always lower than 7%. This adapted method was considered suitable by the study investigators for pharmacokinetic studies of acyclovir.

Tolerability
Tolerability was assessed by monitoring vital signs
(blood pressure, heart rate, body temperature) at
baseline, 3.5, 11.5, and 23.5 hours, and at the end of
each period, laboratory analysis results, and subject
interviews regarding the potential presence of adverse
events (AEs) during the study.
1148

Pharmacokinetic and Statistical Analyses

Using a power analysis ( = 0.2), it was determined
that the power of the analysis of variance (ANOVA) was
>0.8 at a 90% CI, indicating that a total of 24 to 25 subjects would be sufficient for the purposes of each study.
Individual plasma concentrationtime curves were
constructed; Cmax and Tmax were directly obtained from
these curves. AUC from time 0 (baseline) to 24 hours
(AUC024) was calculated using the trapezoidal rule.8,9
From the terminal log-decay phase, elimination rate
constant (ke) was estimated using linear regression,
and t1/2 was estimated using the following equation10:
t1/2 = ln2/ke
where ln was defined as the natural log; and extrapolation of AUC from baseline to infinity (AUC0) was
calculated as follows:
AUC0 = AUC024 + (C24/ke)
where C24 was defined as concentration at 24 hours.
To compare the bioavailability (in accordance with
the criteria for bioequivalence [the rate and extent to
which the active ingredient or active moiety is absorbed from a drug product and becomes available at
the site of action] established by the US Food and Drug
Administration, in vivo bioequivalence guidelines)9 of
the formulations tested, an ANOVA for a 2 2 crossover design for ln-transformed Cmax, AUC from baseline to time t (AUC0t), and AUC0 was carried out for
each study. Ratios of ln Cmax, ln AUC0t, and ln
AUC0 for all formulations were calculated, and 90%
CIs were obtained; ANOVA was performed using the
F test. Probability of exceeding the limits of acceptance
(80%125%) was obtained by the two 1-sided t tests
described by Schuirmann.11 The formulations were
considered bioequivalent if the ln-transformed ratios of
Cmax and AUC were within the predetermined equivalence range of 80% to 125% and if P was 0.05 for
the 90% CIs.8,9 All pharmacokinetic and statistical
analyses were performed using WinNonlin version 5
(Pharsight, Mountain View, California).

RESULTS
A total of 26 subjects were enrolled in the study for the
suspension dosage form. One subject was withdrawn
because he or she did not comply with the dietary requirements established in the corresponding protocol,
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so analyses were performed using data from 25 subjects (13 men, 12 women; mean age, 22.2 years).
Thirteen subjects received the reference formulation
first. A total of 24 subjects (13 men, 11 women; mean
age, 24.7 years) were enrolled in the study for the
tablet dosage form; all of them concluded the study.
Subjects had the following characteristics for the suspension study: age range, 18 to 48 years; weight
range, 46 to 86 kg; and height range, 145 to 179 cm.
Subjects had the following characteristics for the
tablet study: age range, 18 to 49 years; weight range,
46 to 84 kg; and height range, 147 to 185 cm.
Figures 1 and 2 show mean plasma acyclovir concentrations after administration of the 2 formulations.

Pharmacokinetic Parameters
For the suspension study, mean and SD values of
Cmax, Tmax, t1/2, AUC0t, and AUC0 for each formulation are shown in Table I. No period or sequence
effects were observed for the pharmacokinetic properties in the ANOVA.

For the tablet study, mean and SD values of Cmax,

Tmax, t1/2, AUC0t, and AUC0 for each formulation
are shown in Table II. No period or sequence effects
were observed for the pharmacokinetic properties in
the ANOVA. In addition, there was no evidence of
weight-related differences in individual AUC values
and Cmax values.
Table III shows the 90% CIs of the ratios (test/
reference) for the ln-transformed values of Cmax (as an
index of rate of absorption), AUC0t, and AUC0 (as
an index of the extent of absorption); the probability
of exceeding the limits of acceptance (Schuirmanns
two 1-sided t tests); and the power of the test8,9,1113
for acyclovir suspension. The 90% CIs for the corresponding ratios of Cmax, AUC0t, and AUC0 were
within the 80% to 125% range. All P values were
<0.05. Similar results were found for data without a
logarithmic transformation.
Similar data results were observed for acyclovir
tablets (Table IV). The 90% CIs for the corresponding
ratios of Cmax, AUC0t, and AUC0 also were within

Mean (SD) Plasma Acyclovir Concentration

(ng/mL)

Test formulation
Reference formulation

800
700
600
500
400
300
200
100
0
0

10

12

Time After Study Drug Administration (h)

Figure 1. Mean (SD) plasma concentrations of 2 oral formulations of acyclovir 400-mg suspension (Cicloferon,
Laboratorios Liomont, S.A. de C.V., Mexico City, Mexico, test formulation; Zovirax, GlaxoSmithKline
Mexico, S.A. de C.V., Mexico City, Mexico, reference formulation) over 12 hours in healthy adult Mexican
subjects (N = 25).

June 2007

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Clinical Therapeutics

Mean (SD) Plasma Acyclovir Concentration

(ng/mL)

Test formulation
Reference formulation

1000
900
800
700
600
500
400
300
200
100
0
0

10

12

Time After Study Drug Administration (h)

Figure 2. Mean (SD) plasma concentrations of 2 oral formulations of acyclovir 400-mg tablet (Cicloferon,
Laboratorios Liomont, S.A. de C.V., Mexico City, Mexico, test formulation; Zovirax, GlaxoSmithKline,
Research Triangle Park, North Carolina, reference formulation) over 12 hours in healthy adult Mexican
subjects (N = 24).

Table I. Pharmacokinetic properties of 2 oral formulations of single-dose acyclovir 400 mg

(suspension) in healthy subjects (N = 25).* Values are mean (SD).
Property
Cmax, ng/mL
Tmax, h
t1/2, h
AUC0t, ng/mL h
AUC0, ng/mL h

Cicloferon

Zovirax

670.5 (165.1)
1.2 (0.2)
3.8 (1.7)
2649.4 (609.4)
3171.4 (734.0)

599.2 (123.7)
1.4 (0.5)
3.9 (1.2)
2508.4 (609.5)
3015.7 (662.3)

AUC0t = AUC from time 0 (baseline) to time t; AUC0 = AUC from baseline to infinity.
*No significant between-treatment differences were found.
Trademark of Laboratorios Liomont, S.A. de C.V., Mexico City, Mexico (test formulation).
Trademark of GlaxoSmithKline Mexico, S.A. de C.V., Mexico City, Mexico (reference formulation).

80% to 125% range. All P values were <0.05. Again,

similar results were found for data without a logarithmic transformation.

Tolerability
In these single-dose studies, no AEs were reported
and lab abnormalities were not found.
1150

DISCUSSION
Estimated Cmax values for the 2 formulations of the
tablet dosage form were 779 ng/mL (test formulation)
and 800 ng/mL (reference formulation). This was
consistent with respect to those reported by Vergin
et al14 in their crossover study in which they also
compared 2 acyclovir 400-mg tablet formulations in
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J.A. Palma-Aguirre et al.

Table II. Pharmacokinetic properties of 2 oral formulations of single-dose acyclovir

400 mg (tablets) in healthy subjects (N = 24).* Values are mean (SD).
Property
Cmax, ng/mL
Tmax, h
t1/2, h
AUC0t, ng/mL h
AUC0, ng/mL h

Cicloferon

Zovirax

681.1 (281.7)
1.6 (0.8)
2.7 (0.7)
2515.1 (994.7)
2893.2 (1012.6)

704.4 (238.3)
1.4 (0.7)
2.6 (0.6)
2503.3 (978.0)
2874.2 (971.5)

AUC0t = AUC from time 0 (baseline) to time t; AUC0 = AUC from baseline to infinity.
*No significant between-treatment differences were found.
Trademark of Laboratorios Liomont, S.A. de C.V., Mexico City, Mexico (test formulation).
Trademark of GlaxoSmithKline, Research Triangle Park, North Carolina (reference formulation).

Table III. Comparison of 90% CIs of natural log (ln)-transformed Cmax, ln AUC0t, and
ln AUC0, the probability of exceeding the limits of acceptance, and power test
in acyclovir 400 mg (suspension).

Parameter
In Cmax % ratio
In AUC0t % ratio
In AUC0 % ratio

Probability of Exceeding
Limits of Acceptance

Ratio,
% Reference

90% CI

<80%

>125%

Power

111.23
106.01
104.98

106.55116.11
99.29113.19
98.37112.04

<0.001
<0.001
<0.001

<0.001
<0.001
<0.001

1.0000
0.9998
0.9998

AUC0t = AUC from time 0 (baseline) to time t; AUC0 = AUC from baseline to infinity.

Table IV. Comparison of 90% CIs of natural log (ln)-transformed Cmax, ln AUC0t, and ln
AUC0, the probability of exceeding the limits of acceptance, and power test in
acyclovir 400 mg (tablets).

Parameter
In Cmax % ratio
In AUC0t % ratio
In AUC0 % ratio

Probability of Exceeding
Limits of Acceptance

Ratio,
% Reference

90% CI

<80%

>125%

Power

93.01
97.82
98.25

83.46103.64
86.16111.05
87.77109.99

0.013
<0.001
<0.001

<0.001
0.002
<0.001

0.9590
0.8972
0.9463

AUC0t = AUC from time 0 (baseline) to time t; AUC0 = AUC from baseline to infinity.

24 subjects. In the study reported here, mean Cmax

values were 681 and 704 ng/mL for Cicloferon and
Zovirax, respectively. In addition, the mean Tmax
values in this study were 1.6 hours for Cicloferon
and 1.4 hours for Zovirax, whereas the mean Tmax
June 2007

values reported by Vergin et al were 1.5 hours, for

both reference and test formulations. For the suspension study we did not find a comparable reference
study for the comparison of the pharmacokinetic
parameters.
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The results of these 2 studies, obtained from 2 samples of healthy Mexican volunteers, might serve as a
reference for future controlled studies of acyclovir in
the Hispanic population.

6.

CONCLUSIONS
In these 2 studies in healthy Mexican subjects, a single,
400-mg dose of the test formulation of acyclovir
(Cicloferon), administered either in tablet or suspension
form, appeared to be bioequivalent to the reference formulation (Zovirax) based on the rate and extent of absorption in accordance with the definition found in the
US Food and Drug Administrations in vivo bioequivalence guidances. All formulations were well tolerated.

7.

8.

9.

ACKNOWLEDGMENTS
These 2 studies were supported in their entirety by
Laboratorios Liomont, S.A. de C.V., Mexico City,
Mexico. The authors would like to thank Patricia
Andrew (Universidad Nacional Autonoma de
Mexico, FES Acatlan, Mexico) for her help with the
English style and grammar of the manuscript.

10.

11.

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Address correspondence to: Salvador Namur, MS, Privada Jess del Monte
No. 77, Col. Cuajimalpa, 05000 Mexico City, Mexico. E-mail: snamur@
liomont.com.mx
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