Study on the Pharmacokinetics of Fasudil, a selective Rho kinase ...

Zhang SJ et al. Asian Journal of Pharmacodynamics and Pharmacokinetics 2009; 9(3):221-226
Asian Journal of
Pharmacodynamics and
Pharmacokinetics
ISSN 1608-2281
Copyright by Hong Kong Medical Publisher
Publisher Homepage: www.hktmc.com
<strong>Study</strong> on the Pharmacokinetics of Fasudil, a selective Rho kinase
inhibitor
Shi-Jun Zhang, Jing Gao, Ying Huang, and Wei-Ren Xu
Research Center for New Drug Evaluation, Tianjin Institute of Pharmaceutical Research
Tianjin 300193, China
Abstract
Key words
Aim To study the pharmacokinetics of Fasudil. Design and Method The HPLC methods with
UV detection were used for measurement of Fasudil concentration in biological samples,
respectively. Results After intravenous dose at 3, 6 and 12 mg•kg -1 of Fasudil in rats, C-T
data were fitted to tow-compartment models of intravenous, the pharmacokinetic parameters
of Fasudil were as follows: the serum concentration at time 0 (C 0 ) were 3.301.05, 5.10
and 18.82 µ • -1 , and the areas under the serum concentration-time
curves(AUC) were 52.54and 325.79µ•• -1 , the
terminal half- life (t 1/2 ) were 24.9±4.5, 41.1±27.5 and 41.3±21.1 min, respectively. After
a single dose of 6 mg•kg -1 Fasudil intravenous in rats, the drug concentration were observed
higher in lung, kidney, spleen and intestine at 5 and 20 min after dosing. The drug
concentration levels decreased at 60min after dosing in most tissues. After a single dose of 6
mg•kg -1 Fasudil intravenous in rats, the excretion of Fasudil in urine, feces and bile amounted
to 41.1%, 6.4% and 38.4% of dose, respectively. The plasma protein binding ratio of Fasudil
with rat serum was 53.2Conclusion The HPLC methods used in this study is
specific, sensitive, precise and accurate, completely meeting the requirements of
pharmacokinetic study of Fasudil. Fasudil is distributed extracellularly and very quickly from
central to peripheral compartment and eliminates from the body rapidly; the pharmacokinetic
behavior of the drug in rats complies with linear kinetics.
Fasudil; high performance liquid chromatography; Pharmacokinetics; rat;
Article history Received 12 November 2008; Accepted 24 March 2009
Publication data Pages: 7; Tables:3; Figures:3; References: 15; Paper ID 1608-2281-2009-09030221-06
*Corresponding author Mrs. Shi-Jun Zhang, Research Center for New Drug Evaluation, Tianjin Institute of
Pharmaceutical Research, Tianjin 300193, China. E-mail: zsj1121@163.com
Introduction
Fasudil hexahydro-1-(5-isoquinolinesu- lfonyl)-
1H-1,4-diazeoine), (Fig 1), a selective Rho-kinase
inhibitor which attenuates many vasoconstrictions
[1-3] , increases hemodynamic function [4-6] , and
regulates important features of inflammatory
reactions [7-9] . Fasudil is well- tolerated [10] . An
intravenous form of fasudil has been approved in
Japan since 1995, and used in tens of thousands of
patients with vascular spasms in the brain.
In China, Fasudil has been under preclinical
phase of research and showed powerful effects in
animals. The present study is designed to investigate
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Zhang SJ et al. Asian Journal of Pharmacodynamics and Pharmacokinetics 2009; 9(3):221-226
the pharmacokinetic properties of Fasudil in rats in
order to provide experiment basis for its development
as a new drug.
O
O
S
N
N
NH ·HCl
Fig 1. Chemical structure of Fasudil
Materials and methods
Chemicals and reagents
Fasudil was prepared by the chemical department
of our institute, batch number: 970301. acetonitrile,
HPLC grade, was produced by Tianjin Chemical
Reagent Factory. Other chemicals used were of
analytical grade. Distilled water, prepared from
demineralized water, was used throughout the study.
Animals
Male Wistar rats (weighing 150~200g, Grade I,
Certificate No 001) were produced by the animal
department of our institute.
Apparatus and analytical condition for assay
of Fasudil
The high performance liquid chromatographic
(HPLC) analyses were used. It consisted of Waters
510 pump, 710B injector, Shimadzu SPD-6A
UV-visible detector and a ODS column
(250mm×4.6mm I.D., 5µm Particle size). The mobile
phase was water- acetonitrile- ammonia (100:100:1,
v/v) at flow rate of 1.0ml•min -1 . The UV detection
was at 215nm.
Extraction procedure for assay of Fasudil
Biological samples (serum, tissue homogenate,
urine, feces or bile) 0.5mL and 0.5mL trichloroactic
acid were mixed for 20 seconds and centrifuged at
15000r•min -1 for 5min and 20µL of the upper layer
were injected into the liquid chromatograph.
Pharmacokinetic study
According to the requisition on pharmacokinetic
study design [11,12] , three doses of 3, 6 and 12mg•kg -1
in rats were used for pharmacokinetic studies. The
drug was dissolved with 9‰ sodium chloride
solution for injection. The animals were fasted for
15h and the blood samples were collected via eye
vein at 0, 5, 10, 20, 40, 60 and 120min after
intravenous in rats. After centrifugation, the serum
was used for determination of Fasudil.
Distribution
The brain, heart, lung, liver, kidney, spleen,
stomach, intestinal, muscle, fat, testis and uterus
were taken from rats and prepared for tissue
homogenate at 5, 20 and 60min after intravenous of
6mg•kg -1 .
Excretion
Urine and feces excretion 6 of male rats were
placed into metabolic cages, respectively. The urine
and feces were collected before injection and at 0~2,
2~4, 4~8, 8~12, 12~24, 24~36, and 36~48h after
dosing Fasudil of 6 mg•kg -1 . The drug contents in
urine and feces (treated with water) were assayed
with the designed methods, respectively.
Bile excretion Rats were anaesthetized with
uratan and fixed on a stage. A bile fistula was
performed from the common bile duct with a thin
plastic tube to collect bile. The bile was collected
before dosing and at 0~2, 2~6, 6~8, 8~12 and
12~24h after injection Fasudil of 6 mg•kg -1 .
Plasma protein binding
Tree concentration of Fasudil (2.5, 5 and
10µg•mL -1 were used for the plasma protein binding
test using a filtering method.
Pharmacokinetic analysis
Using the 3P97 Program (version 1997)
developed by Mathematical Pharmacological
Committee, Chinese Pharmacological Society, the
compartment model of plasma concentration – time
curves was fitted and the pharmacokinetic parameters
including K (first-order rate constant), t 1/2β (half-life),
V d (apparent volume of distribution), CL (total body
clearance), AUC (area under drug plasma
concentration-time curve) were estimated.
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Zhang SJ et al. Asian Journal of Pharmacodynamics and Pharmacokinetics 2009; 9(3):221-226
Statistical analysis
Data are presented as arithmetic mean ±
standard deviation (SD).
Results
Analytical method validation
According to the requisition for
pharmacokinetic studies, the analytical method was
validated. [13-15]
Chromatographic behavior
In the conditions as described above, the
retention time of Fasudile was about 5 min (Fig1).
The endogenous substances in serum did not
interfered separation.
A B C D
Fig 2. Chromatograms of Fasudil
A. Standard containing Fasudil (F: Fasudil) B. Drug-free serum
C. Serum spiked with Fasudil D. Serum 5min after intravenous of Fasudil 6mg•kg -1 in rat.
Calibration curves
The linearity was evaluated by constructing a
calibration curve in the various biological media. The
linearity was tested by linear regression of the peak
areas (Y) versus the concentration of the drug (X).
The calibration curve of Fasudil in rat serum
(concentration at the ranges of 0-10g•mL -1 )was
Y=0.1862+0.00005634X (r=0.9996).
The calibration curves of Fasudil in rat tissue
homogenate were
for heart Y=0.23+0.00008X (r=0.999),
for liver Y=0.23+0.00009X (r=0.997),
for kidney Y=-0.353+0.00008X (r=0.999),
for brain Y=0.315+0.00008X (r=0.999),
for muscle Y=0.194+0.00008X (r=0.999).
The calibration curves of Fasudil in rat urine,
feces and bile were
for urine Y=0.489+0.0001X (r=0.999),
for feces Y=1.219+0.0001X (r=0.994),
for bile Y=0.31+0.001X (r=0.999).
The limit of detection was 0.2g•mL -1 , the
recovery were 80.9%, 86.0% and 88.2%, the relative
standard deviation (RSD) were 2.8%, 3.3% and 3.6%
for within-day assay and 6.7%, 5.3% and 5.2% for
between-day at Fasudil concentrations of 1, 2 and
5g•mL -1 , respectively.
Pharmacokinetic study
The concentration-time data of Fasudil after
intravenous of three doses in rats were shown in
Table 1 and Fig 2. The results indicated that the
plasma concentration after intravenous administration
decayed biexponentially with time with an extremely
rapid initial phase and followed by a relatively slow
elimination phase. The two-compartment model for
Fasudil was further confirmed by Wss, r, R 2 , AIC and
F test. The main pharmacokinetic parameters were
shown in Table 2 after intravenous Fasudil in rats.
There were good correlation between C 0 , AUC and
doses.
Sensitivity, recovery and precision
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Zhang SJ et al. Asian Journal of Pharmacodynamics and Pharmacokinetics 2009; 9(3):221-226
Table 1. Comparison of Fasudil serum concentrations after intravenous of Fasudil
3, 6 and 12 mg•kg -1 in rats (n=5, meanSD)
Time Serum concentration(g•mL -1 )
(min) 3 mg•kg -1 6 mg•kg -1 12 mg•kg -1
0
5
10
20
40
60
120
Fig2. Mean serum concentration-time curves of Fasudil after single intravenous
doses of Fasudil 3, 6 and 12 mg•kg -1 in rats
Table 2 The main pharmacokinetic parameters of Fasudil after intravenous Fasudil
3, 6 and 12mg•kg -1 in rats
Parameters 3 mg•kg -1 6 mg•kg -1 12 mg•kg -1
C 0 (g•mL -1 ) 3.30±1.05 5.10±0.96 18.82±5.91
T 1/2 24.9±4.5 41.1±27.5 41.3±21.1
V (C) (mL) 0.94±0.26 1.17±0.24 0.72±0.20
CL (s) (mL•min) 0.052±0.009 0.061±0.027 0.037±0.008
AUC (0~Tn) (g•min •mL -1 ) 52.5±8.0 102.9±43.0 325.8±73.0
MRT (min) 15.8±2.6 23.5±9.8 26.3±6.0
Tissue distribution
The drug could be widely distributed into tissues
after intravenous 6 mg•kg -1 in rats (Table 3). As
shown in Fig 3, the concentrations of Fasudil were
observed in lung, kidney, spleen and intestine higher
at 5 and 20min after dosing.
Excretion
Urine and feces excretion
41.1%, 6.4% of administered dose were
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Zhang SJ et al. Asian Journal of Pharmacodynamics and Pharmacokinetics 2009; 9(3):221-226
excreted The results in the urine and the feces
indicated that Fasudil in the urine and could
distribute widely and rapidly to tissues and excreted
gradually from tissues.
As shown in Fig 3, the concentrations of Fasudil
eces during 0~48 h after intravenous dosing of
Fasudil 6 mg•kg -1 in rats. The results indicated that
the drug was mainly excreted via urine.
Bile excretion 38.4% of administered dose was
excreted in the bile during 0~24 h after intravenous
dosing of Fasudil 6 mg•kg -1 in rats.
in most tissues were higher at 5 and 20min, lower at
60min after dosing. The results indicated that Fasudil
could distribute widely and rapidly to tissues and
excreted gradually from tissues.
Protein Binding
At the concentration of Fasudil 2.5, 5 and
10µg•mL -1 , the plasma protein binding ratios were
55.2±5.8%, 54.7±1.3% and 49.6±3.3%, respectively.
Table 3 Tissue distribution of Fasudil
Tissue
Drug concentrations (µg•g-1)
5min 20min 60min
Heart 2.80±1.80 4.16±2.42 2.00±1.61
Liver 4.44±3.60 2.59±1.29 2.91±3.26
Spleen 8.40±3.16 11.73±9.41 3.85±3.42
Lung 18.92±11.72 10.93±13.33 2.62±2.14
Kidney 12.56±11.84 9.91±3.10 5.68±5.80
Brain 3.80±2.29 2.45±0.74 2.20±1.02
Stomach 4.41±3.94 3.85±1.95 1.54±1.52
Intestine 6.21±5.05 5.29±1.22 1.76±1.71
Muscle 4.06±1.40 2.99±2.39 1.91±1.41
Fat 2.49±0.94 2.09±1.05 1.61±0.53
Uterus 1.17±0.81 2.10±0.87 0.60±0.00
Testis 4.82±6.16 1.51±0.46 1.45±0.33
Serum 3.22±1.01 1.39±0.45 0.46±0.32
hear
lung
kidney
brain
fat
testicle
liver
spleen
uterus
stomach
intestine
muscle
Discussion and Conclusion
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Zhang SJ et al. Asian Journal of Pharmacodynamics and Pharmacokinetics 2009; 9(3):221-226
In this study pharmacokinetics in rats of
Fasudil was investigated. The results showed that the
assay method utilized by us is very sensitive, high
specific, precise so as to completely meet the
requirements of Fasudil pharmacokinetic study [13-15] .
The data shows that fasudil is distributed
extracellularly and transferred very quickly from
central to peripheral compartment and eliminates
from the body rapidly; the pharmacokinetic behavior
of the drug in rats complies with linear kinetics.
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