Pharmacokinetics of chlorzoxazone in rats with diabetes: Induction of CYP2E1 on 6-hydroxychlorzoxazone formation

Article abstract of DOI:10.1002/jps.20698

Pharmacokinetic parameters of chlorzoxazone (CZX) and its main metabolite, 6-hydroxychlorzoxazone (OH-CZX), were compared after intravenous (20 mg/kg) and oral (50 mg/kg) administration of CZX in rat model of diabetes induced by alloxan (DMIA) or streptozotocin (DMIS), and their respective control rats. In both rat models of diabetes, the expression and mRNA level of CYP2E1 increased, and CZX was metabolized to OH-CZX via CYP2E1 in rats. Hence, it could be expected that formation of OH-CZX increased in both rat models of diabetes. As expected, after intravenous (80.5% and 74.4% increase in rat models of DMIA and DMIS, respectively) and oral (55.6% and 70.5% increase, respectively) administration of CZX, the AUC of OH-CZX was significantly greater than their respective control rats. Since, CZX is an intermediate hepatic extraction ratio drug, the greater AUC values of OH-CZX (the significantly faster CL_NR of CZX) in both rat models of diabetes could be supported by significantly faster CL_int for the formation of OH-CZX (75.9% and 129% increase for rat models of DMIA and DMIS, respectively) and significantly greater free fractions of CZX in plasma (51.9% and 58.9% increase, respectively). Also it was reported that hepatic blood flow rate was faster in male Wister rat model of DMIS.

Full text of DOI:10.1002/jps.20698

Pharmacokinetics of Chlorzoxazone in Rats with Diabetes:
Induction of CYP2E1 on 6-Hydroxychlorzoxazone Formation
1
2
2
1
HYE W. BAEK, SOO K. BAE, MYUNG G. LEE, YOUNG T. SOHN
1
College of Pharmacy, Duksung Women’s University, Seoul, South Korea
2
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, South Korea
Received 4 January 2006; revised 5 April 2006; accepted 18 April 2006
Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20698
ABSTRACT: Pharmacokinetic parameters of chlorzoxazone (CZX) and its main
metabolite, 6-hydroxychlorzoxazone (OH-CZX), were compared after intravenous
(
20 mg/kg) and oral (50 mg/kg) administration of CZX in rat model of diabetes induced
by alloxan (DMIA) or streptozotocin (DMIS), and their respective control rats. In both rat
models of diabetes, the expression and mRNA level of CYP2E1 increased, and CZX was
metabolized to OH-CZX via CYP2E1 in rats. Hence, it could be expected that formation of
OH-CZX increased in both rat models of diabetes. As expected, after intravenous (80.5%
and 74.4% increase in rat models of DMIA and DMIS, respectively) and oral (55.6%
and 70.5% increase, respectively) administration of CZX, the AUC of OH-CZX was
significantly greater than their respective control rats. Since, CZX is an intermediate
hepatic extraction ratio drug, the greater AUC values of OH-CZX (the significantly faster
CLNR of CZX) in both rat models of diabetes could be supported by significantly faster
CLint for the formation of OH-CZX (75.9% and 129% increase for rat models of DMIA and
DMIS, respectively) and significantly greater free fractions of CZX in plasma (51.9% and
58.9% increase, respectively). Also it was reported that hepatic blood flow rate was faster
in male Wister rat model of DMIS. ß 2006 Wiley-Liss, Inc. and the American Pharmacists
Association J Pharm Sci 95:2452–2462, 2006
Keywords: CZX and OH-CZX; rat model of diabetes induced by alloxan (DMIA) or
streptozotocin (DMIS); pharmacokinetics; CYP2E1; rats; disease state; CYP enzymes;
metabolism; intrinsic clearance; protein binding
2
4–8
INTRODUCTION
Chlorzoxazone [5-chloro-2(3H)-benzoxazolone;
P450 (CYP) 2E1 in humans and rats.
The use
of OH-CZX as a chemical probe has been sug-
gested to assess the activity of CYP2E1 in vitro
and in vivo owing to the good correlation between
the formation rate of OH-CZX and CYP2E1
activity in humans and rats. The formation of
OH-CZX via CYP2E1 in male Sprague–Dawley
rats was proven in rats with protein–calorie
malnutrition (4 weeks fed on 5% casein diet),
water deprivation for 72 h (our unpublished
data), and acute renal failure induced by uranyl
CZX], once used as a skeletal muscle relaxant
1
for the treatment of painful muscle spasms,
9
4
primarily undergoes hydroxylation to form
6-hydroxychlorzoxazone (OH-CZX). OH-CZX is
then rapidly glucuronidated and excreted in
2
,3
5
urine. The formation of OH-CZX is catalyzed
mainly by the hepatic microsomal cytochrome
6
–8
nitrate.
In rats with diabetes mellitus induced by
alloxan (rat model of DMIA) or streptozotocin
Correspondence to: Young T. Sohn (Telephone: þ82-2-901-
8
385; Fax: þ82-2-901-8386; E-mail: ytsohn@duksung.ac.kr)
(rat model of DMIS), some physiological changes,
Journal of Pharmaceutical Sciences, Vol. 95, 2452–2462 (2006)
ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association
for example, decrease in bile flow rate and altered
2452
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 11, NOVEMBER 2006

INDUCTION OF CYP2E1 ON OH-CZX FORMATION IN DIABETIC RATS
11
2453
1
0
bile compositions,
kidney function,
hepatotoxicity,
impaired
Korea). The rats were randomly divided into four
1
2,13
and disorders of the gastro-
groups; rat models of DMIA and DMIS, and their
respective control rats. All rats were provided
with food (Sam Yang Company, Seoul, South
Korea) and water ad libitum and maintained in a
light-controlled room (light: 0700–1900, dark:
1900–0700) kept at a temperature of 22 ꢀ 28C
and a relative humidity of 55 ꢀ 5% (Animal
Center for Pharmaceutical Research, College of
Pharmacy, Seoul National University, Seoul,
South Korea). Rats were housed in metabolic
cages (Tecniplast, Varese, Italy) under the supply
of filtered pathogen-free air. The protocol of this
study was approved by the Animal Center and
Use Committee of the College of Pharmacy, Seoul
National University.
intestinal tract and reductions in protein
binding of drugs due to elevated plasma fatty acid
12
level and/or glycosylation of plasma proteins
have been reported. Glucuronidation and
sulfation were also profoundly affected in rat
1
4
15
model of DMIS. Recently, it was reported that
the expressions and mRNA levels of CYP1A2, 2B1/
2
, 2E1, and 3A1(23) increased in both male
Sprague–Dawley rat models of DMIA and
DMIS as compared to their respective controls.
However, the expression and mRNA level of
CYP2C11 decreased in both rat models of
diabetes as compared to their respective con-
1
5
trols.
Hence, it could be expected that the
formation of OH-CZX increases in both rat
models of diabetes, since OH-CZX was formed via
Induction of Diabetes Mellitus in Rats
by Alloxan or Streptozotocin Injection
4
–8
CYP2E1 in rats
and the expression and mRNA
level of CYP2E1 increased in both rat models of
1
5
diabetes.
Freshly prepared alloxan (40 mg/kg) was admi-
nistered (total injection volume of ꢁ0.25 mL) in
the overnight-fasted rats via the tail vein for
The purpose of this paper is to report
the increase in the formation of OH-CZX after
intravenous and oral administration of CZX in
both male Sprague–Dawley rat models of DMIA
and DMIS due to increase in the expression and
mRNA level of CYP2E1 in both rat models of
15,16
two consecutive days.
An equal volume of
0.9% NaCl-injectable solution was injected in
controls. Freshly prepared streptozotocin (45 mg/
kg) was administered (total injection volume
of ꢁ0.3 mL) once in the overnight-fasted rats via
1
5
diabetes.
1
3,15
the tail vein.
An equal volume of a citrate
buffer of pH 4.5 was injected in controls. On the
fourth day after the first alloxan administration
EXPERIMENTAL
Chemicals
(rat model of DMIA) or 0.9% NaCl-injectable
solution (control for rat model of DMIA), and on
the seventh day after intravenous administration
of streptozotocin (rat model of DMIS) or a citrate
buffer (control for rat model of DMIS), blood
glucose levels were measured using the Medi-
sense Optium Kit (Abbott Laboratories, Bedford,
MA) and rats with blood glucose levels higher
than 250 mg/dL were chosen as rat models of
diabetes mellitus.
CZX, OH-CZX, reduced form of b-nicotinamide
adenine dinucleotide phosphate (NADPH; as a
tetrasodium salt), tris(hydroxymethyl)amino-
methane (Tris)-buffer, ethylenediamine tetraace-
tatic acid (EDTA), 3-aminophenyl sulfone (an
internal standard of high-performance liquid
chromatographic, HPLC, assay for CZX and OH-
CZX), b-glucuronidase (Type H-1; from Helixa
pomatia), alloxan, and streptozotocin were pro-
ducts from Sigma-Aldrich Corporation (St. Louis,
MO). Ketamine hydrochloride was a product from
the Yuhan Corporation (Seoul, South Korea).
Other chemicals were of reagent grade or HPLC
grade.
Measurement of Vmax, K , and CL for the
m
int
Formation of OH-CZX in Hepatic Microsomes
The procedures were similar to the reported
6–8
methods.
The livers of rat models of DMIA
and DMIS, and their control rats (n ¼ 5; each)
were homogenized (Ultra-Turrax T25; Janke and
Kunkel, IKA-Labortechnik, Staufeni, Germany)
in an ice-cold buffer of 0.154 M KCl/50 mM
Tris-HCl in 1 mM EDTA, pH 7.4. The homogenate
was centrifuged at 9000g for 30 min and the
supernatant fraction was further centrifuged at
Animals
Male Sprague–Dawley rats (weighing 220–250 g)
of 7-week-old were purchased from the Charles
River Company Korea (Orient, Seoul, South
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 11, NOVEMBER 2006

2454
BAEK ET AL.
100000g for 90 min. The microsomal pellet
Intravenous Study
was resuspended in a buffer of 0.154 M KCl/50
mM Tris-HCl in 1 mM EDTA, pH 7.4. Protein
content was measured using the reported
CZX (dissolved in a minimum amount of 10 N
NaOH) at a dose of 20 mg/kg was infused (total
infusion volume of ꢁ0.6 mL) over 1-min via the
jugular vein of each group of rats (n ¼ 9, 9, 8, and 7
for rat model of DMIA and their controls, and rat
model of DMIS and their controls, respectively).
An approximately 0.12-mL aliquot of blood sam-
ple was collected via the carotid artery at 0 (to
serve as a control), 1 (at the end of the infusion), 5,
1
7
method.
The Vmax (the maximum velocity)
and Km (the Michaelis–Menten constant; the
concentration at which the rate is one-half
of Vmax
)
for the formation of OH-CZX
were determined after incubating the above
microsomal fraction (equivalent to 0.2 mg pro-
tein), a 10-mL aliquot of CZX (dissolved in a
minimum amount of 10 N NaOH to have
substrate concentrations of 2.5, 5, 10, 20, 50,
1
5, 30, 45, 60, 90, 120, and 180 min after
intravenous administration of CZX. Blood sam-
ples were centrifuged immediately and a 50-mL
aliquot of each plasma sample was stored in a
ꢂ708C freezer (Revco ULT 1490 D-N-S; Western
Mednics, Asheville, NC) until the HPLC analysis
100, and 200 mM), and a 50-mL (1 mM) aliquot of
NADPH in a final volume of 1 mL with 0.1 M
phosphate buffer, pH 7.4, in a water-bath shaker
kept at 378C and at a rate of 50 oscillations per
minute (opm). All of the above microsomal
incubation conditions were linear. The reaction
was terminated by the addition of a 1-mL aliquot
of ethylether after 20-min incubation. The OH-
CZX formed was measured by the HPLC
1
8
of CZX and OH-CZX. The heparinized 0.9%
NaCl-injectable solution (20 U/mL), 0.3 mL, was
used to flush the cannula immediately after each
blood sampling to prevent blood clotting. At the
end of experiment (24 h), each metabolic cage
was rinsed with 20 mL of distilled water and
the rinsings were combined with 24-h urine
sample. After measuring the exact volume of 24-
h urine output and combined urine sample, two
1
8
method. The kinetic constants (Km and Vmax
)
for the formation of OH-CZX were calculated
1
9
using the nonlinear regression method. The
intrinsic clearance for the formation of OH-CZX
5
0-mL aliquots of the combined urine sample were
(CLint) was calculated by dividing the respective
stored in a ꢂ708C freezer until the HPLC analysis
Vmax by the respective Km.
18
of CZX and OH-CZX. At the same time (24 h), as
much blood as possible was collected via the
carotid artery and each rat was sacrificed by
cervical dislocation. And then, the entire gastro-
intestinal tract (including its contents and feces)
was removed, transferred into a beaker contain-
ing 50 mL of 0.1 N NaOH (to facilitate the
extraction of CZX and OH-CZX) and cut into
small pieces using scissors. After stirring with a
glass rod for 1-min, a 50-mL aliquot of the
supernatant were collected from each beaker
and stored in a ꢂ708C freezer until the HPLC
Pretreatment of Rats
In the early morning one day before the com-
mencement of experiment, the jugular vein (for
drug administration for intravenous administra-
tion only) and the carotid artery (for blood
sampling) of each rat were cannulated with
polyethylene tube (Clay Adams, Parsippany, NJ)
while each rat was under ketamine anesthesia
[ketamine hydrochloride at a dose of 100 mg/kg
was administered (total injection volume of 1 mL/
kg) intramuscularly]. Both cannulas were exter-
iorized to the dorsal side of the neck where
each cannula terminated with long Silastic tube
1
8
analysis of CZX and OH-CZX.
Oral Study
(
Dow Corning, Midland, MI). Both Silastic
The CZX (the same solution that was used in the
intravenous study) at a dose of 50 mg/kg was
administered orally (total oral volume of ꢁ1.0 mL)
using a feeding tube in each group of rats (n ¼ 12,
9, 8, and 8 for rat model of DMIA and their
controls, and rat model of DMIS and their
controls, respectively). Blood samples were col-
lected at 0, 5, 15, 30, 45, 60, 90, 120, 180, 240, 360,
and 480 min after oral administration of CZX.
Other procedures were similar to those of the
intravenous study.
tubes were inserted into a wire sheath to allow
free movement of the rat. Each rat was
housed individually in a rat metabolic cage
(Daejong Scientific Company, Seoul, South
Korea). Ketamine was employed instead of ether
to minimize the effect of CYP2E1 on the formation
of OH-CZX; it was reported that ether anesthesia
alone increased the expression of CYP2E1 by 40%
as determined by p-nitrophenol hydroxylase
2
0
activity.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 11, NOVEMBER 2006
DOI 10.1002/jps

INDUCTION OF CYP2E1 ON OH-CZX FORMATION IN DIABETIC RATS
2455
Plasma Protein Binding of CZX using
an Equilibrium Dialysis Technique
coefficients of variation of the assay (within- and
between-day) were below 8.2%.
Plasma protein binding of CZX in additional rat
models of DMIA and DMIS, and their respective
control rats (n ¼ 5; each) was measured using an
Pharmacokinetic Analysis
The total area under the plasma concentration–
time curve from time zero to time infinity (AUC)
was calculated by the trapezoidal rule-extrapola-
tion method; this method uses the logarithmic
trapezoidal rule for the calculation of the area
2
1
equilibrium dialysis technique. One milliliter of
plasma was dialyzed against 1 mL of isotonic
Sørensen phosphate buffer (pH ¼ 7.4) containing
3
1
% (w/v) dextran to minimize volume shift with a
-mL dialysis cell (Fisher Scientific, Fair Lawn,
22
during the declining plasma-level phase and the
linear trapezoidal rule for the rising plasma-level
phase. The area from the last datum point to time
infinity was estimated by dividing the last
measured plasma concentration by the terminal
rate constant.
NJ) and Spectra/Por 4 membrane (mol. wt. cutoff
of 12000–14000; Spectrum Medical Industries,
Inc., Los Angeles, CA). The spiked dialysis cell
was incubated for 24 h in a water-bath shaker
kept at 378C and at a rate of 50 opm. After a 24-h
incubation, two 50-mL aliquots were removed from
each compartment and stored in a ꢂ708C freezer
2
3
Standard methods were used to calculate the
time-averaged total body (CL), renal (CL ), and
1
8
R
until the HPLC analysis of CZX. The initial
concentration of CZX spiked into the plasma
compartment was 10 mg/mL.
nonrenal (CLNR) clearances, terminal half-life,
total area under the first moment of the plasma
concentration–time curve from time zero to time
infinity (AUMC), mean residence time (MRT),
apparent volume of distribution at steady state
HPLC Analysis of CZX and OH-CZX
(
V ), and extent of absolute oral bioavailability
ss
2
4
The concentrations of CZX and OH-CZX in the
above biological samples were analyzed by the
(F). The peak plasma concentration (Cmax) and
time to reach a Cmax (Tmax) were read directly from
the experimental data.
1
8
reported HPLC method.
Briefly, a 0.1-mL
aliquot of 0.2 M sodium acetate buffer (pH 4.75)
and a 0.1-mL aliquot of isotonic Sørensen phos-
phate buffer (pH 7.4) containing 200 U of b-
glucuronidase were added to a 0.05-mL aliquot of
the biological sample. The mixture was manually
mixed and incubated in a water-bath shaker kept
at 378C and at a rate of 50 opm for 2 h. After
incubation, a 0.05-mL aliquot of methanol con-
taining 10 mg/mL of 3-aminophenyl sulfone (an
internal standard) was added, vortex-mixed,
and then a 1-mL aliquot of diethyl ether was
added. The mixture was shaken for 10 min and
centrifuged at 16000g for 10 min. The upper
organic layer was transferred to a clean tube and
evaporated at 378C under a gentle stream of
nitrogen gas. The residue was reconstituted
with a 0.1-mL aliquot of the mobile phase and a
The harmonic mean method was employed to
25
calculate the mean values of V , terminal half-
ss
2
6
27
life, and each clearance.
Statistical Analysis
A p-value of less than 0.05 was considered to be
statistically significant using an unpaired t-test.
All results are expressed as mean ꢀ SD.
RESULTS
Measurement of Vmax, K , and CL for the
m
int
Formation of OH-CZX in Hepatic Microsomes
The Vmax, K , and CL for the formation of OH-
m
int
0
.05-mL aliquot was injected directly onto the
CZX in hepatic microsomal fractions of all rats
studied are listed in Table 1. In rat models of
DMIA and DMIS, the Vmax for the formation of
OH-CZX was significantly faster (94.0% and 157%
increase, respectively) than controls, suggesting
that the maximum velocity for the formation of
OH-CZX was faster than controls. However, the
Km was not significantly different among three
groups of rats, suggesting that the affinity of CZX
to the enzyme(s) (mainly to CYP2E1) was not
reversed-phase (C ) HPLC column. The mobile
1
8
phase, 0.1 M ammonium acetate: acetonitrile:
tetrahydrofuran (72: 22: 5.5; v/v/v) was run at a
flow rate of 1.0 mL/min. An ultraviolet detector
set at 283 nm monitored the column effluent.
The retention times of OH-CZX, 3-aminophenyl
sulfone, and CZX were ꢁ6, 10, and 18 min,
respectively. The detection limits of CZX and
OH-CZX in plasma were both 0.1 mg/mL. The
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 11, NOVEMBER 2006

2456
BAEK ET AL.
Table 1.
m
Vmax, K , and CLint Values for the Formation of OH-CZX by Microsomes
a
Prepared from Livers of Control Rats and Rat Models of DMIA and DMIS
Parameter
Control (n ¼ 5)
DMIA (n ¼ 5)
DMIS (n ¼ 5)
b
V
K
max (nmol/min/mg protein)
(mM)
1.49 ꢀ 0.115
2.89 ꢀ 0.988
3.83 ꢀ 0.877
m
87.7 ꢀ 19.0
93.6 ꢀ 26.3
101 ꢀ 45.4
b
CLint (mL/min/mg protein) 0.0170 ꢀ 0.00206
0.0299 ꢀ 0.00348 0.0389 ꢀ 0.00641
V
max, maximum velocity; K
m
, Michaelis–Menten constant; CLint, intrinsic clearance; OH-CZX,
6
-hydroxychlorzoxazone; DMIA, diabetes mellitus induced by alloxan; DMIS, diabetes mellitus
induced by streptozotocin.
a
Values expressed as mean ꢀ SD.
b
Control was significantly different (p < 0.05) from DMIA and DMIS.
changed considerably in diabetes. As a result, the
intrinsic clearance for the formation of OH-CZX
87.7% increase, respectively), and CLNR (69.4%
and 86.7% increase, respectively) were signifi-
cantly faster, and terminal half-life (29.7% and
38.3% decrease, respectively) and MRT (18.4%
and 49.9% decrease, respectively) were signifi-
cantly shorter than their respective controls. The
percentages of intravenous dose of CZX excreted
in 24-h urine (Ae0–24 h) and recovered from
the gastrointestinal tract at 24 h (GI24 h) as
unchanged drug were almost negligible in all rats
studied.
(CLint) was significantly faster in both rat models
of DMIA and DMIS (75.9% and 129% increase,
respectively), suggesting that the formation of
OH-CZX increased in both rat models of diabetes
due to increase in the expression and mRNA
1
5
levels of CYP2E1. There were no differences in
protein contents in the liver microsomes among
all groups of rats.
After the intravenous administration of CZX,
the formation of OH-CZX was rapid; the OH-CZX
was detected in plasma from the first blood
sampling time (1 min) and Tmax was also rapid;
13.6–21.7 min for all rats studied. In both rat
models of diabetes, the changes in the pharmaco-
kinetic parameters of OH-CZX are as follows: the
AUC was significantly greater (80.5% and 74.4%
increase for rat models of DMIA and DMIS,
respectively), Cmax was significantly higher
(84.2% and 65.2% increase, respectively), and
Ae0–24 h (110% and 99.1% increase, respectively)
and GI24 h (62.1% and 160% increase, respectively)
were significantly greater than their respective
controls.
Plasma Protein Binding of CZX
The plasma protein-binding values of CZX in rat
model of DMIA and their control rats were
4
8.6 ꢀ 4.16% and 64.5 ꢀ 4.39%, respectively; they
were significantly different (p ¼ 0.0014). The
corresponding values in rat model of DMIS
and their control rats were 38.1 ꢀ 10.0% and
6
2.7 ꢀ 5.66%; they were also significantly differ-
ent (p ¼ 0.000366). The decrease in plasma pro-
tein binding of CZX in both rat models of diabetes
could be due to elevated plasma fatty acid
level and/or glycosylation of plasma proteins in
1
2
diabetes.
Pharmacokinetics of CZX and OH-CZX after the
Intravenous Administration of CZX in Rats
Pharmacokinetics of CZX and OH-CZX After the
Oral Administration of CZX in Rats
After the intravenous administration of CZX at a
dose of 20 mg/kg in rat models of DMIA and
DMIS, and their respective controls, the mean
arterial plasma concentration–time profiles of
CZX and OH-CZX are shown in Figure 1 and
some relevant pharmacokinetic parameters are
listed in Table 2. In both rat models of diabetes,
the changes in the pharmacokinetic parameters of
CZX are as follows: the AUC was significantly
smaller (40.7% and 46.3% decrease, for rat models
of DMIA and DMIS, respectively), CL (69.0% and
After the oral administration of CZX at a dose of
50 mg/kg in rat models of DMIA and DMIS, and
their respective control rats, the mean arterial
plasma concentration–time profiles of CZX
and OH-CZX are shown in Figure 2 and some
relevant pharmacokinetic parameters are listed
in Table 3. After the oral administration of CZX,
the absorption of CZX was rapid; the CZX was
detected in plasma from the first blood sampling
time (5 min) and Tmax was also rapid; the values
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 11, NOVEMBER 2006
DOI 10.1002/jps

INDUCTION OF CYP2E1 ON OH-CZX FORMATION IN DIABETIC RATS
2457
Figure 1. Mean arterial plasma concentration–time profiles of CZX (A) and OH-CZX
(
B) after 1-min intravenous infusion of CZX at a dose of 20 mg/kg in male rat model of
DMIA (*; n ¼ 9) and their control rats (*; n ¼ 8) (left panel), and in rat model of DMIS (*;
n ¼ 9) and their control rats (*; n ¼ 8) (right panel). Vertical bars represent standard
deviation.
were 6.25–16.7 min for all groups of rats. In
both rat models of diabetes, the differences in
the pharmacokinetic parameters of CZX are as
follows: the AUC was significantly smaller (44.5%
and 37.2% decrease for rat models of DMIA and
DMIS, respectively) and Cmax was significantly
lower (38.4% and 33.4% decrease, respectively)
than their respective controls. In rat model of
DMIA, the CLR was significantly slower (87.0%
decrease) than their control rats.
respectively), Ae0–24 h was significantly greater
(137% and 164% increase, respectively), and Cmax
was significantly higher (56.6% and 67.7%
increase, respectively) than those of their respec-
28
tive controls. Li and Zhang also reported that
after the oral administration of CZX at a dose of 50
mg/kg in male Sprague–Dawley rat model of
DMIA, the AUC of CZX was significantly smaller
(34% decrease) than control rats. The OH-CZX was
28
not measured in the study.
After the oral administration of CZX, the
formation of OH-CZX was also rapid; the OH-
CZX was detected in plasma from the first blood
sampling time (5 min) and Tmax was also rapid;
DISCUSSION
2
0.0–26.3 min for all rats studied. In both rat
models of diabetes, the differences in the pharma-
cokinetic parameters of OH-CZX are as follows:
the AUC was significantly greater (55.6% and
Induction of diabetes mellitus by injection of
alloxan or streptozotocin was evident based on
the significantly higher blood glucose level,
significantly lager 24-h urine output, and signifi-
cant decrease in body weight gain than those of
their respective controls (Tabs. 2 and 3).
7
0.5% increase for rat models of DMIA and
DMIS, respectively), terminal half-lives were
significantly longer (23.7% and 20.7% increase,
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 11, NOVEMBER 2006

2458
BAEK ET AL.
Table 2. Pharmacokinetic Parameters of CZX and its Main Metabolite, OH-CZX, after the Intravenous
a
Administration of CZX at a Dose of 20 mg/kg in Rat Models of DMIA and DMIS, and Their Respective Control Rats
DMIA Control
Parameter
(n ¼ 9)
DMIA (n ¼ 9)
DMIS Control (n ¼ 7)
DMIS (n ¼ 8)
Initial body weight (g)
Final body weight (g)
Blood glucose (mg/dL)
Urine volume (mL/24 h)
CZX
241 ꢀ 7.41
289 ꢀ 12.1
119 ꢀ 8.04
28.3 ꢀ 4.63
246 ꢀ 7.26
251 ꢀ 1.89
301 ꢀ 18.8
125 ꢀ 10.8
21.4 ꢀ 4.35
248 ꢀ 11.7
243 ꢀ 15.0***
473 ꢀ 36.9***
41.2 ꢀ 11.5**
221 ꢀ 15.9***
451 ꢀ 53.5***
42.6 ꢀ 6.70***
AUC (mg ꢃ min/mL)
Terminal half-life (min)
MRT (min)
CL (mL/min/kg)
R
CL (mL/min/kg)
1770 ꢀ 148
26.9 ꢀ 5.63
1050 ꢀ 241***
18.9 ꢀ 4.07**
23.1 ꢀ 4.89*
1880 ꢀ 378
30.3 ꢀ 5.63
31.4 ꢀ 6.98
10.6 ꢀ 2.20
0.136 ꢀ 0.0710
1010 ꢀ 117***
18.7 ꢀ 2.49***
17.3 ꢀ 4.44***
19.9 ꢀ 2.54***
0.209 ꢀ 0.212
28.3 ꢀ 4.58
11.3 ꢀ 0.980
0.0671 ꢀ 0.203
19.1 ꢀ 4.71***
0.0934 ꢀ 0.24-
0
.246
CLNR (mL/min/kg)
ss (mL/kg)
Ae0–24 h (% of CZX dose)
GI24 h (% of CZX dose)
11.1 ꢀ 1.02
314 ꢀ 56.4
18.8 ꢀ 4.68***
10.5 ꢀ 2.19
329 ꢀ 56.4
19.6 ꢀ 2.51***
326 ꢀ 101
V
431 ꢀ 105**
1.69 ꢀ 1.44
0.172 ꢀ 0.0245
1.35 ꢀ 1.03
1.48 ꢀ 0.678
0.148 ꢀ 0.0812
1.27 ꢀ 0.513
0.230 ꢀ 0.0975
0.134 ꢀ 0.0587
OH-CZX
AUC (mg ꢃ min/mL)
615 ꢀ 138
28.2 ꢀ 7.64
9.72 ꢀ 2.50
18.3 ꢀ 6.61
30.2 ꢀ 8.64
0.975 ꢀ 0.412
1110 ꢀ 317***
30.2 ꢀ 9.19
665 ꢀ 123
35.0 ꢀ 3.82
11.2 ꢀ 2.63
13.6 ꢀ 11.8
31.9 ꢀ 5.56
0.254 ꢀ 0.112
1160 ꢀ 311
Terminal half-life (min)
39.1 ꢀ 4.60
C
T
max (mg/mL)
max (min)
17.9 ꢀ 5.28***
21.7 ꢀ 7.91
18.5 ꢀ 4.76**
18.8 ꢀ 6.94
Ae0–24 h (% of CZX dose)
GI24 h (% of CZX dose)
63.5 ꢀ 19.9***
1.58 ꢀ 1.13**
63.5 ꢀ 12.0***
0.660 ꢀ 0.126***
CZX, chlorzoxazone; OH-CZX, 6-hydroxychlorzoxazone; DMIA, diabetes mellitus induced by alloxan; DMIS, diabetes mellitus
induced by streptozotocin; AUC, total area under the plasma concentration–time curve from time zero to time infinity; MRT, mean
R
residence time; CL, time-averaged total body clearance; CL , time-averaged renal clearance; CLNR, time-averaged nonenal clearance;
Vss, apparent volume of distribution at steady state; Ae0–24 h, total amount excreted in 24-h urine; GI24 h, total amount recovered from
the gastrointestinal tract (including its contents and feces) at 24 h; Cmax, peak plasma concentration, Tmax; time to reach a Cmax
.
a
Values expressed as mean ꢀ SD.
*p < 0.05 compared with their respective control.
**p < 0.01 compared with their respective control.
***p < 0.001 compared with their respective control.
The contribution of CLR to CL of CZX
after the intravenous administration of CZX
was almost negligible; the Ae0–24 h was less than
faster CL of CZX than their respective control
rats (Tab. 2). The faster CL of CZX was due to
significantly faster CLNR of CZX than their
respective control rats since the CLR values of
CZX were comparable between both rat models of
diabetes and their respective controls (Tab. 2).
After the intravenous administration of CZX, the
AUC of OH-CZX in rat models of DMIA and DMIS
was significantly greater than their respective
controls (Tab. 2). This was expected since OH-CZX
1.69% of the intravenous dose for all rats
studied (Tab. 2), indicating that almost all of
the intravenously administered CZX was elimi-
nated via nonrenal route (CLNR). The contribution
of gastrointestinal tract (including biliary) excre-
tion of CZX to CLNR of CZX was also almost
negligible; the GI24 h was less than 0.230% of the
intravenous dose for all rats studied (Tab. 2). The
above data indicated that the CLNR of CZX listed in
Table 2 could represent the metabolic clearance
of CZX. Hence, the changes of CLNR of CZX
could represent the changes in metabolism of
CZX in rats.
4
–8
was formed via CYP2E1 in rats
and the
expression and mRNA level of CYP2E1 increased
1
5
in rat models of diabetes. Chlorzoxazone is an
intermediate hepatic extraction ratio drug (the
8
estimated hepatic first-pass effect of ꢁ53.6% ) in
2
9
rats. Therefore, the hepatic clearance of CZX
depends on the hepatic blood flow rate, intrinsic
clearance (CLint), and free (unbound to plasma
After the intravenous administration of CZX,
the significantly smaller AUC of CZX in both rat
models of diabetes could be due to significantly
2
9
proteins) fractions of CZX in plasma.
The
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 11, NOVEMBER 2006
DOI 10.1002/jps

INDUCTION OF CYP2E1 ON OH-CZX FORMATION IN DIABETIC RATS
2459
Figure 2. Mean arterial plasma concentration–time profiles of CZX (A) and OH-CZX
(
B) after the oral administration of CZX at a dose of 50 mg/kg in rat model of
DMIA (*; n ¼ 12) and their control rats (*; n ¼ 9) (left panel), and in rat model of DMIS
(
*; n ¼ 8) and their control rats (*; n ¼ 8) (right panel). Vertical bars represent standard
deviation.
significantly greater AUC of OH-CZX (the signifi-
cantly faster CLNR of CZX) in rat models of
DMIA and DMIS could be supported by signifi-
cantly faster in vitro CLint for the formation of
OH-CZX and significantly greater free fractions of
CZX in plasma than their respective control
rats; the CLint was significantly faster (Tab. 1)
and free fractions of CZX in plasma were signifi-
cantly greater (44.8% and 66.0% increase for rat
models of DMIA and DMIS, respectively) than
fractions in plasma was 0.189, 0.182, 0.365,
and 0.338 mL/min/kg for rat model of DMIA
and their control rats, and rat model of
DMIS and their control rats, respectively, based
on the CLR (Tab. 2) and plasma protein
binding values of CZX as mentioned earlier.
The 0.182–0.365 mL/min/kg was considerably
slower than the reported glomerular filtration
rats of 3.72–4.23 mL/min/kg in rat models of
31
DMIS and their control rats, indicating that CZX
is mainly reabsorbed in renal tubules for all rats
studied.
30
those in respective control rats. Sato et al.
reported that the hepatic blood flow rate was
significantly faster in male Wistar rat model of
DMIS.
In rat models of DMIA, the Vss of CZX was
significantly larger (37.3% increase) than that in
control rats (Tab. 2). This could be due to
significant increase in the free fractions of CZX in
plasma in rat model of DMIA (44.8% increase) as
mentioned above.
After the oral administration of CZX in both rat
models of DMIA and DMIS, the AUC of CZX was
significantly smaller than those of respective
control rats (Tab. 3). This was unlikely due to
decrease in the absorption of CZX from the
gastrointestinal tract of both rat models of dia-
betes. Based on the linear pharmacokinetics, the
mean ‘‘true’’ fractions of oral dose unabsorbed
(Funabs) in this study could be estimated by the
After the intravenous administration of
CZX, the estimated CLR of CZX as free
3
2
following equations:
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 11, NOVEMBER 2006

2460
BAEK ET AL.
Table 3. Pharmacokinetic Parameters of CZX and its Main Metabolite, OH-CZX, after the Oral Administration of
a
CZX at a Dose of 50 mg/kg in Rat Models of DMIA and DMIS, and Their Respective Control Rats
DMIA Control
DMIA
DMIS Control
DMIS
Parameter
(n ¼ 9)
(n ¼ 12)
(n ¼ 8)
(n ¼ 8)
Initial body weight (g)
Final body weight (g)
Blood glucose (mg/dL)
Urine volume (mL/24 h)
CZX
236 ꢀ 7.26
257 ꢀ 17.3
86.6 ꢀ 10.2
20.3 ꢀ 3.16
236 ꢀ 8.56
249 ꢀ 9.26
280 ꢀ 17.3
99.6 ꢀ 17.3
26.3 ꢀ 4.23
251 ꢀ 8.21
225 ꢀ 11.4***
316 ꢀ 97.4***
36.7 ꢀ 6.58***
220 ꢀ 16.0***
363 ꢀ 86.6***
42.8 ꢀ 7.05***
AUC (mg ꢃ min/mL)
Terminal half-life (min)
Cmax (mg/mL)
2810 ꢀ 960
87.3 ꢀ 8.21
31.8 ꢀ 13.1
8.33 ꢀ 5.00
0.0315 ꢀ 0.335
0.0382 ꢀ 0.0407
2.78 ꢀ 3.25
63.5
1560 ꢀ 603**
86.0 ꢀ 32.3
2390 ꢀ 245
91.9 ꢀ 6.80
30.5 ꢀ 8.17
6.25 ꢀ 3.54
0.0600 ꢀ 0.0947
0.467 ꢀ 0.421
1.36 ꢀ 1.26
50.9
1500 ꢀ 437***
94.4 ꢀ 13.4
20.3 ꢀ 4.21**
6.25 ꢀ 3.54
0.104 ꢀ 0.148
0.527 ꢀ 0.354
1.87 ꢀ 1.16
59.4
19.6 ꢀ 8.22*
16.7 ꢀ 15.4
T
max (min)
CL (mL/min/kg)
R
0.00410 ꢀ 0.0151*
0.619 ꢀ 0.848
2.28 ꢀ 4.18
Ae0–24 h (% of CZX dose)
GI24 h (% of CZX dose)
F (%)
59.4
OH-CZX
AUC (mg ꢃ min/mL)
1170 ꢀ 243
71.7 ꢀ 9.16
11.3 ꢀ 2.23
20.0 ꢀ 7.50
24.4 ꢀ 3.45
1.11 ꢀ 1.35
1820 ꢀ 359***
88.7 ꢀ 19.6*
17.7 ꢀ 5.39**
22.5 ꢀ 10.1
1320 ꢀ 155
95.3 ꢀ 8.21
13.3 ꢀ 4.35
21.3 ꢀ 12.7
26.7 ꢀ 11.4
0.691 ꢀ 0.740
2250 ꢀ 385***
115 ꢀ 32.3**
22.3 ꢀ 2.52***
26.3 ꢀ 15.5
Terminal half-life (min)
C
max (mg/mL)
Tmax (min)
Ae0–24 h (% of CZX dose)
GI24 h (% of CZX dose)
57.8 ꢀ 19.8***
0.634 ꢀ 0.857
70.5 ꢀ 19.2***
1.01 ꢀ 0.719
CZX, chlorzoxazone; OH-CZX, 6-hydroxychlorzoxazone; DMIA, diabetes mellitus induced by alloxan; DMIS, diabetes mellitus
induced by streptozotocin; AUC, total area under the plasma concentration–time curve from time zero to time infinity; Cmax, peak
plasma concentration, Tmax; time to reach a Cmax; CL , time-averaged renal clearance; Ae0–24 h, total amount excreted in 24-h urine;
R
GI24 h, total amount recovered from the gastrointestinal tract (including its contents and feces) at 24 h; F, extent of absolute oral
bioavailability.
a
Values expressed as mean ꢀ SD.
*
*
*
p < 0.05 compared with their respective control.
*p < 0.01 compared with their respective control.
**p < 0.001 compared with their respective control.
0
:0278 ¼ Funabs þ ð0:635
ꢄ 0:00172Þ control for rat model of DMIA
values after the oral administration of CZX
were 0.0278, 0.0228, and 0.0136, and 0.0187
(Tab. 3). The Funabs values thus calculated
were 2.67%, 2.20%, 1.28%, and 1.73% for
control for rat model of DMIA, rat model of DMIA,
control for rat model of DMIS, and rat model
of DMIS, respectively. Hence, more than 97% of
oral dose of CZX were absorbed from the gastro-
intestinal tract for all groups of rats. The
significantly smaller AUC of CZX after oral
administration of CZX in both rat models of
diabetes could also be due to increased metabo-
lism of CZX as explained in the intravenous
study.
0
:0228 ¼ Funabs þ ð0:594
ꢄ 0:00134Þ rat model of DMIA
0
:0136 ¼ Funabs þ ð0:509
ꢄ 0:00148Þ control for rat model of DMIS
0
:0187 ¼ Funabs þ ð0:594
ꢄ 0:00230Þ rat model of DMIS
where 0.635 (0.594, 0.509, and 0.594) and
.00172 (0.00134, 0.00148, and 0.00230) are
In conclusion, after both intravenous (Tab. 2)
and oral (Tab. 3) administration of CZX in rat
models of DMIA and DMIS, the AUC of OH-CZX
was significantly greater than their respective
controls, because the expression and mRNA
level of CYP2E1 increased in both rat models of
0
the F (Tab. 3) and GI24 h values after the
intravenous administration (Tab. 2), respectively,
for control for rat model of DMIA (rat model
of DMIA, control for rat model of DMIS, and
rat model of DMIS). The corresponding GI24 h
1
5
diabetes.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 11, NOVEMBER 2006
DOI 10.1002/jps

INDUCTION OF CYP2E1 ON OH-CZX FORMATION IN DIABETIC RATS
2461
REFERENCES
14. Price VF, Jollow DJ. 1986. Strain differences in
susceptibility of normal and diabetic rats to
acetaminophen hepatotoxicity. Biochem Pharmacol
35:687–695.
1
. Chen L, Yang CS. 1996. Effects of cytochrome P450
E1 modulators on the pharmacokinetics of chlor-
2
zoxazone and 6-hydroxychlorzoxazone in rats. Life
Sci 58:1575–1585.
15. Kim YC, Lee AK, Lee JH, Lee I, Lee DC, Kim SH,
Kim SG, Lee MG. 2005. Pharmacokinetics of
theophylline in diabetes mellitus rats: Induction
of CYP1A2 and CYP2E1 on 1,3-dimethyluric acid
formation. Eur J Pharm Sci 26:114–123.
16. Kim SH, Kim WB, Lee MG. 1998. Pharmacoki-
netics of a new carbapenem, DA-1131, after
intravenous administration to rats with alloxan-
induced diabetes mellitus. Biopharm Drug Dispos
19:303–308.
17. Bradford MM. 1976. A rapid and sensitive method
for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye bind-
ing. Anal Biochem 72:248–254.
18. Frye RF, Stiff DD. 1996. Determination of chlor-
zoxazone and 6-hydroxychlorzoxazone in human
plasma and urine by high-performance liquid
chromatography. J Chromatogr B 686:291–296.
19. Duggleby RG. 1995. Analysis of enzyme progress
curves by nonlinear regression. Methods Enzymol
249:61–90.
2
3
4
. Conney AH, Burns JJ. 1960. Physiological disposi-
tion and metabolic fate of chlorzoxazone (Paraflex)
in man. J Pharmacol Exp Ther 128:340–343.
. Desiraju RK, Renzi NL, Jr., Nayak RK, Ng
KT. 1983. Pharmacokinetics of chlorzoxazone in
humans. J Phram Sci 72:991–994.
. Rockich K, Blouin R. 1999. Effect of the acute-phase
response on the pharmacokinetics of chlorzoxazone
and cytochrome P-450 2E1 in vitro activity in rats.
Drug Metab Dispos 27:1074–1077.
. Kim YG, Cho MK, Kwon JW, Kim SG, Chung SJ,
Shim CK, Lee MG. 2002. Effects of cysteine on the
pharmacokinetics of intravenous chlorzoxazone in
rats with protein–calorie malnutrition. Biopharm
Drug Dispos 23:121–129.
. Ahn CY, Kim EJ, Lee I, Kwon JW, Kim WB, Kim
SG, Lee MG. 2003. Effects of glucose on the
pharmacokinetics of intravenous chlorzoxazone in
rats with acute renal failure induced by uranyl
nitrate. J Pharm Sci 92:1604–1613.
. Chung W-S, Kim EJ, Lee I, Kim SG, Lee MG, Kim
SH. 2003. Effects of recombinant human growth
hormone on the pharmacokinetics of intravenous
chlorzoxazone in rats with acute renal failure
induced by uranyl nitrate. Life Sci 73:253–263.
. Moon YJ, Lee AK, Chung HJ, Kim EJ, Kim SH, Lee
DC, Lee I, Kim SG, Lee MG. 2003. Effects of
acute renal failure on the pharmacokinetics of
chlorzoxazone in rats. Drug Metab Dispos 31:776–
5
6
7
8
9
20. Liu PT, Ioannides C, Shavila J, Symons AM, Parke
DV. 1993. Effects of ether anaesthesia and fasting
on various cytochromes P450 of rat liver and
kidney. Biochem Pharmacol 45:871–877.
21. Shim HJ, Lee EJ, Kim SH, Kim SH, Yoo M, Kwon
JW, Kim WB, Lee MG. 2000. Factors influencing
the protein binding of a new phosphodiesterase V
inhibitor, DA-8159, using an equilibrium dialysis
technique. Biopharm Drug Dispos 21:285–291.
22. Chiou WL. 1978. Critical evaluation of potential
error in pharmacokinetic studies using the linear
trapezoidal rule method for the calculation of the
area under the plasma level-time curve. J Pharma-
cokinet Biopharm 6:539–549.
784.
. Peter R, Bocker R, Beaune PH, Iwasaki M,
Guengerich FP, Yang CS. 1990. Hydroxylation of
chlorzoxazone as a specific probe for human liver
cytochrome P-450 2E1. Chem Res Toxicol 3:566–
23. Gibaldi M, Perrier D. 1982. Pharmacokinetics.
(2nd edn) New York: Marcel-Dekker, Inc.
573.
1
1
1
1
0. Carnovale CE, Marinelli RA, Rodriguez–Garay
EA. 1986. Bile flow decrease and altered bile
composition in streptozotocin-treated rats. Bio-
chem Pharmacol 35:2625–2628.
1. Watkins JB, III, Sherman SE. 1992. Long-term
diabetes alters the hepatobiliary clearance of
acetoaminophen, bilirubin and digoxin. J Pharma-
col Exp Ther 260:1337–1343.
2. Nadai M, Yoshizumi H, Kuzuya T, Hasegawa T,
Johno I, Kitazawa S. 1990. Effects of diabetes on
disposition and renal handling of cefazolin in rats.
Drug Metab Dispos 18:565–570.
3. Park JM, Moon CH, Lee MG. 1996. Pharmacoki-
netic changes of methotrexate after intravenous
administration to streptozotocin-induced diabetes
mellitus rats. Res Commun Mol Pathol Pharmacol
24. Kim SH, Choi YM, Lee MG. 1993. Pharmacoki-
netics and pharmacodynamics of furosemide in
protein–calorie malnutrition. J Pharmacokinet
Biopharm 21:1–17.
25. Chiou WL. 1979. New calculation method for mean
apparent drug volume of distribution and applica-
tion to rational dosage regimen. J Pharm Sci 68:
1067–1069.
26. Eatman FB, Colburn WA, Boxenbaum HG, Pos-
manter HN, Weinfeld RE, Ronfeld R, Weissman L,
Moore JD, Gibaldi M, Kaplan SA. 1977. Pharma-
cokinetics of diazepam following multiple dose oral
administration to healthy human subjects. J Phar-
macokinet Biopharm 5:481–494.
27. Chiou WL. 1980. New calculation method of mean
total body clearance of drugs and its application to
rational regimens. J Pharm Sci 69:90–91.
93:343–352.
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 11, NOVEMBER 2006

2462
BAEK ET AL.
2
8. Li L, Zhang Y. 1998. Changes of CYP2E1 activity
31. Bae SK, Yang SH, Lee SJ, Kwon JW, Kim WB, Lee
DC, Lee MG. 2005. Pharmacokinetic changes of
DA-7867, a new oxazolidinone, after intravenous
and oral administration to rats with short-term and
long-term diabetes mellitus indyced by streptozo-
tocin. Eur J Pharm Sci 25:337–345.
32. Lee MG, Chiou WL. 1983. Evaluation of potential
causes for the incomplete bioavailability of furose-
mide: Gastric first-pass metabolism. J Pharmaco-
kinet Biopharm 11:623–640.
in diabetic rat model. Yao Xue Xue Bao 33:891–
895.
2
3
9. Wilkinson GR, Shand DG. 1975. A physiological
approach to hepatic drug clearance. Clin Pharma-
col Ther 18:377–390.
0. Sato H, Terasaki T, Okumura K, Tsuji A. 1991.
Effect of receptor up-regulation on insulin pharma-
cokinetics in streptozotocin-treated diabetic rats.
Pharm Res 8:563–569.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 11, NOVEMBER 2006
DOI 10.1002/jps