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College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul (Y.J.M., A.K.L., H.C.C., E.J.K., S.G.K., M.G.L.); Department of Pharmacology, College of Dentistry and Research Institute of Oral Science, Kangnung National University, Kangnung (S.H.K); Miz Medi Hospital, Seoul (D.C.L.); and Department of Diagnostic Pathology, College of Medicine, University of Ulsan, Asan Foundation, Asan Medical Center, Seoul (I.L.), South Korea
(Received May 30, 2002; accepted March 12, 2003)
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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),
primarily undergoes hydroxylation to form 6-hydroxychlorzoxazone (OH-CZX)
catalyzed mainly by CYP2E1 (Conney and
Burns, 1960; Peter et al.,
1990). OH-CZX is then rapidly glucuronidated and excreted in urine
(Conney and Burns, 1960;
Desiraju et al., 1983). CZX
has been suggested for use as a chemical probe to assess the activity of
CYP2E1 in vitro and in vivo due to the good correlation between formation rate
of OH-CZX and CYP2E1 activity in rats
(Rockich and Blouin,
1999).
The pharmacokinetic changes of many drugs (mainly excreted either via renal
excretion or by hepatic metabolism) have been reported in rats with acute
renal failure induced by uranyl nitrate (U-ARF). For example, the total area
under the plasma concentration-time curve from time 0 to time infinity (AUC)
was significantly greater, and the time-averaged total body (CL), renal
(CLR), and/or nonrenal (CLNR) clearances were
significantly slower after i.v. administration of the following drugs (or
compounds) to rats with U-ARF: methotrexate
(Park et al., 1996);
vancomycin (Engineer et al.,
1981); DA-1131, a new carbapenem
(Kim SH et al., 1998);
azosemide (Park et al., 1998);
diltiazem (Lee et al., 1992);
amiodarone (Fruncillo et al.,
1986); tetraethylammonium bromide and para-aminohippurate
(Lin and Lin, 1988); salicylic
acid (Liu et al., 1996); and
M1, an active metabolite of a new anthracycline, DA-125 (Kim YG et al., 1996).
The pharmacokinetic differences of the above-mentioned drugs (or compounds)
have mainly been explained due to differences in the formation of conjugates
or liver and/or kidney impairments in rats with U-ARF based on plasma and
urine chemistry data and/or tissue microscopy
(Kim SH et al., 1998, and
references therein). However, the AUC and CL of cyclosporin
(Lee and Ku, 1998),
l-propranolol (Lee and Ku,
1999), YJA-20379-8, a new proton pump inhibitor
(Kim HJ et al., 1998),
doxorubicin (Adriamycin) (Lee et al.,
1996), and tacrolimus (Son et
al., 2000) were not significantly different between control rats
and rats with U-ARF.
It has been reported that although CYP2E1 is the major form metabolizing
CZX to OH-CZX, human CYP1A2 and CYP3A4 are involved in CZX hydroxylation in
vitro (Carriere et al., 1993;
Ono et al., 1995;
Gorski et al., 1997;
Shimada et al., 1999). Hence,
the changes of CYP2E1, CYP1A2, CYP2B1/2, CYP2C11, CYP3A2, and CYP3A23 in rats
with U-ARF were evaluated. We report that the increase in formation of OH-CZX
in rats with U-ARF after i.v. administration of CZX at a dose of 20 mg/kg, a
substrate of CYP2E1, could be mainly due to induction of CYP2E1 in the
rats.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-32P]dCTP (3000
mCi/mmol) and Random prime-labeling kit were purchased from PerkinElmer Life
Sciences (Boston, MA) and Promega (Madison, WI), respectively. DDT
[1,1-bis(4-chlorophenyl)2,2,2-trichloroethane], DDE
[2,2-bis(4-chlorophenyl)1,1-dichloroethylene], CZX, NADPH, and
ß-glucuronidase (type H-1, from Helix pomatia) were products
from Sigma-Aldrich (St. Louis, MO). OH-CZX and 3-aminophenyl sulfone [an
internal standard of high-performance liquid chromatography (HPLC) assay for
CZX and OH-CZX] were obtained from Sigma/RBI (Natick, MA) and Aldrich Chemical
Co. (Milwaukee, WI), respectively. Uranyl nitrate was a product from BDH
Chemicals (Poole, Dorset, UK). Other chemicals were of reagent grade or HPLC
grade and, therefore, were used without further purification. Animals. For the molecular biology and pharmacokinetics studies, 8-week-old male Sprague-Dawley rats (weighing 275–330 g) were purchased from Charles River (Atsugi, Japan). The rats were randomly divided into two groups, control rats and rats with U-ARF. All rats were provided with food (Sam Yang Company, Seoul, South Korea) and water ad libitum, and were maintained in a light-controlled room (light, 7:00 AM–7:00 PM; dark, 7:00 PM–7:00 AM) kept at a temperature of 22 ± 2°C and a humidity of 55 ± 5% (Animal Center for Pharmaceutical Research, College of Pharmacy, Seoul National University, Seoul, South Korea). Each rat was individually housed in a metabolic cage (Tecniplast, Varese, Italy) under the supply of filtered pathogen-free air and water ad libitum.
Induction of Acute Renal Failure in Rats by Uranyl Nitrate
Injection. Uranyl nitrate (the uranyl nitrate powder was dissolved in 0.9%
NaCl-injectable solution to make a concentration of 0.5%), 1 ml/kg (5 mg/kg),
was injected once via the tail vein of each rat to induce acute renal failure
(Kim SH et al., 1998, and
references therein). The same volume of 0.9% NaCl-injectable solution was
injected into control rats.
Preparation of Microsomal Proteins. Hepatic microsomes of rats with
U-ARF and rats treated with DDE and DDT and their respective control rats were
prepared by differential centrifugations at 10,000g for 30 min and
then at 100,000g for 90 min. Microsomes were washed in a
pyrophosphate buffer and stored in a 50 mM Tris-acetate buffer (pH 7.4)
containing 1 mM EDTA and 20% glycerol as described previously (Kim SG et al.,
1996). Protein contents were assayed
(Lowry et al., 1951) and the
subcellular preparations were stored in a -70°C freezer (Revco ULT 1490
D-N-S; Kendro Laboratory Products, Asheville, NC) until use.
Immunoblot Analysis. SDS-polyacrylamide gel electrophoresis and immunoblot analyses were performed according to the previously published procedures (Kim SG et al., 1996). Microsomal proteins were separated by a 7.5% gel and electrophoretically transferred to a nitrocellulose paper. The nitrocellular paper was incubated with anti-CYP1A2, -CYP2B1/2, -CYP2C11, -CYP2E1, and -CYP3A23 antibody (Kim SG et al., 1996), followed by incubation with alkaline phosphate-conjugated secondary antibody, and developed using 5-bromo-4-chloro-3-indolylphosphate and 4-nitroblue tetrazolium chloride, or an ECL chemiluminescence detection kit.
Northern Blot Hybridization. Specific cDNA probes for the
CYP1A2, CYP2B1, CYP2B2, CYP2C11, CYP2E1, CYP3A2, and CYP3A23
genes were amplified by reverse transcription-polymerase chain reaction using
the selective primers (Kim and Novak,
1993), cloned in the pGEM+T vector (Promega), as described
previously (Cho et al., 1999).
Total RNA was isolated using the improved single-step method of
thiocyanate-phenol-chloroform RNA extraction according to the method of
Puissant and Houdebine (1990).
Northern blot analysis was carried out according to the procedure described
previously (Kim and Cho,
1996). Briefly, total RNA isolated from the liver was resolved by
electrophoresis in a 1% agarose gel containing 2.2 M formaldehyde and
transferred to a nitrocellulose paper. The nitrocellulose paper was baked in a
vacuum oven at 80°C for 2 h. The blot was hybridized as described
previously (Kim and Cho,
1996). Membranes were washed in 2x standard saline citrate
and 0.1% SDS for 10 min at 42°C, twice, and in 0.1x standard saline
citrate and 0.1% SDS for 10 min at 42°C, twice. Membranes were finally
washed in the solution containing 0.1x standard saline citrate and 0.1%
SDS for 60 min at 55°C. After quantification of mRNA levels, the membranes
were stripped and rehybridized with a labeled probe complementary to 18S rRNA
to quantify the amount of RNA loaded onto the membranes.
Scanning Densitometry. Scanning densitometry of Northern and Western blots was performed with Image Scan and Analysis System (Alpha Innotech Corporation, San Leandro, CA) to assess the expression of CYP1A2, CYP2B1/2, CYP2C11, CYP2E1, CYP3A2, and CYP3A23. Data were analyzed using the SigmaStat program (SPSS Science, Chicago, IL).
Measurement of Vmax, Km, and
CLint for the Formation of OH-CZX in Hepatic Microsomes. On the
5th day, the livers of control rats (n = 4) and rats with U-ARF
(n = 4) were homogenized 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
9700g for 30 min and the supernatant fraction was further centrifuged
at 100,000g for 90 min. The microsomal pellet 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 method (Lowry
et al., 1951). 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 protein), 10 µl of CZX (to have substrate
concentrations of 1, 1.25, 2, 5, 10, 50, 100, 125, 200, and 500 µM), and 1
mM NADPH in a final volume of 1 ml of 0.1 M Tris-HCl, pH 7.4, in a water bath
shaker kept at 37°C and at a rate of 50 oscillations per minute. The
reaction was terminated by the addition of 0.1 ml of ice-cold 20%
trichloroacetic acid after a 20-min incubation. The OH-CZX formed was
determined by HPLC analysis (Frye and
Stiff, 1996). The kinetic constants (Km and
Vmax) for the formation of OH-CZX were calculated using
the Lineweaver-Burk plot (Lineweaver and
Burk, 1934) by linear regression and the method of least squares.
Intrinsic OH-CZX formation clearance (CLint) was calculated by
dividing the Vmax by the Km.
A CYP3A2 inducer, DDT (dissolved in corn oil), 100 mg/kg, was administered
(total oral volume was 0.6 ml) once using a feeding tube
(Sierra-Santoyo et al., 2000),
and a CYP3A1(23) inducer, DDE (dissolved in corn oil), 100 mg/kg/day, was
administered (total oral volume was 0.6 ml) for 7 consecutive days using a
feeding tube (You et al.,
1999). The same volume of vehicle was administered to each control
rat. On the 2nd day (DDT-treated rats, n = 3, and their controls,
n = 5) and 8th day (DDE-treated rats, n = 6, and their
controls, n = 5), hepatic microsomal fractions were collected.
Similar experiments were also performed at CZX concentrations of 2, 5, 10, 50,
and 100 µM.
Pretreatment of Rats. In the early morning on the 4th day after i.v.
administration of uranyl nitrate or 0.9% NaCl-injectable solution, the jugular
vein and the carotid artery of each rat were cannulated with polyethylene
tubing (Clay Adams, Parsippany, NJ) while each rat was under light ether
anesthesia. Both cannulas were exteriorized to the dorsal side of the neck
where each cannula terminated with long Silastic tubes (Dow Corning, Midland,
MI). Both Silastic 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) and was allowed to recover
from light ether anesthesia for 24 h (to minimize the effect of ether on
CYP2E1 activity) before the study began. It has been reported that ether
anesthesia alone increased expression of CYP2E1 by 40% as determined by
p-nitrophenol hydroxylase activity
(Liu et al., 1993), and
altered expression of CYP2E1 by solvents completely returned to that of
control 24 h after solvent withdrawal
(Roberts et al., 1994). Each
rat was not restrained during the experimental period.
Intravenous Study. The following study was performed at the 5th day (n = 5 for each group). The 24-h urine was collected for the measurement of creatinine level with cannulation of blood vessels. Plasma was collected for the measurement of total proteins, albumin, urea nitrogen, creatinine, glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT) levels (analyzed by Green Cross Reference Laboratory, Seoul, South Korea). The whole kidney and liver of each rat were excised, rinsed, or perfused with 0.9% NaCl-injectable solution, blotted dry with tissue paper, and weighed. Small portions of each organ were fixed in 10% neutral phosphate-buffered formalin and then processed for routine histological examination with hema-toxylin-eosin staining.
On the 5th day, CZX (CZX powder was dissolved in a minimum amount of 1 N
NaOH) at a dose of 20 mg/kg was administered by i.v. infusion over 1 min via
the jugular vein (total injection volume was approximately 0.6 ml) of another
set of control rats (n = 9) and rats with U-ARF (n = 9). An
approximately 0.12-ml aliquot of blood sample was collected via the carotid
artery at 0 (to serve as a control), 1 (at the end of the infusion), 5, 15,
30, 45, 60, 90, 120, 180, 240, 300, 360, and 480 min after i.v. administration
of the drug. After centrifugation, a 0.05-ml aliquot of plasma sample was
stored in a -70°C freezer until HPLC analysis of CZX and OH-CZX
(Frye and Stiff, 1996). An
approximately 0.3-ml aliquot of heparinized 0.9% NaCl-injectable solution (20
units/ml) was used to flush the cannula immediately after each blood sampling
to prevent blood clotting. At the end of 8 h, each metabolic cage was rinsed
with 20 ml of distilled water, and the rinses were combined with the 8-h
urine. After measuring the exact volume of the combined urine, two 0.05-ml
aliquots of combined urine samples were stored in a -70°C freezer until
HPLC analysis of CZX (Frye and Stiff,
1996). At the same time, as much blood as possible was collected
via the carotid artery, and each rat was sacrificed by cervical dislocation.
At the same time, the entire gastrointestinal tract (including its contents
and feces) was removed, transferred into a beaker containing 50 ml of 0.1 N
NaOH (to facilitate the extraction of CZX), and cut into small pieces using
scissors. After stirring with a glass rod, two 0.2-ml aliquots of the
supernatant were collected from each beaker and stored in a -70°C freezer
until HPLC analysis of CZX (Frye and
Stiff, 1996).
Tissue Distribution After Intravenous Administration. On the 5th
day, CZX (the same solution as used in the i.v. study) at a dose of 20 mg/kg
was also administered intravenously to control rats (n = 5 for each
time) and rats with U-ARF (n = 5 for each time). At 30 and 120 min
after the end of i.v. infusion of the drug, as much blood as possible was
collected via the abdominal artery, and each rat was sacrificed by cervical
dislocation. Blood samples were centrifuged immediately and plasma was
collected. Approximately 1 g of each kidney, heart, muscle, lung, stomach,
small intestine, liver, mesentery, large intestine, fat, and spleen was
excised, rinsed with cold 0.9% NaCl-injectable solution to minimize blood
remaining in the tissues, blotted dry with paper tissue, and homogenized with
4 volumes of distilled water using a tissue homogenizer (Ultra-Turrax T25
Janke and Kunkel; IKA-Labortecnik, Staufen, Germany). After centrifugation,
two 0.05-ml aliquots of the 9000g supernatant fraction were stored in
the freezer until HPLC analysis of CZX and OH-CZX
(Frye and Stiff, 1996). All
the procedures were conducted at 4°C in an ice-bath.
HPLC Analysis of CZX. The concentrations (or amounts) of CZX and
OH-CZX in the above biological samples of control rats were analyzed by the
reported HPLC method (Frye and Stiff,
1996). Briefly, a 0.1-ml aliquot of 0.2 M sodium acetate buffer
(pH 4.75) and 200 units of ß-glucuronidase dissolved in 0.1 ml of
isotonic Sørensen phosphate buffer (pH 7.4) were added to a 0.05-ml
aliquot of biological sample. The mixture was manually mixed and incubated in
a water bath shaker kept at 37°C and at a rate of 50 oscillations per
minute for 2 h. After incubation, a 0.05-ml aliquot of methanol containing 10
µg/ml 3-aminophenyl sulfone (the internal standard) was added and
vortex-mixed, and a 1-ml aliquot of diethyl ether was added. The mixture was
shaken for 10 min and then centrifuged at 2000g for 10 min. The upper
organic layer was transferred to a clean tube and evaporated at 37°C under
a stream of nitrogen. The residue was reconstituted with a 0.1-ml aliquot of
the mobile phase, and a 0.05-ml aliquot was injected directly onto the HPLC
column. The mobile phase, 0.1 M ammonium acetate/acetonitrile/tetrahydrofuran
(72:22: 5.5, v/v) was run at a flow rate of 1 ml/min. A UV detector set at 283
nm monitored the column effluent. The retention times of OH-CZX, 3-aminophenyl
sulfone, and CZX were approximately 6, 10, and 18 min, respectively. The
detection limits of CZX and OH-CZX in plasma were both 0.1 µg/ml. The
coefficients of variation of the assay (within- and between-day) were
generally low (below 8.2%). For the biological samples from rats with U-ARF,
the mobile phase was changed to 0.05 M ammonium acetate/acetonitrile (4:1,
v/v) to eliminate interference endogenous peak(s). The flow rate of the mobile
phase was 1.0 ml/min. The retention times of OH-CZX, 3-aminophenyl sulfone,
and CZX were approximately 14, 22, and 40 min, respectively. Since the above
biological samples were incubated with ß-glucuronidase, the
concentrations (or amounts) of CZX (or OH-CZX) in the present study represent
free CZX (or OH-CZX) plus its glucuronide conjugates.
Pharmacokinetic Analysis. The AUC was calculated by the trapezoidal
rule-extrapolation method; this method utilized the logarithmic trapezoidal
rule (Chiou, 1978) for the
calculation of the area during the declining plasma-level phase and the linear
trapezoidal rule for the rising plasma-level phase. The area from the last
data point to time infinity was estimated by dividing the last measured plasma
concentration by the terminal rate constant.
Standard methods (Gibaldi and Perrier,
1982) were used to calculate the CL, area under the first moment
of the plasma concentration-time curve, mean residence time (MRT), and
apparent volume of distribution at steady state (Vss)
(Kim et al., 1993). The
harmonic mean method was employed to calculate the mean values of
Vss (Chiou,
1979), terminal half-life
(Eatman et al., 1977), and CL
(Chiou, 1980).
Glomerular filtration rate was calculated by measuring creatinine clearance (CLCR) assuming that kidney function was stable during the 8-hr experimental period. The CLCR was calculated by dividing the total amount of creatinine excreted in 8-h urine by AUC0–8 h of creatinine in plasma.
Statistical Analysis. For the molecular biology studies, one-way analysis of variance was used to assess significant difference (P < 0.05) between two groups of rats. For each significant effect, the Newman-Keuls test was used for comparisons of each group mean. For the pharmacokinetic studies, a P value of less than 0.05 was considered to be statistically significant using the unpaired t test. All data were expressed as mean ± S.D.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Effect of ARF on CYP1A2, CYP2B1/2, CYP2C11, CYP2E1, CYP3A2, and CYP3A23. In rats with U-ARF, the expression of CYP1A2, CYP2B1/2, CYP2C11, CYP2E1, CYP3A2, and CYP3A23 was monitored. Immunoblot analysis showed that expression of CYP2C11 decreased to 20% of control, whereas CYP2E1 and CYP3A23 increased 2.3 and 4 times, respectively, as compared with control (Fig. 1). The expression of CYP1A2 and CYP2B1/2 was not changed by U-ARF (Fig. 1). Northern blot analysis also revealed that in rats with U-ARF, the CYP2E1 mRNA increased three times and the CYP2C11 mRNA decreased to 25% of control; however, those of CYP1A2, CYP2B1, CYP2B2, and CYP3A2 were comparable to control levels (Fig. 2). These results were consistent with those of Western blot analysis. Interestingly, however, the CYP3A23 mRNA was not increased in rats with U-ARF (Fig. 2). Hence, the induction of CYP3A23 by ARF may result from protein stabilization (i.e., a decrease in protein turnover).
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Measurement of Vmax, Km, and CLint for the Formation of OH-CZX in Hepatic Microsomes. In rats with U-ARF, the Vmax (59.7% increase) for the formation of OH-CZX in hepatic microsomal fraction was significantly faster than that in control rats; however, the Km values were comparable between two groups of rats (Table 2). Hence, the CLint for the formation of OH-CZX in the hepatic microsomal fraction was significantly faster (42.3% increase) in rats with U-ARF (Table 2), suggesting that formation of OH-CZX could be faster in rats with U-ARF. In rats treated with DDE, only CLint was significantly faster (30.9% increase) than that in control rats (Table 2). However, in rats treated with DDT, the Vmax, Km, and CLint were comparable to those in control rats (Table 2).
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Pharmacokinetics of CZX and OH-CZX After Intravenous Administration of CZX. After i.v. administration, the plasma concentrations of CZX declined in a polyexponential fashion for both groups of rats with significantly lower levels in rats with U-ARF (Fig. 3A); this resulted in a significantly smaller AUC of CZX (49.3% decrease) than that in control rats (Table 3). The smaller AUC of CZX could be due to significantly faster CL of CZX (98.7% increase) in rats with U-ARF (Table 3). Significantly shorter terminal half-life (41.3% decrease) and MRT (52.7% decrease) of CZX in rats with U-ARF also support the faster CL of CZX in the rats (Table 3). The unchanged CZX excreted in 8-h urine and recovered from gastrointestinal tract at 8 h were below the detection limit for both groups of rats, suggesting that the contribution of CLR to CL of CZX was negligible and contribution of gastrointestinal excretion (including biliary excretion) of CZX to CLNR of CZX was also negligible. CZX was stable for up to a 3-h incubation (in a water bath shaker kept at 37°C and at a rate of 50 oscillations per minute) in human gastric juices obtained from five patients before surgery at Seoul National University Hospital (Seoul, South Korea) having pH values of 6.77, 6.2, 1.8, 2.72, and 5.1 at a CZX concentration of 5 µg/ml (the gastric juice samples were not incubated with ß-glucuronidase); more than 95.2% of the spiked amount of CZX was recovered after a 3-h incubation. Hence, the CLs of CZX listed in Table 3 could represent the metabolic clearances of CZX in rats. Therefore, it could be concluded that the significantly faster CL of CZX in rats with U-ARF could be due to faster metabolism of CZX to form OH-CZX in the rats.
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Formation of OH-CZX after i.v. administration of CZX was fairly rapid for both groups of rats; the OH-CZX was detected in plasma from the first blood sampling time (1 min) and reached its peak at approximately 30 min (Fig. 3B). After reaching its peak, the plasma concentrations of OH-CZX declined in an apparent monoexponential fashion for both groups of rats (Fig. 3B). The plasma concentrations of OH-CZX in rats with U-ARF were significantly higher (Fig. 3B), and this resulted in a significantly greater AUC of OH-CZX (922% increase) than that in control rats (Table 3). In rats with U-ARF, terminal half-life of OH-CZX was significantly longer (242% increase) than those in control rats (Table 2). The percentages of i.v. dose of CZX recovered from gastrointestinal tract at 8 h (due to gastrointestinal excretion including biliary excretion, GI0–8 h) was 0.583 and 1.81% (expressed in terms of i.v. dose of CZX) for control rats and rats with U-ARF, respectively (Table 3). Although the terminal half-life of OH-CZX after i.v. administration of OH-CZX to rats was not measured in the present study, the half-life of OH-CZX after i.v. administration of CZX was relatively short, 39.5 min, in control rats (Table 2). Hence, in control rats, the rate-limiting step for the elimination of CZX could be the formation rate of OH-CZX and its glucuronide conjugates.
Tissue Distribution After Intravenous Administration. The mean tissue-to-plasma (T/P) ratios of CZX and OH-CZX at 30 and 120 min after i.v. administration of CZX at a dose of 20 mg/kg to control rats (n = 5, each time) and rats with U-ARF (n = 5, each time) are shown in Figs. 4 and 5, respectively. In control rats, the T/P ratios of CZX were less than unity in all the tissues studied except liver and kidney at 30 min and liver, kidney, and mesentery at 120 min (Fig. 4), suggesting that affinity of rat tissues studied to CZX was not considerable. In rats with U-ARF, the T/P ratios of CZX were significantly greater in heart, lung, stomach, and large intestine, and significantly smaller in small intestine and fat at 30 min (Fig. 4). Interestingly, in rats with U-ARF, the CZX was below the detection limit at 120 min in all rat tissues studied; however, it was detected in all tissues of control rats at both 30 and 120 min (Fig. 4). The above data suggested that formation of OH-CZX increased in rats with U-ARF compared with that in control rats. In control rats, the OH-CZX was below the detection limit in all rat tissues studied at both 30 and 120 min except kidney, whereas it was detected in all rat tissues studied at both 30 and 120 min in rats with U-ARF (Fig. 5). The above data suggested that formation of OH-CZX increased in rats with U-ARF more than in control rats.
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Discussion |
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Recently, it was found from our laboratories that the concentrations of CZX
and OH-CZX in plasma after i.v. administration of CZX were unconjugated CZX
(not CZX glucuronides) and conjugated OH-CZX, respectively. The percentage of
hepatic extraction ratio (hepatic first-pass effect) was roughly estimated by
dividing the nonrenal clearance (as mentioned earlier, the CL of CZX in
Table 3 could represent the
metabolic clearance of CZX) by the reported hepatic plasma flow in rats
assuming that nonrenal clearance of CZX was attributed solely to the liver
(hence the percentage represents maximal possible value)
(Lee and Chiou, 1983). The
hepatic plasma flow in rats was estimated based on the reported hepatic blood
flow of 30.4 ml/min/kg (Davies and Morris,
1993) and hematocrit of approximately 45%
(Mitruka and Rawnsley, 1981)
in rats. The hepatic extraction ratio of CZX in control rats was approximately
53.6%, indicating that CZX is an intermediate hepatic extraction ratio drug.
Hence, its hepatic clearance depends on CLint, free (unbound in
plasma protein) fraction of CZX, and hepatic blood flow
(Wilkinson and Shand, 1975).
The significantly faster CL of CZX in rats with U-ARF could be due to both
significantly faster CLint (42.3% increase,
Table 3) and greater unbound
fraction (54.5% increase; the plasma protein binding values of CZX were 75.6
and 62.3% for control rats and rats with U-ARF, respectively, based on
equilibrium dialysis technique).
It is well known that CZX is mainly metabolized to OH-CZX primarily by
CYP2E1 in rats (Conney and Burns,
1960; Peter et al.,
1990). Since the mRNA levels of CYP2E1 increased significantly
(three times) in rats with U-ARF (Fig.
2), it was expected that the formation of OH-CZX could increase in
the rats. This was supported by the following results. First, the plasma
concentrations of CZX were significantly lower
(Fig. 3A) and the resultant AUC
of CZX was significantly smaller (Table
3) in rats with U-ARF, and this could be due to significantly
faster CL of CZX (Table 3) in
the rats. The faster CL of CZX in rats with U-ARF was supported by
significantly shorter terminal half-life and MRT of CZX in the rats
(Table 3). Second, the plasma
concentrations of OH-CZX were significantly higher
(Fig. 3B) and the resultant AUC
of OH-CZX was significantly greater (Table
3) in rats with U-ARF. Moreover, the
AUCOH-CZX/AUCCZX fraction increased from 0.374 in
control rats to 7.54 in rats with U-ARF. However, the effect of acute renal
failure-induced accumulation of OH-CZX on the increase in the ratio could not
be totally ruled out, since acute renal failure reduces urinary excretion of
drugs (compounds). Third, in rats with U-ARF, the CZX was below the detection
limit at 120 min in all rat tissues studied, whereas it was detected in all
tissues of control rats at both 30 and 120 min
(Fig. 4). However, in rats with
U-ARF, OH-CZX was detected in all rat tissues studied at both 30 and 120 min,
whereas it was below the detection limit at both 30 and 120 min in all tissues
of control rats except kidney (Fig.
5). Finally, the in vitro CLint in hepatic microsomal
fraction was significantly faster in rats with U-ARF
(Table 2). The above data
indicated that formation of OH-CZX increased in rats with U-ARF due to
increase in CYP2E1 in the rats. It has also been reported
(Chung et al., 2002) that in
rats with U-ARF, CYP2E1 was induced two to four times compared with that in
control rats.
Although CYP2E1 is the major form metabolizing CZX to OH-CZX, it has been
reported (Carriere et al.,
1993; Ono et al.,
1995; Gorski et al.,
1997; Shimada et al.,
1999) that human CYP1A2 and CYP3A4 are involved in CZX
hydroxylation. The effect of CYP1A2 on formation of OH-CZX in rats with U-ARF
could be ruled out since no significant change in CYP1A2 was obtained in the
rats (Figs. 1 and
2). In rats with U-ARF, the
expression of CYP3A23 increased 4-fold
(Fig. 1). Human CYP3A4 and rat
CYP3A1 (CYP3A23) proteins have 73% homology
(Lewis, 1996). It has been
reported that CYP3A1 (CYP3A23) (Mahnke et
al., 1997) or CYP3A2 (Debri et
al., 1995; Hoen et al.,
2001; Rekka et al.,
2002) is the major CYP3A protein in rats. Hence, the role of
CYP3A23 and CYP3A2 on the formation of OH-CZX in rats was measured by
treatment with DDT (an inducer of CYP3A2;
Sierra-Santoyo et al., 2000)
or DDE (an inducer of CYP3A23; You et al.,
1999). In rats treated with DDT, the Vmax,
Km, and CLint for the formation of OH-CZX were
not significantly different compared to those in the control rats
(Table 2), indicating that the
effect of CYP3A2 on the formation of OH-CZX was almost negligible in rats. It
has been reported (Li et al.,
1995) that there was no effect on CYP2E1 in rats treated with DDT.
Based on Western blot analysis, the expression of CYP3A1/2 and CYP2E1 was
comparable as weighed against control (data not shown). Moreover, in rats with
U-ARF, CYP3A2 mRNA level was not changed
(Fig. 2). However, in rats
treated with DDE, the CLint was significantly faster than that in
control rats (Table 2).
Immunoblot analysis showed that the expression of CYP2E1 and CYP3A23 increased
3.18 and 2.98 times, respectively, in rats treated with DDE compared with
control (Fig. 6A). Northern
blot analysis also revealed that in rats treated with DDE, the mRNA level of
CYP2E1, CYP3A2, and CYP3A23 increased 3.30, 3.68, and 3.94 times,
respectively, compared with control (Fig.
6B). The effect of CYP3A2 on the formation of OH-CZX in rats with
U-ARF could be ruled out as explained above. Since CYP2E1 is the major form
for metabolizing CZX to OH-CZX, the increased formation of OH-CZX in rats with
U-ARF could mainly be due to induction of CYP2E1 in rats. However, the effect
of induction of CYP3A23 could not be totally ruled out.
|
We found that either ARF or DDE induced CYP2E1 and CYP3A23. Although CYP2E1
induction by ARF accompanied the increase in its mRNA, CYP3A23 induction in
rats with ARF failed to concomitantly increase in its mRNA. This was in
contrast to the effects of DDE, which we used as a comparative agent that
induces both CYP2E1 and CYP3A23 with increases in their mRNAs. These results
indicate that the induction of CYP3A23 by ARF may be mediated with the
stabilization of the CYP3A23 protein instead of the transcriptional activation
of the gene. In addition, we observed that ARF markedly suppressed the
expression of CYP2C11, which is involved in the metabolism of testosterone as
an endogenous substrate (i.e., testosterone 16,2-hydroxylation)
(Biagini et al., 1999). Thus,
the changes in the expression of P450 isoforms are likely to result in
differences in the metabolic profiles of endogenous substances, therapeutic
agents, and environmental chemicals.
|
Footnotes |
|---|
1 Abbreviations used are: CZX, chlorzoxazone; OH-CZX, 6-hydroxychlorzoxazone;
U-ARF, acute renal failure induced by uranyl nitrate; AUC, area under the
plasma concentration-time curve from time zero to time infinity; CL,
time-averaged total body clearance; CLR, time-averaged renal
clearance; CLNR, time-averaged nonrenal clearance; DDE,
2,2-bis(4-chlorophenyl)1,1-dichloroethylene; HPLC, high-performance liquid
chromatography; GOT, glutamate oxaloacetate transaminase; GPT, glutamate
pyruvate transaminase; CLint, intrinsic OH-CZX formation clearance;
MRT, mean residence time; Vss, apparent volume of
distribution at steady state; CLCR, creatinine clearance; ARF,
acute renal failure; GI0–8 h, percentage of i.v. dose of CZX
recovered from gastrointestinal tract at 8 h; T/P, tissue-to-plasma ratio;
P450, cytochrome P450; DA-1131,
(IR,5S,6S)-(2S,4S)-2-[(E)-3-methansulfonyl
amino-1-propenyl]pyrrolidine-4-ylthiol-6-[(R)-1-hydroxy-ethyl]-1-methyl-1-carbapen-2-em-3-carboxylic
acid; DA-125,
(8S,10S)-8-(3-aminopropanoyloxyacetyl)-10-[(2,6-dideoxy-2-fluro--L-talopyranosyl)oxy]-7,8,9,
10-tetrahydro-6,8,11-trihydroxy-methoxy-5,12-naphthacenedione; YJA-20379-8,
3-butyryl-4-[5-R-(+)-methylbenzylamino]-8-ethoxy-1,7-naphthyridine. 
Address correspondence to: Dr. Myung G. Lee, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, San 56-1, Shinlim-Dong, Kwanak-Gu, Seoul 151-742, South Korea. E-mail: leemg{at}snu.ac.kr
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; n = 9) and rats with U-ARF (
; n
= 9).
