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Pharmacokinetic Parameters of Chlorzoxazone and Its Main Metabolite, 6-Hydroxychlorzoxazone, after Intravenous and Oral Administration of Chlorzoxazone to Liver Cirrhotic Rats with Diabetes Mellitus

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, South Korea (C.Y.A., S.K.B., Y.S.J., Y.C.K., M.G.L., W.G.S.); Department of Bioequivalence, Korean Food & Drug Administration, Seoul, South Korea (C.Y.A.); Department of Clinical Pharmacology, Busan Paik Hospital, Inje University, Busan, South Korea (S.K.B.); and Department of Diagnostic Pathology, College of Medicine, University of Ulsan, Asan Foundation, Asan Medical Center, Seoul, South Korea (I.L.)

(Received June 26, 2007; accepted March 27, 2008)

	Abstract

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Protein expression of the hepatic CYP2E1 has been reported to be increased in diabetic rats. This enzyme is the primary metabolizer of chlorzoxazone (CZX) to 6-hydroxychlorzoxazone (OH-CZX). Although patients with liver cirrhosis have a higher prevalence of diabetes mellitus, there have been no reported studies on the protein expression of CYP2E1 in rats induced to have liver cirrhosis and diabetes mellitus by injection of N-dimethylnitrosamine followed by streptozotocin [liver cirrhosis with diabetes mellitus (LCD) rats]. Thus, in the present study, the pharmacokinetics of CZX and OH-CZX were evaluated in LCD rats. Compared with control rats, LCD rats had significantly decreased (by 62%) total liver protein and significantly increased (by 124%) protein expression of CYP2E1, but the intrinsic clearance (Cl_int; formation of OH-CZX per milligram protein) was comparable in both groups of rats. As a result, the relative Cl_int was also comparable for the two groups. Thus, OH-CZX formation in LCD and control rats was expected to be similar. As expected, after i.v. (20 mg/kg) and p.o. (50 mg/kg) administration of CZX, the area under the curve (AUC) of OH-CZX was comparable in control and LCD rats (i.v., 571 ± 85.8 and 578 ± 413 µg · min/ml, respectively; p.o., 1540 ± 338 and 2170 ± 1070 µg · min/ml, respectively). In LCD rats, the AUC_OH-CZX/AUC_CZX ratio was similar to the value in control rats after i.v. and p.o. administration. These results indicate that OH-CZX can be used as a chemical probe to assess the activity of CYP2E1 in LCD rats.

Kim et al. (2005) reported that induction of diabetes mellitus in male Sprague-Dawley rats by treatment with alloxan or streptozotocin (DMIA or DMIS rats, respectively) also increased their protein expression and mRNA level of CYP2E1. Furthermore, Baek et al. (2006) reported that the increased protein expression of CYP2E1 caused a significant increase in the formation of OH-CZX in both DMIA and DMIS rats. The association between liver disease and diabetes mellitus is well known (Vidal et al., 1994; Kwon, 2003; Moscatiello et al., 2007). Thus, we examined CZX in this study.

Wang et al. (2003) p.o. administered CZX to diabetic patients and found that the total area under the plasma CZX concentration–time curve from time 0 to infinity (AUC) was reduced by 25% in type 1 diabetic patients and by 70% in obese type 2 diabetic patients as compared with that in 20 control volunteers. Furthermore, they found that protein expression of CYP2E1 in peripheral blood mononuclear cells increased in both types of diabetic patients. However, to our knowledge, no studies on the protein expression of CYP2E1 and the pharmacokinetics of CZX and OH-CZX in diabetic rats or humans with liver cirrhosis have yet been reported.

The objectives of the current studies were to evaluate, using a rat model, the effects of diabetes and liver cirrhosis, alone and in combination, on the pharmacokinetics of CZX and OH-CZX. Changes in the protein expression of hepatic CYP2E1 in rats with liver cirrhosis with or without diabetes were also investigated.

	Materials and Methods

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Chemicals. CZX, OH-CZX, 3-aminophenyl sulfone [internal standard for the high-performance liquid chromatography (HPLC) analysis of CZX and OH-CZX], monoclonal anti-β-actin antibody, NADPH (tetrasodium salt), Tris buffer, EDTA (disodium salt), β-glucuronidase (type H-1, from Helix pomatia), streptozotocin, and Kodak X-OMAT film were purchased from Sigma-Aldrich (St. Louis, MO). N-Dimethylnitrosamine was a product from Tokyo Kasei Kogyo Company (Tokyo, Japan), and ketamine hydrochloride was from Yuhan Corporation (Seoul, South Korea). Polyclonal anti-human CYP2E1 antibody was obtained from Detroit R&D (Detroit, MI), and horseradish peroxidase-conjugated goat anti-rabbit antibody was from Bio-Rad (Hercules, CA). Enhanced chemiluminescence reagents were purchased from Amersham Biosciences Corporation (Piscataway, NJ). Other chemicals were of reagent or HPLC grade.

Animals. Protocols for the animal studies were approved by the Animal Center and Use Committee of the College of Pharmacy of Seoul National University, Seoul, South Korea. Male Sprague-Dawley rats (4–5 weeks old, weighing 180–200 g) were purchased from the Charles River Company Korea (Orient, Seoul, South Korea). The rats were randomly divided into three disease groups [liver cirrhosis (LC), diabetes mellitus (DM), and liver cirrhosis with diabetes mellitus (LCD)] and a control group. They were maintained in a clean room (Animal Center for Pharmaceutical Research, College of Pharmacy, Seoul National University) at a temperature of 22 ± 2°C with 12-h light (7:00 AM to 7:00 PM) and dark (7:00 PM to 7:00 AM) cycles and a relative humidity of 55 ± 5%. Rats were housed in metabolic cages (Tecniplast, Varese, Italy) under filtered, pathogen-free air, with food (Sam Yang Company, Pyungtaek, South Korea) and water available ad libitum.

Induction of LC in Rats by N-Dimethylnitrosamine Injection. Freshly prepared N-dimethylnitrosamine (diluted to 0.01 mg/ml in 0.9% NaCl injectable solution) was injected i.p. at a dose of 0.01 mg/kg on three consecutive days per week for 4 weeks (Ohara and Kusano, 2002; Bae et al., 2006). On day 29, citrate buffer (pH 7.4; 1 ml/kg) was injected via the tail vein. On day 36, rats were treated with CZX.

Laboratory rats with N-dimethylnitrosamine-induced LC have clinical features similar to those of humans with LC, such as increased mortality, hepatic parenchymal cell destruction, formation of connective tissue, and nodular regeneration (Kang et al., 2002). LC in the LC rats was evident by histological analysis, which revealed extensive micronodular cirrhosis with regenerative hepatocellular changes. Bile ductular proliferation was also detected (Bae et al., 2006). It has been reported that N-dimethylnitrosamine-induced liver cirrhosis in rats is reproducible (Jenkins et al., 1985; Jezequel et al., 1987).

Induction of DM in Rats by Streptozotocin Injection. A 0.9% NaCl injectable solution was injected i.p. (1 ml/kg) on three consecutive days a week for 4 weeks. On day 29, one dose (45 mg/kg) of freshly prepared streptozotocin (dissolved in citrate buffer, pH 4.5, to 45 mg/ml) was administered via the tail vein (Kim et al., 2005). The rats were treated with CZX on day 36.

Induction of LCD in Rats with N-Dimethylnitrosamine and Streptozotocin Injections. LC was induced by i.p. injection of N-dimethylnitrosamine as described above. Then, on day 29, DM was induced by injection of streptozotocin via the tail vein as described above. The rats were treated with CZX on day 36.

Control Rats. Rats were injected i.p. with 0.9% NaCl injectable solution (1 ml/kg) on three consecutive days a week for 4 weeks. On day 29, one dose (1 ml/kg) of citrate buffer, pH 4.5, was administered via the tail vein. The rats were treated with CZX on day 36.

During the pretreatment, food and water were available ad libitum to all the rats. Immediately before the experiment, blood glucose levels in all the rats were measured using the MediSense Optium Kit (Abbott Laboratories, Bradford, MA), and rats with blood glucose levels greater than 250 mg/dl were selected as diabetic (DM and LCD rats).

Measurement of Liver, Kidney, and Spleen Function. To assess liver, kidney, and spleen function, a 24-h urine sample was collected on day 36 from LC, DM, LCD, and control rats (n = 6, each) for the measurement of creatinine levels. A blood sample was collected from the carotid artery for the measurement of the hematocrit (microprocessor pH/°C meter) (Eutek Cybernetics, Singapore, Singapore). The plasma was measured for the total protein, albumin, urea nitrogen, glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), total bilirubin, direct bilirubin, alkaline phosphatase, lactate dehydrogenase (LDH), and creatinine by the Green Cross Reference Laboratory (Seoul, South Korea). Plasma protein binding of CZX was measured using equilibrium dialysis (Shim et al., 2000).

The whole liver, kidney, and spleen of each rat were excised, rinsed 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 hematoxylin and eosin staining. Each rat was exsanguinated and sacrificed by cervical dislocation.

Preparation of Hepatic Microsomes. The procedures used were similar to those described by Baek et al. (2006). Livers from LC, DM, LCD, and control rats (n = 5, each) were homogenized (Ultra-Turrax T25, Janke and Kunkel; IKA-Labortechnik, Staufeni, Germany) in ice-cold homogenization buffer (0.154 M KCl/50 mM Tris-HCl in 1 mM EDTA, pH 7.4). After the homogenate was centrifuged (10,000g, 30 min), the supernatant fraction was removed and centrifuged at high speed (100,000g, 90 min). The resulting microsomal pellet was resuspended in homogenization buffer and stored at -70°C (Revco ULT 1490 D-N-S; Western Mednics, Asheville, NC) until used. Protein content was measured using the Bradford method (Bradford, 1976).

Western Immunoblot Analysis of CYP2E1. The procedures used were similar to a reported method (Kim et al., 2001). Liver microsomes were resolved by SDS gel electrophoresis on a 7.5% polyacrylamide gel (10 µg protein/lane; n = 3 each). Proteins were transferred to a nitrocellulose membrane (Bio-Rad) that was then blocked for 1 h in 5% milk powder in phosphate-buffered saline containing 0.05% (v/v) Tween 20 (PBS-T). For immunodetection, blots were incubated overnight at 4°C with rabbit anti-human CYP2E1 antibody (diluted 1:10,000 in PBS-T containing 5% bovine serum albumin), followed by incubation for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (diluted 1:10,000 in PBS-T containing 5% milk powder). Protein expression of CYP2E1 was detected by enhanced chemiluminescence on Kodak X-OMAT film and quantitated by densitometry with a microcomputer imaging device (model M1; Imaging Research, St. Catharine's, ON, Canada). The β-actin band was used as a loading control.

Measurement of V_max, K_m, and Intrinsic Clearance for the Formation of OH-CZX in Hepatic Microsomes. Microsomal fractions (equivalent to 0.2 mg of protein) were mixed with 50 µl of 0.1 M phosphate buffer, pH 7.4, containing 1 mM NADPH; 10 µl of CZX dissolved in a minimal amount of 10 N NaOH to make final concentrations of 2.5, 5, 10, 20, 50, 100, 200, 500, and 1000 µM CZX; and 0.1 M phosphate buffer, pH 7.4, sufficient to make a final volume of 0.5 ml. This reaction mixture was incubated in a water-bath shaker (37°C, 50 oscillations/min) for 20 min, at which time the reaction was terminated by addition of 1 ml of ether. The formation of OH-CZX was determined using an HPLC method (Frye and Stiff, 1996). The kinetic parameters (K_m, V_max) for the formation of OH-CZX were determined by fitting the unweighted kinetic data from rat liver microsomes to a single-site Michaelis-Menten equation, V = V_max x [S]/(K_m + [S]), where [S] is the substrate concentration. The best-fit model was selected based on the statistical goodness of fit (Yamaoka et al., 1978); the model with the lowest Akaike information criterion was chosen. Calculations were performed using the WinNonlin software (Pharsight, Mountain View, CA). The intrinsic clearance (Cl_int) for the formation of OH-CZX per milligram protein was calculated by dividing the V_max by the K_m. The relative Cl_int for the formation of OH-CZX based on the whole rat liver was estimated by the protein expression below and expressed as a percentage of the controls (100%): total liver protein (mg) x protein expression of CYP2E1 (% relative to controls) x Cl_int (ml/min/mg protein).

Pretreatment of Rats for i.v. or p.o. Study. Early in the morning on day 36, each rat was anesthetized by i.m. injection of ketamine hydrochloride at a dose of 100 mg/kg. The jugular vein (for drug administration in the i.v. study) and the carotid artery (for blood sampling) were cannulated with a polyethylene tube (Clay Adams, Parsippany, NJ). Both cannulas were exteriorized to the dorsal side of the neck, where each cannula was terminated with a long silastic tube (Dow Corning, Midland, MI). Both silastic tubes were inserted into a wire sheath to allow free movement of the rat. Each rat then was housed individually in a rat metabolic cage (Daejong Scientific Company, Seoul, South Korea) and allowed to recover from anesthesia for 4 to 5 h before beginning the experiment. Thus, the rats were not restrained in the present study. Ketamine was used instead of ether to minimize the effect on CYP2E1 because Liu et al. (1993) reported that ether anesthesia alone increased the protein expression of CYP2E1 by 40%, as determined by assaying p-nitrophenol hydroxylase activity.

Intravenous Study. CZX (dissolved in a minimum amount of 10 N NaOH) at a dose of 20 mg/kg was infused (total infusion volume of 2 ml/kg) over 1 min via the jugular vein to rats in each group (n = 9, 7, 7, and 8 for LC, DM, LCD, and control rats, respectively). A blood sample (approximately 0.12 ml) was collected via the carotid artery at 0 (control), 1 (at the end of the infusion), 5, 15, 30, 45, 60, 90, 120, and 180 min after the start of the i.v. infusion of CZX. A heparinized 0.9% NaCl injectable solution (20 units/ml; 0.3 ml) was used to flush the cannula immediately after each blood sampling to prevent clotting.

Each blood sample was immediately centrifuged, and a 50-µl aliquot of plasma was stored at -70°C for later analysis of CZX and OH-CZX by HPLC (Frye and Stiff, 1996). At the end of the experiment (24 h after CZX treatment), each metabolic cage was rinsed with 20 ml of distilled water, and the rinse water was combined with the 24-h urine sample. The volume of the combined urine sample was determined, and two 50-µl aliquots were stored at -70°C for later analysis. 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. The abdomen then was opened, and the entire gastrointestinal tract (including its contents and feces) of each rat was removed, transferred to a beaker containing 50 ml of 0.1 N NaOH (to facilitate the extraction of CZX and OH-CZX), and cut into small pieces with scissors. After stirring with a glass rod for 1 min, two 50-µl aliquots of the supernatant were collected from each beaker and stored at -70°C for later analysis.

Oral Study. CZX (the same solution used in the i.v. study) at a dose of 50 mg/kg was administered p.o. (total p.o. volume of 3 ml/kg) using a feeding tube to rats in each group (n = 8, 8, 7, and 7 for LC, DM, LCD, and controls, respectively). Blood samples were collected at 0, 5, 15, 30, 45, 60, 90, 120, 180, 240, 360, and 480 min after p.o. administration of CZX. Other procedures were similar to those described above for the i.v. study.

Measurement of Rat Plasma Protein Binding of CZX Using Equilibrium Dialysis. Binding of CZX to protein in fresh plasma from LC, DM, LCD, and control rats (n = 5 each) was measured using equilibrium dialysis (Shim et al., 2000). Plasma (1 ml) was dialyzed against 1 ml of isotonic Sørensen phosphate buffer, pH 7.4, containing 3% (w/v) dextran to minimize volume shift (Boudinot and Jusko, 1984) in a 1-ml dialysis cell (Fisher Scientific, Fair Lawn, NJ) fitted with a Spectra/Por 4 membrane (molecular mass cutoff of 12–14 kDa; Spectrum Medical Industries Inc., Los Angeles, CA). The initial concentrations of CZX spiked into the plasma compartment were 1, 10, and 50 µg/ml. After a 24-h incubation, two 50-µl aliquots were removed from each compartment and stored at -70°C for later HPLC analysis of CZX.

HPLC Analysis of CZX and OH-CZX. Concentrations of CZX and OH-CZX in the samples were determined using an HPLC method (Frye and Stiff, 1996). 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 phosphate buffer, pH 7.4, containing 200 units of β-glucuronidase were added to 50 µl of sample. The mixture was mixed manually and incubated in a water-bath shaker (50 oscillations/min) for 2 h at 37°C. A 50-µl aliquot of methanol containing 10 mg/ml of 3-aminophenyl sulfone (internal standard) was then added. After the mixture was vortexed, 1 ml of diethyl ether was added, and the mixture was shaken for 10 min. After centrifugation (16,000g, 10 min), the upper organic layer was transferred to a clean tube and dried (Dry Thermobath; Eyela, Tokyo, Japan) under a gentle stream of nitrogen gas at 37°C.

The residue was reconstituted in 0.1 ml of mobile phase [0.1 M ammonium acetate/acetonitrile/tetrahydrofuran (72:22.5:5.5, v/v/v)], and a 50-µl aliquot was directly injected onto a reversed-phase (C₁₈) HPLC column. The mobile phase was run at a flow rate of 1.0 ml/min. An ultraviolet detector at 283 nm was used to monitor the column eluent. Unconjugated concentrations of OH-CZX were also measured in the urine samples without incubation with β-glucuronidase. The retention times of OH-CZX, 3-aminophenyl sulfone (internal standard), and CZX were approximately 6, 10, and 18 min, respectively. The detection limit for CZX and OH-CZX in the rat plasma and urine samples was all 0.05 µg/ml. The coefficients of variation of the assay (within- and between-day) were less than 8.2%.

View larger version (10K): [in this window] [in a new window]   FIG. 1. Hepatic protein expression of CYP2E1 in LC, DM, LCD, and control rats was quantitated by Western immunoblotting and densitometry. A, immunoblot of gel loaded with 10 µg of microsomal protein per lane. β-Actin was used as a loading control. CYP2E1 was detected by enhanced chemiluminescence on Kodak X-OMAT film. B, protein expression of CYP2E1 was quantitated by densitometry. Results are shown relative to protein expression of CYP2E1 in the control rats (control = 100%). Error bars represent S.D. *, p < 0.05 compared with the controls; each value was significantly different.

View larger version (12K): [in this window] [in a new window]   FIG. 2. Kinetics for the formation rate of OH-CZX. CZX was incubated at the indicated concentrations (2.5–1000 µM) with liver microsomes from LC (), DM (), LCD (), and control () rats (n = 5, each) at 37°C for 20 min. The kinetic data were fit to a simple Michaelis-Menten equation. Error bars represent S.D.

Standard methods (Gibaldi and Perrier, 1982) were used to calculate the following pharmacokinetic parameters using a noncompartmental analysis (WinNonlin; Pharsight Corporation): the time-averaged total body, renal, and nonrenal clearances (Cl, Cl_r, and Cl_nr, respectively), the terminal half-life (t_1/2), the first moment of AUC, the mean residence time (MRT), the apparent volume of distribution at steady state (V_ss), and the extent of absolute oral bioavailability (F). The peak plasma concentration (C_max) and the time to reach C_max (T_max) were directly read from the experimental data.

Statistical Analysis. A p value <0.05 was deemed to be statistically significant using an unpaired t test or a Duncan's multiple range test, with the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL) posteriori analysis of variance among the four means for the unpaired data. All the data are expressed as the mean ± S.D., with the exception of T_max, which is expressed as the median (range).

	Results

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Liver, Kidney, and Spleen Function. Body weight, blood glucose level, hematocrit, 24-h urine output, plasma chemistry data, Cl_cr, and relative organ weights for the four rat groups are listed in Table 1. For comparison, literature values from normal (albino) rats (Mitruka and Rawnsley, 1981; Davies and Morris, 1993) are also shown.

Compared with the control rats, the LC rats had significantly decreased 24-h urine volume and plasma levels of total protein and albumin; significantly increased plasma levels of GOT, GPT, total bilirubin, direct bilirubin, alkaline phosphatase, and total cholesterol; significantly decreased relative liver weight; and significantly increased relative spleen weight. In DM rats, the blood glucose level, the 24-h urine output, the plasma levels of GPT, alkaline phosphatase, urea nitrogen, and total cholesterol, and the relative kidney weight were significantly increased compared with the control rats. In LCD rats, the blood glucose level, the plasma levels of total protein and albumin, the 24-h urine output, and the relative liver weight were significantly decreased compared with the control rats, whereas the plasma levels of GOT, GPT, total bilirubin, direct bilirubin, alkaline phosphatase, LDH, urea nitrogen, and total cholesterol and the relative kidney and spleen weights were significantly increased. However, the Cl_cr did not differ significantly among the four groups.

These findings suggest that in LC and LCD rats, neither kidney nor spleen function was seriously impaired, whereas liver function was somewhat impaired. Consistent with this result, no significant histological findings were detected in the liver, kidney, or spleen in any rats, except that extensive hepatocellular degeneration with bridging fibrosis (precirrhotic change) was detected in the livers of LC and LCD rats.

Protein Expression of CYP2E1. Compared with the control rats, the protein expression of CYP2E1 increased (by 258%) in the DM rats, decreased (by 22.0%) in the LC rats, and increased (by 124%) in the LCD rats (Fig. 1).

V_max, K_m, and Cl_int for Formation of OH-CZX in Hepatic Microsomes. The rates of OH-CZX formation in liver microsomes from LC, DM, LCD, and control rats treated with varying concentrations of CZX are shown in Fig. 2, and the V_max, K_m, Cl_int, relative liver weight, total protein, and relative Cl_int for each group are listed in Table 2. For the LCD rats, the V_max for OH-CZX formation was significantly lower than that of the DM rats (by 39.7%) and higher than that of the LC rats (by 289%) but was similar to the value observed in control rats. The changes observed in the V_max reflect the changes observed in the amount of CYP2E1 (Table 2). This result suggests that in LCD rats, the maximum velocity for the formation of OH-CZX was similar to the value observed in control rats. However, the K_m for the formation of OH-CZX was comparable (not significantly different) among the four groups of rats, indicating that the affinity of the enzyme(s) for CZX was not changed. As a result, in LCD rats, the Cl_int for the formation of OH-CZX per milligram protein was significantly slower than in DM rats (by 53.6%) and faster than in LC rats (by 197%) but was similar to the value observed in control rats. The total protein was significantly lower in LCD rats than in control or DM rats, but it was comparable with that in LC rats.

As discussed above (Fig. 1), the protein expression of CYP2E1 differed significantly among the four groups. Because the relative liver weight, total protein per whole liver, and protein contents of CYP2E1 were not comparable among the four groups, the relative Cl_int for the formation of OH-CZX, based on total liver protein, was calculated; in LCD rats, the value was considerably higher (by 353%) than that in LC rats and lower (by 82.4%) than that in DM rats but was similar to the value observed in control rats. This result suggests that in LCD rats, the formation of OH-CZX, based on the whole liver, may be comparable with that in control rats and that the V_max, Cl_int per milligram protein, and relative Cl_int, based on the whole liver, were similar to values observed in control rats.

View larger version (9K): [in this window] [in a new window]   FIG. 3. Mean arterial plasma concentration-time profiles of CZX (A) and OH-CZX (B) after i.v. infusion of CZX (20 mg/kg over 1 min) to LC (; n = 9), DM (; n = 7), LCD (; n = 7), and control (; n = 8) rats. Error bars represent S.D.

Rat Plasma Protein Binding of CZX. The values for CZX binding of proteins in fresh plasma from the four groups of rats were 73.0 ± 4.96% (control), 62.5 ± 8.53% (LC), 67.1 ± 2.44% (DM), and 72.3 ± 2.23% (LCD), respectively; the value in LC rats was significantly lower than that in control and LCD rats. Protein binding of CZX to plasma from control rats (n = 3 each) was constant for CZX concentrations of 1, 10, and 50 µg/ml, which yielded values of 68.3 ± 3.51, 69.6 ± 1.91, and 67.3 ± 5.44%, respectively. Thus, a CZX concentration of 10 µg/ml was arbitrarily chosen for the plasma protein binding studies.

Pharmacokinetics of CZX and OH-CZX after i.v. Administration of CZX. The mean arterial plasma CZX concentration–time profiles for i.v. administration of CZX (20 mg/kg) to LC, DM, LCD, and control rats are shown in Fig. 3A, and the relevant pharmacokinetic parameters are listed in Table 3. In LC rats, the AUC of CZX was significantly greater; the terminal t_1/2 and MRT were significantly longer; and the Cl, Cl_r, and Cl_nr were significantly lower than those in control rats. In DM rats, the AUC was significantly smaller; the terminal t_1/2 was significantly shorter; and the Cl, Cl_r, and Cl_nr were significantly faster than those in control rats. Interestingly, the AUC, MRT, Cl, Cl_r, and Cl_nr of CZX were similar between LCD and control rats. The contribution of the Cl_r to the Cl of CZX was almost negligible; the values were less than 3.63% in all the rats studied. However, the V_ss of CZX and the percentage of the i.v. dose of CZX excreted in the 24-h urine as unchanged drug (Ae_{0-24 h}) were not significantly different among the four groups. CZX was undetectable (under the detection limit) in the gastrointestinal tract at 24 h (GI_{24 h}) in all the rats. Thus, the contribution of changes in the Cl_r of CZX to other pharmacokinetic changes of CZX may also be almost negligible.

For the i.v. administration of CZX to DM, LC, LCD, and control rats, the mean arterial plasma OH-CZX concentration–time profiles are shown in Fig. 3B, and the relevant pharmacokinetic parameters are shown in Table 3. Formation of OH-CZX was rapid; for all four groups of rats, OH-CZX was detected in plasma at the first blood sampling time (1 min), and it rapidly reached T_max within 5 to 60 min. In LC rats, the AUC of OH-CZX was significantly smaller, the C_max was significantly lower, the T_max was significantly longer, the Ae_{0-24 h} of total OH-CZX was significantly smaller, and the AUC_OH-CZX/AUC_CZX ratio was significantly smaller than those in controls. In DM rats, the AUC of OH-CZX was significantly greater, the terminal t_1/2 was significantly longer, the C_max was significantly higher, the Ae_{0-24 h} of both total and free OH-CZX was significantly larger, and the AUC_OH-CZX/AUC_CZX ratio was significantly greater than those in controls. Interestingly, in LCD rats, the AUC, C_max, Ae_{0-24 h} of total OH-CZX, and AUC_OH-CZX/AUC_CZX ratio were similar to those in control rats. OH-CZX was also undetectable in GI_{24 h} for all the rats studied.

The ratios of Ae_{0-24 h, conjugated OH-CZX} to Ae_{0-24 h, total OH-CZX} were 0.638, 0.439, 0.577, and 0.405 for the control, LC, DM, and LCD rats, respectively, suggesting that formation of conjugated OH-CZX decreased considerably in LC and LCD rats compared with control and DM rats. CZX was excreted in the 24-h urine samples as the free (unconjugated) form.

View larger version (10K): [in this window] [in a new window]   FIG. 4. Mean arterial plasma concentration–time profiles of CZX (A) and OH-CZX (B) after p.o. administration of CZX (50 mg/kg) to LC (; n = 7), DM (; n = 8), LCD (; n = 8), and control (; n = 7) rats. Error bars represent S.D.

The mean arterial plasma OH-CZX concentration–time profiles for the p.o. administration of CZX are shown in Fig. 4B, and the relevant pharmacokinetic parameters are listed in Table 4. OH-CZX formed rapidly after p.o. administration of CZX; in all four groups of rats, it was detected in plasma at the first blood sampling time (5 min) and rapidly reached T_max within 5 to 90 min. In LC rats, the terminal t_1/2 of OH-CZX was significantly longer, the C_max was significantly lower, the T_max was significantly longer, the Ae_{0-24 h} of both total and free OH-CZX was significantly smaller, and the AUC_OH-CZX/AUC_CZX ratio was significantly smaller than those in controls. In DM rats, the AUC of OH-CZX was significantly greater, the C_max was significantly higher, the Ae_{0-24 h} of both total and free OH-CZX was significantly greater, and the AUC_OH-CZX/AUC_CZX ratio was significantly greater than those in controls. Interestingly, in LCD rats, the AUC, terminal t_1/2, C_max, T_max, and Ae_{0-24 h} of total OH-CZX and the AUC_OH-CZX/AUC_CZX ratio were similar to those in the control rats.

The ratios of Ae_{0-24 h, conjugated OH-CZX} to Ae_{0-24 h, total OH-CZX} were 0.561, 0.553, 0.562, and 0.458 for the control, LC, DM, and LCD rats, respectively, suggesting that the formation of conjugates of OH-CZX decreased considerably in LCD rats compared with the other rats. CZX was also excreted in the 24-h urine sample as the free (unconjugated) form.

	Discussion

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The presence of LC and DM in LCD rats was apparent by their significantly decreased body weight gain; significantly higher blood glucose level; significantly larger 24-h urine output; significantly higher plasma levels of GOT, GPT, alkaline phosphatase, and LDH; and significantly lighter and heavier relative liver and kidney weights, respectively, compared with control rats (Tables 1, 3, and 4). Liver cirrhosis was also proven, based on histology, as explained under Results.

Baek et al. (2006) reported that the Cl_nr of CZX could represent the metabolic clearance of the drug in rats. Additionally, the Cl_nr of CZX listed in Table 3 could represent the hepatic metabolic clearance of CZX. Thus, changes in the Cl_nr of CZX could represent changes in hepatic metabolism of CZX via CYP2E1 in rats.

Pathological conditions such as diabetes (Fig. 1) (Kim et al., 2005) and starvation (Johansson et al., 1990) induce CYP2E1. Pathological production of ketone bodies may be responsible for this induction of CYP2E1, presumably as the result of an adaptive response (Tu et al., 1983; Lieber, 1997). Nevertheless, ketone body production does not completely account for CYP2E1 induction. A previous study showed that, in the absence of insulin, ketone bodies at concentrations up to 10 mM failed to affect or produced a decrease in mRNA levels of CYP2E1 (Woodcroft et al., 2002), which supports the concept that the induction of CYP2E1 in diabetes or during fasting is not the result of elevated circulating ketone bodies levels. Another study indicated that alterations in energy metabolism (e.g., mitochondrial dysfunction) were associated with induction of CYP2E1 (Chung et al., 2001). We found that the hepatic CYP2E1 level was moderately greater in LCD rats than in control rats (Fig. 1). However, the relative Cl_int of CZX was comparable for the two groups (Table 2), which may be because of the accumulation of extracellular matrix in combination with a decrease in liver parenchymal cells.

After i.v. administration of CZX, the AUC of CZX was significantly greater in LC rats (57.0% increase) than in the control rats, possibly as a result of the significantly slower Cl of CZX (38.2% decrease) in the LC rats (Table 3). The slower Cl was attributable to a significantly slower Cl_nr of CZX (37.5% decrease) in the LC rats because the two groups had comparable Cl_r values (Table 3). The AUC of OH-CZX was significantly smaller (70.6% decrease) in LC rats than in controls (Table 3). These results could have been caused by a significantly slower (96.9% decrease) relative Cl_int for the formation of OH-CZX, based on total liver, because both the content and total liver protein of CYP2E1 were considerably decreased (by 78 and 67.6%, respectively, compared with the controls) (Table 2; Fig. 1). The significantly smaller formation (AUC) of OH-CZX in LC rats could also be supported by their smaller AUC_OH-CZX/AUC_CZX ratio (82.0% decrease) (Table 3).

In contrast to LC rats, DM rats exhibited a significant (57%) decrease in AUC of CZX after i.v. administration of CZX compared with the controls, possibly because the Cl in LC rats was significantly faster (by 121%) than in the controls (Table 3). The faster Cl was attributable to a significantly faster (by 119%) Cl_nr of CZX than in the controls (Table 3). Although Cl_r of CZX was significantly faster (by 146%) in DM rats than in the control rats, the contribution of the Cl_r to the Cl of CZX was almost negligible, constituting only 3.24% in DM rats (Table 3). The AUC of OH-CZX was significantly greater (by 75.1%) in DM rats than in the controls (Table 3). These results could have been caused by an increased (by 541%) relative Cl_int for the formation of OH-CZX, based on total liver, which could have been the result of the significantly higher (by 258%) content of CYP2E1 in the DM rats, despite their significantly lower (by 35.9%) total liver protein, compared with the controls (Table 2; Fig. 1). Thus, the contribution of the increased content of CYP2E1 to CZX metabolism and OH-CZX formation was greater than that of the decreased total liver protein. The significantly greater formation of OH-CZX (AUC) in DM rats could also be supported by the significantly greater (by 282%) AUC_OH-CZX/AUC_CZX ratio compared with that in the control rats (Table 3).

Similar results for the pharmacokinetics of CZX and OH-CZX and for Cl_int per milligram protein have been reported for other rat studies (Baek et al., 2006). Wang et al. (2003) also reported that following p.o. administration of 500 mg of CZX to patients with type 1 diabetes or obese, type 2 diabetes, the AUC of CZX was reduced by 25 and 70%, respectively, compared with that in healthy volunteers. However, the urinary recovery of CZX did not differ significantly among the three groups. Wang et al. (2003) also reported increased mRNA levels for CYP2E1 in peripheral blood mononuclear cells in both types of diabetes.

Protein expression of CYP2E1 was significantly increased in DM and LCD rats and was decreased in LC rats compared with the controls (Fig. 1). Although the protein expression of hepatic CYP2E1 was increased (by 124%) in the LCD rats, compared with that in the control rats (Fig. 1), the total liver protein in the LCD rats was significantly reduced (by 61.7%) (Table 2). As a result, the relative Cl_int for the formation of OH-CZX, based on whole liver, in LCD rats was similar to that of the control rats, with a difference of only 13% (Table 2). Thus, some pharmacokinetic parameters of CZX and OH-CZX would be expected to be similar in LCD and control rats. As expected, the AUC, MRT, Cl, Cl_r, and Cl_nr of CZX, and the AUC and C_max of OH-CZX, the Ae_{0–24 h} of total OH-CZX, and the AUC_OH-CZX/AUC_CZX ratio did not differ significantly between control and LCD rats (Table 3).

After p.o. administration of CZX, the AUC of CZX was significantly greater in LC rats and smaller in DM rats than in control or LCD rats (Table 4). However, this finding was not likely the result of an increase or decrease in gastrointestinal absorption of CZX found in LC and DM rats, respectively, compared with the control and LCD rats because the GI_{24 h} values were undetectable for both groups after i.v. and p.o. administration of CZX (Tables 3 and 4). CZX was stable in rat gastric and intestinal fluids (Baek et al., 2006). Thus, CZX was almost completely absorbed in all the groups of rats. Similar results were obtained from our i.v. studies (Table 3), especially for the AUC values of CZX and OH-CZX (Table 4). The F of CZX in LC rats was considerably greater than in the control, DM, and LCD rats (by 45.3, 63.6, and 28.5%, respectively; Table 4). This result could have primarily been because of the decreased hepatic metabolism of CZX in LC rats.

Although CYP2E1 is the major enzyme that metabolizes CZX to OH-CZX, human CYP1A1/2 and/or CYP3A4 (Carriere et al., 1993; Shimada et al., 1993; Gorski et al., 1997; Ono et al., 1997) and rat CYP1A1 and CYP3A1/2 (Jayyosi et al., 1995) have also been reported to carry out CZX hydroxylation. The role of CYP3A2 in the formation of OH-CZX in rats was measured by treatment with DDT (an inducer of CYP3A2) (Sierra-Santoyo et al., 2000). The V_max, K_m, and Cl_int for the formation of OH-CZX were not significantly different for the DDT-treated versus the control rats, indicating that the effect of CYP3A2 on the formation of OH-CZX was almost negligible (Sierra-Santoyo et al., 2000). Li et al. (1995) reported that treatment of rats with DDT had no effect on CYP2E1. We recently found (our unpublished data) that the protein expression of both CYP1A and CYP3A was increased in DM rats and decreased in LC rats, but in LCD rats, CYP1A was increased, whereas CYP3A was decreased.

In summary, after i.v. (Table 3) and p.o. (Table 4) administration of CZX, the AUC of OH-CZX was significantly smaller in LC rats than in control rats because protein expression of CYP2E1 and total liver protein were both decreased in the LC rats (Fig. 1). However, the AUC of OH-CZX was significantly greater in DM rats than in control rats because of the increased protein expression of CYP2E1 in DM rats (Fig. 1). The LCD and control rats had comparable values for AUC of OH-CZX, which may have resulted from the decrease in total liver protein in LCD rats despite their increase in protein expression of CYP2E1; as a result, the relative Cl_int for the formation of OH-CZX, based on total liver, was comparable with that in the controls. These results suggest that OH-CZX could be used as a chemical probe to assess the activity of hepatic CYP2E1 in LC, DM, and LCD rats.

	Footnotes

 
This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Korea (A050376).

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.107.017442.

ABBREVIATIONS: CZX, chlorzoxazone; OH-CZX, 6-hydroxychlorzoxazone; P450, cytochrome P450; AUC, total area under the plasma concentration–time curve from time zero to time infinity; HPLC, high-performance liquid chromatography; LC, liver cirrhosis; DM, diabetes mellitus; LCD, liver cirrhosis with diabetes mellitus; GOT, glutamate oxaloacetate transaminase; GPT, glutamate pyruvate transaminase; LDH, lactate dehydrogenase; PBS-T, phosphate-buffered saline/Tween 20; Cl_int, intrinsic clearance; Cl, time-averaged total body clearance; Cl_r, time-averaged renal clearance; Cl_nr, time-averaged non-renal clearance; Cl_cr, creatinine clearance; MRT, mean residence time; V_ss, apparent volume of distribution at steady state; F, extent of absolute oral bioavailability; Ae_{0–24 h}, percentage of the dose excreted in the 24-h urine; GI_{24 h}, percentage of the dose recovered from the entire gastrointestinal tract (including its contents and feces) at 24 h.

Address correspondence to: Wan Gyoon Shin, 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: wgshin{at}snu.ac.kr

	References

Top Abstract Materials and Methods Results Discussion References

 


Bae SK, Lee SJ, Kim T, Kim JW, Lee I, Kim SG, and Lee MG (2006) Pharmacokinetics and therapeutic effects of oltipraz after consecutive or intermittent oral administration in rats with liver cirrhosis induced by dimethylnitrosamine. J Pharm Sci 95: 985-997.[CrossRef][Medline]

Baek HW, Bae SK, Lee MG, and Shon YT (2006) Pharmacokinetics of chlorzoxazone in rats with diabetes: induction of CYP2E1 on 6-hydroxychlorzoxazone formation. J Pharm Sci 95: 2452-2462.[CrossRef][Medline]

Boudinot FD and Jusko WJ (1984) Fluid shifts and other factors affecting plasma protein binding of prednisolone by equilibrium dialysis. J Pharm Sci 73: 774-780.[CrossRef][Medline]

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.[CrossRef][Medline]

Carriere V, Goasduff T, Ratanasavanh D, Morel F, Gautier JC, Guillouza A, Geaune P, and Berthou F (1993) Both cytochromes P450 2E1 and 1A1 are involved in the metabolism of chlorzoxazone. Chem Res Toxicol 6: 852-857.[CrossRef][Medline]

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 Pharmacokinet Biopharm 6: 539-549.[CrossRef][Medline]

Chung HC, Kim SH, Lee MG, Cho CK, Kim TH, Lee DH, and Kim SG (2001) Mitochondrial dysfunction by gamma-irradiation accompanies the induction of cytochrome P450 2E1 (CYP2E1) in rat liver. Toxicology 161: 79-91.[CrossRef][Medline]

Conney AH and Burns JJ (1960) Physiological disposition and metabolic fate of chlorzoxazone (Paraflex) in man. J Pharmacol Exp Ther 128: 340-343.[Abstract/Free Full Text]

Davies B and Morris T (1993) Physiological parameters in laboratory animals and humans. Pharm Res 10: 1093-1095.[CrossRef][Medline]

Desiraju RK, Renzi NL Jr, Nayak RK, and Ng KT (1983) Pharmacokinetics of chlorzoxazone in humans. J Pharm Sci 72: 991-994.[CrossRef][Medline]

Frye RF and Stiff DD (1996) Determination of chlorzoxazone and 6-hydroxychlorzoxazone in human plasma and urine by high-performance liquid chromatography. J Chromatogr B 686: 291-296.[CrossRef][Medline]

Gibaldi M and Perrier D (1982) Pharmacokinetics, 2nd ed, Marcel-Dekker, Inc., New York.

Gorski JC, Jones DR, Wrighton SA, and Hall SD (1997) Contribution of human CYP3A subfamily members to the 6-hydroxylation of chlorzoxazone. Xenobiotica 27: 243-256.[CrossRef][Medline]

Jayyosi Z, Knoble D, Muc M, Erick J, Thomas PE, and Kelley M (1995) Cytochrome P-450 2E1 is not the sole catalyst of chlorzoxazone hydroxylation in rat liver microsomes. J Pharmacol Exp Ther 273: 1156-1161.[Abstract/Free Full Text]

Jenkins SA, Grandison A, Baxter JN, Day DW, Taylor I, and Shields R (1985) A dimethylnitrosamine-induced model of cirrhosis and portal hypertension in the rat. J Hepatol 1: 489-499.[CrossRef][Medline]

Jezequel AM, Mancini R, Rinaldesi ML, Macarri G, Venturini C, and Orlandi F (1987) A morphological study of the early stages of hepatic fibrosis induced by low doses of dimethylnitrosamine in the rat. J Hepatol 6: 174-181.

Johansson I, Lindros KO, Eriksson K, and Ingelman-Sundberg M (1990) Transcriptional control of CYP2E1 in the perivenous liver region and during starvation. Biochem Biophys Res Commun 173: 331-338.[CrossRef][Medline]

Kang KW, Kim YG, Cho MK, Bae SK, Kim CW, Lee MG, and Kim SG (2002) Oltipraz regenerates cirrhotic liver through CCAAT/enhancer binding protein-mediated stellate cell inactivation. FASEB J 16: 1988-1990.[Abstract/Free Full Text]

Kim SG, Kim EJ, Kim YG, and Lee MG (2001) Expression of cytochrome P-450s and glutathione S-transferases in the rat liver during water deprivation: effects of glucose supplementation. J Appl Toxicol 21: 123-129.[CrossRef][Medline]

Kim YC, Lee AK, Lee JH, Lee I, Lee DC, Kim SH, Kim SG, and Lee MG (2005) Pharmacokinetics of theophylline in diabetes mellitus rats: induction of CYP2E1 on 1,3-dimethyluric acid formation. Eur J Pharm Sci 26: 114-123.[CrossRef][Medline]

Kwon SY (2003) Prevalence and clinical significance of diabetes mellitus in patients with liver cirrhosis. Taehan Kan Hakhoe Chi 9: 205-211.[Medline]

Li HC, Dehal SS, and Kupfer D (1995) Induction of the hepatic CYP2B and CYP3A enzymes by the proestrogenic pesticide methoxychlor and by DDT in the rats. Effect on methoxychlor metabolism. J Biochem Toxicol 10: 51-61.[Medline]

Lieber CS (1997) Cytochrome P-4502E1: its physiological and pathological role. Physiol Rev 77: 517-544.[Abstract/Free Full Text]

Liu PT, Ioannides C, Shavila J, Symons AM, and Parke DV (1993) Effects of ether anaesthesia and fasting on various cytochromes P450 of rat liver and kidney. Biochem Pharmacol 45: 871-877.[CrossRef][Medline]

Mitruka BM and Rawnsley HM (1981) Clinical Biomedical and Hematological Reference Values in Normal Experimental Animals and Normal Humans, 2nd ed, Masson Publishing U S A Inc., New York.

Mizuno D, Tanaka E, Tanno K, and Misawa S (2000) Chlorzoxazone: a probe drug whose metabolism can be used to monitor toluene exposure in rats. Arch Toxicol 74: 139-144.[CrossRef][Medline]

Moon YJ, Lee AK, Chung HJ, Kim EJ, Kim SH, Lee DC, Lee I, Kim SG, and Lee MG (2003) Effects of acute renal failure on the pharmacokinetics of chlorzoxazone in rats. Drug Metab Dispos 31: 776-784.[Abstract/Free Full Text]

Moscatiello S, Manini R, and Marchesini G (2007) Diabetes and liver disease: an ominous association. Nutr Metab Cardiovasc Dis 17: 63-70.[CrossRef][Medline]

Ohara K and Kusano M (2002) Anti-transforming growth factor-β₁ antibody improves survival rate following partial hepatectomy in cirrhotic rats. Hepatol Res 24: 174-183.[CrossRef][Medline]

Ono S, Hatanaka T, Hotta H, Tsutsui M, Satoh T, and Gonzalez FJ (1995) Chlorzoxazone is metabolized by human CYP1A2 as well as by human CYP2E1. Pharmacogenetics 5: 143-150.[Medline]

Peter R, Bocker R, Beaune PH, Iwasaki M, Guengerich FP, and Yang CS (1990) Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-450 2E1. Chem Res Toxicol 3: 566-573.[CrossRef][Medline]

Rockich K and 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.[Abstract/Free Full Text]

Shim HJ, Lee EJ, Kim SH, Kim SH, Yoo M, Kwon JW, Kim WB, and 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.[CrossRef][Medline]

Shimada T, Tsumura F, and Tamazaki H (1999) Prediction of human liver microsomal oxidation of 7-ethoxycoumarin and chlorzoxazone with kinetic parameters of recombinant cytochrome P-450 enzymes. Drug Metab Dispos 27: 1274-1280.[Abstract/Free Full Text]

Sierra-Santoyo A, Hernadez M, Albores A, and Cebrian ME (2000) Sex-dependent regulation of hepatic cytochrome P-450 by DDT. Toxicol Sci 54: 81-87.[Abstract/Free Full Text]

Tu YY, Peng R, Chang ZF, and Yang CS (1983) Induction of a high affinity nitrosamine demethylase in rat liver microsomes by acetone and isopropanol. Chem Biol Interact 44: 247-260.[CrossRef][Medline]

Vidal J, Ferrer JP, Esmatjes E, Salmeron JM, Gonzalez-Clemente JM, Gomis R, and Rodes J (1994) Diabetes mellitus in patients with liver cirrhosis. Diabetes Res Clin Pract 25: 19-25.[CrossRef][Medline]

Wang Z, Hall SD, Maya JF, Li L, Asghar A, and Gorski JC (2003) Diabetes mellitus increases the in vivo activity of cytochrome P450 2E1 in humans. Br J Clin Pharmacol 55: 77-85.[CrossRef][Medline]

Woodcroft KJ, Hafner MS, and Novak RF (2002) Insulin signaling in the transcriptional and posttranscriptional regulation of CYP2E1 expression. Hepatology 35: 263-273.[CrossRef][Medline]

Yamaoka K, Nakagawa T, and Uno T (1978) Application of the Akaike's information criterion in the evaluation of linear pharmacokinetic equations. J Pharmacokinet Biopharm 6: 165-175.[CrossRef][Medline]

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