Effects of glucose supplementation on the pharmacokinetics of intravenous chlorzoxazone in rats with water deprivation for 72 h

Life Sciences 79 (2006) 2179 – 2186 www.elsevier.com/locate/lifescie

Effects of glucose supplementation on the pharmacokinetics of intravenous chlorzoxazone in rats with water deprivation for 72 h Yu Chul Kim a , Inchul Lee b , Sang Geon Kim a , Seong-Hee Ko c , Myung Gull Lee a , So Hee Kim c,⁎ a College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, South Korea Department of Diagnostic Pathology, College of Medicine, University of Ulsan, Asan Foundation, Asan Medical Center, Seoul, South Korea Department of Pharmacology, College of Dentistry and Research Institute of Oral Science, Kangnung National University, 123, Chibyon-Dong, Kangnung 210-702, South Korea

b c

Received 23 December 2005; accepted 13 July 2006

Abstract It was reported that in rats with water deprivation for 72 h with food (dehydration rat model), the expression of CYP2E1 was 3-fold induced with an increase in mRNA level and glucose supplementation instead of food during 72-h water deprivation (dehydration rat model with glucose supplementation) inhibited the CYP2E1 induction in dehydration rat model. It was also reported that chlorzoxazone (CZX) is metabolized to 6hydroxychlorzoxazone (OH–CZX) mainly via CYP2E1 in rats. Hence, the effects of glucose supplementation on the pharmacokinetics of CZX and OH–CZX were investigated after intravenous administration of CZX at a dose of 25 mg/kg to control male Sprague–Dawley rats and dehydration rat model and dehydration rat model with glucose supplementation. Based on the above mentioned results of CYP2E1, it could be expected that increased formation of OH–CZX in dehydration rat model could decrease in dehydration rat model with glucose supplementation. This was proven by the following results. In dehydration rat model with glucose supplementation, the AUC of OH–CZX was significantly smaller (1900 versus 1050 μg min/ml), AUCOH–CZX/AUCCZX ratio was considerably smaller (105 versus 34.3%), Cmax was significantly lower (20.6 versus 8.08 μg/ml), total amount excreted in 24-h urine as unchanged OH–CZX was significantly smaller (62.3 versus 42.7% of intravenous dose of CZX), and in vitro Vmax (2.18 versus 1.20 nmol/min/mg protein) and CLint (0.0285 versus 0.0171 ml/min/mg protein) were significantly slower than those in dehydration rat model. © 2006 Elsevier Inc. All rights reserved. Keywords: Chlorzoxazone; Dehydration; Pharmacokinetics; Glucose supplementation; CYP2E1; Rats

Introduction Chlorzoxazone [5-chloro-2(3H)-benzoxazolone; CZX], once used as a skeletal muscle relaxant for the treatment of painful muscle spasms (Chen and Yang, 1996), primarily undergoes hydroxylation to form 6-hydroxychlorzoxazone (OH–CZX). OH–CZX is then rapidly glucuronidated and excreted in the urine (Conney and Burns, 1960; Desiraju et al., 1983). The formation of OH–CZX is catalyzed mainly via the hepatic microsomal cytochrome P450 (CYP) 2E1 in humans (Conney and Burns, 1960) and rats (Rockich and Blouin, 1999; Kim et al., 2002b; Ahn et al., 2003; Chung et al., 2003; Moon et ⁎ Corresponding author. Tel.: +82 33 640 2462; fax: +82 33 648 2862. E-mail address: [email protected] (S.H. Kim). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.07.016

al., 2003). The use of OH–CZX as a chemical probe has been suggested 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 (Peter et al., 1990) and rats (Rockich and Blouin, 1999). The formation of OH– CZX via CYP2E1 in male Sprague–Dawley rats was proven in rats with protein-calorie malnutrition (rat model of PCM; 4 weeks fed on 5% casein diet; Kim et al., 2002b) and acute renal failure induced by uranyl nitrate (rat model of U-ARF; Ahn et al., 2003; Chung et al., 2003; Moon et al., 2003). Dehydration of the body occurs as a result of water deprivation, excessive sweating, and various disease states, such as polyurea, severe diarrhea, and hyperthermia (Bakar and Niazi, 1983). Water deprivation causes significant hormonal, physiological (including impaired liver and/or kidney function),

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and biochemical changes in the body (Kim et al., 2002a and references therein). In addition, water deprivation causes reduction in food intake (Tanaka et al., 1986; Kim et al., 2001, 2003) to prevent elevations in extracellular fluid osmolarity and sodium concentration (Kim et al., 2001). The changes in the expressions and/or mRNA levels of CYP isozymes in male Sprague–Dawley rats with water deprivation for 72 h with food (dehydration rat model) were reported; the expressions of CYP1A2, 2B1/2, 2C11, and 3A1/2 were not changed, but the expression and mRNA level of CYP2E1 increased compared with controls (Kim et al., 2001). The CYP2E1 induction in dehydration rat model resulted from less food intake but not from dehydration per se (Kim et al., 2001). Dehydration rat model with glucose supplementation instead of food during 72-h water deprivation (dehydration rat model with glucose supplementation) inhibited the increase in the expression and mRNA level of CYP2E1 in dehydration rat model. Hence, it could be expected that formation of OH–CZX increased in dehydration rat model compared with controls and decreased in dehydration rat model with glucose supplementation compared with that in dehydration rat model. The aim of this paper is to report whether the formation of OH–CZX in dehydration rat model with glucose supplementation decreased compared with that in dehydration rat model with respect to CYP2E1 changes (Kim et al., 2001) after intravenous administration of CZX at a dose of 25 mg/kg. Materials and methods Chemicals CZX, OH–CZX, 3-aminophenyl sulfone (an internal standard of the high-performance liquid chromatographic, HPLC, analysis), D-(+)-glucose (dextrose; corn sugar; minimum, 99.5%), ethylenediamine tetraacetic acid (EDTA), reduced form of β-nicotinamide adenine dinucleotide phosphate (NADPH as a tetrasodium salt), tris(hydroxymethyl) aminomethane (Tris®)-buffer, and β-glucuronidase (Type H-1 from Helix pomatia) were purchased from Sigma-Aldrich Corporation (St. Louis, MO). Other chemicals were of reagent grade or HPLC grade. Rats Male Sprague–Dawley rats (weighing 295–315 g) purchased from the Charles River Company Korea (Orient, Seoul, South Korea) were housed in a light controlled room (light: 0700–1900, dark: 1900–0700) kept at a temperature of 22 ± 2 °C and a relative humidity of 55 ± 5% (Animal Center for Pharmaceutical Research, College of Pharmacy, Seoul National University, Seoul, South Korea). Rats were randomly divided into 3 groups; control, dehydration rat model, and dehydration rat model with glucose supplemented groups. For control group, water and food were supplied ad libitum for 72 h; for dehydration rat model, water was deprived for 72 h with free access to food; and for dehydration rat model with glucose supplemented group, water was deprived for 72 h with free

access to glucose. Water was added to glucose powder and pellet was prepared. After drying, the pellet was supplied instead of food. Based on the previous study (Kim et al., 2001), the maximum effect on CYP2E1 occurred in 48–72 h water deprivation with food and glucose supplementation effect was observed after 72 h water deprivation. Hence, 72-h water deprivation was chosen in the present study. Food or glucose intake and body weight were recorded daily for four days (one day before water deprivation and the first, second, and third days after water deprivation) for each group of rats. Formation of OH–CZX in rat tissue homogenates The procedures were similar (Kim et al., 1999) to those reported previously (Litterst et al., 1975). Approximately 1 g of each liver, kidney, lung, heart, small intestine, and stomach of control rats (n = 5), dehydration rat model (n = 5), and dehydration rat model with glucose supplementation (n = 6) was excised after cervical dislocation, rinsed with cold 0.9% NaCl-injectable solution, blotted dry with tissue paper, and weighed. Metabolic activity was initiated by adding a 150-μl aliquot of 9000 g supernatant fractions of each tissue homogenate (each tissue was homogenized with 4 vol of 0.25 M sucrose; Ultra-Turrax T25; Janke and Kunkel, IKA-Labortechnik, Staufeni, Germany) to an eppendorf tube that contained a 240-μl aliquot of Tris–HCl buffer (pH 7.4), a 10-μl (5 μg; the same solution that was used in the intravenous study) aliquot of CZX, a 50-μl (1 mM) aliquot of NADPH in Tris–HCl buffer, and a 50-μl (5 mM) aliquot of MgCl2 in Tris–HCl buffer. A 50-μl aliquot of methanol that contained an internal standard (3-aminophenyl sulfone; 40 μg/ ml) and a 1-ml aliquot of diethylether (to terminate the enzyme activity) were added after 30-min incubation in a thermomixer (Thermomixer 5436; Eppendorf, Hamburg, Germany) kept at 37 °C and at a rate of 500 oscillations per min (opm). The concentrations of CZX and OH–CZX were measured by the reported HPLC method (Frye and Stiff, 1996). Measurement of Vmax, Km, and CLint for the formation of OH– CZX in hepatic microsomal fractions The procedures were similar to the reported methods (Ahn et al., 2003; Chung et al., 2003; Moon et al., 2003). The livers of control rats (n = 4), dehydration rat model (n = 4), and dehydration rat model with glucose supplementation (n = 5) 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 10,000 g for 30 min and the supernatant fraction was further centrifuged at 100,000 g for 90 min. The microsomal pellet was resuspended in the buffer of 0.154 M KCl/50 mM Tris–HCl in 1 mM EDTA, pH 7.4. Protein contents were measured by the reported method (Bradford, 1976). The Vmax (the maximum velocity) and Km (the Michaelis–Menten constant; the concentration of CZX 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), a 10-μl aliquot of CZX (the same solution that was used in the intravenous study to have substrate concentrations of

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5, 7.5, 10, 15, 20, 100, 200, and 400 μM), a 50-μl (1 mM) aliquot of NADPH (dissolved in 0.1 M Tris–HCl, pH 7.4), and a 50-μl (5 mM) aliquot of MgCl2 (dissolved in 0.1 M Tris–HCl, pH 7.4) in a final volume of 300 μl with 0.1 M Tris–HCl, pH 7.4, in a thermomixer kept at 37 °C and at a rate of 500 opm. All of the above microsomal incubation conditions were linear. A 50-μl aliquot of methanol that contained 40 μg/ml of 3aminophenyl sulfone and a 1-ml aliquot of diethylether (to terminate the enzyme activity) were added after 20-min incubation. The concentrations of OH–CZX were measured by the HPLC method (Frye and Stiff, 1996). The kinetic constants (Km and Vmax) for the formation of OH–CZX were calculated using the nonlinear regression method (Duggleby, 1995). The intrinsic clearance (CLint) for the formation of OH– CZX was calculated by dividing the respective Vmax by the respective Km.

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combined with the 24-h urine. After measuring the exact volume of the combined urine sample, an aliquot of the combined urine sample was stored in a −70 °C freezer until measurement of creatinine level and the HPLC analysis of CZX and OH–CZX (Frye and Stiff, 1996). Other procedures were similar to those reported previously (Kim et al., 2003; Ahn et al., 2003; Chung et al., 2003; Moon et al., 2003). At the same time, as much blood as possible was collected via the carotid artery and then each rat was sacrificed through cervical dislocation. After centrifugation of blood sample, the plasma sample was stored in a − 70 °C freezer for the measurement of creatinine and urea nitrogen levels (analyzed by the Green Cross Reference Lab., Seoul, South Korea). At the same time (24 h), the weight of each liver and kidney was recorded. Small portions of liver and kidney were fixed in a 10% neutral phosphate-buffered formalin and then processed for routine histological examination with hematoxylin–eosin staining.

Pretreatment of rats HPLC analysis of CZX and OH–CZX In the early morning on the fourth day after dehydration, the jugular vein (for drug administration) and the carotid artery (for blood sampling) of each rat were cannulated with polyethylene tube (Clay Adams, Parsippany, NJ) under ketamine anaesthesia [ketamine hydrochloride at dose of 50 mg (1 ml)/kg was administered intramuscularly]. Both cannulas were exteriorized to the dorsal side of the neck where each cannula terminated with a long silastic tube (Dow Corning, Midland, MI). Both silastic tubes were inserted into a wire coil to allow free movement of the rats. After the exposed areas were surgically sutured, each rat was housed individually in a rat metabolic cage (Daejong Scientific Company, Seoul, South Korea) and allowed to recover from anaesthesia for 4–5 h before the study began. They were not restrained during the experimental period. Ketamine was employed instead of diehtylether to minimize the effect of CYP2E1 on the formation of OH–CZX; it has been reported that diehtylether anesthesia alone increased the expression of CYP2E1 by 40% as determined by p-nitrophenol hydroxylase activity (Liu et al., 1993). Intravenous study CZX (dissolved in 0.1 N NaOH and filtered through a 0.45-μm filter) at a dose of 25 mg/kg was administered over 1min via the jugular vein of control rats (n = 9), dehydration rat model (n = 6), and dehydration rat model with glucose supplementation (n = 7). Total injection volume was approximately 1 ml. 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, and 180 min after intravenous administration of CZX. After centrifugation of blood sample, a 50-μl aliquot of each plasma sample was stored in a − 70 °C freezer (Revco ULT 1490 D-N-S; Western Mednics, Asheville, NC) until HPLC analysis of CZX and OH–CZX (Frye and Stiff, 1996). Urine sample was collected between 0 and 24 h. At the end of 24 h, each metabolic cage was rinsed with 20 ml of distilled water and the rinses were

The concentrations of CZX and OH–CZX were analyzed by the reported HPLC method (Frye and Stiff, 1996). Briefly, a 100-μl aliquot of 0.2 M sodium acetate buffer (pH 4.75) that contained 1000 U of β-glucuronidase was added to a 50-μl aliquot of sample. The mixture was vortex-mixed and incubated at 37 °C for 3 h. After incubation, a 1-ml aliquot of diethylether and a 50-μl aliquot of methanol that contained 40 μg/ml of 3-aminophenyl sulfone were added to the mixture. After vortex-mixing for 5 min and centrifugation at 3000 rpm for 5 min, the organic phase was transferred into a clean eppendorf tube and evaporated under a gentle stream of nitrogen gas at 30 °C. The residue was reconstituted in a 150μl aliquot of the mobile phase and a 100-μl aliquot was injected directly onto the HPLC column. The mobile phase, 0.1 M ammonium acetate : acetonitrile : tetrahydrofuran (72 : 22.5 : 5.5; v/v/v), was run at a flow rate of 1 ml/min and the column effluent was monitored by an ultraviolet detector set at 283 nm. Separation was achieved using a reversed-phase (C18) HPLC column (250 mm, l. × 4.6 mm, i. d.; particle size, 5 μm; Symmetry, Waters, Milford, MA). 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 (withinand between-day) were below 8.23%. Pharmacokinetic analysis The total area under the plasma concentration–time curve from time zero to time infinity (AUC) was calculated by the trapezoidal rule-extrapolation method; this method uses 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 datum point to time infinity was estimated by dividing the last measured plasma concentration by the terminal phase rate constant.

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Standard methods (Gibaldi and Perrier, 1982) were used to calculate the following pharmacokinetic parameters using noncompartmental analysis (WinNonlin, Pharsight Corporation, Mountain View, CA); the time-averaged total body (CL) and renal (CLR) 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), and apparent volume of distribution at steady state (Vss) (Kim et al., 1993). The peak plasma concentration (Cmax) and time to reach a Cmax (Tmax) of OH–CZX were read directly from the experimental data. Glomerular filtration rate (GFR) was measured by calculating the creatinine clearance (CLcr) assuming that the kidney function was stable during the experimental period. The CLcr was calculated by dividing the total amount of unchanged creatinine excreted in 24-h urine by the AUC0–24 h of creatinine in plasma. The mean values of each clearance (Chiou, 1980), Vss (Chiou, 1979), and terminal half-life (Eatman et al., 1977) were calculated by the harmonic mean method. Statistical analysis A P value of less than 0.05 was considered to be statistically significant using a Duncan's multiple range test of Statistical Package of Social Sciences (SPSS) a posteriori analysis of

variance (ANOVA) among the three means for the unpaired data. All results were expressed as mean ± SD. Results Body weight, food or glucose intake, and kidney and liver function In control rats, body weight increased with days (mean body weights of 307, 323, 337, and 349 g for before and the first, second, and third days, respectively), but in dehydration rat model, dehydration caused a significant decrease in body weight (the corresponding values of 302, 288, 269, and 252 g). By glucose supplementation (in dehydration rat model with glucose supplementation), mean body weight was not maintained in control level but were similar to those of dehydration rat model (the corresponding values of 300, 284, 269, and 255 g). In control rats, daily food intake (or calorie intake) was almost constant; mean values were 25.8, 26.5, and 28.5 g (93.5, 96.2, and 103 kcal, respectively) for the first, second, and third days, respectively. In dehydration rat model, food intake decreased with days; mean food intakes (or calorie intake) were 17.1, 7.50, and 5.63 g (62.0, 27.2, and 19.0 kcal, respectively) for the first, second, and third days, respectively. In dehydration rat model with glucose supplementation, glucose

Table 1 Mean (±SD) body weight, organ weight, plasma chemistry data, and pharmacokinetic parameters of CZX and OH–CZX after intravenous administration of CZX at a dose of 25 mg/kg to control rats, dehydration rat model, and dehydration rat model with glucose supplementation Parameter

Body weight (g) Initial a Final b Liver weight (% of body weight) Kidney weight (% of body weight) Plasma chemistry Creatinine (mg/dl) Urea nitrogen (mg/dl) CLcr (ml/min/kg) CZX AUC (μg min/ml) Terminal half-life (min) MRT (min) CL (ml/min/kg) Vss (ml/kg) AeCZX, 0–24 h (% of dose of CZX) OH–CZX AUC (μg min/ml) Terminal half-life (min) Cmax (μg/ml) Tmax (min) CLR (ml/min/kg) AeOH–CZX, 0–24 h (% of dose of CZX) a b c d e f

Control rats (n = 9)

Dehydration rat model (n = 6)

Dehydration rat model with glucose supplementation (n = 7)

307 ± 6.18 349 ± 4.86 c 3.33 ± 0.158 0.804 ± 0.0373 c

302 ± 6.83 252 ± 11.7 3.42 ± 0.0940 0.900 ± 0.0647

300 ± 4.08 255 ± 6.45 3.51 ± 0.182 0.942 ± 0.0729

0.589 ± 0.0928 15.7 ± 4.56 2.65 ± 0.642

0.529 ± 0.168 20.8 ± 8.83 3.86 ± 1.78

0.491 ± 0.188 18.8 ± 7.16 2.89 ± 1.32

2990 ± 713 32.6 ± 8.74 e 48.6 ± 11.2 8.37 ± 3.01 404 ± 73.0 0.519 ± 0.756

1810 ± 372 d 25.2 ± 6.67 29.3 ± 5.58 d 13.8 ± 2.90 d 399 ± 104 0.275 ± 0.166

3060 ± 907 44.9 ± 6.54 59.5 ± 12.2 8.15 ± 2.17 482 ± 106 0.142 ± 0.100

1900 ± 187 48.8 ± 11.7 20.6 ± 2.55 d 35.0 ± 22.9 7.55 ± 0.485 c 62.3 ± 14.7 f

1050 ± 163 56.2 ± 11.5 8.08 ± 1.66 41.3 ± 7.50 10.7 ± 1.71 42.7 ± 6.02

733 ± 182 e 49.7 ± 8.60 6.20 ± 1.56 21.0 ± 8.22 16.5 ± 7.52 53.9 ± 9.80

Measured just before starting the 72-h water deprivation. Measured just before starting the experiment. Control rats was significantly different (P < 0.05) from dehydration rat model and dehydration rat model with glucose supplementation. Dehydration rat model was significantly different (P < 0.05) from control rats and dehydration rat model with glucose supplementation. Each value was significantly different (P < 0.05). Dehydration rat model was significantly different (P < 0.05) from dehydration rat model with glucose supplementation.

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intakes (or calorie intake) were 18.3, 10.8, and 9.17 g (73.3, 43.3, and 36.7 kcal, respectively) for the first, second, and third days, respectively. The above data indicate that glucose supplementation did not restore body weight to the control level (Table 1). Although kidney weights were significantly heavier in dehydration rat model and dehydration rat model with glucose supplementation than controls, the plasma levels of creatinine and urea nitrogen, and CLcr were similar (not significantly different) among three groups of rats (Table 1), suggesting that the kidney function seemed not to be impaired considerably by dehydration. This was also supported by kidney microscopy; no significant changes were observed in kidneys of three groups of rats. However, the liver function was impaired in dehydration rat model based on liver microscopy; multifocal hepatocellular necrosis was present in the centrilobular zone. However, no significant findings were observed in livers of control rats and dehydration rat model with glucose supplementation. This data suggest that glucose supplementation could inhibit liver damage caused by dehydration rat model and maintained the liver function to the control level. Formation of OH–CZX in rat tissue homogenates The percentages of the spiked amounts of CZX remaining and OH–CZX formed after incubation of CZX with each rat tissue homogenates are listed in Table 2. Only liver had considerable metabolic activities for the formation of OH–CZX and other rat tissues studied did not have considerable metabolic activities (less than 10% of the spiked amounts of CZX disappeared) for the formation of OH–CZX. The formation of OH–CZX in liver increased significantly in dehydration rat model and dehydration rat model with glucose supplementation compared with controls; the percentages of OH–CZX formed

Table 2 Mean (±SD) percentages of the spiked amount of CZX (5 μg) remaining and OH–CZX formed after 30-min incubation with 9000 g supernatant fraction of the liver, kidney, lung, heart, small intestine, and stomach homogenates in control rats, dehydration rat model, and dehydration rat model with glucose supplementation in the presence of NADPH Tissue

Control rats (n = 5)

Dehydration rat model (n = 5)

Dehydration rat model with glucose supplementation (n = 6)

Liver

56.4 ± 7.64 (30.4 ± 10.9)b 93.3 ± 5.59 94.6 ± 6.54 99.4 ± 0.838 96.3 ± 3.48 94.4 ± 3.06

31.7 ± 7.67a (66.3 ± 15.5) 91.1 ± 5.39 93.9 ± 6.54 95.3 ± 4.15 98.0 ± 4.13 92.6 ± 7.26

47.5 ± 8.16 (55.9 ± 4.92) 92.0 ± 4.44 97.2 ± 4.67 99.9 ± 0.193 97.1 ± 4.30 93.3 ± 2.50

Kidney Lung Heart Small intestine Stomach

The numbers in parentheses represent the formation of OH–CZX expressed in terms of percentages of spiked amount of CZX. a Dehydration rat model was significantly different (P < 0.05) from control rats and dehydration rat model with glucose supplementation. b Control rats was significantly different (P < 0.05) from dehydration rat model and dehydration rat model with glucose supplementation.

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Table 3 Mean (±SD) Km, Vmax, and CLint for the formation of OH–CZX by microsomes prepared from the livers of control rats, dehydration rat model, and dehydration rat model with glucose supplementation Parameter

Control rats (n = 4)

Dehydration rat model (n = 4)

Dehydration rat model with glucose supplementation (n = 5)

1.33 ± 0.272 2.18 ± 0.495 a 1.20 ± 0.373 Vmax (nmol/min/ mg protein) Km (μM) 88.9 ± 20.2 84.5 ± 37.2 70.0 ± 14.3 CLint 0.0150 ± 0.000940 0.0285 ± 0.00888 a 0.0171 ± 0.00325 (ml/min/ mg protein) a Dehydration rat model was significantly different (P < 0.05) from control rats and dehydration rat model with glucose supplementation.

(expressed in terms of spiked amount of CZX) per g liver after 30-min incubation of 5 μg of CZX were 30.4, 66.3, and 55.9% for control rats, dehydration rat model, and dehydration rat model with glucose supplementation, respectively. OH–CZX was only detected in the liver and was below the detection limit in other rat tissues studied, suggesting that the formation of OH– CZX in rat tissues studied was almost negligible except liver. Measurement of Vmax, Km, and CLint for the formation of OH– CZX in hepatic microsomal fractions The Vmax, Km, and CLint for the formation of OH–CZX in microsomes prepared from the livers of control rats, dehydration rat model, and dehydration rat model with glucose supplementation are listed in Table 3. The Vmax in dehydration rat model was significantly faster than those in control rats and dehydration rat model with glucose supplementation; the value in dehydration rat model was 63.9 and 81.7% increased compared with those in control rats and dehydration rat model with glucose supplementation, respectively. The Vmax values were not significantly different between control rats and dehydration rat model with glucose supplementation. The Km values were not significantly different among three groups of rats. As a result, the CLint in dehydration rat model was significantly faster than those in control rats and dehydration rat model with glucose supplementation; the value in rats with dehydration with food was 90.0 and 66.7% increased compared with those in control rats and dehydration rat model with glucose supplementation, respectively. The CLint values were not significantly different between control rats and dehydration rat model with glucose supplementation. The above data suggest that the formation of OH–CZX in dehydration rat model was significantly faster than those in control rats and dehydration rat model with glucose supplementation. Pharmacokinetics of CZX and OH–CZX after intravenous administration of CZX The mean arterial plasma concentration–time profiles of CZX and OH–CZX after intravenous administration of CZX

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at a dose of 25 mg/kg to control rats (n = 9), dehydration rat model (n = 6), and dehydration rat model with glucose supplementation (n = 7) are shown in Fig. 1, and some relevant pharmacokinetic parameters are also listed in Table 1. After intravenous administration of CZX in dehydration rat model, the AUC of CZX was significantly smaller (39.5 and 40.8% decrease than those in control rats and dehydration rat model with glucose supplementation, respectively), CL of CZX was significantly faster (64.9 and 69.3% increase, respectively), and MRT of CZX was significantly shorter (39.7 and 50.8% decrease, respectively) than those in control rats and dehydration rat model with glucose supplementation; the values were not significantly different between control rats and dehydration rat model with glucose supplementation. The terminal half-life of CZX in dehydration rat model was significantly shorter than those in control rats and dehydration rat model with glucose supplementation; the values were significantly different among three groups of rats. After intravenous administration of CZX, the formation of OH–CZX was rapid for all three groups of rats; OH–CZX was detected in plasma from the second blood sampling time (5 min) and reached its peak (Cmax) rapidly at 21–41 min (Tmax). The AUC of OH–CZX in dehydration rat model was significantly greater than those in control rats (159% increase) and dehydration rat model with glucose supplementation (81.0% increase); each group was significantly different. The AeOH–CZX, 0–24 h in dehydration rat model was greater than that in control rats (15.6% increase, P < 0.207) and significantly greater than that in dehydration rat model with glucose supplementation (45.9% increase). However, the CLR of OH–CZX in dehydration rat model was significantly slower than that in control rats (54.2% decrease) due to significantly greater AUC of OH–CZX in the rats.

Discussion It was reported that the expression and mRNA level of CYP2E1 increased in dehydration rat model compared with controls, and this might result from the decrease in food consumption, but not from dehydration per se (Kim et al., 2001, 2003). However, dehydration rat model with glucose supplementation inhibited the induction of CYP2E1 in dehydration rat model (Kim et al., 2001). CZX was metabolized to OH–CZX catalyzed mainly via the CYP2E1 in rats (Rockich and Blouin, 1999; Ahn et al., 2003; Chung et al., 2003; Moon et al., 2003). Hence, it could be expected that the formation of OH–CZX in dehydration rat model increases compared with controls. This was proven by the following results. In dehydration rat model, the AUC of OH–CZX was significantly greater, AUCOH–CZX/ AUCCZX ratio was considerably greater (105 versus 24.5%), Cmax of OH–CZX was significantly higher, AeOH–CZX, 0–24 h was significantly greater (Table 1), and Vmax and CLint for the formation of OH–CZX were significantly faster (Table 3) than controls. Moreover, it could also be expected that the formation of OH–CZX in dehydration rat model with glucose supplementation decreases compared with that in dehydration rat model. This was proven by the following results. In dehydration rat model with glucose supplementation, the AUC of OH–CZX was significantly smaller, AUCOH–CZX/AUCCZX ratio was considerably smaller (34.3 versus 105%), Cmax of OH–CZX was significantly lower, AeOH–CZX, 0–24 h was significantly smaller (Table 1), and Vmax and CLint for the formation of OH–CZX were significantly slower (Table 3) than those in dehydration rat model. The formation of OH–CZX was considerable in rat liver tissue homogenates, however, was almost negligible in other rat tissue homogenates studied (Table 2). It was also reported (Rockich and Blouin, 1999; Ahn et al., 2003; Chung et al., 2003; Moon et al.,

Fig. 1. Mean arterial plasma concentration–time profiles of CZX (A) and OH–CZX (B) after 1-min intravenous administration of CZX at a dose of 25 mg/kg to control rats (●, n = 9), dehydration rat model (▴, n = 6), and dehydration rat model with glucose supplementation (○, n = 7). Bar represents SD.

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2003) that OH–CZX was formed mainly via the CYP2E1 in rats. Hence, it could be expected that rat liver is a main organ for the formation of OH–CZX. The hepatic extraction ratio (the hepatic first-pass effect) of CZX was roughly estimated in control rats by dividing the CL of CZX (the CL of CZX is almost equal to the CLNR of CZX) by the reported hepatic plasma flow rate in rats assuming that the CL of CZX was attributed solely to the liver (Lee and Chiou, 1983). Hence, the estimated hepatic extraction ratio represents the maximal possible value of liver (Lee and Chiou, 1983). The hepatic plasma flow rate in rats was estimated based on the reported hepatic blood flow rate of 30.4 ml/min/kg (Davies and Morris, 1993) and hematocrit of 45.2% in control rats (Kim et al., 2003). The estimated hepatic extraction ratio of CZX in control rats was approximately 50.2% and the similar value, 53.6%, was also estimated in other rat studies (Moon et al., 2003). Since, CZX is an intermediate hepatic extraction ratio drug, the hepatic clearance of CZX depends on the intrinsic clearance (CLint), free (unbound to plasma proteins) fractions of CZX in plasma, and hepatic blood flow rate (Wilkinson and Shand, 1975). In dehydration rat model, the significantly greater AUC of OH– CZX (significantly faster CL of CZX) (Table 1) could be supported by the significantly faster CLint (90.0% increase; Table 3) and greater free fractions of CZX (15.3% increase; Kim et al., 2003), since hepatic blood flow rate was slower in dehydration rat model (Bencsath and Takacs, 1966). Although CYP2E1 is the major form metabolizing CZX to OH–CZX, it was reported (Carriere et al., 1993; Ono et al., 1995; Gorski et al., 1997) that human CYP1A2 and 3A4 are involved in CZX hydroxylation. In dehydration rat model, no significant changes in the expression of CYP1A2 and 3A1/2 were observed (Kim et al., 2001). Human CYP3A4 and rat CYP3A1 proteins have 73% homology (Lewis, 1996). Hence, the role of CYP1A2 and 3A1/2 on the increased formation of OH–CZX in dehydration rat model could be negligible. Other rat studies showed that glucose and growth hormone in association with insulin seemed to be involved in the regulation of expression of CYP2E1 (Son et al., 2000). CYP2E1 was induced by hypophysectomy. Hypophysectomyzed rats showed a 20% reduction in plasma glucose level and glucose supplementation prevented the induction of CYP2E1. Human growth hormone treatment also inhibited the increase in the expression of CYP2E1 in hypophysectomyzed rats. However, human growth hormone failed in the restoration of the expression of CYP2E1 in starving hypophysectomyzed rats, while glucose supplementation abolished the induction of CYP2E1 in those rats. Expression of major CYP isozymes was not affected by water deprivation, whereas CYP2E1 induction observed in this study results from the reduction in food intake, but not from dehydration per se (Kim et al., 2001). Glucose supplementation restored CYP2E1 expression during water deprivation. CYP2E1 was induced with a paralleled elevation of the mRNA level (Kim et al., 2001; Hong et al., 1987). Induction of CYP2E1 by water deprivation would be due to reduction of food intake and presumably due to a subsequent decrease in glucose utilization. This was supported by the observation that glucose feeding prevented CYP2E1 induction during water deprivation. Other factors such as growth hormones would also be involved in the

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regulation of expression of CYP2E1 (Peng and Coon, 1998). The prevention of induction of CYP2E1 by human growth hormone is due to improved glucose utilization. Thus, glucose utilization is an important factor in the expression of CYP2E1. In summary, the formation of OH–CZX decreased significantly in dehydration rat model with glucose supplementation compared with that in dehydration rat model due to inhibition of the induction of CYP2E1 by glucose supplementation. The pharmacokinetic studies of OH–CZX in control rats and dehydration rat model are required to confirm the present pharmacokinetic differences of OH–CZX after intravenous administration of CZX to control rats and rat model of dehydration. Acknowledgement This study was supported by Research Fund by Kangnung National University in 2002. References Ahn, C.Y., Kim, E.J., Lee, I., Kwon, J.W., Kim, S.G., Lee, M.G., 2003. Effect of glucose on the pharmacokinetics of intravenous chlorzoxazone in rats with acute renal failure induced by uranyl nitrate. Journal of Pharmaceutical Sciences 92 (8), 1604–1613. Bakar, S.K., Niazi, S., 1983. Effect of water deprivation on aspirin disposition kinetics. Journal of Pharmaceutical Sciences 72 (9), 1030–1034. Bencsath, P., Takacs, L., 1966. Effect of dehydration on cardiac output and organ flow in anaesthetized and unanaesthetized rats. Acta Medica Academiae Scientiarum Hungaricae 22 (3), 275–282. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Carriere, V., Goasduff, T., Ratanasavanh, D., Morel, F., Gautier, J.C., Guillouza, A., Beaune, P., Berthou, F., 1993. Both cytochrome P450 2E1 and 1A1 are involved in the metabolism of chlorzoxazone. Chemical Research in Toxicology 6 (6), 852–857. Chen, L., Yang, C.S., 1996. Effects of cytochrome P450 2E1 modulators on the pharmacokinetics of chlorzoxazone and 6-hydroxychlorazoxazone in rats. Life Sciences 58 (18), 1575–1585. Chiou, W.L., 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. Journal of Pharmacokinetics and Biopharmaceutics 6 (6), 539–546. Chiou, W.L., 1979. New calculation method of mean apparent drug volume of distribution and application to rational dosage regimen. Journal of Pharmaceutical Sciences 68 (8), 1067–1069. Chiou, W.L., 1980. New calculation method of mean total body clearance of drugs and its application to dosage regimens. Journal of Pharmaceutical Sciences 69 (1), 90–91. Chung, W.-S., Kim, E.J., Lee, I., Kim, S.G., Lee, M.G., Kim, S.H., 2003. Effects of recombinant human growth hormone on the pharmacokinetics of intravenous chlorzoxazone in rats with acute renal failure induced by uranyl nitrate. Life Sciences 73 (3), 253–263. Conney, A.H., Burns, J.J., 1960. Physiological disposition and metabolic fate of chlorzoxazone (Paraflex) in man. Journal of Pharmacology and Experimental Therapeutics 128, 340–343. Davies, B., Morris, T., 1993. Physiological parameters in laboratory animals and humans. Pharmaceutical Research 10 (7), 1093–1095. Desiraju, R.K., Renzi Jr., N.L., Nayak, R.K., Ng, K.T., 1983. Pharmacokinetics of chlorzoxazone in humans. Journal of Pharmaceutical Sciences 72 (9), 991–994. Duggleby, R.G., 1995. Analysis of enzyme progress curves by nonlinear regression. Methods in Enzymology 249, 61–90.

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