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Effects of Glucose on the Pharmacokinetics of Intravenous Chlorzoxazone in Rats with Acute Renal Failure Induced by Uranyl Nitrate
Effects of Glucose on the Pharmacokinetics of Intravenous Chlorzoxazone in Rats with Acute Renal Failure Induced by Uranyl Nitrate CHOONG Y. AHN,1 EUN J. KIM,1 INCHUL LEE,2 JONG W. KWON,3 WON B. KIM,3 SANG G. KIM,1 MYUNG G. LEE1 1
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, South Korea
2
Department of Diagnostic Pathology, College of Medicine, University of Ulsan, Asan Foundation, Asan Medical Center, Seoul, South Korea
3
Research Laboratory, Dong-A Pharmaceutical Company, Ltd., Yongin, South Korea
Received 28 January 2003; accepted 8 March 2003
ABSTRACT: The effects of glucose on CYP2E1 expression in rats with acute renal failure induced by uranyl nitrate (U-ARF) have been reported. CYP2E1 was significantly induced (2.3-fold) in rats with U-ARF compared with that in control rats. In contrast, CYP2E1 expression was significantly decreased in rats with U-ARF supplied with glucose (dissolved in tap water to make 10%, w/v) in their drinking water for 5 days (U-ARFG) compared with that in rats with U-ARF. However, CYP2E1 in rats with U-ARFG was significantly greater than that in control rats. Chlorzoxazone (CZX) primarily undergoes hydroxylation, catalyzed mainly by CYP2E1, to form 6-hydroxychlorzoxazone (OH-CZX) rats. Hence, it could be expected that in rats with U-ARFG, formation of OH-CZX could significantly decrease and increase compared with those in rats with U-ARF and control rats, respectively. This expectation is proven by the following results of a study of intravenous administration of CZX at a dose 20 mg/kg to control rats and rats with U-ARF and U-ARFG. First, the total area under the plasma concentration–time curve from time zero to 8 h (AUC0–8 h) of OH-CZX in rats with U-ARFG (8730 mg min/mL) was significantly greater than that in control rats (414 mg min/mL) and significantly smaller than that in rats with U-ARF (11500 mg min/mL). Second, the AUC0–8 h, OH-CZX/AUCCZX ratio in rats with U-ARFG (10.0) was significantly greater than that in control rats (0.252) and significantly smaller than that in rats with U-ARF (17.5). Finally, the in vitro intrinsic OH-CZX formation clearance (CLint) in rats with U-ARFG (27.9 mL/min/mg protein) was significantly slower than that in rats with U-ARF (36.7 mL/min/mg protein) and significantly faster than that in control rats (17.7 mL/min/mg protein). ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1604–1613, 2003
Keywords:
CZX; OH-CZX; U-ARF; glucose; pharmacokinetics; rats
INTRODUCTION Chlorzoxazone [5-chloro-2(3H)-benzoxazolone, CZX], once used as a skeletal muscle relaxant for the treatment of painful muscle spasms,1 Correspondence to: Myung G. Lee (Telephone: 82-2-8807855 (7877); Fax: 82-2-889-8693; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 1604–1613 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association
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primarily undergoes hydroxylation [catalyzed mainly by hepatic microsomal cytochrome P450 (CYP) 2E12,3] to form 6-hydroxychlorzoxazone (OH-CZX). OH-CZX is then rapidly glucuronidated and excreted in urine.2,4 CZX has been suggested for use as a chemical probe to assess the activity of CYP2E1 in vitro and in vivo because of the good correlation between the formation rate of OH-CZX and the CYP2E1 activity in rats.5
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It has been reported from our laboratories6 that in rats with acute renal failure induced by uranyl nitrate (U-ARF), CYP2C11 decreased to 20% of control, CYP2E1 and CYP3A23 increased 2.3 and 4 times, respectively, compared with control, and CYP1A2 and CYP2B1/2 were not changed compared with control based on Western and Northern blot analyses. However, the production of CYP2E1 was reduced in rats given glucose (dissolved in tap water to make 10%, w/v) in the drinking water for 5 days with U-ARF (U-ARFG) compared with that in rats with U-ARF alone. The CYP2E1 protein and mRNA level in rats with U-ARFG were significantly higher than those in control rats, but significantly lower than those in rats with U-ARF.7 It has also been reported that the induced CYP2E1 in rats with hypophysectomy8 and rats with 72-h dehydration9 was reduced significantly by treatment with glucose. Based on these CYP2E1 results, it could be expected that in rats with UARFG, formation of OH-CZX could decrease significantly compared with that in rats with U-ARF, but increase significantly compared with that in control rats. Changes in the pharmacokinetics of many drugs (either mainly eliminated via renal excretion or by hepatic metabolism) have been reported in rats with U-ARF. For example, the total area under the plasma concentration–time curve from time zero 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 an intravenous administration of the following drugs (or compounds) to rats with U-ARF;6 methotrexate, vancomycin, DA-1131 (a new carbapenem), M1 (an active component of a new anthracycline, DA-125), salicylic acid, azosemide, diltiazem, amiodarone, tetraethylammonium bromide, and para-aminohippurate. Pharmacokinetic differences of the aforementioned drugs (or compounds) were mainly explained by impaired kidney and/or liver function,6 or increased formation of conjugates10 in rats with U-ARF. However, the changes in the pharmacokinetics of drugs (except CZX) in rats with U-ARF are not explained with regard to CYP isozyme changes.6 Moreover, the effects of glucose on the pharmacokinetics of drugs in rats with U-ARF have not been published. The aim of this study is to report whether formation of OH-CZX in rats with U-ARFG increased significantly compared with that in control rats and decreased significantly compared with that in rats with UARF with respect to CYP2E1 changes.
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MATERIALS AND METHODS Chemicals Chlorzoxazone [CZX; 5-chloro-2(3H)-benzoxazolone], the reduced form of b-nicotinamide adenine dinucleotide phosphate (NADPH, as a tetrasodium salt), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer, and b-glucuronidase (Type H-1, from Helix pomatia) were purchased from Sigma Chemical Company (St. Louis, MO). OH-CZX and 3-aminophenyl sulfone (an internal standard of high-performance liquid chromatographic [HPLC] assay for CZX and OHCZX) were obtained from Research Biomedical International (Natick, MA) and Aldrich Chemical Company (Milwaukee, WI), respectively. Uranyl nitrate was a product from BDH Chemicals (Poole, England). Ketamine hydrochloride was purchased from Yuhan Research Center of Yuhan Corporation (Kunpo, South Korea). Other chemicals were of reagent grade or HPLC grade, and therefore, were used without further purification. Animals Male Sprague–Dawley rats (weighing 245–280 g) were purchased from Charles River Company Korea (Biogenomics, Seoul, South Korea). Rats were housed in metabolic cages (Tecniplast, Varese, Italy) under the supply of filtered pathogen-free air. The rats were randomly divided into three groups; control rats and rats with U-ARF without (U-ARF) or with (U-ARFG) a supply of glucose for 5 days. All rats were provided with food (Sam Yang Company, Seoul, South Korea) and water ad libitum and maintained in a lightcontroled room (light: 0700–1900, dark: 1900– 0700) kept at a temperature of 22 28C and a relative humidity of 55 5% (Animal Center for Pharmaceutical Research, College of Pharmacy, Seoul National University, Seoul, South Korea). 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%) at a dose of 5 mg/ kg was injected once via the tail vein of each rat (total injection volume was 1 mL/kg) to induce acute renal failure.6,11 Control rats were injected with the same volume of 0.9% NaCl-injectable solution. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
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Supply of Glucose in Rats with U-ARF (Rats with U-ARFG) Rats with U-ARF were divided into two groups, with (U-ARFG) or without (U-ARF) glucose supplementation. Rats with U-ARFG received glucose (dissolved in tap water to make 10%, w/v) in drinking water for 5 days. Measurement of Vmax, Km, and CLint for the Formation of OH-CZX in Hepatic Microsomes On the fifth day, the livers of control rats (n ¼ 5) and rats with U-ARF (n ¼ 4) and U-ARFG (n ¼ 5) were homogenized in an ice-cold, pH 7.4 buffer of 0.154 M KCl/50 mM Tris-HCl in 1 mM ethylenediamine tetraacetate (EDTA). The homogenate was centrifuged at 9700 g for 30 min, and the supernatant fraction was further centrifuged at 100,000 g for 90 min. The microsomal pellet was resuspended in a pH 7.4 buffer of 0.154 M KCl/50 mM Tris-HCl in 1 mM EDTA. Protein content was measured using the reported method.12 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 aforementioned microsomal fraction (equivalent to 0.5 mg protein), a 10-mL aliquot of CZX (to have substrate concentrations of 1, 1.25, 2, 5, 10, 50, 100, and 200 mM), and 1 mM of NADPH in a final volume of 1 mL by adding 0.1 M Tris-HCl, pH 7.4, in a water-bath shaker kept at 378C and at a rate of 50 oscillations per min (opm). All of the aforementioned microsomal incubation conditions were linear. The reaction was terminated by the addition of 1 mL of tert-butyl methyl ether after a 20-min incubation. The OH-CZX formed was determined by HPLC analysis.13 The kinetic constants (Km and Vmax) for the formation of OH-CZX were calculated using the Lineweaver–Burk plot14 by the linear regression and the method of least squares. Intrinsic OH-CZX formation clearance (CLint) was calculated by dividing the Vmax by the Km. Pretreatment of Rats In the early morning on the fifth day after a single intravenous administration of uranyl nitrate or 0.9% NaCl-injectable solution, the jugular vein (for drug administration) and the carotid artery (for blood sampling) of each rat were cannulated with polyethylene tubing (Clay Adams, ParsipJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
pany, NJ) while each rat was under ketamine hydrochloride anesthesia [ketamine hydrochloride at a dose of 100 mg/kg was administered (total injection volume was 1 mL/kg) intramuscularly]. Ketamine was employed instead of ether to minimize the effect of CYP2E1 on the formation of OH-CZX; it has been reported15 that ether anesthesia alone increased expression of CYP2E1 by 40% as determined by p-nitrophenol hydroxylase activity. Both cannulae were exteriorized to the dorsal side of the neck, where each cannula terminated with long silastic tubing (Dow Corning, Midland, MI). Both silastic tubings were inserted into a wire coil to allow free movement of the rat. Each rat was not restrained during the experimental period. After surgical suture, each rat was housed individually in a rat metabolic cage (Daejong Scientific Company, Seoul, South Korea). Experimentation was started after 5–6 h of recovery from the anesthesia. Intravenous Study CZX (the CZX powder was dissolved in a minimum amount of 1 N NaOH) at a dose of 20 mg/kg was administered by an intravenous infusion over a 1-min via the jugular vein (total injection volume was 0.6 mL) of control rats (n ¼ 11) and rats with U-ARF (n ¼ 16) and U-ARFG (n ¼ 15). An 0.22-mL aliquot sample of blood was collected via the carotid artery at 0 (to serve as a control), 1 (at the end of the infusion), 5, 15, 30, 60, 120, 180, 240, 360, and 480 min after an intravenous administration of the drug. After centrifugation, two 0.05-mL aliquots samples of plasma (each plasma sample was analyzed with and without addition of b-glucuronidase) were stored in a 708C freezer (Revco ULT 1490 D-N-S; Western Mednics, Asheville, NC) until HPLC analysis of CZX and OH-CZX.13 A 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 24 h, as much blood as possible was collected via the carotid artery and each rat was sacrificed by cervical dislocation. Plasma samples were stored in a 708C freezer for the measurement of urea nitrogen, glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), creatinine, and glucose levels (analyzed by Green Cross Reference Lab., Seoul, South Korea) and plasma protein binding. The whole kidney and liver of each rat were excised, rinsed with 0.9% NaCl-injectable
GLUCOSE EFFECT ON CHLORZOXAZONE PHARMACOKINETICS IN RATS
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-eosin staining. Urine samples were collected between 0 and 24 h. Each metabolic cage was rinsed with 10 mL of distilled water at the end of 24 h, and the rinsings were combined with the urine samples. After measuring the exact volume of combined urine samples, two 0.05-mL aliquots of the combined urine samples (each urine sample was also analyzed with and without addition of b-glucuronidase) were stored in the freezer until HPLC analysis of CZX and OH-CZX.13
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the column effluent. The retention times of OHCZX, 3-aminophenyl sulfone, and CZX were 6, 10, and 18 min, respectively. The detection limits of CZX and OH-CZX in plasma were both 0.1 mg/ mL. The coefficients of variation of the assay (within- and between-day) were generally low (<8.2%). For the biological samples of rats with U-ARF and U-ARFG, the mobile phase was changed to 0.05 M ammonium acetate/acetonitrile (4:1, v/v) to eliminate endogenous interference peak(s) in HPLC chromatogram from the biological sample. The flow rate of the mobile phase was 1 mL/min. The retention times of OH-CZX, 3-aminophenyl sulfone, and CZX were 14, 22, and 40 min, respectively. In the present study, plasma and urine samples were analyzed with and without addition of b-glucuronidase.
Measurement of Plasma Protein Binding The binding of CZX (at a CZX concentration of 10 mg/mL) to plasma protein of control rats and rats with U-ARF and U-ARFG was determined by an equilibrium dialysis technique.16 After 24 h of incubation, a 0.1-mL aliquot was removed from each compartment and stored in a 708C freezer until HPLC analysis of CZX.13
HPLC Analysis of CZX and OH-CZX The concentrations of CZX and OH-CZX in the aforementioned biological samples of control rats were analyzed by the reported HPLC method.13 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 b-glucuronidase were added to a 0.05-mL aliquot of biological sample. The mixture was manually mixed and incubated for 2 h in a water-bath shaker kept at 378C and at a rate of 50 opm. After incubation, a 0.05-mL aliquot of methanol containing 40 mg/mL of 3-aminophenyl sulfone (the internal standard) was added and mixed by vortex, and a 1-mL aliquot of tert-butyl methyl ether was added. The mixture was shaken for 10 min and then centrifuged at 2000 g for 10 min. The upper organic layer was transferred to a clean tube and evaporated at 378C under a stream of nitrogen. The residue was reconstituted in a 100-mL aliquot of the mobile phase, and a 50-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/v), was run at a flow rate of 1 mL/min. An ultraviolet (UV) detector set at 283 nm monitored
Pharmacokinetic Analysis The AUC of CZX and AUC0–8 h of OH-CZX were calculated by the trapezoidal rule method; this method utilized the logarithmic trapezoidal rule17 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 (for the measurement of AUC of CZX) was estimated by dividing the last measured plasma concentration by the terminal rate constant. Standard methods18 were used to calculate the CL, CLR, CLNR, area under the first moment of the plasma concentration–time curve (AUMC), mean residence time (MRT), and apparent volume of distribution at steady state (VSS).19 The harmonic mean method was used to calculate the each mean value of VSS,20 terminal half-life,21 and each clearance.22 Glomerular filtration rate was calculated by measuring the creatinine clearance (CLCR), assuming that kidney function was stable during 24 h. The CLCR was calculated by dividing the total amount of creatinine excreted in 24-h urine by AUC0–24 h of creatinine in plasma.
Statistical Analysis A p value of <0.05, determined with a Duncan’s multiple range test of SPSS posteriori analysis of variance (ANOVA), was considered to be statistically significant. All results were expressed as mean standard deviation (SD). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
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RESULTS Induction of Acute Renal Failure in Rats Impaired kidney function was observed in rats with U-ARF; serum levels of urea nitrogen (7.59 times increase) and creatinine (10.2 times increase) were significantly higher, kidney weight (% of body weight) was significantly heavier (24.8% increase), and CLCR was significantly slower (99.9% decrease) than those in control rats (Table 1). Impaired kidney function in rats with U-ARF was also supported by kidney microscopy; there was extensive tubular necrosis involving distal tubules. Impaired kidney function in rats with U-ARF has also been reported elsewhere.6,11 However, no significant findings were indicated by kidney microscopy from control rats. Note that the aforementioned parameters were not significantly different between rats with U-ARF and U-ARFG (Table 1), suggesting that kidney function in rats with U-ARF was not recovered to control by treatment with glucose for 5 days. Impaired kidney function in rats with U-ARFG was also supported by kidney microscopy; there was extensive tubular necrosis involving distal tubules, and the differences between rats with U-ARF and U-ARFG were not significant. Unlike kidney function, liver function seemed not to be impaired considerably by treatment with uranyl nitrate; the plasma level of GOT was significantly higher (87.5% increase) in rats with U-ARF than that in control rats, however, the values were not significantly different between U-ARF and
U-ARFG (Table 1). This result could also be supported by liver microscopy; no significant findings were found for the three groups of rats. Note that body weight gain decreased significantly in rats with U-ARF (from 268 to 249 g) and U-ARFG (from 264 to 248 g) compared with that in control rats (from 260 to 294 g; Table 1). This result indicates that 5 days supply with glucose did not increase the body weight gain reduced by U-ARF.
Measurement of Vmax, Km, and CLint for the Formation of OH-CZX in Hepatic Microsomes In rats with U-ARF, the Vmax (104% 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 intrinsic OH-CZX formation clearance (CLint) in hepatic microsomal fraction was significantly faster (107% increase) in rats with UARF than that in control rats (Table 2). In rats with U-ARFG, CLint was significantly faster (57.6% increase) and slower (24.0% decrease) than those in control rats and rats with U-ARF, respectively (Table 2). These data suggest that in rats with U-ARFG, formation of OH-CZX could increase and decrease compared with those in control rats and rats with U-ARF, respectively. The liver weight and protein content were not significantly different among three groups of rats (Table 2).
Table 1. Plasma Values and Organ Weight in Control Rats and Rats with U-ARF and U-ARFGa Parameter
Control (n ¼ 11)
U-ARF (n ¼ 16)
Plasma Urea nitrogen (mg/dL) GOT (IU/L) GPT (IU/L) Creatinine (mg/dL) Glucose (mg/dL) CLCR (mL/min/kg) Initial body weightc (g) Final body weightd (g) Kidney weight (% of body weight) Liver weight (% of body weight)
17.0 3.78b 128 37.8b 50.1 11.8 0.491 0.114b 148 23.8 4.20 0.633b 260 17.6 294 14.2b 0.795 0.0528b 3.79 0.341
146 29.3 240 38.9 51.9 13.0 5.51 1.42 146 29.3 0.00438 0.0103 268 8.50 249 13.1 0.992 0.109 3.34 0.589
U-ARFG (n ¼ 15) 151 39.6 182 58.2 40.4 10.4 5.81 1.55 167 43.5 0.0156 0.0669 264 7.45 248 23.9 1.07 0.0149 3.17 0.129
a Values expressed as mean SD. Abbreviations: U-ARF, acute renal failure induced by uranyl nitrate; U-ARFG, U-ARF with glucose supplementation; GOT, glutamate oxaloacetate transaminase; GPT, glutamate pyruvate transaminase; IU, international unit; CLCR, creatinine clearance. b Control group was significantly different (p < 0.05) from U-ARF and U-ARFG groups. c Measured just before the injection of uranyl nitrate or 0.9% NaCl-injectable solution. d Measured just before the experiment.
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Table 2. Km, Vmax, and CLint Values for the Formation of OH-CZX by Microsomes Prepared from Livers of Control Rats and Rats with U-ARF and U-ARFGa Parameter Vmax (nmol/min/mg protein) Km (mM) CLint (mL/min/mg protein) Liver weight (% of body weight) Protein content (mg/g liver)
Control (n ¼ 5)
U-ARF (n ¼ 4)
U-ARFG (n ¼ 5)
2.47 1.27b 146 87.4 17.7 4.33c 4.66 0.161 29.6 3.72
5.04 1.51 150 76.4 36.7 6.01 3.54 0.319 29.0 2.31
3.55 2.12 121 67.8 27.9 4.05 3.55 0.957 31.3 3.25
a
Values expressed as mean SD. Abbreviations: Vmax, maximum velocity; Km, Michaelis–Menten constant, the concentration at which the rate is one-half of Vmax; CLint, intrinsic OH-CZX formation clearance; U-ARF, acute renal failure induced by uranyl nitrate; U-ARFG, U-ARF with glucose supplementation. b Control group was significantly different (p < 0.05) from U-ARF group. c Each group was significantly different (p < 0.05).
Pharmacokinetics after Intravenous Administration of CZX The mean arterial plasma concentration–time profiles of CZX and OH-CZX after intravenous administration of CZX at a dose of 20 mg/kg to three groups of rats are shown in Figure 1 and some relevant pharmacokinetic parameters are listed in Table 3. After intravenous administration of CZX, the plasma concentrations of CZX declined in a polyexponential fashion for all three groups of rats (Figure 1A), with mean terminal
half-lives of 24.8, 15.4, and 17.5 min for control rats and rats with U-ARF and U-ARFG, respectively; the value in control rats was significantly longer than those in rats with U-ARF and UARFG (Table 3). Similar trends were also obtained for MRTs (Table 3). The plasma concentrations of CZX were the highest and lowest for control rats and rats with U-ARF, respectively (Figure 1A). As a result, the AUC was the greatest and lowest for control rats and rats with U-ARF, respectively; the value in control rats (1640 mg min/mL) was significantly greater than those in rats with U-
Table 3. Pharmacokinetic Parameters of CZX and OH-CZX after Intravenous Administration of CZX at a Dose of 20 mg/kg to Control Rats and Rats with U-ARF and U-ARFGa Parameter CZX AUC (mg min/mL) Terminal half-life (min) MRT (min) CL (mL/min/kg) CLR (mL/min/kg) CLNR (mL/min/kg) VSS (mL/kg) Plasma protein binding (%) Ae0–24 h (% of CZX dose) OH-CZX AUC0–8 h (mg min/mL) CLR (mL/min/kg) Ae0–24 h (% of CZX dose) Cmax (mg/mL) Tmax (min)
Control (n ¼ 11) 1640 631b 24.8 5.67b 32.4 12.1b 12.2 6.05c 0.0948 0.737 11.4 6.27d 307 301 68.5 6.86 4.96 5.04 414 97.3c 20.8 14.2b 47.9 13.1b 4.34 1.30c 19.5 8.79b
U-ARF (n ¼ 16) 659 140 15.4 3.27 12.7 3.20 30.3 7.72 NM 366 165 71.8 3.01 BD 11500 3380 0.00805 0.176 10.9 11.1 40.5 10.5 72.0 25.3
U-ARFG (n ¼ 15) 872 327 17.5 6.14 15.8 4.25 22.9 7.61 0.0307 0.264 23.3 5.50 339 153 63.4 7.05 0.638 0.738 8730 2310 0.135 0.139 7.86 8.40 28.1 4.76 64.0 15.5
a
Values expressed as mean SD. Abbreviations: CZX, chlorzoxazone; OH-CZX, 6-hydroxychlorzoxazone; U-ARF, acute renal failure induced by uranyl nitrate; U-ARFG, U-ARF with glucose suplementation; AUC, area under the plasma concentration–time curve from time zero to time infinity; MRT, mean residence time; CL, time-averaged total body clearance; CLR, time-averaged renal clearance; CLNR, time-averaged nonrenal clearance; VSS, apparent volume of distribution at steady state; Ae0–24 h, total amount excreted in urine as unchanged drug from time zero to 24 h; AUC0–8 h, total area under the plasma concentration– time curve from time zero to 8 h; Cmax, maximum plasma concentration; Tmax, time to reach Cmax; NM, not measurable; BD, below detection limit. b Control group was significantly different (p < 0.05) from U-ARF and U-ARFG groups. c Each group was significantly different (p < 0.05). d Control group was significantly different (p < 0.05) from U-ARFG. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
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peaks at 19.5–72.0 min (Figure 1B). The peak plasma concentration of OH-CZX was maintained for up to 8 h in rats with U-ARF and U-ARFG (Figure 1B). However, in control rats, the peak plasma concentration of OH-CZX reached its peak at 19.5 min and decreased for up to 4 h, and the decreased concentrations were maintained for up to 8 h (Figure 1B). The plasma concentrations and Cmax of OH-CZX were the highest and lowest for rats with U-ARF and control rats, respectively (Figure 1B). As a result, the values of AUC0–8 h of OH-CZX were significantly different among three groups of rats; the value in rats with U-ARFG was significantly greater (20.1 times increase) and smaller (24.1% decrease) than those in control rats and rats with U-ARF, respectively (Table 3). The CLR of OH-CZX was significantly faster in control rats (20.8 mL/min/kg) than those in rats with U-ARF (0.00805 mL/min/kg) and U-ARFG (0.135 mL/min/kg). Similar results were also obtained for the total amount of unchanged OHCZX excreted in a 24-h urine sample (Ae0–24 h) (Table 3).
DISCUSSION
Figure 1. Mean arterial plasma concentration–time profiles of (A) CZX and (B) OH-CZX after intravenous administration of CZX at a dose of 20 mg/kg to control rats (*, n ¼ 11) and rats with U-ARF (*, n ¼ 16) and U-ARFG (~, n ¼ 15). Bars represent standard deviation.
ARF (659 mg min/mL) and U-ARFG (872 mg min/ mL; Table 3). The CLs were significantly different among three groups of rats; the CL was the fastest and slowest for rats with U-ARF (30.3 mL/min/kg) and control rats (12.2 mL/min/kg), respectively (Table 3). The CLNR in rats with U-ARFG was significantly faster (104% increase) than that in control rats (Table 3). The VSS was not significantly different among three groups of rats, which could be expected because plasma protein binding values were not significantly different among three groups of rats (Table 3). In rats with UARF, CZX was below the detection limit in urine (Table 3). After intravenous administration of CZX, OHCZX was formed quickly for all three groups of rats; OH-CZX was detected in plasma from the 5-min blood sampling and reached respective JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
As mentioned earlier, the changes in the reported pharmacokinetic parameters of many drugs (or compounds) in rats with U-ARF are explained mainly by impaired kidney and/or liver function or formation of conjugates. However, the relationship between pharmacokinetic changes of drugs (except CZX6) and changes in CYP isozymes in rats with U-ARF seem not to be published. In the present study, the CZX in plasma and urine was unconjugated CZX (not CZX glucuronides). After intravenous administration of CZX at a dose 20 mg/kg, the CLs (12.2–30.3 mL/min/kg based on plasma data, Table 3) were considerably slower than the reported cardiac output of 295 mL/ min/kg in rats based on blood data.23 This difference suggests that the first-pass effect of CZX in the lung and heart could be almost negligible, if any, in the three groups of rats. The plasma protein binding values of CZX (at a CZX concentration of 10 mg/mL) were 68.5, 71.8, and 63.4% for control rats and rats with U-ARF and U-ARFG, respectively, as determinied by an equilibrium dialysis technique (Table 3). The estimated CLR values of CZX based on free (unbound in plasma proteins) fraction were 0.301 and 0.0839 mL/min/kg for control rats and rats with U-ARFG, respectively; the values were considerably slower than the
GLUCOSE EFFECT ON CHLORZOXAZONE PHARMACOKINETICS IN RATS
reported glomerular filtration rate of 5.24 mL/min/ kg in rats.23 These data indicate that CZX is mainly reabsorbed in rat renal tubules. Considering the CLR values of CZX (Table 3), reported kidney blood flow rate of 36.8 mL/min/kg,23 and hematocrit of 45%24 in rats, the estimated renal extraction ratios (CLR of CZX/kidney plasma flow rate, only for urinary excretion of unchanged CZX) were 0.468 and 0.152% for control rats and rats with U-ARFG, respectively. These data indicate that CZX was excreted poorly via rat kidney. The hepatic extraction ratio (hepatic first-pass effect) of CZX was roughly estimated by dividing the CLNR of CZX by the reported hepatic plasma flow rate in rats and assuming that nonrenal clearance of CZX was attributed solely to the liver (hence the ratio represents the maximal possible value of liver).25 The hepatic plasma flow rate in rats was estimated based on the reported hepatic blood flow rate of 30.4 mL/min/kg23 and hematocrit of 45%24 in rats. The hepatic extraction ratio of CZX in control rats was 68.2%, indicating that CZX is an intermediate to high hepatic extraction ratio drug. Because CZX is an intermediate-tohigh hepatic extraction ratio drug, its hepatic clearance depends more on hepatic blood flow than CLint and free (unbound in plasma protein) fraction of CZX.26 The significantly faster CL of CZX in rats with U-ARF could be due to significantly faster CLint (107% increase, Table 2) because it has been reported27 that cardiac output, renal blood flow rate, and hepatic blood flow rate decreased significantly in rats with U-ARF induced by glycerol compared with those parameters in control rats. A similar explanation could also be applied to rats with U-ARFG; in these rats, the CLint was significantly faster (57.6% increase) than that in control rats. Northern and Western blot analyses revealed that CYP2E1 was induced 2.3-fold in rats with UARF.6 CZX primarily undergoes metabolism that is catalyzed mainly via CYP2E1 to form OHCZX.4,5 Hence, it was expected that formation of OH-CZX could increase in rats with U-ARF compared with that of control rats. This expectation was supported by the following results. First, in rats with U-ARF, AUC0–8 h of OH-CZX was significantly greater (26.7 times increase) and AUC of CZX was significantly smaller (59.8% decrease) than those parametersin control rats (Table 3). Moreover, in rats with U-ARF, the Cmax of OHCZX reached at 72 min and the peak concentration was maintained for up to 8 h, but in control rats, the Cmax reached at 19.5 min, declined for up to 4 h
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(the mean plasma concentration of OH-CZX at 4 h in control rats was 1.14% of that in rats with U-ARF), and the low concentrations were maintained for up to 8 h (Figure 1B). Second, in rats with U-ARF, the AUC0–8 h, OH-CZX/AUCCZX was 68.2 times greater than that in control rats (Table 2). However, the effect of acute renal failure-induced accumulation of OH-CZX on the increase in the ratio could not be totally ruled out because acute renal failure reduces urinary excretion of drugs (compounds). It has also been reported6 that in rats with UARFG, the CYP2E1 level was significantly greater and smaller than those in control rats and in rats with U-ARF, respectively. Hence, it was expected that in rats with U-ARFG, formation of OH-CZX could be significantly greater and smaller than those in control rats and in rats with U-ARF, respectively. This expectation was also supported by the following results. First, in rats with UARFG, AUC0–8 h of OH-CZX was significantly greater (20.1 times increase) and smaller (24.1% decrease) than those in control rats and in rats with U-ARF (Table 3), respectively. Second, the AUC0–8 h, OH-CZX/AUCCZX in rats with U-ARFG was greater (38.7 times increase) and smaller (42.7% decrease) than those in control rats and in rats with U-ARF (Table 3), respectively. Finally, the CLint in rats with U-ARFG was significantly faster (57.6% increase) and slower (24.0%) than those in control rats and in rats with U-ARF (Table 2), respectively. Although CYP2E1 is the major form metabolizing CZX to OH-CZX, it has been reported,28–31 that human CYP1A2 and CYP3A4 are involved in CZX hydroxylation. The effect of CYP1A2 on the formation of OH-CZX in rats with U-ARF could be ruled out because no significant change in CYP1A2 was obtained in the rats.6 In rats with U-ARF, the expression of CYP3A23 increased 4-fold.6 Human CYP3A4 and rat CYP3A1 (CYP3A23) proteins have 73% homology.32 It has been reported that CYP3A1 (CYP3A23)33 or CYP3A234–36 is the major CYP3A protein in rats. Hence, the role of CYP3A23 and CYP3A2 in the formation of OH-CZX in rats was measured by treatment with DDT, an inducer of CYP3A237 or DDE, an inducer of CYP3A23.38 It has been concluded6 that OH-CZX is formed mainly via CYP2E1 in rats with U-ARF. In summary, the CYP2E1 protein and mRNA level in rats with U-ARFG were greater than those in control rats, but significantly lower than those in rats with U-ARF. OH-CZX is mainly metabolized via CYP2E1 in rats.1 Hence, it could be JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
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expected that the formation of OH-CZX in rats with U-ARFG would be greater than that in control rats but smaller than that in rats with UARF. This expectation was proven by AUC0–8 h values of OH-CZX; the values were 414, 11500, and 8730 mg min/mL for control rats and rats with U-ARF and U-ARFG, respectively; each value was significantly different among three groups (Table 3). These data indicate that the formation of OH-CZX increased significantly in rats with U-ARFG compared with that in control rats, but decreased significantly compared with that in rats with U-ARF due to CYP2E1 changes.
ACKNOWLEDGMENTS This study was supported in part by 2002 BK21 Project for Medicine, Dentistry, and Pharmacy.
REFERENCES 1. Chen L, Yang CS. 1996. Effects of cytochrome P450 2E1 modulators on the pharmacokinetics of chlorzoxazone and 6-hydroxychlorzoxazone in rats. Life Sci 58:1575–1585. 2. Conney AH, Burns JJ. 1960. Physiological disposition and metabolic fate of chlorzoxazone (Paraflex) in man. J Pharmacol Exp Ther 128:340–343. 3. Peter R, Bocker R, Beaune PH, Iwasaki M, Guengerich FP, Yang CS. 1990. Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-450 2E1. Chem Res Toxicol 3:566–573. 4. Desiraju RK, Renzi NL Jr, Nayak RK, Ng KT. 1983. Pharmacokinetics of chlorzoxazone in humans. J Pharm Sci 72:991–994. 5. Rockich K, Blouin R. 1999. Effect of the acute-phase response on the pharmacokinetics of chlorzoxazone and cytochrome P-450 2E1 in vitro activity in rats. Drug Metab Dispos 27:1074–1077. 6. Moon YJ, Lee AK, Chung HC, Kim EJ, Kim SH, Lee I, Kim SG, Lee MG. 2003. Effects of acute renal failure on the pharmacokintics of chlorzoxazone in rats, submitted for publication to Drug Metab Dispos, in press. 7. Chung HC, Kim SH, Lee MG, Kim SG. 2002. Increase in urea in conjunction with L-arginine metabolism in the liver leads to induction of cytochrome P450 2E1 (CYP2E1): The role of urea in CYP2E1 induction by acute renal failure. Drug Metab Dispos 30:739–746. 8. Son MH, Kang KW, Kim EJ, Ryu JH, Cho H, Kim SH, Kim WB, Kim SG. 2000. Role of glucose utilization in the restoration of hypophysectomyinduced hepatic cytochrome P450 by growth hormone in rats. Chem Biol Interact 127:13–28.
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9. Kim SG, Kim EJ, Kim YG, Lee MG. 2001. Expression of cytochrome P450s and glutathione S-transferases in the rat liver during water deprivation: The effects of glucose supplementation. J Appl Toxicol 21:123–129. 10. Liu JH, Malone RS, Stallings H, Smith PC. 1996. Influence of renal failure in rats on the disposition of salicyl acyl glucuronide and covalent binding of salicylate to plasma proteins. J Pharmacol Exp Ther 278:277–283. 11. Kim SH, Shim HJ, Kim WB, Lee MG. 1998. Pharmacokinetics of a new carbapenem, DA-1131, after intravenous administration to rats with uranyl nitrate-induced acute renal failure. Antimicrobial Agents Chemother 42:1217–1221. 12. Lowry OH, Rosebrough NG, Farr AL, Randall RJ. 1951. Protein measurement with Folin-phenol reagent. J Biol Chem 193:265–275. 13. Frye RF, Stiff DD. 1996. Determination of chlorzoxazone and 6-hydroxychorzoxazone in human plasma and urine by high-performance liquid chromatography. J Chromatogr B 686:291–296. 14. Lineweaver H, Burk D. 1934. The determination of enzyme dissociation constants. J Am Chem Soc 56: 658–666. 15. Liu PT, Ioannides C, Shavila J, Symons AM, Parke DV. 1993. Effects of ether anaesthesia and fasting on various cytochromes P450 of rat liver and kidney. Biochem Pharmacol 45:871–877. 16. Shim HJ, Lee EJ, Kim SH, Kim SH, Yoo M, Kwon JW, Kim WB, Lee MG. 2000. Factors influencing the protein binding of a new phosphodiesterase V inhibitor, DA-8159, using an equilibrium dialysis technique. Biopharm Drug Dispos 21:285–291. 17. Chiou WL. 1978. Critical evaluation of potential error in pharmacokinetics studies using the linear trapezoidal method for the calculation of the area under the plasma level–time curve. J Pharmacokinet Biopharm 6:539–549. 18. Gibaldi M, Perrier D. 1982. Pharmacokinetics, 2nd ed. New York: Marcel Dekker. 19. Kim SH, Choi YM, Lee MG. 1993. Pharmacokinetics and pharmacodynamics of furosemide in protein–calorie malnutrition. J Pharmacokinet Biopharm 21:1–17. 20. Chiou WL. 1979. New calculation method for mean apparent drug volume of distribution and application to rational dosage regimens. J Pharm Sci 68: 1067–1069. 21. Eatman FB, Colburn WA, Boxenbaum HG, Posmanter HN, Weinfeld RE, Ronfeld R, Weissman L, Moore JD, Gibaldi M, Kaplan SA. 1977. Pharmacokinetics of diazepam following multiple dose oral administration to healthy human subjects. J Pharmacokinet Biopharm 5:481–494. 22. Chiou WL. 1980. New calculation method of mean total body clearance of drugs and its application to dosage regimens. J Pharm Sci 69:90–91.
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23. Davies B, Morris T. 1993. Physiological parameters in laboratory animals and humans. Pharm Res 10: 1093–1095. 24. Mitruka BM, Rawnsley HM. 1982. Clinical Biochemical and Hematological Reference Values in Normal Experimental Animal and Normal Humans. 2nd ed. New York: Masson Publishing USA. 25. Lee MG, Chiou WL. 1983. Evaluation of potential causes for the incomplete bioavailability of furosemide: Gastric first-pass metabolism. J Pharmacokinet Biopharm 11:623–640. 26. Wilkinson GR, Shand DG. 1975. Commentary: A physiological approach to hepatic drug clearance. Clin Pharmacol Ther 18:377–390. 27. Kashimoto T, Sakamoto W, Nakatani T, Ito T, Iwai K, Kim T, Abe Y. 1989. Cardiac output, renal blood flow and hepatic blood flow in rats with glycerolinduced acute renal failure. Nephron 53:353–357. 28. Carriere V, Goasduff T, Ratanasavanh D, Morel F, Gautier JC, Guillouza A, Beaune P, Berthou F. 1993. Both cytochromes P450 2E1 and 1A1 are involved in the metabolism of chlorzoxazone. Chem Res Toxicol 6:852–857. 29. Ono S, Hatanaka T, Hotta H, Tsutsui M, Satoh T, Gonzalez FJ. 1995. Chlorzoxazone is metabolized by human CYP1A2 as well as by human CYP2E1. Pharmacogenetics 5:143–150. 30. Gorski JC, Jones DR, Wrighton SA, Hall SD. 1997. Contribution of human CYP3A subfamily members to the 6-hydroxylation of chlorzoxazone. Xenobiotica 27:243–256. 31. Shimada T, Tsumura F, Tamazaki H. 1999. Prediction of human liver microsomal oxidation of 7-ethoxycoumarin and chlorzoxazone with kinetic
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parameters of recombinant cytochrome P-450 enzymes. Drug Metab Dispos 27:1274–1280. Lewis DFV. 1996. Substrate specificity and metabolism, in Cytochromes P450 Structures, Function and Mechanism, Bristol, PA: Taylor & Francis, p. 123. Mahnke A, Strotkamp D, Roos PH, Hanstein WG, Chabot GG, Nef P. 1997. Expression and inducibility of cytochrom P450 3A9 (CYP3A9) and other members of the CYP3A subfamily in rat liver. Arch Biochem Biophys 337:62–68. Debri K, Boobis AR, Davies DS, Edwards RJ. 1995. Distribution and induction of CYP3A1 and CYP 3A2 in rat liver and extrahepatic tissues. Biochem Pharmacol 50:2047–2056. Hoen PA, Bijsterbosch MK, van Berkel TJ, Vermeulen NPE, Commandeur JNM. 2001. Midazolam is a phenobarbital-like cytochrome P450 inducer in rats. J Pharmacol Exp Ther 299:921–927. Rekka E, Evdokimova E, Eeckhoudt S, Labar G, Calderon PB. 2002. Role of temperature on protein and mRNA cytochrome P450 3A (CYP3A) isozymes expression and midazolam oxidation by cultured rat precision-cut liver slices. Biochem Pharmacol 64:633–643. Sierra-Santoyo A, Hernandez M, Albores A, Cebrian ME. 2000. Sex-dependent regulation of hepatic cytochrome P-450 by DDT. Toxicol Sci 54:81–87. You L, Chan SK, Bruce JM, Archibeque-Engle S, Casanova M, Corton JC, Heck H. 1999. Modulation of testosterone-metabolizing hepatic cytochrome P450 enzymes in developing Sprague–Dawley rats following in utero exposure to p,p’-DDE. Toxicol Appl Pharmacol 158:197–205.
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College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, South Korea
2
Department of Diagnostic Pathology, College of Medicine, University of Ulsan, Asan Foundation, Asan Medical Center, Seoul, South Korea
3
Research Laboratory, Dong-A Pharmaceutical Company, Ltd., Yongin, South Korea
Received 28 January 2003; accepted 8 March 2003
ABSTRACT: The effects of glucose on CYP2E1 expression in rats with acute renal failure induced by uranyl nitrate (U-ARF) have been reported. CYP2E1 was significantly induced (2.3-fold) in rats with U-ARF compared with that in control rats. In contrast, CYP2E1 expression was significantly decreased in rats with U-ARF supplied with glucose (dissolved in tap water to make 10%, w/v) in their drinking water for 5 days (U-ARFG) compared with that in rats with U-ARF. However, CYP2E1 in rats with U-ARFG was significantly greater than that in control rats. Chlorzoxazone (CZX) primarily undergoes hydroxylation, catalyzed mainly by CYP2E1, to form 6-hydroxychlorzoxazone (OH-CZX) rats. Hence, it could be expected that in rats with U-ARFG, formation of OH-CZX could significantly decrease and increase compared with those in rats with U-ARF and control rats, respectively. This expectation is proven by the following results of a study of intravenous administration of CZX at a dose 20 mg/kg to control rats and rats with U-ARF and U-ARFG. First, the total area under the plasma concentration–time curve from time zero to 8 h (AUC0–8 h) of OH-CZX in rats with U-ARFG (8730 mg min/mL) was significantly greater than that in control rats (414 mg min/mL) and significantly smaller than that in rats with U-ARF (11500 mg min/mL). Second, the AUC0–8 h, OH-CZX/AUCCZX ratio in rats with U-ARFG (10.0) was significantly greater than that in control rats (0.252) and significantly smaller than that in rats with U-ARF (17.5). Finally, the in vitro intrinsic OH-CZX formation clearance (CLint) in rats with U-ARFG (27.9 mL/min/mg protein) was significantly slower than that in rats with U-ARF (36.7 mL/min/mg protein) and significantly faster than that in control rats (17.7 mL/min/mg protein). ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1604–1613, 2003
Keywords:
CZX; OH-CZX; U-ARF; glucose; pharmacokinetics; rats
INTRODUCTION Chlorzoxazone [5-chloro-2(3H)-benzoxazolone, CZX], once used as a skeletal muscle relaxant for the treatment of painful muscle spasms,1 Correspondence to: Myung G. Lee (Telephone: 82-2-8807855 (7877); Fax: 82-2-889-8693; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 1604–1613 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association
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primarily undergoes hydroxylation [catalyzed mainly by hepatic microsomal cytochrome P450 (CYP) 2E12,3] to form 6-hydroxychlorzoxazone (OH-CZX). OH-CZX is then rapidly glucuronidated and excreted in urine.2,4 CZX has been suggested for use as a chemical probe to assess the activity of CYP2E1 in vitro and in vivo because of the good correlation between the formation rate of OH-CZX and the CYP2E1 activity in rats.5
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It has been reported from our laboratories6 that in rats with acute renal failure induced by uranyl nitrate (U-ARF), CYP2C11 decreased to 20% of control, CYP2E1 and CYP3A23 increased 2.3 and 4 times, respectively, compared with control, and CYP1A2 and CYP2B1/2 were not changed compared with control based on Western and Northern blot analyses. However, the production of CYP2E1 was reduced in rats given glucose (dissolved in tap water to make 10%, w/v) in the drinking water for 5 days with U-ARF (U-ARFG) compared with that in rats with U-ARF alone. The CYP2E1 protein and mRNA level in rats with U-ARFG were significantly higher than those in control rats, but significantly lower than those in rats with U-ARF.7 It has also been reported that the induced CYP2E1 in rats with hypophysectomy8 and rats with 72-h dehydration9 was reduced significantly by treatment with glucose. Based on these CYP2E1 results, it could be expected that in rats with UARFG, formation of OH-CZX could decrease significantly compared with that in rats with U-ARF, but increase significantly compared with that in control rats. Changes in the pharmacokinetics of many drugs (either mainly eliminated via renal excretion or by hepatic metabolism) have been reported in rats with U-ARF. For example, the total area under the plasma concentration–time curve from time zero 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 an intravenous administration of the following drugs (or compounds) to rats with U-ARF;6 methotrexate, vancomycin, DA-1131 (a new carbapenem), M1 (an active component of a new anthracycline, DA-125), salicylic acid, azosemide, diltiazem, amiodarone, tetraethylammonium bromide, and para-aminohippurate. Pharmacokinetic differences of the aforementioned drugs (or compounds) were mainly explained by impaired kidney and/or liver function,6 or increased formation of conjugates10 in rats with U-ARF. However, the changes in the pharmacokinetics of drugs (except CZX) in rats with U-ARF are not explained with regard to CYP isozyme changes.6 Moreover, the effects of glucose on the pharmacokinetics of drugs in rats with U-ARF have not been published. The aim of this study is to report whether formation of OH-CZX in rats with U-ARFG increased significantly compared with that in control rats and decreased significantly compared with that in rats with UARF with respect to CYP2E1 changes.
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MATERIALS AND METHODS Chemicals Chlorzoxazone [CZX; 5-chloro-2(3H)-benzoxazolone], the reduced form of b-nicotinamide adenine dinucleotide phosphate (NADPH, as a tetrasodium salt), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer, and b-glucuronidase (Type H-1, from Helix pomatia) were purchased from Sigma Chemical Company (St. Louis, MO). OH-CZX and 3-aminophenyl sulfone (an internal standard of high-performance liquid chromatographic [HPLC] assay for CZX and OHCZX) were obtained from Research Biomedical International (Natick, MA) and Aldrich Chemical Company (Milwaukee, WI), respectively. Uranyl nitrate was a product from BDH Chemicals (Poole, England). Ketamine hydrochloride was purchased from Yuhan Research Center of Yuhan Corporation (Kunpo, South Korea). Other chemicals were of reagent grade or HPLC grade, and therefore, were used without further purification. Animals Male Sprague–Dawley rats (weighing 245–280 g) were purchased from Charles River Company Korea (Biogenomics, Seoul, South Korea). Rats were housed in metabolic cages (Tecniplast, Varese, Italy) under the supply of filtered pathogen-free air. The rats were randomly divided into three groups; control rats and rats with U-ARF without (U-ARF) or with (U-ARFG) a supply of glucose for 5 days. All rats were provided with food (Sam Yang Company, Seoul, South Korea) and water ad libitum and maintained in a lightcontroled room (light: 0700–1900, dark: 1900– 0700) kept at a temperature of 22 28C and a relative humidity of 55 5% (Animal Center for Pharmaceutical Research, College of Pharmacy, Seoul National University, Seoul, South Korea). 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%) at a dose of 5 mg/ kg was injected once via the tail vein of each rat (total injection volume was 1 mL/kg) to induce acute renal failure.6,11 Control rats were injected with the same volume of 0.9% NaCl-injectable solution. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
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Supply of Glucose in Rats with U-ARF (Rats with U-ARFG) Rats with U-ARF were divided into two groups, with (U-ARFG) or without (U-ARF) glucose supplementation. Rats with U-ARFG received glucose (dissolved in tap water to make 10%, w/v) in drinking water for 5 days. Measurement of Vmax, Km, and CLint for the Formation of OH-CZX in Hepatic Microsomes On the fifth day, the livers of control rats (n ¼ 5) and rats with U-ARF (n ¼ 4) and U-ARFG (n ¼ 5) were homogenized in an ice-cold, pH 7.4 buffer of 0.154 M KCl/50 mM Tris-HCl in 1 mM ethylenediamine tetraacetate (EDTA). The homogenate was centrifuged at 9700 g for 30 min, and the supernatant fraction was further centrifuged at 100,000 g for 90 min. The microsomal pellet was resuspended in a pH 7.4 buffer of 0.154 M KCl/50 mM Tris-HCl in 1 mM EDTA. Protein content was measured using the reported method.12 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 aforementioned microsomal fraction (equivalent to 0.5 mg protein), a 10-mL aliquot of CZX (to have substrate concentrations of 1, 1.25, 2, 5, 10, 50, 100, and 200 mM), and 1 mM of NADPH in a final volume of 1 mL by adding 0.1 M Tris-HCl, pH 7.4, in a water-bath shaker kept at 378C and at a rate of 50 oscillations per min (opm). All of the aforementioned microsomal incubation conditions were linear. The reaction was terminated by the addition of 1 mL of tert-butyl methyl ether after a 20-min incubation. The OH-CZX formed was determined by HPLC analysis.13 The kinetic constants (Km and Vmax) for the formation of OH-CZX were calculated using the Lineweaver–Burk plot14 by the linear regression and the method of least squares. Intrinsic OH-CZX formation clearance (CLint) was calculated by dividing the Vmax by the Km. Pretreatment of Rats In the early morning on the fifth day after a single intravenous administration of uranyl nitrate or 0.9% NaCl-injectable solution, the jugular vein (for drug administration) and the carotid artery (for blood sampling) of each rat were cannulated with polyethylene tubing (Clay Adams, ParsipJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
pany, NJ) while each rat was under ketamine hydrochloride anesthesia [ketamine hydrochloride at a dose of 100 mg/kg was administered (total injection volume was 1 mL/kg) intramuscularly]. Ketamine was employed instead of ether to minimize the effect of CYP2E1 on the formation of OH-CZX; it has been reported15 that ether anesthesia alone increased expression of CYP2E1 by 40% as determined by p-nitrophenol hydroxylase activity. Both cannulae were exteriorized to the dorsal side of the neck, where each cannula terminated with long silastic tubing (Dow Corning, Midland, MI). Both silastic tubings were inserted into a wire coil to allow free movement of the rat. Each rat was not restrained during the experimental period. After surgical suture, each rat was housed individually in a rat metabolic cage (Daejong Scientific Company, Seoul, South Korea). Experimentation was started after 5–6 h of recovery from the anesthesia. Intravenous Study CZX (the CZX powder was dissolved in a minimum amount of 1 N NaOH) at a dose of 20 mg/kg was administered by an intravenous infusion over a 1-min via the jugular vein (total injection volume was 0.6 mL) of control rats (n ¼ 11) and rats with U-ARF (n ¼ 16) and U-ARFG (n ¼ 15). An 0.22-mL aliquot sample of blood was collected via the carotid artery at 0 (to serve as a control), 1 (at the end of the infusion), 5, 15, 30, 60, 120, 180, 240, 360, and 480 min after an intravenous administration of the drug. After centrifugation, two 0.05-mL aliquots samples of plasma (each plasma sample was analyzed with and without addition of b-glucuronidase) were stored in a 708C freezer (Revco ULT 1490 D-N-S; Western Mednics, Asheville, NC) until HPLC analysis of CZX and OH-CZX.13 A 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 24 h, as much blood as possible was collected via the carotid artery and each rat was sacrificed by cervical dislocation. Plasma samples were stored in a 708C freezer for the measurement of urea nitrogen, glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), creatinine, and glucose levels (analyzed by Green Cross Reference Lab., Seoul, South Korea) and plasma protein binding. The whole kidney and liver of each rat were excised, rinsed with 0.9% NaCl-injectable
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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-eosin staining. Urine samples were collected between 0 and 24 h. Each metabolic cage was rinsed with 10 mL of distilled water at the end of 24 h, and the rinsings were combined with the urine samples. After measuring the exact volume of combined urine samples, two 0.05-mL aliquots of the combined urine samples (each urine sample was also analyzed with and without addition of b-glucuronidase) were stored in the freezer until HPLC analysis of CZX and OH-CZX.13
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the column effluent. The retention times of OHCZX, 3-aminophenyl sulfone, and CZX were 6, 10, and 18 min, respectively. The detection limits of CZX and OH-CZX in plasma were both 0.1 mg/ mL. The coefficients of variation of the assay (within- and between-day) were generally low (<8.2%). For the biological samples of rats with U-ARF and U-ARFG, the mobile phase was changed to 0.05 M ammonium acetate/acetonitrile (4:1, v/v) to eliminate endogenous interference peak(s) in HPLC chromatogram from the biological sample. The flow rate of the mobile phase was 1 mL/min. The retention times of OH-CZX, 3-aminophenyl sulfone, and CZX were 14, 22, and 40 min, respectively. In the present study, plasma and urine samples were analyzed with and without addition of b-glucuronidase.
Measurement of Plasma Protein Binding The binding of CZX (at a CZX concentration of 10 mg/mL) to plasma protein of control rats and rats with U-ARF and U-ARFG was determined by an equilibrium dialysis technique.16 After 24 h of incubation, a 0.1-mL aliquot was removed from each compartment and stored in a 708C freezer until HPLC analysis of CZX.13
HPLC Analysis of CZX and OH-CZX The concentrations of CZX and OH-CZX in the aforementioned biological samples of control rats were analyzed by the reported HPLC method.13 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 b-glucuronidase were added to a 0.05-mL aliquot of biological sample. The mixture was manually mixed and incubated for 2 h in a water-bath shaker kept at 378C and at a rate of 50 opm. After incubation, a 0.05-mL aliquot of methanol containing 40 mg/mL of 3-aminophenyl sulfone (the internal standard) was added and mixed by vortex, and a 1-mL aliquot of tert-butyl methyl ether was added. The mixture was shaken for 10 min and then centrifuged at 2000 g for 10 min. The upper organic layer was transferred to a clean tube and evaporated at 378C under a stream of nitrogen. The residue was reconstituted in a 100-mL aliquot of the mobile phase, and a 50-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/v), was run at a flow rate of 1 mL/min. An ultraviolet (UV) detector set at 283 nm monitored
Pharmacokinetic Analysis The AUC of CZX and AUC0–8 h of OH-CZX were calculated by the trapezoidal rule method; this method utilized the logarithmic trapezoidal rule17 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 (for the measurement of AUC of CZX) was estimated by dividing the last measured plasma concentration by the terminal rate constant. Standard methods18 were used to calculate the CL, CLR, CLNR, area under the first moment of the plasma concentration–time curve (AUMC), mean residence time (MRT), and apparent volume of distribution at steady state (VSS).19 The harmonic mean method was used to calculate the each mean value of VSS,20 terminal half-life,21 and each clearance.22 Glomerular filtration rate was calculated by measuring the creatinine clearance (CLCR), assuming that kidney function was stable during 24 h. The CLCR was calculated by dividing the total amount of creatinine excreted in 24-h urine by AUC0–24 h of creatinine in plasma.
Statistical Analysis A p value of <0.05, determined with a Duncan’s multiple range test of SPSS posteriori analysis of variance (ANOVA), was considered to be statistically significant. All results were expressed as mean standard deviation (SD). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
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RESULTS Induction of Acute Renal Failure in Rats Impaired kidney function was observed in rats with U-ARF; serum levels of urea nitrogen (7.59 times increase) and creatinine (10.2 times increase) were significantly higher, kidney weight (% of body weight) was significantly heavier (24.8% increase), and CLCR was significantly slower (99.9% decrease) than those in control rats (Table 1). Impaired kidney function in rats with U-ARF was also supported by kidney microscopy; there was extensive tubular necrosis involving distal tubules. Impaired kidney function in rats with U-ARF has also been reported elsewhere.6,11 However, no significant findings were indicated by kidney microscopy from control rats. Note that the aforementioned parameters were not significantly different between rats with U-ARF and U-ARFG (Table 1), suggesting that kidney function in rats with U-ARF was not recovered to control by treatment with glucose for 5 days. Impaired kidney function in rats with U-ARFG was also supported by kidney microscopy; there was extensive tubular necrosis involving distal tubules, and the differences between rats with U-ARF and U-ARFG were not significant. Unlike kidney function, liver function seemed not to be impaired considerably by treatment with uranyl nitrate; the plasma level of GOT was significantly higher (87.5% increase) in rats with U-ARF than that in control rats, however, the values were not significantly different between U-ARF and
U-ARFG (Table 1). This result could also be supported by liver microscopy; no significant findings were found for the three groups of rats. Note that body weight gain decreased significantly in rats with U-ARF (from 268 to 249 g) and U-ARFG (from 264 to 248 g) compared with that in control rats (from 260 to 294 g; Table 1). This result indicates that 5 days supply with glucose did not increase the body weight gain reduced by U-ARF.
Measurement of Vmax, Km, and CLint for the Formation of OH-CZX in Hepatic Microsomes In rats with U-ARF, the Vmax (104% 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 intrinsic OH-CZX formation clearance (CLint) in hepatic microsomal fraction was significantly faster (107% increase) in rats with UARF than that in control rats (Table 2). In rats with U-ARFG, CLint was significantly faster (57.6% increase) and slower (24.0% decrease) than those in control rats and rats with U-ARF, respectively (Table 2). These data suggest that in rats with U-ARFG, formation of OH-CZX could increase and decrease compared with those in control rats and rats with U-ARF, respectively. The liver weight and protein content were not significantly different among three groups of rats (Table 2).
Table 1. Plasma Values and Organ Weight in Control Rats and Rats with U-ARF and U-ARFGa Parameter
Control (n ¼ 11)
U-ARF (n ¼ 16)
Plasma Urea nitrogen (mg/dL) GOT (IU/L) GPT (IU/L) Creatinine (mg/dL) Glucose (mg/dL) CLCR (mL/min/kg) Initial body weightc (g) Final body weightd (g) Kidney weight (% of body weight) Liver weight (% of body weight)
17.0 3.78b 128 37.8b 50.1 11.8 0.491 0.114b 148 23.8 4.20 0.633b 260 17.6 294 14.2b 0.795 0.0528b 3.79 0.341
146 29.3 240 38.9 51.9 13.0 5.51 1.42 146 29.3 0.00438 0.0103 268 8.50 249 13.1 0.992 0.109 3.34 0.589
U-ARFG (n ¼ 15) 151 39.6 182 58.2 40.4 10.4 5.81 1.55 167 43.5 0.0156 0.0669 264 7.45 248 23.9 1.07 0.0149 3.17 0.129
a Values expressed as mean SD. Abbreviations: U-ARF, acute renal failure induced by uranyl nitrate; U-ARFG, U-ARF with glucose supplementation; GOT, glutamate oxaloacetate transaminase; GPT, glutamate pyruvate transaminase; IU, international unit; CLCR, creatinine clearance. b Control group was significantly different (p < 0.05) from U-ARF and U-ARFG groups. c Measured just before the injection of uranyl nitrate or 0.9% NaCl-injectable solution. d Measured just before the experiment.
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Table 2. Km, Vmax, and CLint Values for the Formation of OH-CZX by Microsomes Prepared from Livers of Control Rats and Rats with U-ARF and U-ARFGa Parameter Vmax (nmol/min/mg protein) Km (mM) CLint (mL/min/mg protein) Liver weight (% of body weight) Protein content (mg/g liver)
Control (n ¼ 5)
U-ARF (n ¼ 4)
U-ARFG (n ¼ 5)
2.47 1.27b 146 87.4 17.7 4.33c 4.66 0.161 29.6 3.72
5.04 1.51 150 76.4 36.7 6.01 3.54 0.319 29.0 2.31
3.55 2.12 121 67.8 27.9 4.05 3.55 0.957 31.3 3.25
a
Values expressed as mean SD. Abbreviations: Vmax, maximum velocity; Km, Michaelis–Menten constant, the concentration at which the rate is one-half of Vmax; CLint, intrinsic OH-CZX formation clearance; U-ARF, acute renal failure induced by uranyl nitrate; U-ARFG, U-ARF with glucose supplementation. b Control group was significantly different (p < 0.05) from U-ARF group. c Each group was significantly different (p < 0.05).
Pharmacokinetics after Intravenous Administration of CZX The mean arterial plasma concentration–time profiles of CZX and OH-CZX after intravenous administration of CZX at a dose of 20 mg/kg to three groups of rats are shown in Figure 1 and some relevant pharmacokinetic parameters are listed in Table 3. After intravenous administration of CZX, the plasma concentrations of CZX declined in a polyexponential fashion for all three groups of rats (Figure 1A), with mean terminal
half-lives of 24.8, 15.4, and 17.5 min for control rats and rats with U-ARF and U-ARFG, respectively; the value in control rats was significantly longer than those in rats with U-ARF and UARFG (Table 3). Similar trends were also obtained for MRTs (Table 3). The plasma concentrations of CZX were the highest and lowest for control rats and rats with U-ARF, respectively (Figure 1A). As a result, the AUC was the greatest and lowest for control rats and rats with U-ARF, respectively; the value in control rats (1640 mg min/mL) was significantly greater than those in rats with U-
Table 3. Pharmacokinetic Parameters of CZX and OH-CZX after Intravenous Administration of CZX at a Dose of 20 mg/kg to Control Rats and Rats with U-ARF and U-ARFGa Parameter CZX AUC (mg min/mL) Terminal half-life (min) MRT (min) CL (mL/min/kg) CLR (mL/min/kg) CLNR (mL/min/kg) VSS (mL/kg) Plasma protein binding (%) Ae0–24 h (% of CZX dose) OH-CZX AUC0–8 h (mg min/mL) CLR (mL/min/kg) Ae0–24 h (% of CZX dose) Cmax (mg/mL) Tmax (min)
Control (n ¼ 11) 1640 631b 24.8 5.67b 32.4 12.1b 12.2 6.05c 0.0948 0.737 11.4 6.27d 307 301 68.5 6.86 4.96 5.04 414 97.3c 20.8 14.2b 47.9 13.1b 4.34 1.30c 19.5 8.79b
U-ARF (n ¼ 16) 659 140 15.4 3.27 12.7 3.20 30.3 7.72 NM 366 165 71.8 3.01 BD 11500 3380 0.00805 0.176 10.9 11.1 40.5 10.5 72.0 25.3
U-ARFG (n ¼ 15) 872 327 17.5 6.14 15.8 4.25 22.9 7.61 0.0307 0.264 23.3 5.50 339 153 63.4 7.05 0.638 0.738 8730 2310 0.135 0.139 7.86 8.40 28.1 4.76 64.0 15.5
a
Values expressed as mean SD. Abbreviations: CZX, chlorzoxazone; OH-CZX, 6-hydroxychlorzoxazone; U-ARF, acute renal failure induced by uranyl nitrate; U-ARFG, U-ARF with glucose suplementation; AUC, area under the plasma concentration–time curve from time zero to time infinity; MRT, mean residence time; CL, time-averaged total body clearance; CLR, time-averaged renal clearance; CLNR, time-averaged nonrenal clearance; VSS, apparent volume of distribution at steady state; Ae0–24 h, total amount excreted in urine as unchanged drug from time zero to 24 h; AUC0–8 h, total area under the plasma concentration– time curve from time zero to 8 h; Cmax, maximum plasma concentration; Tmax, time to reach Cmax; NM, not measurable; BD, below detection limit. b Control group was significantly different (p < 0.05) from U-ARF and U-ARFG groups. c Each group was significantly different (p < 0.05). d Control group was significantly different (p < 0.05) from U-ARFG. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
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peaks at 19.5–72.0 min (Figure 1B). The peak plasma concentration of OH-CZX was maintained for up to 8 h in rats with U-ARF and U-ARFG (Figure 1B). However, in control rats, the peak plasma concentration of OH-CZX reached its peak at 19.5 min and decreased for up to 4 h, and the decreased concentrations were maintained for up to 8 h (Figure 1B). The plasma concentrations and Cmax of OH-CZX were the highest and lowest for rats with U-ARF and control rats, respectively (Figure 1B). As a result, the values of AUC0–8 h of OH-CZX were significantly different among three groups of rats; the value in rats with U-ARFG was significantly greater (20.1 times increase) and smaller (24.1% decrease) than those in control rats and rats with U-ARF, respectively (Table 3). The CLR of OH-CZX was significantly faster in control rats (20.8 mL/min/kg) than those in rats with U-ARF (0.00805 mL/min/kg) and U-ARFG (0.135 mL/min/kg). Similar results were also obtained for the total amount of unchanged OHCZX excreted in a 24-h urine sample (Ae0–24 h) (Table 3).
DISCUSSION
Figure 1. Mean arterial plasma concentration–time profiles of (A) CZX and (B) OH-CZX after intravenous administration of CZX at a dose of 20 mg/kg to control rats (*, n ¼ 11) and rats with U-ARF (*, n ¼ 16) and U-ARFG (~, n ¼ 15). Bars represent standard deviation.
ARF (659 mg min/mL) and U-ARFG (872 mg min/ mL; Table 3). The CLs were significantly different among three groups of rats; the CL was the fastest and slowest for rats with U-ARF (30.3 mL/min/kg) and control rats (12.2 mL/min/kg), respectively (Table 3). The CLNR in rats with U-ARFG was significantly faster (104% increase) than that in control rats (Table 3). The VSS was not significantly different among three groups of rats, which could be expected because plasma protein binding values were not significantly different among three groups of rats (Table 3). In rats with UARF, CZX was below the detection limit in urine (Table 3). After intravenous administration of CZX, OHCZX was formed quickly for all three groups of rats; OH-CZX was detected in plasma from the 5-min blood sampling and reached respective JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
As mentioned earlier, the changes in the reported pharmacokinetic parameters of many drugs (or compounds) in rats with U-ARF are explained mainly by impaired kidney and/or liver function or formation of conjugates. However, the relationship between pharmacokinetic changes of drugs (except CZX6) and changes in CYP isozymes in rats with U-ARF seem not to be published. In the present study, the CZX in plasma and urine was unconjugated CZX (not CZX glucuronides). After intravenous administration of CZX at a dose 20 mg/kg, the CLs (12.2–30.3 mL/min/kg based on plasma data, Table 3) were considerably slower than the reported cardiac output of 295 mL/ min/kg in rats based on blood data.23 This difference suggests that the first-pass effect of CZX in the lung and heart could be almost negligible, if any, in the three groups of rats. The plasma protein binding values of CZX (at a CZX concentration of 10 mg/mL) were 68.5, 71.8, and 63.4% for control rats and rats with U-ARF and U-ARFG, respectively, as determinied by an equilibrium dialysis technique (Table 3). The estimated CLR values of CZX based on free (unbound in plasma proteins) fraction were 0.301 and 0.0839 mL/min/kg for control rats and rats with U-ARFG, respectively; the values were considerably slower than the
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reported glomerular filtration rate of 5.24 mL/min/ kg in rats.23 These data indicate that CZX is mainly reabsorbed in rat renal tubules. Considering the CLR values of CZX (Table 3), reported kidney blood flow rate of 36.8 mL/min/kg,23 and hematocrit of 45%24 in rats, the estimated renal extraction ratios (CLR of CZX/kidney plasma flow rate, only for urinary excretion of unchanged CZX) were 0.468 and 0.152% for control rats and rats with U-ARFG, respectively. These data indicate that CZX was excreted poorly via rat kidney. The hepatic extraction ratio (hepatic first-pass effect) of CZX was roughly estimated by dividing the CLNR of CZX by the reported hepatic plasma flow rate in rats and assuming that nonrenal clearance of CZX was attributed solely to the liver (hence the ratio represents the maximal possible value of liver).25 The hepatic plasma flow rate in rats was estimated based on the reported hepatic blood flow rate of 30.4 mL/min/kg23 and hematocrit of 45%24 in rats. The hepatic extraction ratio of CZX in control rats was 68.2%, indicating that CZX is an intermediate to high hepatic extraction ratio drug. Because CZX is an intermediate-tohigh hepatic extraction ratio drug, its hepatic clearance depends more on hepatic blood flow than CLint and free (unbound in plasma protein) fraction of CZX.26 The significantly faster CL of CZX in rats with U-ARF could be due to significantly faster CLint (107% increase, Table 2) because it has been reported27 that cardiac output, renal blood flow rate, and hepatic blood flow rate decreased significantly in rats with U-ARF induced by glycerol compared with those parameters in control rats. A similar explanation could also be applied to rats with U-ARFG; in these rats, the CLint was significantly faster (57.6% increase) than that in control rats. Northern and Western blot analyses revealed that CYP2E1 was induced 2.3-fold in rats with UARF.6 CZX primarily undergoes metabolism that is catalyzed mainly via CYP2E1 to form OHCZX.4,5 Hence, it was expected that formation of OH-CZX could increase in rats with U-ARF compared with that of control rats. This expectation was supported by the following results. First, in rats with U-ARF, AUC0–8 h of OH-CZX was significantly greater (26.7 times increase) and AUC of CZX was significantly smaller (59.8% decrease) than those parametersin control rats (Table 3). Moreover, in rats with U-ARF, the Cmax of OHCZX reached at 72 min and the peak concentration was maintained for up to 8 h, but in control rats, the Cmax reached at 19.5 min, declined for up to 4 h
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(the mean plasma concentration of OH-CZX at 4 h in control rats was 1.14% of that in rats with U-ARF), and the low concentrations were maintained for up to 8 h (Figure 1B). Second, in rats with U-ARF, the AUC0–8 h, OH-CZX/AUCCZX was 68.2 times greater than that in control rats (Table 2). However, the effect of acute renal failure-induced accumulation of OH-CZX on the increase in the ratio could not be totally ruled out because acute renal failure reduces urinary excretion of drugs (compounds). It has also been reported6 that in rats with UARFG, the CYP2E1 level was significantly greater and smaller than those in control rats and in rats with U-ARF, respectively. Hence, it was expected that in rats with U-ARFG, formation of OH-CZX could be significantly greater and smaller than those in control rats and in rats with U-ARF, respectively. This expectation was also supported by the following results. First, in rats with UARFG, AUC0–8 h of OH-CZX was significantly greater (20.1 times increase) and smaller (24.1% decrease) than those in control rats and in rats with U-ARF (Table 3), respectively. Second, the AUC0–8 h, OH-CZX/AUCCZX in rats with U-ARFG was greater (38.7 times increase) and smaller (42.7% decrease) than those in control rats and in rats with U-ARF (Table 3), respectively. Finally, the CLint in rats with U-ARFG was significantly faster (57.6% increase) and slower (24.0%) than those in control rats and in rats with U-ARF (Table 2), respectively. Although CYP2E1 is the major form metabolizing CZX to OH-CZX, it has been reported,28–31 that human CYP1A2 and CYP3A4 are involved in CZX hydroxylation. The effect of CYP1A2 on the formation of OH-CZX in rats with U-ARF could be ruled out because no significant change in CYP1A2 was obtained in the rats.6 In rats with U-ARF, the expression of CYP3A23 increased 4-fold.6 Human CYP3A4 and rat CYP3A1 (CYP3A23) proteins have 73% homology.32 It has been reported that CYP3A1 (CYP3A23)33 or CYP3A234–36 is the major CYP3A protein in rats. Hence, the role of CYP3A23 and CYP3A2 in the formation of OH-CZX in rats was measured by treatment with DDT, an inducer of CYP3A237 or DDE, an inducer of CYP3A23.38 It has been concluded6 that OH-CZX is formed mainly via CYP2E1 in rats with U-ARF. In summary, the CYP2E1 protein and mRNA level in rats with U-ARFG were greater than those in control rats, but significantly lower than those in rats with U-ARF. OH-CZX is mainly metabolized via CYP2E1 in rats.1 Hence, it could be JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
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expected that the formation of OH-CZX in rats with U-ARFG would be greater than that in control rats but smaller than that in rats with UARF. This expectation was proven by AUC0–8 h values of OH-CZX; the values were 414, 11500, and 8730 mg min/mL for control rats and rats with U-ARF and U-ARFG, respectively; each value was significantly different among three groups (Table 3). These data indicate that the formation of OH-CZX increased significantly in rats with U-ARFG compared with that in control rats, but decreased significantly compared with that in rats with U-ARF due to CYP2E1 changes.
ACKNOWLEDGMENTS This study was supported in part by 2002 BK21 Project for Medicine, Dentistry, and Pharmacy.
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