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Effects of Normothermic Hepatic Ischemia–Reperfusion Injury on the In Vivo, Isolated Perfused Liver, and Microsomal Disposition of Chlorzoxazone, a Cytochrome P450 2E1 Probe, in Rats
PHARMACOKINETICS, PHARMACODYNAMICS AND DRUG METABOLISM Effects of Normothermic Hepatic Ischemia–Reperfusion Injury on the In Vivo, Isolated Perfused Liver, and Microsomal Disposition of Chlorzoxazone, a Cytochrome P450 2E1 Probe, in Rats IMAM H. SHAIK, REZA MEHVAR Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106 Received 26 March 2011; revised 28 June 2011; accepted 28 June 2011 Published online 20 July 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22708 ABSTRACT: In vitro studies have shown that the activities of cytochrome P450 (P450) enzymes may be altered after hepatic ischemia–reperfusion (IR) injury. Here, we investigated the effects of 1 h of partial ischemia, followed by 3 (IR3) or 24 (IR24) h of in vivo reperfusion, on the in vivo, isolated perfused rat liver (IPRL), and microsomal disposition of chlorzoxazone (CZX) and its cytochrome P450 2E1 (CYP2E1)-mediated metabolite, 6-hydroxychlorzoxazone (HCZX), in rats. Although IR3 caused a 30% reduction in the in vivo clearance of CZX, the area under the plasma concentration-time curve of HCZX was not affected. IPRL experiments showed that IR3, in addition to a 30% reduction in the clearance of CZX, causes a 70% decrease in the biliary clearance of HCZX. Microsomal data revealed a 50% decline in the intrinsic clearance of HCZX formation due to an IR3-induced significant decline in maximum velocity. Although IR3 did not affect the microsomal CYP2E1 protein, it caused approximately 30% reduction in the cytochrome P450 reductase activity. IR24 did not have any effect on the disposition of CZX or HCZX. In conclusion, metabolism of xenobiotics and endogenous compounds that are substrates for CYP2E1, and possibly other P450 isoenzymes, may be reduced shortly after surgical procedures that require transient interruption of the hepatic blood flow. © 2011 WileyLiss, Inc. and the American Pharmacists Association J Pharm Sci 100:5281–5292, 2011 Keywords: hepatic ischemia–reperfusion injury; pharmacokinetics; isolated perfused rat liver; hepatobiliary disposition; microsomes; cytochrome P450 2E1; cytochrome P450 reductase; chlorzoxazone; hepatic metabolism; metabolite kinetics
INTRODUCTION Warm (normothermic) hepatic ischemia–reperfusion (IR) injury occurs in a number of emergency clinical settings, such as trauma, shock, and liver retrieval for transplantation, and in nonemergency elective liver surgery such as removal of cancerous tissue.1,2 In most cases, the injury is due to a deliberate clamping of the portal triad by the surgeon to avoid excessive bleeding during liver resection or from the cut hepatic surface.2 This transient interruption of blood supply to the liver, followed by the reinstatement of blood flow after ischemia, may induce significant damages to the organ, potentially leading to liver failure and systemic inflammatory response syndrome.2–4 It Correspondence to: Reza Mehvar (Telephone: +806-356-4015; Fax: +806-356-4034; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 100, 5281–5292 (2011) © 2011 Wiley-Liss, Inc. and the American Pharmacists Association
is believed that one of the hallmarks of IR injury is an overproduction of reactive oxygen species, resulting in lipid peroxidation, DNA oxidation and fragmentation, and protein oxidation.5 In addition to cellular damage and progression of the IR injury, these changes, and in particular oxidation of proteins and lipids, may potentially affect the transport and metabolism of drugs. Therefore, it is necessary to determine the effects of hepatic IR on the pharmacokinetics, pharmacodynamics, and toxicity of different drugs that are used in this population. Several in vitro studies6–11 have shown that warm hepatic IR significantly affects the protein and/or activities of cytochrome P450 (P450) enzymes, which are responsible for the metabolism of a large number of xenobiotics and endogenous compounds. Generally, these studies report that warm hepatic IR causes a significant decrease in the total P450 content of the liver and activities of a number of P450 isoenzymes,
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including rat CYP3A, CYP2A, and CYP2B.6,11 However, the effects of warm IR on the cytochrome P450 2E1 (CYP2E1) activity and/or protein are not clear, as both induction8,10 and inhibition9,12 of CYP2E1 activity have been reported. Although important, the in vitro microsomal studies may be affected by factors, such as preparation, handling, and storage of microsomes, which might have contributed to the disagreements among in vitro reports with regard to the effects of hepatic IR on the CYP2E1 in vitro activity. Therefore, it is necessary to study the effects of warm hepatic IR injury on the in vivo metabolism and pharmacokinetics of specific substrates for various P450 isoenzymes such as CYP2E1. Cytochrome P450 2E1 is an important P450 enzyme responsible for the metabolism of a number of xenobiotics, ethanol, and environmental chemicals.13 Commonly used probes for CYP2E1 activity are chlorzoxazone (CZX), p-nitrophenol, aniline, and Nnitrosodimethylamine. Among these, CZX, a centrally acting muscle relaxant, is most widely used as a CYP2E1 probe for in vivo studies.14–18 In both rats and humans, the major metabolite of CZX is 6-hydroxychlorzoxazone (HCZX), which is mostly formed via CYP2E1. Additionally, in both species, HCZX is completely metabolized by glucuronidation without undergoing any other metabolic pathway.19,20 Therefore, we used CZX as an in vivo probe to study the effects of warm hepatic IR injury on the CYP2E1 activity. Our hypothesis was that warm hepatic IR reduces CYP2E1 in vivo activity in rats, resulting in altered pharmacokinetics of CZX and its CYP2E1mediated metabolite, HCZX. In addition to the in vivo studies, the hepatobiliary dispositions of CZX and HCZX were also determined in an ex vivo model of isolated perfused rat liver (IPRL) preparation. These studies, supplemented with additional in vitro microsomal experiments, depict a relatively clear picture of the effects of warm hepatic IR on the hepatobiliary disposition of CZX and HCZX and expression/activity of CYP2E1.
Massachusetts). All other reagents were of analytical grade and obtained from commercial sources. Animals Male Sprague–Dawley rats weighing 200–250 g were purchased from Charles River Laboratories, Inc. (Wilmington, Massachusetts). Animals were maintained on a 12-h light/dark cycle with free access to food and water. The procedures involving animals were approved by the Texas Tech University Health Sciences Center Animal Care and Use Committee and were consistent with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996, Washington, District of Columbia). Experimental Groups A total of 60 rats were used for in vivo, IPRL, and in vitro microsomal studies. Animals were subjected to 1 h of partial (70%) ischemia (IR) or sham operation (Sham), followed by 3 or 24 h of in vivo reperfusion, resulting in four groups of Sham (3 h), IR (3 h), Sham (24 h), and IR (24 h). IR Surgery
MATERIALS AND METHODS
The normothermic hepatic IR procedure was similar to that described by us previously.11 Briefly, after an overnight fast, rats were anaesthetized with an intramuscular injection of ketamine–xylazine (80:8 mg/ kg), and ischemia was induced for 1 h by occluding the left branches of the portal vein, hepatic artery, and bile duct using a microvascular clamp. This procedure results in ischemia to the left and median lobes of the liver, while leaving blood flow to the right and caudate lobes intact to prevent intestinal congestion. The animal body temperature was closely monitored and maintained at 37◦ C by a combination of a heating plate and a heating lamp connected to an automatic thermoregulated temperature controller. After 1 hour, the clamp was removed, and livers were allowed to reperfuse in vivo for 3 or 24 h. Animals in the Sham group underwent an identical surgical manipulation but without occlusion of the vasculature.
Chemicals and Reagents
In Vivo Studies
Aspartate aminotransferase (AST) assay kit was from Teco Diagnostics (Anaheim, California). CZX, HCZX, 7-hydroxycoumarin (umbelliferone), $-glucuronidase (type-II from limpets), magnesium chloride, and cytochrome c were obtained from Sigma–Aldrich (St. Louis, Missouri). Nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from Calzyme Laboratories (San Luis Obispo, California). Mouse anti-rat primary antibody for CYP2E1 was obtained from Lifespan Biosciences (Seattle, Washington). Anti-mouse horseradish peroxidase secondary antibody was purchased from PerkinElmer Inc. (Boston,
After an overnight fast, under a ketamine–xylazine (80:8 mg/kg) anesthesia, the right jugular veins of the rats were cannulated by using a silicone-tipped polyethylene tubing, followed by ischemia or sham operation as described above. After reperfusion (3 or 24 h), rats were administered a single intravenous dose (∼20 mg/kg) of CZX injected over 2 min. CZX was dissolved in normal saline with the aid of NaOH.21 Briefly, 10 mg of CZX was added to 1 mL of a saline solution containing 0.06 M NaOH, and the mixture was vortexed vigorously. The dosing solution was then centrifuged before injection to remove any possible
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insoluble CZX, and the actual strength of the solution was determined by high-performance liquid chromatography (HPLC). The pH of the final solution was approximately 10, and the actual dose administered to rats (2 mL/kg) ranged from 17 to 20 mg/ kg. Blood samples (∼250 :L) were collected from the jugular vein catheter at 0 (before drug injection), 5, 15, 30, 60, 90, 120, 150, and 180 min into heparinized microcentrifuge tubes, and plasma was separated. Additionally, total urine output was collected for 24 h. At the end of experiments, animals were euthanized using CO2 , and liver was collected and snap-frozen by immersing in liquid nitrogen. All the samples were stored at −80◦ C until further analysis. The number of animals in the Sham (3 h), IR (3 h), Sham (24 h), and IR (24 h) groups were 10, 9, 7, and 8, respectively. IPRL Studies In these experiments, 3 or 24 h after IR or sham operation, rats were used as liver donors for ex vivo IPRL studies. The procedures for the isolation and perfusion of the livers were similar to those reported before.19,22,23 In brief, after opening the abdominal wall, bile duct, portal vein (inlet), and suprahepatic vena cava (outlet) were cannulated. The livers were then transferred to a water jacketed (37◦ C), all glass perfusion system (Radnoti Glass Technology Inc., Monrovia, California) and perfused in a singlepass manner at a constant flow rate of 30 mL/min [∼3–4 mL/(min g) liver]. The perfusate was a Krebs– Henseleit bicarbonate buffer supplemented with 1.2 g/L glucose, 4.75 mg/L sodium taurocholate, which was oxygenated with 95% oxygen and 5% carbon dioxide. The viability of the liver was confirmed through different indices as described previously,22 such as overall macroscopic appearance of the liver, wet liver weights of less than 4% of body weight at the end of perfusion, low and stable inlet pressure, and relatively stable bile flow over the entire perfusion period (60 min). After a stabilization period, livers were infused with a constant concentration (∼1 :g/mL or 6 :M) of CZX for 1 h. This concentration of CZX in the proteinfree perfusate was selected based on the observed plasma concentrations of CZX in our in vivo studies and the reported14 free fraction of the drug in rat plasma. Inlet perfusate samples (0.5 mL) were collected at 5, 30, and 60 min, and outlet perfusate samples (1 mL) were collected at 0 (before drug injection), 5, 10, 15, 20, 30, 40, 50, and 60 min. Bile samples were collected in preweighed microcentrifuge tubes at 15 min intervals. At the end of perfusion, the livers were blotted dry, weighed, and snap-frozen by immersing in liquid nitrogen. All the samples were stored at −80◦ C until further analysis. The number of animals in the Sham (3 h), IR (3 h), Sham (24 h), and IR (24 h) groups were 4, 4, 5, and 5, respectively. DOI 10.1002/jps
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In Vitro Studies In an independent study, rats were subjected to 1 h of ischemia (n = 4) or sham operation (n = 4), followed by 3 h of reperfusion, and the livers were used for preparation of microsomes and in vitro metabolism of CZX. Additionally, livers collected at the end of the IPRL studies from the Sham (3 h), IR (3 h), Sham (24 h), and IR (24 h) groups were used for preparation of microsomes and determination of the Michaelis–Menten parameters of formation of HCZX from CZX in vitro. Measurement of Liver Injury Marker (AST) in Plasma Undiluted or 20-fold diluted (3 h IR group only) plasma samples were used to quantitate AST levels spectrophotometrically using a kit from Teco Diagnostics. Microsomal Preparation, Cytochrome b5 Content, and Cytochrome P450 Reductase Activity Ischemic lobes (left and median) were used for preparation of liver microsomes using an established ultracentrifugation method as described previously.11 Protein concentrations were measured by the Bradford method. Cytochrome b5 content was determined based on the method of Omura and Sato.24 Cytochrome P450 reductase (CPR) activity was measured by its ability to reduce cytochrome c, as described previously.25 Microsomal Metabolism of CZX The CZX hydoxylating activity of liver microsomes was measured according to previous methods at a single26 or multiple27 concentrations of CZX. For the single concentration, CZX (200 :M) was incubated for 20 min with 400 :g/mL microsomal protein in Tris buffer (pH 7.4) in the presence of 5 mM MgCl2 , and the reaction was started by the addition of NADPH (1 mM). For determination of Michaelis–Menten values, the microsomal protein concentration and incubation time were 800 :g/mL and 15 min, respectively, and the substrate (CZX) concentrations were 5, 10, 25, 50, 100, 250, 500, and 1000 :M. The reactions (250 :L) were terminated by the addition of 5 :L of 70% perchloric acid, and the samples were used for determination of CZX and HCZX concentrations by HPLC. Western Blot Analysis of Microsomal CYP2E1 The microsomal content of CYP2E1 protein was determined by Western immunoblot analysis according to standard methods described before.12 Briefly, equal amounts of proteins (5 :g per lane) were resolved by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a nitrocellulose JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
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membrane, and incubated with mouse monoclonal antibody against rat CYP2E1. Immunoreactive bands were visualized after incubation with the horseradish peroxidase secondary antibody using an enhanced chemiluminescent Western blotting system according to the manufacturers’ instructions (Pierce Biotechnology, Inc., Rockford, Illinois). Calreticulin, which is an endoplasmic reticulum marker, was used as a loading control. The rabbit polyclonal antibody to calreticulin was obtained from Santa Cruz Biotechnology (Santa Cruz, California). Sample Analysis Before analysis, the urine and bile samples were diluted with deionized water by fourfold and 100fold, respectively. The concentrations of CZX and HCZX (both before and after hydrolysis with $glucuronidase) in the plasma, diluted urine, inlet and outlet perfusate, and diluted bile samples were analyzed according to a reported HPLC method28 with slight modifications. Briefly, for measurement of CZX and total (free plus glucuronidated) HCZX, 100 :L of each sample was mixed with 100 :L of 2000 U/ mL $-glucuronidase and 200 :L of 0.2 M sodium acetate buffer (pH 4.6), followed by incubation at 37◦ C for 3 h. After incubation, 5 :L of perchloric acid, 100 :L of umbelliferone (internal standard) in methanol (10 :g/mL), and 3 mL of diethyl ether were added. The samples were vortex-mixed for 2 min and centrifuged at 2000g for 4 min. The upper organic layer was transferred to a clean glass tube containing 100 :L of glacial acetic acid and dried under a nitrogen stream. The residues were reconstituted in 200 :L of 0.1% acetic acid: acetonitrile (70:30), and 50–150 :L aliquots were injected into the HPLC system. For measurement of the free (unconjugated) HCZX, samples were analyzed similarly but without the incubation with $-glucuronidase. The concentrations of CZX and HCZX in the microsomal samples were determined using a direct protein precipitation method as described before.26 Briefly, after stopping the reactions (250 :L) by the addition of 5 :L of 70% perchloric acid and addition of internal standard (100 :L of 10 :g/mL umbelliferone in methanol), samples were vortex mixed, centrifuged, and the supernatant was used for the HPLC analysis. Data Analysis For the in vivo studies, noncompartmental pharmacokinetic parameters for CZX and HCZX were estimated using WinNonlin program (Pharsight, Mountain View, California). The estimated pharmacokinetic parameters were area under the plasma concentration–time curve from time zero to infinity (AUC0–∞ ), total body clearance (CL), renal clearance JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
(CLR ), volume of distribution at steady state (Vss ), and plasma half-life (t1/2 ). The latter parameter was calculated from the slope of the log-linear terminal portion of the plasma concentration–time profile. Additionally, linear trapezoidal method was used for calculation of AUC. The total amount of the drug or metabolite excreted in the urine (Au ) was estimated by multiplying the concentration of the analyte in the urine sample collected from zero to 24 h after the drug administration by the volume of the urine. For the IPRL studies, the steady-state outlet concentration (Cout ) of the CZX and HCZX were estimated by the average outlet concentrations at the last four sampling points. For the inlet concentrations (Cin ), the average of the inlet samples taken at 5, 30, and 60 min was used. The hepatic extraction ratio of CZX (E) was calculated according to the following equation: E=
Cin − COut Cin
(1)
The hepatic (CLh ) and intrinsic (CLint ) clearances of CZX and biliary clearance (CLb ) of HCZX glucuronide were then calculated using the following equations according to the well-stirred model: CLh = E × Q
(2)
CLh 1−E
(3)
Abile AUCPerfusate
(4)
CLint = CLb =
where Q is the perfusate flow rate (30 mL/min) and Abile and AUCPerfusate are the amount of HCZX glucuronide eliminated in the bile and the AUC of HCZX glucuronide in the outlet perfusate, respectively, during the 60 min of perfusion. AUCPerfusate was estimated using linear trapezoidal rule. The amount of HCZX glucuronide recovered in the perfusate (APerfusate ) was estimated by multiplying the AUCPerfusate by the perfusate flow rate. The total amount of HCZX recovered (Atotal ) was calculated by adding Abile to APerfusate . For the in vitro microsomal studies, nonlinear regression analysis using WinNonlin program was employed for the estimation of Michaelis–Menten parameters of the metabolism of CZX to HCZX using the following equation: L=
Vmax × [s] km + [s]
(5)
where υ, Vmax , and km are velocity, maximum velocity, and Michaelis–Menten constant, respectively, and [s] DOI 10.1002/jps
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is the concentration of CZX in the microsomal reaction mixture. The in vitro CLint value was then calculated by dividing Vmax by km . The statistical differences among experimental groups were tested using analysis of variance (ANOVA), followed by Bonferroni’s post-test analysis of means between Sham (3 h) and IR (3 h), Sham (24 h) and IR (24 h), Sham (3 h) and Sham (24 h), and IR (3 h) and IR (24 h) groups. When two groups were available only, an unpaired, two-tailed t-test was used. A p value of less than 0.05 was considered significant. The results are presented as mean ± SD.
RESULTS IR Injury The plasma concentrations of the hepatic injury marker AST are shown in Figure 1 for both in vivo (Fig. 1a) and IPRL (Fig. 1b) studies. The plasma AST levels at 3 h after reperfusion were significantly (p < 0.001) higher than those in the respective shamoperated rats in both studies. However, there were no significant differences between the Sham and IR groups at 24 h following reperfusion. Extent of Glucuronidation of HCZX During In Vivo and IPRL Studies A previous report19 showed that HCZX is rapidly and completely glucuronidated in one passage through the rat liver. In agreement with this report, preliminary analysis of samples obtained during the in vivo and IPRL studies before and after glucuronidase treatment showed that HCZX was present almost entirely in the conjugated form. Therefore, except for the microsomal studies, all the samples were subjected to glucuronidase treatment before the analysis, and HCZX data presented in the subsequent sections for the in vivo and IPRL studies indeed are related to HCZX glucuronide.
In Vivo Studies The plasma concentration–time profiles of CZX and HCZX in the Sham and IR rats at 3 or 24 h after in vivo reperfusion are presented in Figure 2. Additionally, a summary of the major pharmacokinetic parameters of CZX and HCZX are presented in Table 1. In the 3 h reperfusion group, IR caused a 31% decrease (p < 0.05) in the CL of CZX, resulting in an increase in its plasma concentrations, as reflected in a 41% increase (p < 0.05) in the AUC0–∞ value (Fig. 2 and Table 1). The Vss of CZX, however, was not affected by 1 h of ischemia, followed by 3 h of reperfusion (Table 1). Although the decrease in CL in the IR (3 h) group, compared with the Sham (3 h) group, also caused a 47% increase in the terminal t1/2 of the DOI 10.1002/jps
Figure 1. Plasma concentrations of AST in the in vivo (a) and IPRL (b) studies. Rats were subjected to 1 h of partial hepatic ischemia (IR) or sham operation (Sham), followed by 3 or 24 h of in vivo reperfusion. Rats in the in vivo study (a) were also subjected to jugular vein cannulation. Columns and bars represent the mean and SD values, respectively. ∗ p < 0.001, compared with the Sham (3 h) group; ∗∗ p < 0.001, compared with the IR (3 h) group.
drug, the change in t1/2 did not reach statistical significance (Table 1). Despite the decrease in the CL of CZX, the plasma concentration–time profiles (Fig. 2a) and pharmacokinetic parameters of HCZX (Table 1), such as AUC0–∞ , Au , or CLR , were not affected by the IR (3 h) injury. The IR (3 h)-induced in vivo decrease in the CL of CZX (Table 1) was further confirmed in a separate study, which showed (Fig. 3) a 50% decrease (p < 0.01) in the formation rate of HCZX from CZX in the microsomes of rats subjected to IR (3 h). In contrast to the 3 h reperfusion groups, the plasma concentration–time profiles (Fig. 2b) and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
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Figure 2. Plasma concentration–time courses of CZX and HCZX. Rats were subjected to 1 h of partial hepatic ischemia (IR) or sham operation (Sham), followed by 3 (a) or 24 (b) h of in vivo reperfusion, when a single 20 mg/kg dose of CZX was administered intravenously. Symbols and bars represent the mean and SD values, respectively. Number of animals was 10, 9, 7, or 8 in the Sham (3 h), IR (3 h), Sham (24 h), or IR (24 h), respectively.
pharmacokinetic parameters (Table 1) of both CZX and HCZX were similar in the Sham and IR groups after 24 h of reperfusion. However, there were significant differences between the 3 h and 24 h groups in some pharmacokinetic parameters of CZX and HCZX (Table 1). Although the Vss of CZX was significantly lower, resulting in shorter t1/2 , in the 24 h groups, the CLR of HCZX was substantially (∼twofold) higher in the 24 h group, as compared with the 3 h groups
(Table 1). The twofold higher CLR of HCZX in the 24 h groups also contributed to the 2.5-fold to 3.0fold lower AUC0–∞ of the metabolite in these groups (Table 1). Only negligible amounts (<0.5%) of the unchanged parent drug were found in the urine of rats, regardless of the treatment. Therefore, the urinary excretion data are not presented here for the parent drug.
Table 1. Major Pharmacokinetic Parameters of CZX and HCZX After Administration of a Single Intravenous Dose (20 mg/kg) of CZX to Rats Reperfusion Time 3h
n CZX AUC0–∞ [(:g min)/mL. CL [mL/(min kg)] Vss (L/kg) t1/2 (min) HCZX AUC0–∞ [(:g min)/mL. Au (% dose) CLR [mL/(min kg)]
24 h
Sham
IR
Sham
IR
10
9
7
8
1650 ± 507 11.5 ± 3.8 0.597 ± 0.153 31.9 ± 13.2
2320 ± 750a 7.97 ± 1.97a 0.563 ± 0.162 46.8 ± 32.5
2030 ± 261 8.64 ± 1.16 0.340 ± 0.068b 22.2 ± 7.4
1990 ± 394 8.96 ± 1.86 0.323 ± 0.043c 20.2 ± 4.4d
740 ± 360 36.9 ± 8.5 13.0 ± 9.3
758 ± 224 42.0 ± 5.8 10.7 ± 3.8
245 ± 57b 35.1 ± 3.7 25.6 ± 5.2e
304 ± 66c 38.9 ± 6.0 22.6 ± 4.4c
Rats were subjected to sham operation or partial hepatic ischemia (1 h), followed by 3 or 24 h of reperfusion when CZX was administered. Data are presented as mean ± SD. a Significantly different from the Sham group at 3 h: p < 0.05, ANOVA, followed by Bonferroni’s multiple comparison test. b Significantly different from the Sham group at 3 h: p < 0.001, ANOVA, followed by Bonferroni’s multiple comparison test. c Significantly different from the IR group at 3 h: p < 0.01, ANOVA, followed by Bonferroni’s multiple comparison test. d Significantly different from the IR group at 3 h: p < 0.05, ANOVA, followed by Bonferroni’s multiple comparison test. e Significantly different from the Sham group at 3 h: p < 0.01, ANOVA, followed by Bonferroni’s multiple comparison test.
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Figure 3. In vitro activity of CYP2E1. Activity was determined by the formation rate of HCZX from CZX in the liver microsomes from the rats subjected to 1 h of partial hepatic ischemia (IR) or sham operation (Sham), followed by 3 h of in vivo reperfusion. Columns and bars represent the mean and SD values, respectively. ∗∗ p < 0.01, compared with the Sham group.
IPRL Studies The concentration–time profiles of CZX and HCZX in the inlet and outlet perfusate samples of the livers infused with approximately 1 :g/mL (6 :M) CZX are presented in Figure 4 for the four experimental groups. As expected, the inlet concentrations of CZX remained relatively constant during the entire perfusion period for all the groups. Additionally, both CZX and HCZX reached relatively stable concentrations in the outlet soon after the start of the ex vivo perfusion. IR caused a significant change in the relative perfusate concentration–time profiles of CZX and HCZX in the 3 h groups (Fig. 4a and Fig. 4b); whereas the mean steady-state concentrations of CZX were lower than those of the metabolite in the Sham (3 h) livers (Fig. 4a), the opposite was true for the IR (3 h) group (Fig. 4b). However, the concentration–time profiles of both CZX and HCZX were very similar for the 24 h Sham and IR groups (Fig. 4c and Fig. 4d). The bile flow rates and hepatobiliary disposition parameters of CZX and HCZX in IPRL preparations are presented in Table 2. Bile flow rates were relatively high in all groups, except for the IR (3 h) group, which showed a 40% reduction (p < 0.01), compared with the Sham (3 h) livers. In the IR (3 h) group, the hepatic extraction ratio and clearance of CZX were significantly (p < 0.05) reduced by approximately 30%, when compared with the values in the Sham (3 h) group. The reduced E was due to approximately 70% reduction (p < 0.001) in the CLint of CZX (Table 2). The IR (3 h)-induced decrease in the CLint , E, and CLh of CZX was also reflected in a 42% decrease (p < 0.01) in the total (bile plus perfusate) recovery of the metabolite. In addition to the decreased metabolism of the parent drug to HCZX, 3 h IR also caused approximately 75% reduction (p < 0.001) in the bilDOI 10.1002/jps
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iary excretion and CLbile of the metabolite HCZX (Table 2). In contrast to the 3 h groups, the hepatobiliary disposition parameters of CZX or HCZX were the same in the 24 h Sham and IR groups (Table 2). However, the CLint (p < 0.001), E (p < 0.01), and CLh (p < 0.001) of CZX in the Sham (24 h) group were significantly lower than those in the Sham (3 h) group (Table 2). Similarly, CLbile (p < 0.001), Abile (p < 0.001), and Atotal (p < 0.05) of HCZX in the Sham (24 h) group were all significantly lower than those in the Sham (3 h) group (Table 2).
In Vitro Studies The microsomes isolated from the livers after IPRL studies were used to determine the effects of IR on the kinetics of in vitro metabolism of CZX to HCZX. Michaelis–Menten plots depicting the kinetics of HCZX formation from CZX are shown in Figure 5, and the kinetic parameters obtained from these plots are presented in Table 3. The Vmax of the formation of HCZX in the IR (3 h) group was approximately 40% less than that in the Sham (3 h) microsomes (p < 0.05) (Fig. 5 and Table 3). However, there was no effect of IR on the km values. The reduction in Vmax was associated with approximately 50% reduction in CLint (p < 0.05) (Table 3). In contrast, the Michaelis–Menten parameters of HCZX formation were similar in the IR (24 h) and Sham (24 h) groups (Fig. 5 and Table 3). In addition to the IR injury, the Vmax and CLint values in the Sham (24 h) group were significantly (p < 0.05) lower than those in the Sham (3 h) group (Table 3). Table 3 also lists the CPR activity and cytochrome b5 content of the microsomes. Similar to Vmax , CPR activity was significantly (p < 0.05) reduced in the IR (3 h) group, compared with the Sham (3 h) microsomes. However, a 25% reduction in the cytochrome b5 content in the IR (3 h) microsomes did not attain statistical significance. The CYP2E1 protein contents in the Sham (3 h) and IR (3 h) microsomes were measured by Western immunoblot analysis (Fig. 6). There was no difference in the CYP2E1 protein contents between the two groups.
DISCUSSION Despite several in vitro evidence that warm hepatic IR alters the protein and/or activities of drug metabolizing enzymes and transporters, the information on the effects of the injury on the in vivo pharmacokinetics of drugs is scarce.29 In the present report, we studied the effects of warm IR injury on the in vivo pharmacokinetics of CZX and its metabolite HCZX in rats. Our results (Fig. 2 and Table 1) showed that although the pharmacokinetics of CZX and HCZX were not affected 24 h after IR injury, IR (3 h) significantly JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
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Table 2.
Bile Flow Rates and Hepatobiliary Disposition Parameters of CZX and HCZX in IPRL Preparations Reperfusion Time 3h
n Bile flow rate [:L/(min g)] CZX E CLh [mL/(min g)] CLint [mL/(min g)] HCZX CLbile [mL/(min g)] APerfusate (% dose) Abile (% dose) Atotal (% dose)
24 h
Sham
IR
Sham
IR
4 1.80 ± 0.19
4 1.02 ± 0.40a
5 1.69 ± 0.25
5 1.89 ± 0.28b
0.838 ± 0.021 3.18 ± 0.15 19.9 ± 2.5
0.592 ± 0.099c 2.13 ± 0.56c 5.63 ± 2.50d
0.535 ± 0.109a 1.68 ± 0.40d 3.89 ± 1.49d
0.526 ± 0.132 1.77 ± 0.54 4.38 ± 2.88
2.96 ± 0.70 21.9 ± 4.9 16.8 ± 3.6 38.7 ± 6.4
0.791 ± 0.381d 18.5 ± 4.8 3.95 ± 1.46d 22.4 ± 5.2a
1.25 ± 0.32d 16.8 ± 3.8 6.99 ± 3.3d 23.8 ± 6.3c
0.930 ± 0.165 19.0 ± 6.1 5.09 ± 0.73 24.1 ± 6.6
Livers were collected from rats subjected to sham operation or partial hepatic ischemia (1 h), followed by 3 or 24 h of reperfusion and perfused ex vivo with a constant concentration (∼1 :g/mL or 6 :M) of CZX for 60 min. Data are presented as mean ± SD. a Significantly different from the Sham group at 3 h: p < 0.01, ANOVA, followed by Bonferroni’s multiple comparison test. b Significantly different from the IR group at 3 h: p < 0.01, ANOVA, followed by Bonferroni’s multiple comparison test. c Significantly different from the Sham group at 3 h: p < 0.05, ANOVA, followed by Bonferroni’s multiple comparison test. d Significantly different from the Sham group at 3 h: p < 0.001, ANOVA, followed by Bonferroni’s multiple comparison test.
Figure 4. Inlet and outlet perfusate concentration–time courses of CZX and outlet perfusate concentration–time courses of HCZX in isolated perfused livers. Samples were collected from rats subjected to 1 h of partial hepatic ischemia (IR) or sham operation (Sham), followed by 3 (a and b) or 24 (c and d) h of in vivo reperfusion. Symbols and bars represent the mean and SD values, respectively. Number of animals was 4, 4, 5, or 5 in the Sham (3 h), IR (3 h), Sham (24 h), or IR (24 h), respectively.
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Table 3.
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In Vitro Microsomal Data for Metabolism of CZX to HCZX Reperfusion Time 3h
n CZX metabolism Vmax [nmol/(min mg)] km (:M) CLint [:L/(min mg) CPR activity [nmol/(min mg)] Cytochrome b5 (nmol/mg)
24 h
Sham
IR
Sham
IR
4
4
5
5
1.70 ± 0.30 143 ± 12 11.9 ± 2.2 154 ± 17 0.204 ± 0.018
1.01 ± 0.42a 177 ± 28 6.09 ± 3.48a 107 ± 21a 0.152 ± 0.040
1.07 ± 0.18a 200 ± 122 6.61 ± 2.94a 151 ± 8 0.215 ± 0.032
1.02 ± 0.26 165 ± 15 6.16 ± 1.39 152 ± 32b 0.217 ± 0.042b
Microsomes were prepared from the livers subjected to sham operation or partial hepatic ischemia (1 h), followed by 3 or 24 h of in vivo reperfusion and 1 h of ex vivo perfusion. Data are presented as mean ±SD. a Significantly different from the Sham group at 3 h: p < 0.05, ANOVA, followed by Bonferroni’s multiple comparison test. b Significantly different from the IR group at 3 h: p < 0.05, ANOVA, followed by Bonferroni’s multiple comparison test.
reduced the CL of CZX, suggesting a significant decrease in the CYP2E1 in vivo activity and formation of HCZX as a result of IR (3 h). However, to our surprise, the AUC of HCZX in the IR (3 h) and Sham (3 h) groups were very similar (Fig. 2a and Table 1). An independent in vitro study (Fig. 3) suggested that the IR (3 h)-induced in vivo reduction in the CL of CZX (Table 1) is indeed due to a reduction in the formation of the CYP2E1-mediated metabolic pathway, thus confirming the discrepancy. The AUC of HCZX is a function of both formation and elimination processes of the metabolite. Therefore, the lack of the effects of IR (3 h) on the AUC of HCZX, despite a decrease in its formation, might have been due to a decreased elimination of HCZX in the IR (3 h) group. However, the CLR of HCZX was similar in both IR (3 h) and Sham (3 h) groups (Table 1). Therefore, further studies were needed to explain the discrepancy
between the IR (3 h)-induced lower CL of CZX and similar AUCs of HCZX in the IR (3 h) and Sham (3 h) groups. A previous work19 in an IPRL model similar to that used in the current study showed that after administration into the portal vein, HCZX is rapidly and completely converted to its glucuronide metabolite, which is excreted into the bile. This finding suggests that in addition to CLR , biliary excretion of HCZX may have significantly contributed to the in vivo clearance of the metabolite in rats. Therefore, we hypothesized that the discrepancy between the effects of IR on the CL of CZX and AUC of HCZX, observed in our in vivo study (Table 1), is due to an IR-induced reduction in the CLb of HCZX. To test this hypothesis, further studies were conducted in IPRL preparations, which could allow determination of hepatobiliary disposition of CZX and HCZX. Indeed, the IPRL studies (Table 2) clearly
Figure 5. Michaelis–Menten plots for formation of HCZX from CZX in liver microsomes. Microsomes were obtained from the rats subjected to 1 h of partial hepatic ischemia (IR) or sham operation (Sham), followed by 3 (a) or 24 (b) h of in vivo reperfusion. The livers were also perfused for 1 h ex vivo before preparation of microsomes. Symbols and bars represent the mean and SD values, respectively. Number of animals was 4, 4, 5, or 5 in the Sham (3 h), IR (3 h), Sham (24 h), or IR (24 h), respectively.
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Figure 6. Western blot analysis of CYP2E1 protein content in liver microsomes. Microsomes were obtained from the rats subjected to 1 h of partial hepatic ischemia (IR) or sham operation (Sham), followed by 3 h of in vivo reperfusion. The livers were also perfused for 1 h ex vivo before preparation of microsomes. a and b represent the Western blots and densitometric analysis of immunoblots, expressed as a percentage of Sham, respectively. For densitometric analysis, the CYP2E1 values were corrected for the corresponding loading control calreticulin. Columns and bars represent the mean and SD values, respectively.
showed that IR (3 h) causes a substantial reduction in the biliary excretion and clearance of HCZX. Therefore, we suggest that the lack of the effect of IR (3 h) on the AUC of HCZX, despite a reduction in its formation, observed in our in vivo studies (Fig. 2 and Table 1), is due to a simultaneous IR (3 h)-induced reduction in the CLb of the metabolite. Considering the plasma CZX concentrations of 0.4–40 :g/mL, observed in our in vivo study (Fig. 2), and a free fraction of 0.1 in rat plasma,14 the expected free (unbound) concentration of CZX in our plasma samples were approximately 0.04–4 :g/mL. Therefore, we chose an inlet CZX concentration of 1 :g/mL (6 :M) for our IPRL studies, which did not use any protein in the perfusate, to be within the observed free plasma concentration range of CZX. Indeed, the dispositions of CZX and HCZX in IPRLs (Fig. 4 and Table 2) were in complete agreement with the in vivo data (Fig. 2 and Table 1). Although the hepatobiliary disposition of CZX and HCZX were similar in the IR and Sham groups at 24 h after reperfusion, substantial effects of IR was evident at 3 h of reperfusion. Additionally, although the outlet perfusate concentrations of CZX in the IR (3 h) group were substantially higher than those in the Sham (3 h) group, the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
perfusate concentrations of the metabolite were similar in both groups (Fig. 4 and Table 2), a situation similar to that observed in vivo (Fig. 2). However, the IPRL data also provided a direct evidence for the IR (3 h)-induced reduction of the formation of HCZX; an IR (3 h)-induced reduction in the CLh and E of CZX was also associated with a significant reduction in the total amount of HCZX recovered in the bile and perfusate in this group (Table 2). The kinetics of the metabolism of CZX to HCZX in the liver microsomes (Table 3 and Fig. 5) were consistent with the in vivo (Table 1 and Fig. 2) and IPRL (Table 2 and Fig. 4) data. However, in contrast to the decrease in the microsomal CLint of CZX (Table 3), the microsomal CYP2E1 protein content remained unaffected by IR (3 h) (Fig. 6). A similar observation (unchanged CYP2E1 protein content despite a reduction in CYP2E1 activity) was also made by us in an earlier study,12 where CYP2E1 activity was measured using a probe (p-nitrophenol) different than CZX. Additional data obtained from the microsomal studies (Table 3) suggest that a significant decrease in the activity of CPR, which is the rate-limiting enzyme for P450-catalyzed reactions, may have been responsible for the observed reduction in the CYP2E1 activity. In agreement with this postulate, previous studies30 using recombinant CYP2E1 and CPR in lipid vesicles have shown that decreasing the CPR contents in the presence of fixed amounts of CYP2E1 significantly decreases the CYP2E1 activity measured by p-nitrophenol or CZX hydroxylation. Although it has been reported31 that, in addition to CYP2E1, CYP3A may also contribute to the metabolism of CZX to HCZX in rat liver microsomes, our current (Fig. 5) and previous27 data in rat liver microsomes were best described by a singleenzyme kinetic model. More importantly, a previous IPRL study19 using CZX inlet concentrations of up to 200 :M also showed that the steady-state formation rate of HCZX could only be described by a singleenzyme kinetic model with a km value of 8 :M, which is close to the inlet concentration used in the current study (Fig. 4). Nevertheless, the contribution of CYP3A to the formation of HCZX cannot be ruled out in our study. In addition to the effects of IR on CYP2E1, the reduction in the CLb of HCZX glucuronide, observed in our studies (Table 2), suggests that IR may also affect the pharmacokinetics of drugs via CLb mechanisms. The CLb of glucuronides, such as HCZX glucuronide, is expected to occur through the ABC transporter multidrug resistance-related protein type 2 (Mrp2).32 Therefore, a reduction in the CLb of HCZX glucuronide in the IR (3 h) group may be due to an IRinduced reduction in the function of Mrp2. Although our study is the first to suggest an IR-induced decrease in the function of Mrp2, previous studies33,34 DOI 10.1002/jps
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have shown that warm hepatic IR reduces the mRNA expression and protein content of this transporter in the rat livers. Nevertheless, more studies are needed to confirm the role of IR on the function of liver transporters and the pharmacokinetics of drugs that are subjected to these transporters. In addition to the effects of IR, our data also detected an influence of the time of study (3 vs. 24 h) on the disposition parameters of CZX and HCZX in all of our studies (in vivo, IPRL, and in vitro) (Table 1–Table 3). Generally, the CL, CLh , and Vmax of CZX in the Sham (24 h) group were lower than those in the Sham (3 h) group (Table 1–Table 3), although the data for the in vivo CL was not significant. Additionally, Vss of CZX was lower in the 24 h groups, compared with that in the 3 h groups (Table 1). As for the metabolite, although the CLb of HCZX glucuronide was lower at 24 h (Table 2), its CLR was significantly higher at 24 h (Table 1), compared with the 3 h data. These differences are due, most likely, to a time-dependent effect of sham surgery and jugular vein cannulation on the activity of the liver enzymes and biliary and renal excretion pathways. Indeed, in agreement with our data, several studies35–37 have shown that jugular vein cannulation causes a timedependent decrease in the metabolic clearance and volume of distribution of drugs. For instance, it was shown36 that the hepatic CLint and the free fraction of propranolol at 48 h following jugular vein cannulation were, respectively, fourfold and twofold lower than those at 2 h after implantation. Interestingly, the propranolol and antipyrine metabolizing activities of hepatocytes obtained at 2 h following catheter implantation were not different from those obtained from the control animals without catheters.36 As for CLR , it has been reported38 that surgery and anesthesia cause severe depression of renal blood flow and glomerular filtration rate, within 2–3 h of surgery. The twofold lower CLR value of HCZX glucuronide observed in our 3 h groups, compared with the values in the 24 h groups (Table 1), is in agreement with this report. Aside from our present study, we are aware of only one report29 that investigated the effects of warm hepatic IR injury on the in vivo pharmacokinetics of drugs. That study showed that 1 h of partial ischemia, followed by 12 h of reperfusion in rats caused a decrease in the oral absorption of cyclosporine A due to IR-induced increases in the CYP3A activity and P-glycoprotein content in the upper segments of the small intestine. Therefore, the effects of hepatic IR are not limited to the P450 enzymes in the liver and may involve remote organs. Further studies are needed to fully characterize the impact of hepatic IR on the pharmacokinetics of drugs used following IR.
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CONCLUSIONS In conclusion, in vivo, ex vivo (IPRLs), and in vitro (liver microsomes) studies showed that the CYP2E1 activity is reduced shortly (3 h) after 1 h of partial hepatic ischemia in rats. This effect of IR was short lived as 24 h following ischemia, no effect of IR could be detected on the CYP2E1 activity. The 3 h IR-induced reduction in the CYP2E1 activity was accompanied with no change in the liver microsomal protein content of the enzyme. It is suggested that a significant IR-induced reduction in the activity of CPR, which is the rate-limiting enzyme for all microsomal P450catalyzed reactions, is responsible for the observed reduction in the CYP2E1 activity after IR. Therefore, the metabolism of xenobiotics and endogenous compounds that are substrates for CYP2E1, and possibly other P450 isoenzymes, may be reduced shortly after surgical procedures that require transient interruption of the hepatic blood flow.
ACKNOWLEDGMENTS This research was financially supported by the Vascular Drug Research Center at the Texas Tech School of Pharmacy.
REFERENCES 1. Klune JR, Tsung A. 2010. Molecular biology of liver ischemia/ reperfusion injury: Established mechanisms and recent advancements. Surg Clin North Am 90:665–677. 2. Abu-Amara M, Yang SY, Tapuria N, Fuller B, Davidson B, Seifalian A. 2010. Liver ischemia/reperfusion injury: Processes in inflammatory networks—A review. Liver Transpl 16:1016–1032. 3. Lichtman SN, Lemasters JJ. 1999. Role of cytokines and cytokine-producing cells in reperfusion injury to the liver. Semin Liver Dis 19:171–187. 4. Jaeschke H, Lemasters JJ. 2003. Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion injury. Gastroenterology 125:1246–1257. 5. Toledo-Pereyra LH, Lopez-Neblina F, Toledo AH. 2004. Reactive oxygen species and molecular biology of ischemia/ reperfusion. Ann Transplant 9:81–83. 6. Lindstrom TD, Hanssen BR, Bendele AM. 1990. Effects of hepatic ischemia–reperfusion injury on the hepatic mixed function oxidase system in rats. Mol Pharmacol 38:829–835. 7. Izuishi K, Hamamoto I, Ichikawa Y, Okano K, Akram HM, Maeba T, Tanaka S. 1994. Effect of liver warm ischemia on the hepatic microsomal cytochrome P-450 monooxygenase system. Transplant Proc 26:2417–2419. 8. Lee SM, Clemens MG. 1992. Effect of alpha-tocopherol on hepatic mixed function oxidases in hepatic ischemia/reperfusion. Hepatology 15:276–281. 9. Pahan K, Smith BT, Singh AK, Singh I. 1997. Cytochrome P-450 2E1 in rat liver peroxisomes: Downregulation by ischemia/reperfusion-induced oxidative stress. Free Radic Biol Med 23:963–971. 10. Eum HA, Lee SM. 2004. Effects of Trolox on the activity and gene expression of cytochrome P450 in hepatic ischemia/ reperfusion. Br J Pharmacol 142:35–42.
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11. Shaik IH, Mehvar R. 2010. Cytochrome P450 induction by phenobarbital exacerbates warm hepatic ischemia–reperfusion injury in rat livers. Free Radic Res 44:441–453. 12. Shaik IH, George JM, Thekkumkara TJ, Mehvar R. 2008. Protective effects of diallyl sulfide, a garlic constituent, on the warm hepatic ischemia–reperfusion injury in a rat model. Pharm Res 25:2231–2242. 13. Lieber CS. 1997. Cytochrome P-4502E1: Its physiological and pathological role. Physiol Rev 77:517–544. 14. 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. 15. Emery MG, Jubert C, Thummel KE, Kharasch ED. 1999. Duration of cytochrome P-450 2E1 (CYP2E1) inhibition and estimation of functional CYP2E1 enzyme half-life after single-dose disulfiram administration in humans. J Pharmacol Exp Ther 291:213–219. 16. Ahn CY, Bae SK, Jung YS, Lee I, Kim YC, Lee MG, Shin WG. 2008. Pharmacokinetic parameters of chlorzoxazone and its main metabolite, 6-hydroxychlorzoxazone, after intravenous and oral administration of chlorzoxazone to liver cirrhotic rats with diabetes mellitus. Drug Metab Dispos 36:1233–1241. 17. Ahn CY, Kim EJ, Lee I, Kwon JW, Kim WB, Kim SG, Lee MG. 2003. Effects of glucose on the pharmacokinetics of intravenous chlorzoxazone in rats with acute renal failure induced by uranyl nitrate. J Pharm Sci 92:1604–1613. 18. Chalasani N, Gorski JC, Asghar MS, Asghar A, Foresman B, Hall SD, Crabb DW. 2003. Hepatic cytochrome P450 2E1 activity in nondiabetic patients with nonalcoholic steatohepatitis. Hepatology 37:544–550. 19. Mehvar R, Vuppugalla R. 2006. Hepatic disposition of the cytochrome P450 2E1 marker chlorzoxazone and its hydroxylated metabolite in isolated perfused rat livers. J Pharm Sci 95:1414–1424. 20. Conney AH, Burns JJ. 1960. Biochemical pharmacological considerations of zoxazolamine and chlorzoxazone metabolism. Ann N Y Acad Sci 86:167–177. 21. Chen L, Yang CS. 1996. Effects of cytochrome P450 2E1 modulators on the pharmacokinetics of chlorzoxazone and 6hydroxychlorzoxazone in rats. Life Sci 58:1575–1585. 22. Mehvar R, Zhang XP. 2002. Development and application of an isolated perfused rat liver model to study the stimulation and inhibition of tumor necrosis factor-alpha production ex vivo. Pharm Res 19:47–53. 23. Vuppugalla R, Mehvar R. 2006. Selective effects of nitric oxide on the disposition of chlorzoxazone and dextromethorphan in isolated perfused rat livers. Drug Metab Dispos 34:1160–1166. 24. Omura T, Sato R. 1964. The carbon monoxide-binding pigment of the liver microsomes. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378. 25. Guengerich FP. 1994. Analysis and characterization of enzymes. In Principles and methods of toxicology; Hayes AW, Ed. 3rd ed.New York: Raven Press, pp 1259–1313.
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26. Vuppugalla R, Mehvar R. 2004. Hepatic disposition and effects of nitric oxide donors: Rapid and concentration-dependent reduction in the cytochrome P450-mediated drug metabolism in isolated perfused rat livers. J Pharmacol Exp Ther 310:718– 727. 27. Vuppugalla R, Mehvar R. 2005. Enzyme-selective effects of nitric oxide on affinity and maximum velocity of various rat cytochromes P450. Drug Metab Dispos 33:829– 836. 28. Frye RF, Stiff DD. 1996. Determination of chlorzoxazone and 6-hydroxychlorzoxazone in human plasma and urine by highperformance liquid chromatography. J Chromatogr B Biomed Appl 686:291–296. 29. Ikemura K, Urano K, Matsuda H, Mizutani H, Iwamoto T, Okuda M. 2009. Decreased oral absorption of cyclosporine A after liver ischemia–reperfusion injury in rats: The contribution of CYP3A and P-glycoprotein to the first-pass metabolism in intestinal epithelial cells. J Pharmacol Exp Ther 328:249– 255. 30. Kim HR, Lee GH, Yi Cho E, Chae SW, Ahn T, Chae HJ. 2009. Bax inhibitor 1 regulates ER-stress-induced ROS accumulation through the regulation of cytochrome P450 2E1. J Cell Sci 122:1126–1133. 31. Jayyosi Z, Knoble D, Muc M, Erick J, Thomas PE, Kelley M. 1995. Cytochrome P-450 2E1 is not the sole catalyst of chlorzoxazone hydroxylation in rat liver microsomes. J Pharmacol Exp Ther 273:1156–1161. 32. Hoffmann U, Kroemer HK. 2004. The ABC transporters MDR1 and MRP2: Multiple functions in disposition of xenobiotics and drug resistance. Drug Metab Rev 36:669–701. 33. Tanaka Y, Chen C, Maher JM, Klaassen CD. 2006. Kupffer cell-mediated downregulation of hepatic transporter expression in rat hepatic ischemia–reperfusion. Transplantation 82:258–266. 34. Tanaka Y, Chen C, Maher JM, Klaassen CD. 2008. Ischemia– reperfusion of rat livers decreases liver and increases kidney multidrug resistance associated protein 2 (Mrp2). Toxicol Sci 101:171–178. 35. Terao N, Shen DD. 1983. Alterations in serum protein binding and pharmacokinetics of l-propranolol in the rat elicited by the presence of an indwelling venous catheter. J Pharmacol Exp Ther 227:369–375. 36. Chindavijak B, Belpaire FM, De Smet F, Bogaert MG. 1988. Alteration of the pharmacokinetics and metabolism of propranolol and antipyrine elicited by indwelling catheters in the rat. J Pharmacol Exp Ther 246:1075–1079. 37. Torres-Molina F, Aristorena JC, Garcia-Carbonell C, Granero L, Chesa-Jimenez J, Pla-Delfina J, Peris-Ribera JE. 1992. Influence of permanent cannulation of the jugular vein on pharmacokinetics of amoxycillin and antipyrine in the rat. Pharm Res 9:1587–1591. 38. Walker LA, Buscemi-Bergin M, Gellai M. 1983. Renal hemodynamics in conscious rats: Effects of anesthesia, surgery, and recovery. Am J Physiol 245:F67–F74.
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INTRODUCTION Warm (normothermic) hepatic ischemia–reperfusion (IR) injury occurs in a number of emergency clinical settings, such as trauma, shock, and liver retrieval for transplantation, and in nonemergency elective liver surgery such as removal of cancerous tissue.1,2 In most cases, the injury is due to a deliberate clamping of the portal triad by the surgeon to avoid excessive bleeding during liver resection or from the cut hepatic surface.2 This transient interruption of blood supply to the liver, followed by the reinstatement of blood flow after ischemia, may induce significant damages to the organ, potentially leading to liver failure and systemic inflammatory response syndrome.2–4 It Correspondence to: Reza Mehvar (Telephone: +806-356-4015; Fax: +806-356-4034; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 100, 5281–5292 (2011) © 2011 Wiley-Liss, Inc. and the American Pharmacists Association
is believed that one of the hallmarks of IR injury is an overproduction of reactive oxygen species, resulting in lipid peroxidation, DNA oxidation and fragmentation, and protein oxidation.5 In addition to cellular damage and progression of the IR injury, these changes, and in particular oxidation of proteins and lipids, may potentially affect the transport and metabolism of drugs. Therefore, it is necessary to determine the effects of hepatic IR on the pharmacokinetics, pharmacodynamics, and toxicity of different drugs that are used in this population. Several in vitro studies6–11 have shown that warm hepatic IR significantly affects the protein and/or activities of cytochrome P450 (P450) enzymes, which are responsible for the metabolism of a large number of xenobiotics and endogenous compounds. Generally, these studies report that warm hepatic IR causes a significant decrease in the total P450 content of the liver and activities of a number of P450 isoenzymes,
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including rat CYP3A, CYP2A, and CYP2B.6,11 However, the effects of warm IR on the cytochrome P450 2E1 (CYP2E1) activity and/or protein are not clear, as both induction8,10 and inhibition9,12 of CYP2E1 activity have been reported. Although important, the in vitro microsomal studies may be affected by factors, such as preparation, handling, and storage of microsomes, which might have contributed to the disagreements among in vitro reports with regard to the effects of hepatic IR on the CYP2E1 in vitro activity. Therefore, it is necessary to study the effects of warm hepatic IR injury on the in vivo metabolism and pharmacokinetics of specific substrates for various P450 isoenzymes such as CYP2E1. Cytochrome P450 2E1 is an important P450 enzyme responsible for the metabolism of a number of xenobiotics, ethanol, and environmental chemicals.13 Commonly used probes for CYP2E1 activity are chlorzoxazone (CZX), p-nitrophenol, aniline, and Nnitrosodimethylamine. Among these, CZX, a centrally acting muscle relaxant, is most widely used as a CYP2E1 probe for in vivo studies.14–18 In both rats and humans, the major metabolite of CZX is 6-hydroxychlorzoxazone (HCZX), which is mostly formed via CYP2E1. Additionally, in both species, HCZX is completely metabolized by glucuronidation without undergoing any other metabolic pathway.19,20 Therefore, we used CZX as an in vivo probe to study the effects of warm hepatic IR injury on the CYP2E1 activity. Our hypothesis was that warm hepatic IR reduces CYP2E1 in vivo activity in rats, resulting in altered pharmacokinetics of CZX and its CYP2E1mediated metabolite, HCZX. In addition to the in vivo studies, the hepatobiliary dispositions of CZX and HCZX were also determined in an ex vivo model of isolated perfused rat liver (IPRL) preparation. These studies, supplemented with additional in vitro microsomal experiments, depict a relatively clear picture of the effects of warm hepatic IR on the hepatobiliary disposition of CZX and HCZX and expression/activity of CYP2E1.
Massachusetts). All other reagents were of analytical grade and obtained from commercial sources. Animals Male Sprague–Dawley rats weighing 200–250 g were purchased from Charles River Laboratories, Inc. (Wilmington, Massachusetts). Animals were maintained on a 12-h light/dark cycle with free access to food and water. The procedures involving animals were approved by the Texas Tech University Health Sciences Center Animal Care and Use Committee and were consistent with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996, Washington, District of Columbia). Experimental Groups A total of 60 rats were used for in vivo, IPRL, and in vitro microsomal studies. Animals were subjected to 1 h of partial (70%) ischemia (IR) or sham operation (Sham), followed by 3 or 24 h of in vivo reperfusion, resulting in four groups of Sham (3 h), IR (3 h), Sham (24 h), and IR (24 h). IR Surgery
MATERIALS AND METHODS
The normothermic hepatic IR procedure was similar to that described by us previously.11 Briefly, after an overnight fast, rats were anaesthetized with an intramuscular injection of ketamine–xylazine (80:8 mg/ kg), and ischemia was induced for 1 h by occluding the left branches of the portal vein, hepatic artery, and bile duct using a microvascular clamp. This procedure results in ischemia to the left and median lobes of the liver, while leaving blood flow to the right and caudate lobes intact to prevent intestinal congestion. The animal body temperature was closely monitored and maintained at 37◦ C by a combination of a heating plate and a heating lamp connected to an automatic thermoregulated temperature controller. After 1 hour, the clamp was removed, and livers were allowed to reperfuse in vivo for 3 or 24 h. Animals in the Sham group underwent an identical surgical manipulation but without occlusion of the vasculature.
Chemicals and Reagents
In Vivo Studies
Aspartate aminotransferase (AST) assay kit was from Teco Diagnostics (Anaheim, California). CZX, HCZX, 7-hydroxycoumarin (umbelliferone), $-glucuronidase (type-II from limpets), magnesium chloride, and cytochrome c were obtained from Sigma–Aldrich (St. Louis, Missouri). Nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from Calzyme Laboratories (San Luis Obispo, California). Mouse anti-rat primary antibody for CYP2E1 was obtained from Lifespan Biosciences (Seattle, Washington). Anti-mouse horseradish peroxidase secondary antibody was purchased from PerkinElmer Inc. (Boston,
After an overnight fast, under a ketamine–xylazine (80:8 mg/kg) anesthesia, the right jugular veins of the rats were cannulated by using a silicone-tipped polyethylene tubing, followed by ischemia or sham operation as described above. After reperfusion (3 or 24 h), rats were administered a single intravenous dose (∼20 mg/kg) of CZX injected over 2 min. CZX was dissolved in normal saline with the aid of NaOH.21 Briefly, 10 mg of CZX was added to 1 mL of a saline solution containing 0.06 M NaOH, and the mixture was vortexed vigorously. The dosing solution was then centrifuged before injection to remove any possible
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insoluble CZX, and the actual strength of the solution was determined by high-performance liquid chromatography (HPLC). The pH of the final solution was approximately 10, and the actual dose administered to rats (2 mL/kg) ranged from 17 to 20 mg/ kg. Blood samples (∼250 :L) were collected from the jugular vein catheter at 0 (before drug injection), 5, 15, 30, 60, 90, 120, 150, and 180 min into heparinized microcentrifuge tubes, and plasma was separated. Additionally, total urine output was collected for 24 h. At the end of experiments, animals were euthanized using CO2 , and liver was collected and snap-frozen by immersing in liquid nitrogen. All the samples were stored at −80◦ C until further analysis. The number of animals in the Sham (3 h), IR (3 h), Sham (24 h), and IR (24 h) groups were 10, 9, 7, and 8, respectively. IPRL Studies In these experiments, 3 or 24 h after IR or sham operation, rats were used as liver donors for ex vivo IPRL studies. The procedures for the isolation and perfusion of the livers were similar to those reported before.19,22,23 In brief, after opening the abdominal wall, bile duct, portal vein (inlet), and suprahepatic vena cava (outlet) were cannulated. The livers were then transferred to a water jacketed (37◦ C), all glass perfusion system (Radnoti Glass Technology Inc., Monrovia, California) and perfused in a singlepass manner at a constant flow rate of 30 mL/min [∼3–4 mL/(min g) liver]. The perfusate was a Krebs– Henseleit bicarbonate buffer supplemented with 1.2 g/L glucose, 4.75 mg/L sodium taurocholate, which was oxygenated with 95% oxygen and 5% carbon dioxide. The viability of the liver was confirmed through different indices as described previously,22 such as overall macroscopic appearance of the liver, wet liver weights of less than 4% of body weight at the end of perfusion, low and stable inlet pressure, and relatively stable bile flow over the entire perfusion period (60 min). After a stabilization period, livers were infused with a constant concentration (∼1 :g/mL or 6 :M) of CZX for 1 h. This concentration of CZX in the proteinfree perfusate was selected based on the observed plasma concentrations of CZX in our in vivo studies and the reported14 free fraction of the drug in rat plasma. Inlet perfusate samples (0.5 mL) were collected at 5, 30, and 60 min, and outlet perfusate samples (1 mL) were collected at 0 (before drug injection), 5, 10, 15, 20, 30, 40, 50, and 60 min. Bile samples were collected in preweighed microcentrifuge tubes at 15 min intervals. At the end of perfusion, the livers were blotted dry, weighed, and snap-frozen by immersing in liquid nitrogen. All the samples were stored at −80◦ C until further analysis. The number of animals in the Sham (3 h), IR (3 h), Sham (24 h), and IR (24 h) groups were 4, 4, 5, and 5, respectively. DOI 10.1002/jps
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In Vitro Studies In an independent study, rats were subjected to 1 h of ischemia (n = 4) or sham operation (n = 4), followed by 3 h of reperfusion, and the livers were used for preparation of microsomes and in vitro metabolism of CZX. Additionally, livers collected at the end of the IPRL studies from the Sham (3 h), IR (3 h), Sham (24 h), and IR (24 h) groups were used for preparation of microsomes and determination of the Michaelis–Menten parameters of formation of HCZX from CZX in vitro. Measurement of Liver Injury Marker (AST) in Plasma Undiluted or 20-fold diluted (3 h IR group only) plasma samples were used to quantitate AST levels spectrophotometrically using a kit from Teco Diagnostics. Microsomal Preparation, Cytochrome b5 Content, and Cytochrome P450 Reductase Activity Ischemic lobes (left and median) were used for preparation of liver microsomes using an established ultracentrifugation method as described previously.11 Protein concentrations were measured by the Bradford method. Cytochrome b5 content was determined based on the method of Omura and Sato.24 Cytochrome P450 reductase (CPR) activity was measured by its ability to reduce cytochrome c, as described previously.25 Microsomal Metabolism of CZX The CZX hydoxylating activity of liver microsomes was measured according to previous methods at a single26 or multiple27 concentrations of CZX. For the single concentration, CZX (200 :M) was incubated for 20 min with 400 :g/mL microsomal protein in Tris buffer (pH 7.4) in the presence of 5 mM MgCl2 , and the reaction was started by the addition of NADPH (1 mM). For determination of Michaelis–Menten values, the microsomal protein concentration and incubation time were 800 :g/mL and 15 min, respectively, and the substrate (CZX) concentrations were 5, 10, 25, 50, 100, 250, 500, and 1000 :M. The reactions (250 :L) were terminated by the addition of 5 :L of 70% perchloric acid, and the samples were used for determination of CZX and HCZX concentrations by HPLC. Western Blot Analysis of Microsomal CYP2E1 The microsomal content of CYP2E1 protein was determined by Western immunoblot analysis according to standard methods described before.12 Briefly, equal amounts of proteins (5 :g per lane) were resolved by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a nitrocellulose JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
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membrane, and incubated with mouse monoclonal antibody against rat CYP2E1. Immunoreactive bands were visualized after incubation with the horseradish peroxidase secondary antibody using an enhanced chemiluminescent Western blotting system according to the manufacturers’ instructions (Pierce Biotechnology, Inc., Rockford, Illinois). Calreticulin, which is an endoplasmic reticulum marker, was used as a loading control. The rabbit polyclonal antibody to calreticulin was obtained from Santa Cruz Biotechnology (Santa Cruz, California). Sample Analysis Before analysis, the urine and bile samples were diluted with deionized water by fourfold and 100fold, respectively. The concentrations of CZX and HCZX (both before and after hydrolysis with $glucuronidase) in the plasma, diluted urine, inlet and outlet perfusate, and diluted bile samples were analyzed according to a reported HPLC method28 with slight modifications. Briefly, for measurement of CZX and total (free plus glucuronidated) HCZX, 100 :L of each sample was mixed with 100 :L of 2000 U/ mL $-glucuronidase and 200 :L of 0.2 M sodium acetate buffer (pH 4.6), followed by incubation at 37◦ C for 3 h. After incubation, 5 :L of perchloric acid, 100 :L of umbelliferone (internal standard) in methanol (10 :g/mL), and 3 mL of diethyl ether were added. The samples were vortex-mixed for 2 min and centrifuged at 2000g for 4 min. The upper organic layer was transferred to a clean glass tube containing 100 :L of glacial acetic acid and dried under a nitrogen stream. The residues were reconstituted in 200 :L of 0.1% acetic acid: acetonitrile (70:30), and 50–150 :L aliquots were injected into the HPLC system. For measurement of the free (unconjugated) HCZX, samples were analyzed similarly but without the incubation with $-glucuronidase. The concentrations of CZX and HCZX in the microsomal samples were determined using a direct protein precipitation method as described before.26 Briefly, after stopping the reactions (250 :L) by the addition of 5 :L of 70% perchloric acid and addition of internal standard (100 :L of 10 :g/mL umbelliferone in methanol), samples were vortex mixed, centrifuged, and the supernatant was used for the HPLC analysis. Data Analysis For the in vivo studies, noncompartmental pharmacokinetic parameters for CZX and HCZX were estimated using WinNonlin program (Pharsight, Mountain View, California). The estimated pharmacokinetic parameters were area under the plasma concentration–time curve from time zero to infinity (AUC0–∞ ), total body clearance (CL), renal clearance JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
(CLR ), volume of distribution at steady state (Vss ), and plasma half-life (t1/2 ). The latter parameter was calculated from the slope of the log-linear terminal portion of the plasma concentration–time profile. Additionally, linear trapezoidal method was used for calculation of AUC. The total amount of the drug or metabolite excreted in the urine (Au ) was estimated by multiplying the concentration of the analyte in the urine sample collected from zero to 24 h after the drug administration by the volume of the urine. For the IPRL studies, the steady-state outlet concentration (Cout ) of the CZX and HCZX were estimated by the average outlet concentrations at the last four sampling points. For the inlet concentrations (Cin ), the average of the inlet samples taken at 5, 30, and 60 min was used. The hepatic extraction ratio of CZX (E) was calculated according to the following equation: E=
Cin − COut Cin
(1)
The hepatic (CLh ) and intrinsic (CLint ) clearances of CZX and biliary clearance (CLb ) of HCZX glucuronide were then calculated using the following equations according to the well-stirred model: CLh = E × Q
(2)
CLh 1−E
(3)
Abile AUCPerfusate
(4)
CLint = CLb =
where Q is the perfusate flow rate (30 mL/min) and Abile and AUCPerfusate are the amount of HCZX glucuronide eliminated in the bile and the AUC of HCZX glucuronide in the outlet perfusate, respectively, during the 60 min of perfusion. AUCPerfusate was estimated using linear trapezoidal rule. The amount of HCZX glucuronide recovered in the perfusate (APerfusate ) was estimated by multiplying the AUCPerfusate by the perfusate flow rate. The total amount of HCZX recovered (Atotal ) was calculated by adding Abile to APerfusate . For the in vitro microsomal studies, nonlinear regression analysis using WinNonlin program was employed for the estimation of Michaelis–Menten parameters of the metabolism of CZX to HCZX using the following equation: L=
Vmax × [s] km + [s]
(5)
where υ, Vmax , and km are velocity, maximum velocity, and Michaelis–Menten constant, respectively, and [s] DOI 10.1002/jps
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is the concentration of CZX in the microsomal reaction mixture. The in vitro CLint value was then calculated by dividing Vmax by km . The statistical differences among experimental groups were tested using analysis of variance (ANOVA), followed by Bonferroni’s post-test analysis of means between Sham (3 h) and IR (3 h), Sham (24 h) and IR (24 h), Sham (3 h) and Sham (24 h), and IR (3 h) and IR (24 h) groups. When two groups were available only, an unpaired, two-tailed t-test was used. A p value of less than 0.05 was considered significant. The results are presented as mean ± SD.
RESULTS IR Injury The plasma concentrations of the hepatic injury marker AST are shown in Figure 1 for both in vivo (Fig. 1a) and IPRL (Fig. 1b) studies. The plasma AST levels at 3 h after reperfusion were significantly (p < 0.001) higher than those in the respective shamoperated rats in both studies. However, there were no significant differences between the Sham and IR groups at 24 h following reperfusion. Extent of Glucuronidation of HCZX During In Vivo and IPRL Studies A previous report19 showed that HCZX is rapidly and completely glucuronidated in one passage through the rat liver. In agreement with this report, preliminary analysis of samples obtained during the in vivo and IPRL studies before and after glucuronidase treatment showed that HCZX was present almost entirely in the conjugated form. Therefore, except for the microsomal studies, all the samples were subjected to glucuronidase treatment before the analysis, and HCZX data presented in the subsequent sections for the in vivo and IPRL studies indeed are related to HCZX glucuronide.
In Vivo Studies The plasma concentration–time profiles of CZX and HCZX in the Sham and IR rats at 3 or 24 h after in vivo reperfusion are presented in Figure 2. Additionally, a summary of the major pharmacokinetic parameters of CZX and HCZX are presented in Table 1. In the 3 h reperfusion group, IR caused a 31% decrease (p < 0.05) in the CL of CZX, resulting in an increase in its plasma concentrations, as reflected in a 41% increase (p < 0.05) in the AUC0–∞ value (Fig. 2 and Table 1). The Vss of CZX, however, was not affected by 1 h of ischemia, followed by 3 h of reperfusion (Table 1). Although the decrease in CL in the IR (3 h) group, compared with the Sham (3 h) group, also caused a 47% increase in the terminal t1/2 of the DOI 10.1002/jps
Figure 1. Plasma concentrations of AST in the in vivo (a) and IPRL (b) studies. Rats were subjected to 1 h of partial hepatic ischemia (IR) or sham operation (Sham), followed by 3 or 24 h of in vivo reperfusion. Rats in the in vivo study (a) were also subjected to jugular vein cannulation. Columns and bars represent the mean and SD values, respectively. ∗ p < 0.001, compared with the Sham (3 h) group; ∗∗ p < 0.001, compared with the IR (3 h) group.
drug, the change in t1/2 did not reach statistical significance (Table 1). Despite the decrease in the CL of CZX, the plasma concentration–time profiles (Fig. 2a) and pharmacokinetic parameters of HCZX (Table 1), such as AUC0–∞ , Au , or CLR , were not affected by the IR (3 h) injury. The IR (3 h)-induced in vivo decrease in the CL of CZX (Table 1) was further confirmed in a separate study, which showed (Fig. 3) a 50% decrease (p < 0.01) in the formation rate of HCZX from CZX in the microsomes of rats subjected to IR (3 h). In contrast to the 3 h reperfusion groups, the plasma concentration–time profiles (Fig. 2b) and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
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Figure 2. Plasma concentration–time courses of CZX and HCZX. Rats were subjected to 1 h of partial hepatic ischemia (IR) or sham operation (Sham), followed by 3 (a) or 24 (b) h of in vivo reperfusion, when a single 20 mg/kg dose of CZX was administered intravenously. Symbols and bars represent the mean and SD values, respectively. Number of animals was 10, 9, 7, or 8 in the Sham (3 h), IR (3 h), Sham (24 h), or IR (24 h), respectively.
pharmacokinetic parameters (Table 1) of both CZX and HCZX were similar in the Sham and IR groups after 24 h of reperfusion. However, there were significant differences between the 3 h and 24 h groups in some pharmacokinetic parameters of CZX and HCZX (Table 1). Although the Vss of CZX was significantly lower, resulting in shorter t1/2 , in the 24 h groups, the CLR of HCZX was substantially (∼twofold) higher in the 24 h group, as compared with the 3 h groups
(Table 1). The twofold higher CLR of HCZX in the 24 h groups also contributed to the 2.5-fold to 3.0fold lower AUC0–∞ of the metabolite in these groups (Table 1). Only negligible amounts (<0.5%) of the unchanged parent drug were found in the urine of rats, regardless of the treatment. Therefore, the urinary excretion data are not presented here for the parent drug.
Table 1. Major Pharmacokinetic Parameters of CZX and HCZX After Administration of a Single Intravenous Dose (20 mg/kg) of CZX to Rats Reperfusion Time 3h
n CZX AUC0–∞ [(:g min)/mL. CL [mL/(min kg)] Vss (L/kg) t1/2 (min) HCZX AUC0–∞ [(:g min)/mL. Au (% dose) CLR [mL/(min kg)]
24 h
Sham
IR
Sham
IR
10
9
7
8
1650 ± 507 11.5 ± 3.8 0.597 ± 0.153 31.9 ± 13.2
2320 ± 750a 7.97 ± 1.97a 0.563 ± 0.162 46.8 ± 32.5
2030 ± 261 8.64 ± 1.16 0.340 ± 0.068b 22.2 ± 7.4
1990 ± 394 8.96 ± 1.86 0.323 ± 0.043c 20.2 ± 4.4d
740 ± 360 36.9 ± 8.5 13.0 ± 9.3
758 ± 224 42.0 ± 5.8 10.7 ± 3.8
245 ± 57b 35.1 ± 3.7 25.6 ± 5.2e
304 ± 66c 38.9 ± 6.0 22.6 ± 4.4c
Rats were subjected to sham operation or partial hepatic ischemia (1 h), followed by 3 or 24 h of reperfusion when CZX was administered. Data are presented as mean ± SD. a Significantly different from the Sham group at 3 h: p < 0.05, ANOVA, followed by Bonferroni’s multiple comparison test. b Significantly different from the Sham group at 3 h: p < 0.001, ANOVA, followed by Bonferroni’s multiple comparison test. c Significantly different from the IR group at 3 h: p < 0.01, ANOVA, followed by Bonferroni’s multiple comparison test. d Significantly different from the IR group at 3 h: p < 0.05, ANOVA, followed by Bonferroni’s multiple comparison test. e Significantly different from the Sham group at 3 h: p < 0.01, ANOVA, followed by Bonferroni’s multiple comparison test.
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Figure 3. In vitro activity of CYP2E1. Activity was determined by the formation rate of HCZX from CZX in the liver microsomes from the rats subjected to 1 h of partial hepatic ischemia (IR) or sham operation (Sham), followed by 3 h of in vivo reperfusion. Columns and bars represent the mean and SD values, respectively. ∗∗ p < 0.01, compared with the Sham group.
IPRL Studies The concentration–time profiles of CZX and HCZX in the inlet and outlet perfusate samples of the livers infused with approximately 1 :g/mL (6 :M) CZX are presented in Figure 4 for the four experimental groups. As expected, the inlet concentrations of CZX remained relatively constant during the entire perfusion period for all the groups. Additionally, both CZX and HCZX reached relatively stable concentrations in the outlet soon after the start of the ex vivo perfusion. IR caused a significant change in the relative perfusate concentration–time profiles of CZX and HCZX in the 3 h groups (Fig. 4a and Fig. 4b); whereas the mean steady-state concentrations of CZX were lower than those of the metabolite in the Sham (3 h) livers (Fig. 4a), the opposite was true for the IR (3 h) group (Fig. 4b). However, the concentration–time profiles of both CZX and HCZX were very similar for the 24 h Sham and IR groups (Fig. 4c and Fig. 4d). The bile flow rates and hepatobiliary disposition parameters of CZX and HCZX in IPRL preparations are presented in Table 2. Bile flow rates were relatively high in all groups, except for the IR (3 h) group, which showed a 40% reduction (p < 0.01), compared with the Sham (3 h) livers. In the IR (3 h) group, the hepatic extraction ratio and clearance of CZX were significantly (p < 0.05) reduced by approximately 30%, when compared with the values in the Sham (3 h) group. The reduced E was due to approximately 70% reduction (p < 0.001) in the CLint of CZX (Table 2). The IR (3 h)-induced decrease in the CLint , E, and CLh of CZX was also reflected in a 42% decrease (p < 0.01) in the total (bile plus perfusate) recovery of the metabolite. In addition to the decreased metabolism of the parent drug to HCZX, 3 h IR also caused approximately 75% reduction (p < 0.001) in the bilDOI 10.1002/jps
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iary excretion and CLbile of the metabolite HCZX (Table 2). In contrast to the 3 h groups, the hepatobiliary disposition parameters of CZX or HCZX were the same in the 24 h Sham and IR groups (Table 2). However, the CLint (p < 0.001), E (p < 0.01), and CLh (p < 0.001) of CZX in the Sham (24 h) group were significantly lower than those in the Sham (3 h) group (Table 2). Similarly, CLbile (p < 0.001), Abile (p < 0.001), and Atotal (p < 0.05) of HCZX in the Sham (24 h) group were all significantly lower than those in the Sham (3 h) group (Table 2).
In Vitro Studies The microsomes isolated from the livers after IPRL studies were used to determine the effects of IR on the kinetics of in vitro metabolism of CZX to HCZX. Michaelis–Menten plots depicting the kinetics of HCZX formation from CZX are shown in Figure 5, and the kinetic parameters obtained from these plots are presented in Table 3. The Vmax of the formation of HCZX in the IR (3 h) group was approximately 40% less than that in the Sham (3 h) microsomes (p < 0.05) (Fig. 5 and Table 3). However, there was no effect of IR on the km values. The reduction in Vmax was associated with approximately 50% reduction in CLint (p < 0.05) (Table 3). In contrast, the Michaelis–Menten parameters of HCZX formation were similar in the IR (24 h) and Sham (24 h) groups (Fig. 5 and Table 3). In addition to the IR injury, the Vmax and CLint values in the Sham (24 h) group were significantly (p < 0.05) lower than those in the Sham (3 h) group (Table 3). Table 3 also lists the CPR activity and cytochrome b5 content of the microsomes. Similar to Vmax , CPR activity was significantly (p < 0.05) reduced in the IR (3 h) group, compared with the Sham (3 h) microsomes. However, a 25% reduction in the cytochrome b5 content in the IR (3 h) microsomes did not attain statistical significance. The CYP2E1 protein contents in the Sham (3 h) and IR (3 h) microsomes were measured by Western immunoblot analysis (Fig. 6). There was no difference in the CYP2E1 protein contents between the two groups.
DISCUSSION Despite several in vitro evidence that warm hepatic IR alters the protein and/or activities of drug metabolizing enzymes and transporters, the information on the effects of the injury on the in vivo pharmacokinetics of drugs is scarce.29 In the present report, we studied the effects of warm IR injury on the in vivo pharmacokinetics of CZX and its metabolite HCZX in rats. Our results (Fig. 2 and Table 1) showed that although the pharmacokinetics of CZX and HCZX were not affected 24 h after IR injury, IR (3 h) significantly JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
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Table 2.
Bile Flow Rates and Hepatobiliary Disposition Parameters of CZX and HCZX in IPRL Preparations Reperfusion Time 3h
n Bile flow rate [:L/(min g)] CZX E CLh [mL/(min g)] CLint [mL/(min g)] HCZX CLbile [mL/(min g)] APerfusate (% dose) Abile (% dose) Atotal (% dose)
24 h
Sham
IR
Sham
IR
4 1.80 ± 0.19
4 1.02 ± 0.40a
5 1.69 ± 0.25
5 1.89 ± 0.28b
0.838 ± 0.021 3.18 ± 0.15 19.9 ± 2.5
0.592 ± 0.099c 2.13 ± 0.56c 5.63 ± 2.50d
0.535 ± 0.109a 1.68 ± 0.40d 3.89 ± 1.49d
0.526 ± 0.132 1.77 ± 0.54 4.38 ± 2.88
2.96 ± 0.70 21.9 ± 4.9 16.8 ± 3.6 38.7 ± 6.4
0.791 ± 0.381d 18.5 ± 4.8 3.95 ± 1.46d 22.4 ± 5.2a
1.25 ± 0.32d 16.8 ± 3.8 6.99 ± 3.3d 23.8 ± 6.3c
0.930 ± 0.165 19.0 ± 6.1 5.09 ± 0.73 24.1 ± 6.6
Livers were collected from rats subjected to sham operation or partial hepatic ischemia (1 h), followed by 3 or 24 h of reperfusion and perfused ex vivo with a constant concentration (∼1 :g/mL or 6 :M) of CZX for 60 min. Data are presented as mean ± SD. a Significantly different from the Sham group at 3 h: p < 0.01, ANOVA, followed by Bonferroni’s multiple comparison test. b Significantly different from the IR group at 3 h: p < 0.01, ANOVA, followed by Bonferroni’s multiple comparison test. c Significantly different from the Sham group at 3 h: p < 0.05, ANOVA, followed by Bonferroni’s multiple comparison test. d Significantly different from the Sham group at 3 h: p < 0.001, ANOVA, followed by Bonferroni’s multiple comparison test.
Figure 4. Inlet and outlet perfusate concentration–time courses of CZX and outlet perfusate concentration–time courses of HCZX in isolated perfused livers. Samples were collected from rats subjected to 1 h of partial hepatic ischemia (IR) or sham operation (Sham), followed by 3 (a and b) or 24 (c and d) h of in vivo reperfusion. Symbols and bars represent the mean and SD values, respectively. Number of animals was 4, 4, 5, or 5 in the Sham (3 h), IR (3 h), Sham (24 h), or IR (24 h), respectively.
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Table 3.
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In Vitro Microsomal Data for Metabolism of CZX to HCZX Reperfusion Time 3h
n CZX metabolism Vmax [nmol/(min mg)] km (:M) CLint [:L/(min mg) CPR activity [nmol/(min mg)] Cytochrome b5 (nmol/mg)
24 h
Sham
IR
Sham
IR
4
4
5
5
1.70 ± 0.30 143 ± 12 11.9 ± 2.2 154 ± 17 0.204 ± 0.018
1.01 ± 0.42a 177 ± 28 6.09 ± 3.48a 107 ± 21a 0.152 ± 0.040
1.07 ± 0.18a 200 ± 122 6.61 ± 2.94a 151 ± 8 0.215 ± 0.032
1.02 ± 0.26 165 ± 15 6.16 ± 1.39 152 ± 32b 0.217 ± 0.042b
Microsomes were prepared from the livers subjected to sham operation or partial hepatic ischemia (1 h), followed by 3 or 24 h of in vivo reperfusion and 1 h of ex vivo perfusion. Data are presented as mean ±SD. a Significantly different from the Sham group at 3 h: p < 0.05, ANOVA, followed by Bonferroni’s multiple comparison test. b Significantly different from the IR group at 3 h: p < 0.05, ANOVA, followed by Bonferroni’s multiple comparison test.
reduced the CL of CZX, suggesting a significant decrease in the CYP2E1 in vivo activity and formation of HCZX as a result of IR (3 h). However, to our surprise, the AUC of HCZX in the IR (3 h) and Sham (3 h) groups were very similar (Fig. 2a and Table 1). An independent in vitro study (Fig. 3) suggested that the IR (3 h)-induced in vivo reduction in the CL of CZX (Table 1) is indeed due to a reduction in the formation of the CYP2E1-mediated metabolic pathway, thus confirming the discrepancy. The AUC of HCZX is a function of both formation and elimination processes of the metabolite. Therefore, the lack of the effects of IR (3 h) on the AUC of HCZX, despite a decrease in its formation, might have been due to a decreased elimination of HCZX in the IR (3 h) group. However, the CLR of HCZX was similar in both IR (3 h) and Sham (3 h) groups (Table 1). Therefore, further studies were needed to explain the discrepancy
between the IR (3 h)-induced lower CL of CZX and similar AUCs of HCZX in the IR (3 h) and Sham (3 h) groups. A previous work19 in an IPRL model similar to that used in the current study showed that after administration into the portal vein, HCZX is rapidly and completely converted to its glucuronide metabolite, which is excreted into the bile. This finding suggests that in addition to CLR , biliary excretion of HCZX may have significantly contributed to the in vivo clearance of the metabolite in rats. Therefore, we hypothesized that the discrepancy between the effects of IR on the CL of CZX and AUC of HCZX, observed in our in vivo study (Table 1), is due to an IR-induced reduction in the CLb of HCZX. To test this hypothesis, further studies were conducted in IPRL preparations, which could allow determination of hepatobiliary disposition of CZX and HCZX. Indeed, the IPRL studies (Table 2) clearly
Figure 5. Michaelis–Menten plots for formation of HCZX from CZX in liver microsomes. Microsomes were obtained from the rats subjected to 1 h of partial hepatic ischemia (IR) or sham operation (Sham), followed by 3 (a) or 24 (b) h of in vivo reperfusion. The livers were also perfused for 1 h ex vivo before preparation of microsomes. Symbols and bars represent the mean and SD values, respectively. Number of animals was 4, 4, 5, or 5 in the Sham (3 h), IR (3 h), Sham (24 h), or IR (24 h), respectively.
DOI 10.1002/jps
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Figure 6. Western blot analysis of CYP2E1 protein content in liver microsomes. Microsomes were obtained from the rats subjected to 1 h of partial hepatic ischemia (IR) or sham operation (Sham), followed by 3 h of in vivo reperfusion. The livers were also perfused for 1 h ex vivo before preparation of microsomes. a and b represent the Western blots and densitometric analysis of immunoblots, expressed as a percentage of Sham, respectively. For densitometric analysis, the CYP2E1 values were corrected for the corresponding loading control calreticulin. Columns and bars represent the mean and SD values, respectively.
showed that IR (3 h) causes a substantial reduction in the biliary excretion and clearance of HCZX. Therefore, we suggest that the lack of the effect of IR (3 h) on the AUC of HCZX, despite a reduction in its formation, observed in our in vivo studies (Fig. 2 and Table 1), is due to a simultaneous IR (3 h)-induced reduction in the CLb of the metabolite. Considering the plasma CZX concentrations of 0.4–40 :g/mL, observed in our in vivo study (Fig. 2), and a free fraction of 0.1 in rat plasma,14 the expected free (unbound) concentration of CZX in our plasma samples were approximately 0.04–4 :g/mL. Therefore, we chose an inlet CZX concentration of 1 :g/mL (6 :M) for our IPRL studies, which did not use any protein in the perfusate, to be within the observed free plasma concentration range of CZX. Indeed, the dispositions of CZX and HCZX in IPRLs (Fig. 4 and Table 2) were in complete agreement with the in vivo data (Fig. 2 and Table 1). Although the hepatobiliary disposition of CZX and HCZX were similar in the IR and Sham groups at 24 h after reperfusion, substantial effects of IR was evident at 3 h of reperfusion. Additionally, although the outlet perfusate concentrations of CZX in the IR (3 h) group were substantially higher than those in the Sham (3 h) group, the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
perfusate concentrations of the metabolite were similar in both groups (Fig. 4 and Table 2), a situation similar to that observed in vivo (Fig. 2). However, the IPRL data also provided a direct evidence for the IR (3 h)-induced reduction of the formation of HCZX; an IR (3 h)-induced reduction in the CLh and E of CZX was also associated with a significant reduction in the total amount of HCZX recovered in the bile and perfusate in this group (Table 2). The kinetics of the metabolism of CZX to HCZX in the liver microsomes (Table 3 and Fig. 5) were consistent with the in vivo (Table 1 and Fig. 2) and IPRL (Table 2 and Fig. 4) data. However, in contrast to the decrease in the microsomal CLint of CZX (Table 3), the microsomal CYP2E1 protein content remained unaffected by IR (3 h) (Fig. 6). A similar observation (unchanged CYP2E1 protein content despite a reduction in CYP2E1 activity) was also made by us in an earlier study,12 where CYP2E1 activity was measured using a probe (p-nitrophenol) different than CZX. Additional data obtained from the microsomal studies (Table 3) suggest that a significant decrease in the activity of CPR, which is the rate-limiting enzyme for P450-catalyzed reactions, may have been responsible for the observed reduction in the CYP2E1 activity. In agreement with this postulate, previous studies30 using recombinant CYP2E1 and CPR in lipid vesicles have shown that decreasing the CPR contents in the presence of fixed amounts of CYP2E1 significantly decreases the CYP2E1 activity measured by p-nitrophenol or CZX hydroxylation. Although it has been reported31 that, in addition to CYP2E1, CYP3A may also contribute to the metabolism of CZX to HCZX in rat liver microsomes, our current (Fig. 5) and previous27 data in rat liver microsomes were best described by a singleenzyme kinetic model. More importantly, a previous IPRL study19 using CZX inlet concentrations of up to 200 :M also showed that the steady-state formation rate of HCZX could only be described by a singleenzyme kinetic model with a km value of 8 :M, which is close to the inlet concentration used in the current study (Fig. 4). Nevertheless, the contribution of CYP3A to the formation of HCZX cannot be ruled out in our study. In addition to the effects of IR on CYP2E1, the reduction in the CLb of HCZX glucuronide, observed in our studies (Table 2), suggests that IR may also affect the pharmacokinetics of drugs via CLb mechanisms. The CLb of glucuronides, such as HCZX glucuronide, is expected to occur through the ABC transporter multidrug resistance-related protein type 2 (Mrp2).32 Therefore, a reduction in the CLb of HCZX glucuronide in the IR (3 h) group may be due to an IRinduced reduction in the function of Mrp2. Although our study is the first to suggest an IR-induced decrease in the function of Mrp2, previous studies33,34 DOI 10.1002/jps
EFFECTS OF LIVER ISCHEMIA-REPERFUSION ON THE DISPOSITION OF CZX IN RATS
have shown that warm hepatic IR reduces the mRNA expression and protein content of this transporter in the rat livers. Nevertheless, more studies are needed to confirm the role of IR on the function of liver transporters and the pharmacokinetics of drugs that are subjected to these transporters. In addition to the effects of IR, our data also detected an influence of the time of study (3 vs. 24 h) on the disposition parameters of CZX and HCZX in all of our studies (in vivo, IPRL, and in vitro) (Table 1–Table 3). Generally, the CL, CLh , and Vmax of CZX in the Sham (24 h) group were lower than those in the Sham (3 h) group (Table 1–Table 3), although the data for the in vivo CL was not significant. Additionally, Vss of CZX was lower in the 24 h groups, compared with that in the 3 h groups (Table 1). As for the metabolite, although the CLb of HCZX glucuronide was lower at 24 h (Table 2), its CLR was significantly higher at 24 h (Table 1), compared with the 3 h data. These differences are due, most likely, to a time-dependent effect of sham surgery and jugular vein cannulation on the activity of the liver enzymes and biliary and renal excretion pathways. Indeed, in agreement with our data, several studies35–37 have shown that jugular vein cannulation causes a timedependent decrease in the metabolic clearance and volume of distribution of drugs. For instance, it was shown36 that the hepatic CLint and the free fraction of propranolol at 48 h following jugular vein cannulation were, respectively, fourfold and twofold lower than those at 2 h after implantation. Interestingly, the propranolol and antipyrine metabolizing activities of hepatocytes obtained at 2 h following catheter implantation were not different from those obtained from the control animals without catheters.36 As for CLR , it has been reported38 that surgery and anesthesia cause severe depression of renal blood flow and glomerular filtration rate, within 2–3 h of surgery. The twofold lower CLR value of HCZX glucuronide observed in our 3 h groups, compared with the values in the 24 h groups (Table 1), is in agreement with this report. Aside from our present study, we are aware of only one report29 that investigated the effects of warm hepatic IR injury on the in vivo pharmacokinetics of drugs. That study showed that 1 h of partial ischemia, followed by 12 h of reperfusion in rats caused a decrease in the oral absorption of cyclosporine A due to IR-induced increases in the CYP3A activity and P-glycoprotein content in the upper segments of the small intestine. Therefore, the effects of hepatic IR are not limited to the P450 enzymes in the liver and may involve remote organs. Further studies are needed to fully characterize the impact of hepatic IR on the pharmacokinetics of drugs used following IR.
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CONCLUSIONS In conclusion, in vivo, ex vivo (IPRLs), and in vitro (liver microsomes) studies showed that the CYP2E1 activity is reduced shortly (3 h) after 1 h of partial hepatic ischemia in rats. This effect of IR was short lived as 24 h following ischemia, no effect of IR could be detected on the CYP2E1 activity. The 3 h IR-induced reduction in the CYP2E1 activity was accompanied with no change in the liver microsomal protein content of the enzyme. It is suggested that a significant IR-induced reduction in the activity of CPR, which is the rate-limiting enzyme for all microsomal P450catalyzed reactions, is responsible for the observed reduction in the CYP2E1 activity after IR. Therefore, the metabolism of xenobiotics and endogenous compounds that are substrates for CYP2E1, and possibly other P450 isoenzymes, may be reduced shortly after surgical procedures that require transient interruption of the hepatic blood flow.
ACKNOWLEDGMENTS This research was financially supported by the Vascular Drug Research Center at the Texas Tech School of Pharmacy.
REFERENCES 1. Klune JR, Tsung A. 2010. Molecular biology of liver ischemia/ reperfusion injury: Established mechanisms and recent advancements. Surg Clin North Am 90:665–677. 2. Abu-Amara M, Yang SY, Tapuria N, Fuller B, Davidson B, Seifalian A. 2010. Liver ischemia/reperfusion injury: Processes in inflammatory networks—A review. Liver Transpl 16:1016–1032. 3. Lichtman SN, Lemasters JJ. 1999. Role of cytokines and cytokine-producing cells in reperfusion injury to the liver. Semin Liver Dis 19:171–187. 4. Jaeschke H, Lemasters JJ. 2003. Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion injury. Gastroenterology 125:1246–1257. 5. Toledo-Pereyra LH, Lopez-Neblina F, Toledo AH. 2004. Reactive oxygen species and molecular biology of ischemia/ reperfusion. Ann Transplant 9:81–83. 6. Lindstrom TD, Hanssen BR, Bendele AM. 1990. Effects of hepatic ischemia–reperfusion injury on the hepatic mixed function oxidase system in rats. Mol Pharmacol 38:829–835. 7. Izuishi K, Hamamoto I, Ichikawa Y, Okano K, Akram HM, Maeba T, Tanaka S. 1994. Effect of liver warm ischemia on the hepatic microsomal cytochrome P-450 monooxygenase system. Transplant Proc 26:2417–2419. 8. Lee SM, Clemens MG. 1992. Effect of alpha-tocopherol on hepatic mixed function oxidases in hepatic ischemia/reperfusion. Hepatology 15:276–281. 9. Pahan K, Smith BT, Singh AK, Singh I. 1997. Cytochrome P-450 2E1 in rat liver peroxisomes: Downregulation by ischemia/reperfusion-induced oxidative stress. Free Radic Biol Med 23:963–971. 10. Eum HA, Lee SM. 2004. Effects of Trolox on the activity and gene expression of cytochrome P450 in hepatic ischemia/ reperfusion. Br J Pharmacol 142:35–42.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
5292
SHAIK AND MEHVAR
11. Shaik IH, Mehvar R. 2010. Cytochrome P450 induction by phenobarbital exacerbates warm hepatic ischemia–reperfusion injury in rat livers. Free Radic Res 44:441–453. 12. Shaik IH, George JM, Thekkumkara TJ, Mehvar R. 2008. Protective effects of diallyl sulfide, a garlic constituent, on the warm hepatic ischemia–reperfusion injury in a rat model. Pharm Res 25:2231–2242. 13. Lieber CS. 1997. Cytochrome P-4502E1: Its physiological and pathological role. Physiol Rev 77:517–544. 14. 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. 15. Emery MG, Jubert C, Thummel KE, Kharasch ED. 1999. Duration of cytochrome P-450 2E1 (CYP2E1) inhibition and estimation of functional CYP2E1 enzyme half-life after single-dose disulfiram administration in humans. J Pharmacol Exp Ther 291:213–219. 16. Ahn CY, Bae SK, Jung YS, Lee I, Kim YC, Lee MG, Shin WG. 2008. Pharmacokinetic parameters of chlorzoxazone and its main metabolite, 6-hydroxychlorzoxazone, after intravenous and oral administration of chlorzoxazone to liver cirrhotic rats with diabetes mellitus. Drug Metab Dispos 36:1233–1241. 17. Ahn CY, Kim EJ, Lee I, Kwon JW, Kim WB, Kim SG, Lee MG. 2003. Effects of glucose on the pharmacokinetics of intravenous chlorzoxazone in rats with acute renal failure induced by uranyl nitrate. J Pharm Sci 92:1604–1613. 18. Chalasani N, Gorski JC, Asghar MS, Asghar A, Foresman B, Hall SD, Crabb DW. 2003. Hepatic cytochrome P450 2E1 activity in nondiabetic patients with nonalcoholic steatohepatitis. Hepatology 37:544–550. 19. Mehvar R, Vuppugalla R. 2006. Hepatic disposition of the cytochrome P450 2E1 marker chlorzoxazone and its hydroxylated metabolite in isolated perfused rat livers. J Pharm Sci 95:1414–1424. 20. Conney AH, Burns JJ. 1960. Biochemical pharmacological considerations of zoxazolamine and chlorzoxazone metabolism. Ann N Y Acad Sci 86:167–177. 21. Chen L, Yang CS. 1996. Effects of cytochrome P450 2E1 modulators on the pharmacokinetics of chlorzoxazone and 6hydroxychlorzoxazone in rats. Life Sci 58:1575–1585. 22. Mehvar R, Zhang XP. 2002. Development and application of an isolated perfused rat liver model to study the stimulation and inhibition of tumor necrosis factor-alpha production ex vivo. Pharm Res 19:47–53. 23. Vuppugalla R, Mehvar R. 2006. Selective effects of nitric oxide on the disposition of chlorzoxazone and dextromethorphan in isolated perfused rat livers. Drug Metab Dispos 34:1160–1166. 24. Omura T, Sato R. 1964. The carbon monoxide-binding pigment of the liver microsomes. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378. 25. Guengerich FP. 1994. Analysis and characterization of enzymes. In Principles and methods of toxicology; Hayes AW, Ed. 3rd ed.New York: Raven Press, pp 1259–1313.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011
26. Vuppugalla R, Mehvar R. 2004. Hepatic disposition and effects of nitric oxide donors: Rapid and concentration-dependent reduction in the cytochrome P450-mediated drug metabolism in isolated perfused rat livers. J Pharmacol Exp Ther 310:718– 727. 27. Vuppugalla R, Mehvar R. 2005. Enzyme-selective effects of nitric oxide on affinity and maximum velocity of various rat cytochromes P450. Drug Metab Dispos 33:829– 836. 28. Frye RF, Stiff DD. 1996. Determination of chlorzoxazone and 6-hydroxychlorzoxazone in human plasma and urine by highperformance liquid chromatography. J Chromatogr B Biomed Appl 686:291–296. 29. Ikemura K, Urano K, Matsuda H, Mizutani H, Iwamoto T, Okuda M. 2009. Decreased oral absorption of cyclosporine A after liver ischemia–reperfusion injury in rats: The contribution of CYP3A and P-glycoprotein to the first-pass metabolism in intestinal epithelial cells. J Pharmacol Exp Ther 328:249– 255. 30. Kim HR, Lee GH, Yi Cho E, Chae SW, Ahn T, Chae HJ. 2009. Bax inhibitor 1 regulates ER-stress-induced ROS accumulation through the regulation of cytochrome P450 2E1. J Cell Sci 122:1126–1133. 31. Jayyosi Z, Knoble D, Muc M, Erick J, Thomas PE, Kelley M. 1995. Cytochrome P-450 2E1 is not the sole catalyst of chlorzoxazone hydroxylation in rat liver microsomes. J Pharmacol Exp Ther 273:1156–1161. 32. Hoffmann U, Kroemer HK. 2004. The ABC transporters MDR1 and MRP2: Multiple functions in disposition of xenobiotics and drug resistance. Drug Metab Rev 36:669–701. 33. Tanaka Y, Chen C, Maher JM, Klaassen CD. 2006. Kupffer cell-mediated downregulation of hepatic transporter expression in rat hepatic ischemia–reperfusion. Transplantation 82:258–266. 34. Tanaka Y, Chen C, Maher JM, Klaassen CD. 2008. Ischemia– reperfusion of rat livers decreases liver and increases kidney multidrug resistance associated protein 2 (Mrp2). Toxicol Sci 101:171–178. 35. Terao N, Shen DD. 1983. Alterations in serum protein binding and pharmacokinetics of l-propranolol in the rat elicited by the presence of an indwelling venous catheter. J Pharmacol Exp Ther 227:369–375. 36. Chindavijak B, Belpaire FM, De Smet F, Bogaert MG. 1988. Alteration of the pharmacokinetics and metabolism of propranolol and antipyrine elicited by indwelling catheters in the rat. J Pharmacol Exp Ther 246:1075–1079. 37. Torres-Molina F, Aristorena JC, Garcia-Carbonell C, Granero L, Chesa-Jimenez J, Pla-Delfina J, Peris-Ribera JE. 1992. Influence of permanent cannulation of the jugular vein on pharmacokinetics of amoxycillin and antipyrine in the rat. Pharm Res 9:1587–1591. 38. Walker LA, Buscemi-Bergin M, Gellai M. 1983. Renal hemodynamics in conscious rats: Effects of anesthesia, surgery, and recovery. Am J Physiol 245:F67–F74.
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