Pharmacokinetics of a Cytochrome P 450 2 E 1 Probe , Chlorzoxazone , and its 6-Hydroxy Metabolite in Poloxamer 407-Induced Hyperlipidemic Rats

Purpose. To evaluate the possible changes in CYP2E1 expression and activity in hyperlipidemia (HL), we evaluated the pharmacokinetics of chlorzoxazone (CZX) as a CYP2E1 probe in rats with HL induced by poloxamer 407 (HL rats). Methods. The pharmacokinetics of CZX and its 6hydroxy metabolite (OH-CZX) were evaluated after intravenous administration of 20 mg/kg CZX to both control and HL rats. We also examined changes in the expression of CYP2E1 and its in vitro metabolic activity in hepatic microsomal fractions from HL rats. Results. The total area under the plasma concentration–time curve (AUC) of CZX in the HL rats after its intravenous administration was comparable with that in the controls due to unchanged non-renal clearance (CLNR). The AUC of OH-CZX and AUCOHCZX/AUCCZX ratios in HL rats also remained unchanged. This was primarily due to the comparable hepatic CLint for metabolism of CZX to OH-CZX via CYP2E1 between the control and HL rats as a result of unchanged expression of CYP2E1 in HL rats. Conclusions. This is the first study to evaluate CYP2E1 expression and activity in HL rats and their effects on the pharmacokinetics of a CYP2E1 probe drug. These findings have potential therapeutic implications assuming that the HL rat model qualitatively reflects similar changes in patients with HL. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.


INTRODUCTION
Hyperlipidemia (HL) involves abnormally elevated levels of one or more plasma lipids (cholesterol, cholesteryl esters, triglycerides, or phospholipids) and/or lipoproteins.This elevation in plasma lipids may be caused by a primary genetic defect or may be secondary to diet, drugs, or disease (1).Plasma lipid profile abnormalities potentially lead to changes in the pharmacokinetics of drugs.A significantly reduced time-averaged total body clearance (CL) has been reported for nifedipine (2,3), amiodarone (4,5), nelfinavir (6), clomipramine (7), and docetaxel (8) in rats with HL (HL rats).In HL rats, both the unbound fraction in plasma and the apparent steady-state volume of distribution (V ss ) of some drugs (amiodarone, nelfinavir, clomipramine, and docetaxel) also display a tendency to decrease due to increased plasma lipoprotein levels (5)(6)(7)(8).
A decrease in liver metabolic enzyme expression, including a reduced expression of hepatic microsomal cytochrome P450 (CYP) 3A1/2 (by 51.9% for 3A1 and by 38.4% for 3A2) and 2C11 (by 39.6%), is observed in an HL rat model (4).In primary rat hepatocytes exposed to lipoprotein-rich serum, reductions in mRNA for cytochrome P450 1A1, 3A2, and 2D1, and some transporters are reported (9).We have found that the metabolic clearance of carbamazepine (10) and verapamil (11), substrates of the CYP3A subfamily, is reduced in HL rats.Moreover, 4hydroxylation of tolbutamide, which is mediated via CYP2C11, is also reduced in HL rats (our unpublished data).These results show that reduced expression of hepatic CYP3A1/2 or 2C11 in HL rats causes metabolic deficiency of CYP3A or 2C11 substrates in vivo.
However, to our knowledge, the hepatic CYP2E1 expression level in HL rats has never been reported.CYP2E1 metabolizes important xenobiotics-such as ethanol-and is capable of activating xenobiotics-such as nitrosaminesto hepatotoxic products (11).It also initiates the lipid peroxidation process, produces free radicals, and contributes to oxidative stress resulting in cell damage.Because fatty acids are both substrates and inducers of CYP2E1 (12), alterations of hepatic CYP2E1 expression are likely in HL rats.Therefore, it is important to evaluate the possible changes of CYP2E1 expression and activity in the HL condition.
We examined changes in the expression of CYP2E1 in a well-established poloxamer 407induced HL rat model.We also evaluated the pharmacokinetics of chlorzoxazone (CZX, 5chloro-3H-benzooxazol-2-one) and its major metabolite, 6-hydroxy CZX (OH-CZX) as probes for CYP2E1 activity after intravenous administration of CZX to both control and HL rats.OH-CZX formation has been used as a chemical probe to assess the activity of CYP2E1 both in vitro and in vivo because of its good correlation with CYP2E1 activity in both humans and rats (13,14).The in vitro metabolic activity of CYP2E1 in hepatic microsomal fractions from HL rats was also evaluated with CZX.

Animals
The animal study protocols were approved by the Department of Laboratory Animals, Institutional Animal Care and Use Committee (IACUC) of Sungsim Campus, The Catholic University of Korea, Seoul, South Korea.Male Sprague-Dawley rats (7-8 weeks old, 265-310 g) were purchased from OrientBio (Sung Nam, South Korea).The procedures used for housing and handling of rats were similar to those described previously (10,11).

Induction of HL
Rats were randomly divided into HL and control groups.To induce HL, 400 mg (in 5 mL)/kg of poloxamer 407 (dissolved in cold sterile 0.9% NaCl solution and refrigerated overnight to facilitate its dissolution) was injected intraperitoneally (i.p.) into the rats (10,11).Control rats received the same volume of vehicle alone (cold sterile 0.9% NaCl solution).Pharmacokinetic experiments were conducted 36 h after administration of poloxamer 407 or vehicle (10).

Immunoblot Analysis of CYP2E1
The procedures used for the preparation of hepatic microsomes from control (n = 6) and HL (n = 6) rats were similar to methods reported previously (15).The protein content in hepatic microsomes was measured using the Bradford assay (16).
Hepatic microsomes containing equal amounts of protein (10 g per lane; n = 5, each) were resolved by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes.The membranes were blocked for 1 h in 5% skim milk powder in phosphate buffered 0.9% NaCl solution containing 0.05% (v/v) Tween 20 (PBS-T).For immunodetection, blots were incubated overnight at 4°C with rabbit anti-CYP2E1 antibody (diluted 1:5000 in PBS-T containing 3% skim milk powder), followed by incubation for 1 h at room temperature with anti-rabbit secondary antibody (diluted 1:5000 in PBS-T containing 3% skim milk powder).Western blots were visualized with an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA) using a Chemidoc XRS imager system (Bio-Rad Laboratories, Hercules, CA, USA).The β-actin band was used as a loading control.

Measurement of Formation Kinetics of OH-CZX from CZX in Hepatic Microsomal Fractions
The methods used for the measurement of V max , K m , and CL int for the formation of OH-CZX from CZX in hepatic microsomes were similar to those reported previously (14).The following components were mixed: the above hepatic microsomes from each group (equivalent to 0.2 mg protein), 0.1 M Tris-HCl buffer (pH 7.4), and 5 L of methanol containing CZX (final CZX concentrations of 2.5, 5, 10, 20, 50, 100, 200, 500, and 1000 M).Mixtures were preincubated for 5 min in a thermomixer (Thermomixer 5436; Eppendorf, Hamburg, Germany) at 37°C and 600 rpm.To initiate the reaction, NADPH (in Tris-HCl buffer of pH 7.4 to a final concentration of 1 mM) was added to a final volume of 250 L.After incubation for 20 min, an aliquot of 50 L was transferred to an Eppendorf tube containing 25 L of methanol with 10 g/mL of 4'-HPPH (internal standard for OH-CZX) and 1 ml of methyl tertiary butyl ether (MTBE), and vortexmixed to terminate the reaction.The mixture was then treated following the sample preparation procedure.All of the above microsomal incubation conditions were within the linear range of the reaction rate.The kinetic constants (K m , apparent Michaelis-Menten constant, and V max , maximum velocity) for formation of OH-CZX were calculated using a nonlinear regression method (17).The unweighted kinetic data from microsomes were fitted to a single-site Michaelis-Menten equation: where [S] is the substrate (CZX) concentration.The CL int (intrinsic clearance) for the formation of OH-CZX was calculated by dividing V max by K m .

Measurement of CZX Plasma Protein Binding
Protein binding of CZX to fresh rat plasma (n = 5) was assessed by equilibrium dialysis (10,11).Fresh plasma (0.2 mL) was dialyzed against 0.2 mL of isotonic Sørensen phosphate buffer (pH 7.4) containing 3% (w/v) dextran ("the buffer") to minimize volume shift (18) using a 0.25-mL micro-equilibrium dialyzer (Harvard Apparatus, Holliston, MA) and a regenerated cellulose membrane with a molecular weight cut-off of 10 kDa (Harvard Apparatus).To reduce the equilibrium time of CZX between the buffer and plasma compartments, the drug was spiked into the plasma side (19) to a final concentration of 5 g/mL.Because the protein binding of CZX to fresh rat plasma remained constant at 67.3% to 68.3% for CZX concentrations of 1 to 50 g/mL (20), 5 g/mL of CZX was chosen in this study.The spiked dialysis cell was incubated in a waterbath shaker kept at 37°C at a rate of 50 oscillations per min (opm).After 24 h, two 50-L aliquots were removed from each compartment and stored at -20°C until HPLC analysis.

Intravenous Administration of CZX to Rats
The procedures used for pretreatment of rats, including cannulation of the jugular vein (for drug administration in the intravenous study) and the carotid artery (for blood sampling), were similar to those reported previously (10,11).Zoletil 50 ® (intramuscular injection at a dose of 40 mg/kg [0.8 mL/kg]) was employed instead of inhalation anesthetics to minimize the effect on CYP2E1 (21), because diethyl ether and isoflurane anesthesia reportedly increase CYP2E1 activity (22).The rats were allowed to recover from the anesthetics overnight and were not restrained during the experiment.
For analysis of plasma lipid profiles, 400 L of blood (200 L of plasma) were obtained from each rat immediately before the start of the experiment.CZX, dissolved in distilled water (an adjusted pH of 8.5) with a minimal amount (5 L/ 1 mL) of 10 N NaOH at a dose of 20 mg (in 2 mL)/kg (14,20), was manually infused over 1 min via the jugular vein of control (n = 9) and HL rats (n = 10).Blood samples (approximately 0.12 mL) were collected via the carotid artery at 0 (control), 1 (end of infusion), 5, 15, 30, 45, 60, 90, 120, and 180 min after the start of intravenous infusion.A 0.3 mL of heparinized 0.9% NaClinjectable solution (20 units/mL) was used to flush the cannula immediately after each blood sampling to prevent blood clotting.Based on our previous preliminary study (10), this heparin dose might not influence triglyceride level significantly.Blood samples were immediately centrifuged, and 50 L of plasma were collected in 1.5-mL polyethylene tubes and stored at -20°C until used for HPLC analysis of CZX and OH-CZX.At the end of the experiment (24 h after CZX treatment), each metabolic cage was rinsed with 20 mL of distilled water, and the rinse water was combined with a urine sample.The volume of the combined urine sample was determined, and two 50-L aliquots were stored at -20°C until HPLC analysis.

HPLC Analysis of CZX and OH-CZX
Concentrations of CZX and OH-CZX were determined by HPLC according to previously described methods (14,23) with slight modifications.Briefly, a 0.1-mL aliquot of 0.2 M sodium acetate buffer (pH 4.75), a 0.1-mL aliquot of isotonic Sørensen phosphate buffer (pH 7.4) containing 200 units of β-glucuronidase, 25 L of methanol containing 4'-HPPH (internal standard: 10 g/mL), and 1 mL of MTBE (extraction solvent) were added to 50 L of sample.The resulting mixture was vortex-mixed and centrifuged.The upper organic layer was transferred and evaporated under a gentle stream of nitrogen gas at 50°C.The residue was reconstituted in 100 L of a 1:1 mixture (v/v) of 2 mM ammonium acetate and acetonitrile, and 20 L were directly injected onto a C 18 HPLC column (5 m, 250 × 4.60 mm; Phenomenex, Torrance, CA, USA).The mobile phase (20 mM ammonium acetate:acetonitrile = 64:36 [v/v] for plasma and other samples excluding urine samples and 68:32 [v/v] for urine samples) was run at a flow rate of 1.0 mL/min, and the column eluent was monitored at 283 nm using an ultraviolet detector.Unconjugated concentrations of OH-CZX were also measured in urine samples without incubation with β-glucuronidase.The retention times of OH-CZX, CZX, and 4'-HPPH (internal standard) in plasma and other samples excluding urine samples were approximately 4.6, 10.7, and 5.5 min, respectively.Those in urine samples were 5.1, 13.6, and 6.9 min, respectively.The lower limit of quantification of both OH-CZX and CZX in plasma samples was 50 ng/mL.The values in urine samples were 250 and 50 ng/mL for OH-CZX and CZX, respectively.The relative errors of assay accuracy were below 16.4%, and the coefficients of variation (CVs) of the analysis were below 12.5%.

Pharmacokinetic Analysis
The total area under the plasma concentrationtime curve from time zero to infinity (AUC) was calculated using the trapezoidal rule-extrapolation method (24), which uses the logarithmic trapezoidal rule to calculate the area during the phase of declining plasma level, and the linear trapezoidal rule for the phase of rising plasma level.The area from the last datum point to time infinity was estimated by dividing the last measured plasma concentration by the terminalphase rate constant.
Standard methods (25) were used to calculate the following pharmacokinetic parameters by non-compartmental analysis (WinNonlin; Pharsight Corporation, Mountain View, CA, USA): the time-averaged total body, renal, and non-renal clearances (CL, CL R , and CL NR , respectively); terminal half-life; mean residence time (MRT); and apparent steady-state volume of distribution (V ss ).The peak plasma concentration (C max ) and time to reach C max (T max ) were read directly from the experimental data.

Statistical Analysis
Differences between the two means for unpaired data were analyzed using Student's t-test, and p < 0.05 was taken to indicate statistical significance.All data are presented as means ± SD unless otherwise specified.

Protein Expression of Hepatic CYP2E1
The protein levels of hepatic CYP2E1 within hepatic microsomal fractions from control and HL rats are shown in Figure 1.The protein expression of hepatic CYP2E1 in HL rats was comparable with that of controls.

Formation Kinetics of OH-CZX from CZX in Hepatic Microsomal Fractions
The mean OH-CZX formation velocities in hepatic microsomal fractions from control and HL rats are shown in Figure 2. The V max , K m , and CL int values for the formation of OH-CZX in hepatic microsomal fractions from both groups are listed in Table 1.Although the V max value in HL rats was significantly slower (by 36.9%)than that in controls, the K m and CL int values were comparable between the two groups.This suggests that the hepatic metabolic activity of hydroxylation of CZX in HL rats is comparable with that of control rats.

Plasma Protein Binding of CZX
Plasma protein binding of CZX was comparable between control and HL rats (67.5% ± 16.3% and 65.0% ± 8.67% for control and HL rats, respectively).The recovery of CZX from the dialysis apparatus was almost complete (81.6% ± 6.12%).

Pharmacokinetics of CZX and OH-CZX after Intravenous Administration of CZX
The mean arterial plasma concentration-time profiles of CZX and OH-CZX after intravenous administration of 20 mg/kg CZX to control and HL rats are shown in Figures 3A and 3B, respectively.The relevant pharmacokinetic parameters and plasma lipid profiles of each group are listed in Table 2.The pharmacokinetic parameters of CZX and OH-CZX were comparable between control and HL rats.

DISCUSSION
We chose poloxamer 407 to induce HL in rats because its i.p. administration shows no apparent toxicity (4).Poloxamer 407-induced hypertriglyceri-demia and hypercholesterolemia are well-documented phenomena: triglyceride levels are elevated due to inhibition of lipoprotein lipase (26) and hypercholesterolemia due to the indirect stimulation of the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA)

CYP2E1
Control HL

-actin
reductase, the rate-limiting enzyme in cholesterol biosynthesis, and decreased LDL receptor expression in the liver (27,28).The results of our previous study indicated that hypertriglyceridemia and hypercholesterolemia were induced following i.p. administration of poloxamer 407, with no significant hepatic or kidney toxicities (10).The induction of HL was confirmed based on the significant increase in total cholesterol (>220 mg/dL) and triglyceride (>200 mg/dL) levels following the analysis of the plasma lipid profiles prior to the pharmacokinetic studies (Table 2).CZX dose for this study (20 mg/kg) was in the intravenous dose range of 15 to 50 mg/kg, showed linear disposition of CZX (29).The pharmacokinetic parameters of CZX in control rats (Table 2) were similar to those in previous pharmacokinetic studies on CZX including CL (8.96-12.3mL/min/kg) and V ss (279-391 mL/kg) values (14,20,23,30).
In HL rats, the AUC of intravenous CZX was comparable with that in controls because of a similar CL of CZX (concretely CL NR ) (Table 2).The CL R values of CZX in both groups of rats were comparable and made a negligible contribution (~1%) to the CL of CZX (Table 2).Moreover, considerable recovery of total (free + conjugated) OH-CZX in the urine (86.6% and 102% of CZX dose for control and HL rats, respectively, Table 2) indicates that most of the intravenous CZX dose is eliminated via the 6hydroxylation of CZX followed by conjugation reaction and urinary excretion of the metabolite.Therefore, the CL NR of CZX in HL rats is related to the metabolic clearance of CZX to OH-CZX.CZX has a low to intermediate hepatic extraction ratio in rats (0.087-0.681 in perfused rat liver; 0.317 by estimation based on CL NR of control rats [Table 2] and hepatic plasma flow rate [29.8 mL/min/kg in rats]) (13,31).Therefore, hepatic clearance of CZX depends primarily on its hepatic intrinsic clearance (CL int ) and free fraction in plasma, and possibly on the hepatic blood flow rate (32).In HL rats, the CL int for metabolism of CZX to OH-CZX in the hepatic microsomal fraction was comparable to that in controls (Figure 2, Table 1), suggesting a comparable CYP2E1 activity in HL rats.This is consistent with the unchanged expression level of hepatic CYP2E1 in HL rats (Figure 1).The free fractions of CZX in plasma are comparable between control and HL rats, based on the protein binding study.Although there has been no study of the changes in the hepatic blood flow rate in HL rats, the decreased hepatic blood flow in HL rats as a result of minimal fatty changes of the liver was suggested based on slowed CL NR of verapamil, a drug with a high hepatic extraction ratio, in HL rats (11).We concluded that a comparable CL int for metabolism of CZX to OH-CZX and free fraction of CZX between control and HL rats resulted in a comparable CL NR of CZX.The pharmacokinetic parameters of OH-CZX, including its formation ratio (AUC OH- CZX /AUC CZX ratio), were also comparable between control and HL rats.This is again due to the similar CL int for metabolism of CZX to OH-CZX in the hepatic microsomal fraction from control and HL rats as a result of the comparable CYP2E1 expression and activity between the two groups.Based on the similar urinary recovery of both free and total OH-CZX in control and HL rats, urinary excretion of conjugated OH-CZX was also unchanged in HL rats.This result suggests that the conjugation reaction of OH-CZX, such as its glucuronidation, was also comparable in control and HL rats.
In summary, hepatic metabolism of CZX to OH-CZX was unchanged in HL rats, itself a result of unchanged CYP2E1 expression and activity.As a result, HL rats showed comparable pharmacokinetics of CZX and OH-CZX following intravenous CZX administration.Although possible induction of CYP2E1 in HL rats had been hypothesized based on the fact that fatty acids are substrates and inducers of CYP2E1, our results suggested that HL states did not affect the expression and activity of CYP2E1.This is the first study to evaluate CYP2E1 expression and activity in HL rats and their effects on the pharmacokinetics of a CYP2E1 probe drug, CZX.As is the case for any animal model, caution is necessary in extrapolating these findings to humans.Nevertheless, these findings have potential therapeutic implications assuming that this model qualitatively reflects similar changes in patients with HL.

Figure 1 .Figure 2 .
Figure1.Hepatic protein expression of CYP2E1 in HL and control rats was quantitated by immunoblotting and densitometry of microsomal fraction (10 g of protein) from each rat liver (n = 5 each) (A).The protein expression (mean ± SD) was expressed in terms of percentage of the control rats, 100% (B).The -actin band was used as a loading control.

Table 1 .
In vitro V max , K m , and CL int for the formation of OH-CZX from CZX (mean ± SD) in hepatic microsomal fraction of control and HL rats.
* Significantly different from the control group (p < 0.05, t-test)

Table 2 .
Pharmacokinetic parameters of CZX and OH-CZX (mean ± SD) after intravenous administration of 20 mg/kg CZX to control and HL rats.Significantly different from the control group (p < 0.001, t-test).
b Measured just before experiment.c Median (range) ***