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Vol. 27, Issue 9, 1074-1077, September 1999
Division of Pharmaceutical Sciences, University of Kentucky, College of Pharmacy, Lexington, Kentucky
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results and Discussion
References
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The acute-phase response is known to produce alterations in hepatic cytochrome P-450 (CYP) expression. Lipopolysaccharide (LPS), a well known inducer of acute-phase response decreases hepatic CYP2E1 in vitro activity in rats. This study was designed to determine if LPS administration produced alterations in the pharmacokinetics of chlorzoxazone (CZN), a marker for CYP2E1 expression. Sprague-Dawley rats were administered a single i.p. injection of LPS (5 mg/kg) or saline control approximately 24 h before a single i.v. bolus dose of CZN (15 mg/kg). Serial blood samples were collected over a 120-min period to quantitate CZN plasma concentrations and protein binding. In addition, livers were removed and processed for evaluating in vitro CYP2E1 protein concentrations and activity. Systemic clearance decreased by 35% in LPS-treated rats, whereas half-life and steady-state volume of distribution increased by 167 and 66%, respectively. The plasma free-fraction of CZN increased 2-fold after LPS treatment. The CZN intrinsic clearance decreased in LPS rats by 71% compared with control values. The CYP2E1 liver microsomal activity decreased between 55 and 75% along with a 41% decrease in CYP2E1 protein concentration. The CZN intrinsic clearance was significantly correlated with both the CZN and p-nitrophenol liver microsomal activity (r = 0.97 and r = 0.91, respectively). This study demonstrated that LPS administration produced expected reductions in the in vivo intrinsic clearance of CZN, and these changes were highly correlated with in vitro activity studies. In addition, LPS produced significant increases in the steady-state volume of distribution of CZN secondary to reductions in its plasma protein binding.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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Gram-negative
sepsis is a major cause of morbidity and mortality in the critically
ill patient. Gram-negative sepsis initiates a systemic inflammatory
response syndrome after systemic cytokine release. This systemic
inflammatory reaction often leads to multiple organ dysfunction
syndrome, which contributes to significant morbidity and mortality in
these patients (Beal and Cerra, 1994
).
One of the body's first defenses initiated in response to
Gram-negative sepsis is the acute-phase response
(APR)2. The APR is triggered when resident
macrophages, mainly the Kupffer cells of the liver, circulating
monocytes, and macrophages are stimulated to release factors known as
cytokines. These cytokines are noted to trigger a large inflammatory
response and are also responsible for physiologic changes to include
the release of proteolytic enzymes such as C-reactive protein
from hepatocytes. The known proinflammatory cytokines include
interleukin (IL)-1, IL-6, and tumor necrosis factor. The changes that
occur during the APR have been associated with altering the disposition
and metabolism of certain drugs that are biotransformed through the cytochrome P-450 (CYP) pathway in healthy volunteers (Shedlofsky et al., 1994, 1997). In addition, hospitalized patients with viral infections were observed to have altered theophylline pharmacokinetics (Chang et al., 1978).
To assess the effect of the APR on the CYP-mediated drug metabolism,
animal rodent models have been used. In rats, the administration of
endotoxin [lipopolysaccharide (LPS)] resulted in a suppression of
mRNA levels, apoprotein, and liver microsomal activity (Morgan, 1989,
1993; Sewer et al., 1996; Roe et al., 1998). Also, the administration of the individual cytokines, IL-1, IL-6, and tumor necrosis factor caused a similar decrease in the CYP drug-metabolizing enzymes (Chen et
al., 1992, 1995; Morgan et al., 1994).
Ethanol-inducible CYP2E1 is one of the CYPs that is altered in rats
after LPS administration. Levels of mRNA decreased to 20% of control
6 h after LPS administration to male Sprague-Dawley rats and did
not return to baseline until 48 h (Sewer et al., 1996).
Furthermore, the apoprotein and the liver microsomal activity were both
decreased compared with control and did not return to baseline until
72 h (R.B., unpublished observations).
The changes after LPS administration in the CYP drug-metabolizing enzymes, which includes CYP2E1, is well documented in the rodent model. A very important interpretation of this model to critically ill patients would be to understand how the APR could alter the pharmacokinetics of drugs whose metabolism is mediated by the CYP enzymes. Patients that experience an APR, such as sepsis, trauma, and burn patients, typically receive medications that are metabolized by CYP enzymes. This could result in drug concentrations reaching the toxic range, which would further complicate these already critically ill patients. At present, it has not been determined how the in vitro changes in the CYP enzymes in the rodent model would effect the pharmacokinetic parameters of a drug that is metabolized by the CYP enzymes. Therefore, the main objectives of this study was to determine the pharmacokinetic changes of a probe drug for CYP2E1 after LPS administration and also to determine if there is a correlation between the in vitro and the in vivo changes.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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Male Spague-Dawley rats (body weight, 375-390 g) were obtained
from Harlan (Indianapolis, IN). The animals were maintained on a 12-h
light/dark cycle and were given free access to food (Purina Rodent
Chow) and drinking water. The animals were acclimated for a minimum of
5 days before being randomly assigned to an experimental group. On the
first day of the study, a pair of rats was placed under general
anesthesia with a mixture of a 3:1 ratio of ketamine and xylazine (v/v)
at a dose of 1.25 cc/kg (93.8 and 6.3 mg/kg of ketamine and xylazine,
respectively). A sterile silastic cannula (Dow Corning; i.d., 0.51 mm;
o.d., 0.94 mm) was surgically inserted into the right jugular vein of
each rat. On day 2, animals randomized to the treatment group received
an i.p. injection of LPS at a dose of 5 mg/kg at 9:00 AM and control
animals received an i.p. injection of an equal volume of saline. Food
was removed at 12:00 AM from the animals but free access to water was
permitted. A solution of chlorzoxazone (CZN) was prepared as described
previously (Chen and Yang, 1996). On day 3, a bolus dose of CZN
(15 mg/kg) was administered at 9:00 AM to each animal via the jugular
cannula. Approximately 0.6 ml of blood was taken from the jugular
cannula before and at 10, 20, 30, 60, 90, and 120 min. Plasma was
removed and stored at 
20°C until analysis. Twenty-eight hours
post-LPS administration, animals were placed under light anesthesia
with 3:1 ketamine and xylazine (v/v) and sacrificed by
exsanguination by cardiac stick in which plasma was obtained for CZN
protein-binding analysis.
Materials. Chemicals and reagents were purchased from suppliers as follows: Fort Dodge Laboratories (Fort Dodge, IA) ketamine; Butler Company (Columbus, OH) xylazine; Difco Laboratories (Detroit, MI) LPS B E. coli 055:B5/LD50 = 28.7 mg/kg; Sigma Chemicals (St. Louis, MO) CZN, umbelliferone, butylated hydroxytoluene, and p-nitrophenol; Fischer Scientific (Fairlawn, NJ) acetic acid, acetonitrile, and diethyl ether; Research Biochemicals International (Natick, MA) 6-hydroxy-CZN.
Microsomes were prepared from livers that were excised and placed in cold 0.154 M KCL/0.25 M potassium phosphate buffer to remove blood. Liver samples were homogenized in 4 volumes of cold 0.154 M KCl/0.25 M potassium phosphate, pH 7.4, with the addition of butylated hydroxytoluene (10 µl/g tissue) as an antioxidant before homogenization. Livers were homogenized using a Teflon grinder and spun to separate the microsomal fraction. The resulting microsomal pellet was resuspended in 0.25 M sucrose/0.02 M Tris buffer, pH 7.4, and stored at80.0°C until analysis. Total protein concentration in rat
liver microsomes was determined by the method of Lowry et al. (1951)
using BSA as the standard.
The microsomal fractions were used in the determination of total CYP
concentrations by spectral analysis. The concentration of CYP in these
fractions were determined spectrophotometrically by the method of Omura
and Sato (1964) and based on an extinction coefficient of 91 mM
1 cm1.
The rat plasma concentration of CZN was measured by a modified
HPLC method by Peter et al. (1990). Briefly, the HPLC system was
a Shimadzu SIL-6B auto-injector with UV detection. The mobile phase was
isocratic that consisted of 78% of a 0.25% acetic acid solution and
22% acetonitrile. All solutions were of HPLC grade. The flow rate was
set at 1 ml/min. The UV detector wavelength was set at 287 nm. The CZN
and umbelliferone (internal standard, 0.078 mM) were separated using a
LC-18 reversed phase (5 µm, 15 cm × 4.6 cm) column (Supelco,
Bellefont, PA). The extraction method was modified from Frye and Stiff
(1996). Briefly, 0.1 ml of plasma from each sample was used for
analysis and 1 ml of 10 mM sodium acetate buffer, pH 4.5, was added to
the plasma. Diethyl ether (4 ml) was added to each sample and standard
and vortexed for 1 min. The samples were centrifuged and the organic
phase was evaporated to dryness under a stream of nitrogen. The residue was reconstituted in 200 µl of mobile phase and 20 µl was injected onto the HPLC system.
Protein binding of CZN in rat plasma was determined by the
ultrafiltration technique. A 1.5-ml plasma sample was spiked to give a
final concentration of 50 µg/ml. The samples were incubated for 15 min at 37°C and 1-ml aliquots were placed into an ultrafiltration device (Amicon, Cambridge, MA) equipped with an ultrafiltration membrane. The samples were centrifuged at 3200 rpm at 37°C for 30 min
or until 250 µl filtrate was collected. Concentration of CZN in the
filtrate and the aliquot for total CZN concentration was determined by
the HPLC method described above.
The p-nitrophenol hydroxylase activity was determined
spectrophotometrically by measuring the increase in adsorbance at 546 nm and using the extinction coefficient 
mM = 9.53 mM1 cm1 as
described by Koop (1986).
The formation of 6-OH CZN was determined from the method of Peter et
al. (1990) with modifications from Jayyosi et al. (1995). The
microsomal protein was incubated and 6-OH CZN was measured as described
previously (Warren et al., 1999).
Liver microsomal samples were analyzed for CYP2E1 by noncompetitive
enzyme linked immunosorbent assay. Our laboratory has shown that rat
liver microsomes produce a single band on a Western blot analysis and
comigrated the same distance with purified CYP2E1 (R. B., unpublished
data). Liver microsomes were diluted in phosphate carbonate/bicarbonate buffered saline pH 9.6. Subsequently, 0.25 µg
of total microsomal protein and known concentrations of microsomal standards (Gentest, Woburn, MA) were plated onto a 96-well flat bottom
plate (Corning Glass Works, Corning, NY). The microsomal standards produced a signal that was linear through the concentration range of 62.5 to 1,000 pmol/mg. Proteins were blocked and incubated for
1 h with 50% horse serum and 50% Tris-buffered saline with 0.1%
Tween 20. The plate was washed and incubated with polyclonal goat
anti-rat antibody for CYP2E1 (Gentest, Woburn, MA). Next, the plate was
washed and incubated with alkaline phosphatase-conjugated monoclonal
rabbit anti-goat IgG antibody. After the end of the incubation, the
plate was washed and p-nitrophenol phosphate substrate (ELISA Technologies, Lexington, KY) was added. The plate was analyzed at 405 nm over 30 min at 37°C with a Biotek EL340 microplate reader for color formation. Samples were quantified by extrapolation from the
fitted standard curve.
Data Analysis.
The plasma concentration-time curves for CZN were fitted to a
one-compartment model and were analyzed by nonlinear least-squares regression analysis using the computer program PK Analysis (Micromath Scientific, Salt Lake City, UT).
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(2) |
Statistical Analysis. A two-tailed t test was used assuming equal variances for all comparisons except for the protein binding analysis where unequal variances were assumed. The alpha value was set a priori at p < .05. The correlations were determined by using a nonlinear least-squares regression analysis software program (Prism; GraphPad Software, San Diego, CA.).
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Results and Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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The mean semilog plot of the plasma CZN concentration-versus-time
profile followed a simple monoexponential decline for both the LPS and
control groups (Fig. 1). This clearly
illustrates that the administration of LPS to male Sprague-Dawley rats
significantly alters the pharmacokinetics of CZN. Table
1 lists the pertinent pharmacokinetic
parameters for the control and treated groups. The decrease in liver
microsomal activity as determined by CZN (75%) and
p-nitrophenol (55.3%) plus a decrease in the CYP2E1 protein
(41%) accounted for a decrease of 35 and 71% in Cls and intrinsic
clearance of CZN, respectively. This decrease in Cls agrees with
similar results in which LPS administration to rats significantly
decreased the Cls for antipyrine and metronidazole (Kokwaro et al.,
1993) and also R and S-pindolol (Hasegawa et al.,
1989). However, the liver microsomal activity was not determined in
either of these studies. Because CZN is a restrictively cleared (low E)
drug, it was important to measure the change, if any, in the protein
binding of CZN. In the present study, the free-fraction in the
LPS-treated animals increased compared with control values. Protein
binding parameters in control animals from this study were similar to
what has been shown in the literature (Yasuhara and Levy, 1988). These
results may explain why only a 35% change in the Cls was observed
whereas the liver microsomal activity decreased by 55 to 75%. On the
other hand, the intrinsic clearance of CZN decreased by approximately
71% in the LPS animals compared with control, which falls within the
range of the 55 to 75% decrease in the liver microsomal activity.
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The LPS-treated group had a 66% increase in the Vss of CZN
compared with controls. This may be explained by the 2-fold increase of
the free-fraction in the LPS treatment group. This is supported by the
result in Fig. 2D, which shows a strong
correlation between the Vss and the plasma CZN free-fraction
(r = 0.95, p < .05). It has not been
reported in the literature as to which protein CZN is bound in the
systemic circulation. However, preliminary results from our laboratory
determined that CZN is highly bound to albumin (R.B., unpublished
observation). In the present study, the albumin decreased by 13% in
LPS animals. Although this is a modest decrease in the albumin
concentration, it has been determined that albumin has a decrease in
binding capacity and number of binding sites (n) after LPS
administration (Nadai et al., 1993; Wang et al., 1993). Nadai et al.
(1993) showed that the extracellular fluid of rats does not change
after LPS administration; this suggests that the change in
Vss is a result of a change in binding. The influence of protein binding on the Vss was also
illustrated with R- and S-pindolol, which showed
a decrease in the Vss of both enantiomers along
with a decrease in the free-fraction after LPS administration (Hasegawa
et al., 1989). It is well established that pindolol is mainly bound to
-1 acid glycoprotein, which increased in this study from 13.4 to
75.3 mg/dl after LPS administration to rats (Hasegawa et al., 1989). In
other reports, there was shown to be an increase in the
Vss of enprofylline after LPS administration to
rats without a change in the free-fraction (Nadai et al., 1995).
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Half-life increased over 2.5-fold in the LPS-treated group compared
with controls. This dramatic increase in half-life is the result of the
increase in Vss and decrease in Cls, because half-life is directly proportional to Vss and
indirectly proportional to Cls (Rowland and Tozer, 1980). This is an
excellent illustration of the profound effect that
Vss has on the half-life of a drug because the
change in Cls was not large enough to explain the 2.5-fold increase in
the half-life of CZN for this study.
The in vitro hepatic microsomal CYP changes after LPS administration were as expected. Animals that received LPS demonstrated lower total CYP concentrations [0.29 ± 0.04 nmol/mg microsomal protein (LPS) versus 0.67 ± 0.07 nmol/mg microsomal protein (control), p < .05], lower CYP2E1 protein concentrations [440 ± 93 pmol/mg microsomal protein (LPS) versus 748 ± 145 pmol/mg microsomal protein (control), p < .05], and lower microsomal enzyme activities for CZN [0.44 ± 0.1 nmol/mg/min (LPS) versus 1.78 ± 0.33 nmol/mg/min (control), p < .05] and p-nitrophenol [1.05 ± 0.4 nmol/mg/min (LPS) versus 2.37 ± 0.29 nmol/mg/min (control), p < .05], compared with controls.
One of the main objectives of this study was to determine if there was
a correlation between in vitro and in vivo parameters measured in this
study. When the intrinsic clearance of CZN was plotted against the
liver microsomal activity, it resulted in a statistically significant
correlation (Fig. 2, A and B). The liver microsomal activity measured
by CZN (Fig. 2A; r = 0.97, p < .05)
had a slightly higher correlation than did the activity measured by
p-nitrophenol (Fig. 2B; r = 0.91, p < .05). CYP2E1 protein concentration was also
plotted against the intrinsic clearance of CZN (Fig. 2C); however, it
did not result in a statistically significant correlation
(r = 0.64, p > .05). There are a
couple of reasons why these two values may not have correlated. First, the anti-CYP2E1 antibody recognizes not only the haloprotein but also
the apoprotein, which is inactive. Second, there is strong evidence
that not only CYP2E1 is responsible for CZN's metabolic fate but also
the subfamilies of CYP3A and CYP1A each may play a minor role in the
metabolism of CZN in the rat (Carriere et al., 1993; Jayyosi et al.,
1995; Yamozaki et al., 1995).
In conclusion, this study showed that the degree of down-regulation of the CYP2E1 enzyme activity resulted in a significant change in the pharmacokinetic parameters of CZN. Furthermore, there was a strong correlation between liver microsomal activity and intrinsic clearance of CZN. This latter result may be important to investigators that utilize the animal rodent model to study changes in the CYP drug-metabolizing enzymes. Measurable changes of CYP in liver microsomal preparations, according to our results, may be highly correlated to the intrinsic clearance.
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Footnotes |
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Received January 21, 1999; accepted April 30, 1999.
1 Current address: University of Kentucky, College of Pharmacy, Division of Pharmaceutical Sciences, Lexington, KY 40536-0082.
This work was supported by the Kentucky Spinal Cord Head Injury Research Trust (Grant BB-9502-K).
Send reprint requests to: Robert A. Blouin, Pharm.D., Division of Pharmaceutical Sciences, College of Pharmacy, 907 Rose Street, Lexington, KY 40536-0082. E-mail: rblou1{at}pop.uky.edu
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Abbreviations |
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Abbreviations used are: APR, acute-phase response; LPS, lipopolysaccharide; CYP, cytochrome P-450; CZN, chlorzoxazone; Vss, steady-state volume of distribution; IL, interleukin; Cls, systemic clearance.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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) and LPS (
) treated
rats after an i.v. bolus dose of CZN (15 mg/kg).
