![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 28, Issue 3, 354-359, March 2000
Toxicology Program, University of New Mexico, College of Pharmacy, Albuquerque, New Mexico (M.R.W.); Department of Biochemistry, Mount Sinai School of Medicine, New York, New York (J.M.L.); The Scripps Research Institute, Department of Molecular and Experimental Medicine, La Jolla, California (E.F.J.); and The La Jolla Institute for Experimental Medicine, La Jolla, California (J.L.R.)
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Tolbutamide is a sulfonylurea-type oral hypoglycemic agent whose action is terminated by hydroxylation of the tolylsulfonyl methyl moiety catalyzed by cytochrome P-450 (CYP) enzymes of the human CYP2C subfamily. Although most studies have implicated CYP2C9 as the exclusive catalyst of hepatic tolbutamide hydroxylation in humans, there is evidence that other CYP2C enzymes (e.g., CYP2C19) may also participate. To that end, we used an immunochemical approach to assess the role of individual CYP2Cs in microsomal tolbutamide metabolism. Polyclonal antibodies were raised to CYP2C9 purified from human liver, and were then back-adsorbed against recombinant CYP2C19 coupled to a solid-phase support. Western blotting revealed that the absorbed anti-human CYP2C9 preparation reacted with only recombinant CYP2C9 and the corresponding native protein in hepatic microsomes, and no longer recognized CYP2C19 and CYP2C8. Monospecific anti-CYP2C9 not only retained the ability to inhibit CYP2C9-catalyzed reactions, as evidenced by its marked (90%) inhibition of diclofenac 4'-hydroxylation by purified CYP2C9 and by human liver microsomes, but also exhibited metabolic specificity, as indicated by its negligible (<15%) inhibitory effect on S-mephenytoin 4'-hydroxylation by purified CYP2C19 or hepatic microsomes containing CYP2C19. Monospecific anti-CYP2C9 was also found to inhibit rates of tolbutamide hydroxylation by 93 ± 4 and 78 ± 6% in CYP2C19-deficient and CYP2C19-containing human liver microsomes, respectively. Taken together, our results indicate that both CYP2C9 and CYP2C19 are involved in tolbutamide hydroxylation by human liver microsomes, and that CYP2C19 underlies at least 14 to 22% of tolbutamide metabolism. Although expression of CYP2C19 in human liver is less than that of CYP2C9, it may play an important role in tolbutamide disposition in subjects expressing either high levels of CYP2C19 or a catalytically deficient CYP2C9 enzyme.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Tolbutamide, an
oral hypoglycemic agent, has been used extensively as a metabolic probe
to study human cytochrome P-450
(CYP)3
function. Previous studies led to the assignment of tolbutamide as a specific substrate for CYP2C9, a member of the CYP2C
gene subfamily (Srivastava et al., 1991
; Veronese et al., 1991; Hall et
al., 1994). Although CYP2C9 is indeed a strong
catalyst of tolbutamide methylhydroxylation, we as well as other
investigators have recently shown that a second CYP2C protein expressed
in human liver, CYP2C19, catalyzes the same reaction (Richardson et
al., 1995; Lasker et al., 1998; Venkatakrishnan et al., 1998). In fact, rates of hydroxytolbutamide (OHT) formation by purified CYP2C19 and the
corresponding recombinant enzyme in reconstituted systems were similar
to those exhibited by purified CYP2C9 and its corresponding heterologously expressed enzyme (Lasker et al., 1998). Despite such
observations, it is still not known whether CYP2C19, like CYP2C9, plays
a significant role in tolbutamide hydroxylation by intact human liver
microsomes, as the expression level of CYP2C19 is low relative to
CYP2C9 (Lasker et al., 1998). If CYP2C19 is as active as CYP2C9, then
CYP2C19 would be an important determinant of tolbutamide disposition in
individuals with elevated hepatic levels of this CYP and in individuals
expressing CYP2C9 allelic variants that are catalytically deficient
toward this drug (Sullivan-Klose et al., 1996).
Assessing the contribution of CYP2C19 to hepatic tolbutamide
hydroxylation has been challenging for several reasons. First, CYP2C19
possesses a Km value for tolbutamide
(Lasker et al., 1998; Venkatakrishnan et al., 1998) similar to that of
CYP2C9, thereby obviating kinetic approaches for determining the
contribution of the former enzyme to this microsomal drug-metabolizing
reaction. Second, the development of monospecific CYP2C19 antibodies
for use in immunoinhibition studies has been hampered by the extensive degree of sequence homology (92%) found between this CYP2C protein and
CYP2C9 (Romkes et al., 1991). As polyclonal antibodies produced against
purified CYP2C19 recognize not only the corresponding immunogen but
also CYP2C9 (and CYP2C8), the inhibition of microsomal tolbutamide
hydroxylation elicited by these antibodies (95%) overestimates the
CYP2C19 contribution (Lasker et al., 1998). Chemical inhibitors, such
as sulfaphenazole, have also been used to determine the contribution of
CYP2Cs to this reaction (Newton et al., 1995). However, sulfaphenazole exhibits greater specificity toward CYP2C9 than CYP2C19. Correlation analyses between CYP2C19 and/or CYP2C9 enzyme levels and tolbutamide hydroxylase activity in human liver microsomes have also yielded equivocal results (Forrester et al., 1992; Hall et al., 1994; Inoue et
al., 1997; Lasker et al., 1998). Moreover, the use of such correlation
analyses for determining the metabolic specificity of CYP2C19 and/or
CYP2C9 toward tolbutamide suffers from inherent limitations, including
the inability to reveal strong relationships when allelic variants
exist at a relatively high frequency, but contribute only slightly to
tolbutamide hydroxylation.
We used an immunochemical-based approach to assess the respective roles of CYP2C19 and CYP2C9 in hepatic tolbutamide metabolism. Solid-phase adsorption was used to derive a monospecific anti-CYP2C9 antibody. Recombinant CYP2C19 was used as the back adsorbent, and the resultant anti-CYP2C9 preparation reacted specifically with CYP2C9 on Western blots. Using this specific inhibitory probe, it was established that CYP2C9 catalyzes the majority of tolbutamide hydroxylation in human liver microsomes. However, CYP2C19 was also found to contribute to metabolism of tolbutamide, particularly in subjects expressing high levels of this hepatic CYP2C enzyme.
| |
Experimental Procedures |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Human Liver Specimens.
Liver samples were obtained from organ donors through the Liver Tissue
Procurement and Distribution System (LTPADS, University of Minnesota,
Minneapolis, MN). None of the subjects had a reported history of
alcohol or drug abuse. The livers were removed within 12 h of
death, frozen in liquid nitrogen, and stored at 
80°C until used for
microsomal preparation (Raucy and Lasker, 1991). Protein and CYP
contents were determined using the bicinchoninic acid procedure (Smith
et al., 1985) and the method of Omura and Sato (1964), respectively.
Expression and Purification of Recombinant CYP2C Enzymes.
CYP2C8, CYP2C9, and CYP2C19 were expressed in Escherichia
coli as described previously (Richardson et al., 1995). The
recombinant CYP enzymes were purified from E. coli membranes
as follows. A 3.0-liter culture of E. coli expressing
CYP2C8, CYP2C9, or CYP2C19 was centrifuged at 8000g for 10 min, and the pellet was resuspended in 450 ml of 100 mM Tris buffer (pH
7.8) containing 0.5 mM EDTA and 20% glycerol by stirring for 10 min at
4°C. Lysozyme was added to a final concentration of 0.2 mg/ml, the
solution was diluted 1:1 with distilled water, stirred for an
additional 30 min at 4°C, and then centrifuged at 8000g
for 10 min. The pellet was resuspended in 90 ml of 10 mM Tris-HCl
buffer (pH 7.3) containing 14 mM magnesium acetate, 60 mM potassium
acetate, and 0.1 mM dithiothreitol (DTT), and then frozen at 80°C.
After thawing and the addition of phenylmethylsulfonyl fluoride to a
final concentration of 0.5 mM, the sample was subjected to 10 cycles of
sonication at 100W for 30 s with a 60-s cooling period on ice
between cycles. The sample was again centrifuged at 8000g
for 10 min, and the pellet was resuspended in 2.0 ml of 10 mM
KPO4 buffer (pH 7.4) containing 0.1 mM EDTA and
20% glycerol. The bacterial membranes were then solubilized by
dropwise addition of 10% Lubrol PX to a final concentration of 0.5%
while stirring at 4°C for 60 min. The clarified solution was
centrifuged at 150,000g for 60 min, and the supernatant was applied to a 1.5 × 10 cm column of hydroxylapatite (Hypatite C; Clarkson Chromatography Products, Inc., South Williamsport, PA) that
had been equilibrated with 10 mM KPO4 buffer (pH
7.4) containing 0.5% Lubrol PX, 1 mM DTT, 0.1 mM EDTA, and 20%
glycerol. Bound proteins were eluted from the column by washing with 12 volumes of equilibration buffer, followed by 12 volumes each of
equilibration buffer containing 25 and 50 mM KPO4
buffer (pH 7.4). Lubrol PX was removed from the bound CYP sample by
washing the column with 50 volumes of the same buffer used for
equilibration, but with the detergent omitted. CYP2C proteins were then
eluted from the hydroxylapatite resin with 300 mM
KPO4 buffer (pH 7.4) containing 0.5% cholate, 1 mM DTT, 1 mM EDTA, and 20% glycerol (Raucy and Lasker, 1991). The
final CYP2C8, CYP2C9, and CYP2C19 enzyme preparations were exhaustively
dialyzed against 100 mM KPO4 buffer (pH 7.4) containing 1 mM DTT, 1 mM EDTA, and 20% glycerol to remove cholate, and concentrated by ultrafiltration through a Filtron Omega 30k membrane (Filtron Technology Corp., Northborough, MA). Cytochrome b5 (b5;
specific content = 41.5 nmol/mg protein) and NADPH:P-450 oxidoreductase (P-450 reductase; specific activity = 40,000 U or
11.9 nmol/mg protein) were isolated from human liver microsomes as
described elsewhere (Raucy and Lasker, 1991). One nanomole of P-450
reductase was considered equivalent to 3,370 U of activity; 1 U was
defined as that amount catalyzing the reduction of 1 nmol of
ferrocytochrome c/min at 30°C in 300 mM potassium phosphate buffer
(pH 7.7).
Catalytic Activities.
Hydroxylation of S-mephenytoin, diclofenac, and tolbutamide
by human liver microsomes and purified recombinant CYP2C enzymes were
determined under conditions where product formation was directly proportional to both CYP concentration and time of incubation. Reactions with microsomes contained an amount of protein equivalent to
25 to 150 pmol of aggregate CYP, whereas those with reconstituted systems contained 5 to 25 pmol of purified CYP2C enzyme, 50 to 250 pmol
of P-450 reductase, 1.5 to 7.5 µg of
L-
-dilauroylphosphatidylcholine (DLPC), and 20 to 100 pmol of b5. Other reaction
components are given below. For antibody inhibition studies, microsomes
or reconstituted CYP enzymes were first incubated with anti-CYP2C9 or
preimmune IgG (see below) for 3 min at 37°C, and then for 10 min at
room temperature, followed by the addition of the remaining reaction components. Antibody titration curves contained increasing amounts of
either polyspecific anti-CYP2C9 or monospecific anti-CYP2C9 IgG (0-20
mg of IgG/nmol CYP). The amount of IgG added to the incubation mixtures
was maintained at a constant level by the addition of preimmune IgG.
;
Shimada et al., 1985, 1986; Lasker et al., 1998). Briefly, incubation mixtures (0.25 ml) contained human liver microsomes or reconstituted CYP2Cs, 100 µM [14C]S-mephenytoin
(3.8 µCi/mmol), and 1.0 mM NADPH. Reactions with microsomes and
reconstituted CYPs were performed in 100 mM KPO4 buffer (pH 7.4) and 50 mM HEPES buffer (pH 7.4), respectively. Incubations were terminated after 30 min by the addition of 200 µl of
1 N HCl, followed by extraction with 3 ml of ethyl acetate. The organic
extracts were dried under nitrogen gas at room temperature, and
dissolved in 25 µl of acetonitrile. Formation of 4-hydroxymephenytoin was assessed by injecting the resolubilized residues onto a Versapack C18 column (4.6 × 300 mm; Alltech,
Deerfield, IL) with 26% (v/v) aqueous acetonitrile as the mobile
phase. The flow rate used was 1.0 ml/min, and radioactivity in the
column eluates was detected with a Radiomatic 150TR radio-HPLC detector
(scintillant flow rate of 2.5 ml/min). Under these conditions,
4'-hydroxymephenytoin and mephenytoin exhibited retention times of 7.9 and 19.5 min, respectively.
Diclofenac 4'-hydroxylation was assessed essentially as described by
Leemann et al. (1993). Incubation mixtures (1.0 ml) contained human
liver microsomes or reconstituted CYP2Cs, 10 µM diclofenac, and 1.0 mM NADPH. Reactions with microsomes and reconstituted CYPs were
performed in 100 mM KPO4 buffer (pH 7.4) and 50 mM HEPES buffer (pH 7.4), respectively. Incubations were terminated
after 30 min by the addition of 200 µl of 1 N HCl containing 100 µM chlorpropamide (used as the internal standard), followed by extraction with 3 ml of ethyl acetate. The organic extracts were dried under nitrogen gas at room temperature, and dissolved in 25 µl of
acetonitrile. Formation of 4'-hydroxydiclofenac was assessed by
injecting the resolubilized residues onto a Versapack
C18 column (4.6 × 300 mm; Alltech)
developed with a linear gradient of 45 to 59% aqueous acetonitrile
containing 0.025% phosphoric acid. The flow rate used was 1.0 ml/min,
and eluates were continuously monitored at 282 nm. These HPLC
conditions gave retention times for 4'-hydroxydiclofenac and diclofenac
of 7.5 and 13.3 min, respectively.
Tolbutamide hydroxylation was assessed according to a previously
published procedure (Knodell et al., 1987) but with the following modifications. Incubation mixtures (1.0 ml) contained human liver microsomes (protein equivalent to 50 pmol) or 50 pmol of reconstituted CYP2Cs, 100 mM potassium phosphate buffer (pH 7.4), 0.25 mM
[14C]tolbutamide (µCi/mmol), and 1.0 mM
NADPH. When sulfaphenazole was included, the final concentration ranged
from 50 to 100 µM. Reactions were terminated after 60 min at 37°C
with 200 µl of 1 N HCl, followed by extraction with 3 ml of ethyl
acetate. The ethyl acetate extracts were dried with nitrogen gas at
room temperature, and the residue was resolubilized in 25 µl of
acetonitrile. OHT formation was assessed by injecting the resolubilized
residues onto a Versapack C18 column (4.6 × 300 mm; Alltech) with 40% acetonitrile containing 0.03% phosphoric
acid (pH 2.6) as the mobile phase. The flow rate used was 1.0 ml/min,
and radioactivity in the column eluates was detected with a Radiomatic
150TR radio-HPLC detector (scintillant flow rate of 2.5 ml/min). Under
these conditions, OHT and tolbutamide exhibited retention times of 3.8 and 7.9 min, respectively.
Immunochemical Methods.
Polyclonal antibodies to CYP2C9 were raised in male New Zealand White
rabbits, and the IgG fractions were derived from sera using caprylic
acid/ammonium sulfate precipitation (Lasker et al., 1998). These
polyspecific CYP2C9 antibodies (anti-CYP2C9-Ps), which exhibited
cross-reactivity with CYP2C8 as well as CYP2C19 (Lasker et al., 1998),
were subsequently made monospecific for CYP2C9 by repeated passage over
a column of cross-linked agarose to which purified recombinant CYP2C19
had been covalently coupled. The immunosorbent resin was prepared by
reacting 12 ml of activated Affi-Gel 10 agarose resin (Bio-Rad Labs,
Richmond, CA) with 20 ml of 250 mM sodium bicarbonate buffer (pH 8.6)
containing 1% Lubrol PX, 1 mM EDTA, and 200 nmol recombinant CYP2C19
for 3 h at ambient temperature according to the manufacturer's
instructions. The column was regenerated between anti-CYP2C9
applications by washing with 20 ml of 100 mM glycine-HCl buffer (pH
3.1), followed by rapid neutralization with 50 ml of 100 mM
KPO4 buffer (pH 7.4). Preimmune (control) IgG was
prepared from rabbit sera obtained before immunization.
).
CYP2C9 and CYP2C19 levels in the human liver samples were quantified on
Western blots by staining with polyspecific anti-CYP2C9 and
polyspecific anti-CYP2C19, respectively. The blots were scanned with a
Microtek ScanMaker II HR flat bed scanner interfaced to a computer, and
immunoreactive areas were measured using ImageQuant software (Molecular
Dynamics, Sunnyvale, CA). All immunochemical staining was performed
under conditions where the peroxidase reaction density was directly
proportional to the amount of protein applied to the original
polyacrylamide gels.
Statistical Analysis.
Data was analyzed using repeated-measure ANOVA or by Student's
t test. Levels of significance were set at P 
.05.
Materials. [14C]tolbutamide and [14C]S-mephenytoin were purchased from Amersham (Arlington Heights, IL). Unlabeled S-mephenytoin was obtained from Salford Ultrafine Chemicals (Manchester, England). Unlabeled tolbutamide, diclofenac, sulfaphenazole, cytochrome c, and Lubrol PX were obtained from Sigma Chemical Co. (St. Louis, MO), whereas NADPH was purchased from Boehringer-Mannheim (Indianapolis, IN). Hydroxylapatite (Hypatite C) was obtained from Clarkson Chromatography Products, Inc. (South Williamsport, PA). All other chemicals used were of the highest grade commercially available.
| |
Results |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Preparation and Characterization of Monospecific CYP2C9
Antibodies.
Rabbit polyclonal antibodies to human CYP2C9 were found to recognize
CYP2C9 as well as CYP2C8 and CYP2C19 on immunoblots containing purified
human liver CYP2C enzymes and hepatic microsomes (Lasker et al., 1998).
Preliminary small-scale experiments indicated that monospecific CYP2C9
antibodies (anti-CYP2C9-Ms) could be derived from anti-CYP2C9-Ps by
back-adsorption against heterologously expressed CYP2C19. We expanded
this procedure to derive CYP2C9 IgG preparations that were not only
monospecific on Western blots but were also inhibitory toward only
CYP2C9-catalyzed reactions. To obtain anti-CYP2C9-M, we used
recombinant CYP2C19-based immunoadsorption. Expressed CYP2C19
(Richardson et al., 1995) was purified using a single chromatographic
step, hydroxylapatite chromatography. A yield of 43% (200 of the 470 nmol originally processed) was achieved. The entire preparation of
purified CYP2C19 was then covalently linked to Affigel 10, giving a
solid-phase immunoadsorbent containing 16.7 nmol hemoprotein/ml agarose
resin. Back-adsorption of 30 mg of anti-CYP2C9-P using this
immunosorbent resin resulted in a yield of 15.4 mg of anti-CYP2C9-M.
|
75%) diclofenac
4'-hydroxylation at an IgG/CYP ratio of 5 mg/nmol (Fig. 2B).
|
75%) at IgG/CYP ratios ranging from
2.5 to 10 mg/nmol, whereas the anti-CYP2C9-M preparation was much less
inhibitory (25%) at these same IgG/CYP ratios. Similar results were
obtained with other human liver samples examined (Table
1). As shown in Table 1, anti-CYP2C9-P
completely inhibited microsomal S-mephenytoin 4-hydroxylation whereas anti-CYP2C9-M produced only a mean of 8.5 ± 5.3% inhibition (n = 4). Conversely, both
anti-CYP2C9-P and anti-CYP2C9-M markedly inhibited (89 ± 6 and
89.3 ± 2%, respectively) diclofenac 4'-hydroxylation in the
liver samples (n = 4) (Table 1).
|
|
CYP2C Enzymes Catalyzing Tolbutamide Hydroxylation.
We recently reported that tolbutamide hydroxylation in human liver
microsomes was catalyzed by both CYP2C9 and CYP2C19 (Lasker et al.,
1998). However, the lack of monospecific CYP2C antibodies prevented us
from quantitatively assessing the contribution of CYP2C9 and CYP2C19 to
overall microsomal metabolism of this drug. We thus examined the
utility of anti-CYP2C9-M preparations to differentiate the respective
roles of these CYP2C enzymes in tolbutamide hydroxylation by human
liver samples that either contained CYP2C19 (CYP2C19+) or
were deficient in the enzyme (CYP2C19) (Table 2). Incubation of CYP2C19+
microsomes with optimal amounts of anti-CYP2C9-M produced 78 ± 6% inhibition of tolbutamide hydroxylase activity, whereas the same
amount of anti-CYP2C9-P gave complete (100%) inhibition (Fig.
4). With CYP2C19 microsomes, the
inhibition of tolbutamide metabolism observed with anti-CYP2C9-M
increased to 93 ± 4%. CYP2C9 immunoquantitation performed with
anti-CYP2C9-M revealed a correlation (r = 0.72, P < .02) between CYP2C9 content and anti-CYP2C9-M
inhibited rates of tolbutamide hydroxylation in the seven human samples
examined. A somewhat weaker but still significant (r = 0.66; P < .05) correlation was noted between microsomal CYP2C9 levels and anti-CYP2C9-P inhibited rates of tolbutamide metabolism, which was similar to the correlation found between levels of this CYP enzyme and uninhibited rates of OHT formation.
|
|
). Tolbutamide hydroxylation by recombinant CYP2C9 was inhibited by 75% in the presence of 50 µM sulfaphenazole, whereas this compound had
negligible (4%) effects on the CYP2C19-mediated reaction. With
CYP2C19+ microsomes, sulfaphenazole inhibited rates of OHT
formation by an average of 57% (n = 2), whereas with
CYP2C19 microsomes inhibition (65%, n = 2) was
somewhat greater. Sulfaphenazole at 100 µM produced inhibition that
was similar in CYP2C19 and CYP2C19+ microsomes, indicating a loss of
specificity with increasing concentrations of the inhibitor.
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
In this study, we demonstrated that both CYP2C9 and CYP2C19 participate in metabolism of the antidiabetic agent tolbutamide in human liver. Despite the marked sequence similarity between these CYP2C enzymes, we used an immunochemical approach to estimate the contribution of each CYP toward microsomal tolbutamide hydroxylation. After eliminating the cross-reactivity of CYP2C9 antibodies with CYP2C19 (and CYP2C8), use of the monospecific anti-CYP2C9 preparation indicated that CYP2C9 catalyzed from 78 to 93% of the total tolbutamide hydroxylation occurring in human hepatic microsomes, and the remaining 7 to 22% could be attributed to CYP2C19 catalysis. These findings were subsequently validated in experiments with the specific CYP2C9 chemical inhibitor, sulfaphenazole, which also indicated that CYP2C9 was the major tolbutamide hydroxylase in microsomes from human liver.
Tolbutamide has been used extensively as a probe of CYP function, a
fact that may partly stem from the polymorphism purported to underlie
its oxidative metabolism (Scott and Poffenbarger, 1979). Whereas the
majority of studies have found that tolbutamide is specifically
hydroxylated by CYP2C9, recent reports have suggested that this
specificity may extend to another member of the human CYP2C subfamily,
CYP2C19 (Richardson et al., 1995; Lasker et al., 1998; Venkatakrishnan
et al., 1998). Indeed, we found that rates of tolbutamide hydroxylation
mediated by native and recombinant CYP2C19 were essentially equivalent
to those of purified CYP2C9 and the corresponding recombinant CYP
(Lasker et al., 1998). However, as catalysis of a given
drug-metabolizing reaction by a purified and/or recombinant CYP enzyme
in reconstituted systems does not imply that it contributes extensively
to the reaction in intact liver microsomes (Guengerich et al., 1998),
we attempted to discriminate CYP2C19-catalyzed tolbutamide
hydroxylation from that mediated by CYP2C9. A kinetic approach to this
problem proved unsuccessful as the Michaelis constants for tolbutamide
derived for these two CYP2C enzymes were similar, and different human
liver specimens exhibited monophasic rather than biphasic tolbutamide
hydroxylation kinetics (Lasker et al., 1998). Furthermore, an
immunochemical approach was precluded due to the extensive
cross-reactivity of the CYP2C9 and CYP2C19 antibody preparations used
in our previous study. In fact, the marked (94-96%) inhibition of
microsomal tolbutamide hydroxylation elicited by the latter IgG
preparation grossly overestimated the contribution of CYP2C19 to the reaction.
Herein, we refined the immunochemical-based methodology to discern the respective roles of CYP2C9 and CYP2C19 in hepatic tolbutamide metabolism. In contrast to our previous efforts, we derived a monospecific CYP2C9 antibody from its polyspecific precursor by back-adsorption of the IgG against purified recombinant CYP2C19 coupled to a solid-phase support. Exposure of anti-CYP2C9-P to this immunoadsorbent gave an antibody preparation that: 1) reacted on protein blots with only CYP2C9 and no longer recognized either CYP2C19 or CYP2C8 (compare lanes 1-3 in Fig. 1, A and B); and 2) inhibited CYP2C9-catalyzed but not CYP2C19-catalyzed reactions. For instance, diclofenac 4'-hydroxylation mediated by either recombinant CYP2C9 or intact liver microsomes was inhibited to essentially the same extent by both monospecific and polyspecific CYP2C9 antibodies (Fig. 2, A and B). In contrast, the inhibition of CYP2C19-mediated S-mephenytoin 4'-hydroxylation noted with anti-CYP2C9-P (Fig. 3A) was minimal but nonspecific after back-adsorption of this polyspecific CYP antibody against CYP2C19 (Fig. 3B). To the best of our knowledge, this is the first report of a specific inhibitory CYP2C polyclonal antibody that was originally raised to the corresponding native CYP apoprotein.
Using anti-CYP2C9-M as a specific immunochemical probe for
CYP2C9-mediated catalysis demonstrated that this human CYP enzyme was
the predominant tolbutamide hydroxylase in human liver microsomes, confirming our earlier observations (Lasker et al., 1998). Indeed, antibodies to CYP2C9, even after back-adsorption, retained the capacity
to markedly inhibit (78-93%) microsomal OHT formation (Fig. 4). A
significant correlation (r = 0.72; P < .02) was established between hepatic CYP2C9 content and anti-CYP2C9-M
inhibitable rates of tolbutamide hydroxylation in the seven subjects
studied. Nevertheless, several lines of evidence presented here
indicated that CYP2C19 was also involved in hepatic tolbutamide
metabolism. First, the capacity of anti-CYP2C9-M to inhibit tolbutamide
metabolism was reduced in hepatic microsomes containing CYP2C19
(78 ± 6% inhibition) compared with microsomes deficient in
CYP2C19 (93 ± 4% inhibition) (Fig. 4). Similarly, less average
inhibition of tolbutamide hydroxylase activity with CYP2C19+ liver
microsomes compared with CYP2C19 microsomes (Table 2) was also
observed with sulfaphenazole, a known chemical inhibitor of
CYP2C9-mediated reactions (Newton et al., 1995 and references therein).
Evidence that sulfaphenazole, like anti-CYP2C9-M, was a specific probe
for CYP-mediated tolbutamide metabolism was indicated by its capacity
to inhibit OHT formation only by recombinant CYP2C9 and not by CYP2C19
(Table 2). Despite this specificity, sulfaphenazole proved considerably
less efficacious than anti-CYP2C9-M as an inhibitor of microsomal
tolbutamide metabolism (concentrations higher than 50 µM failed to
elicit additional decreases in metabolic rates), and the results
obtained with this chemical inhibitor were not as clear-cut as those
obtained with the immunochemical reagents. Finally, no clear
relationship (r = 0.62; P = .14) was
found between hepatic CYP2C19 levels and tolbutamide hydroxylase
activity, which was expected because of the low hepatic CYP2C19 content
relative to that of CYP2C9 in the subjects studied (see
Results).
In conclusion, our results indicate that CYP2C9 and CYP2C19
function as the major and minor tolbutamide hydroxylases, respectively, in human liver. When considered together with abundance of hepatic CYP2C9 relative to that of CYP2C19 (Inoue et al., 1997; Lasker et al.,
1998), our findings imply that the latter CYP typically plays a nominal
role in the metabolism of this oral antidiabetic agent. However,
CYP2C19 involvement in hepatic tolbutamide disposition may be much
greater in subjects who express CYP2C9 and CYP2C19 at comparable levels
or in individuals who express a catalytically deficient CYP2C9 enzyme,
such as the CYP2C9V1 allelic variant (Sullivan-Klose et al.,
1996). This CYP2C9 variant exhibits a marked decrease in both
tolbutamide and S-warfarin hydroxylase activity compared
with the wild-type CYP2C9 as a result of an increased
Km and decreased
Vmax for these therapeutic substrates. Clearly, more studies of this type are required to better differentiate the contribution of individual CYP2C enzymes to the oxidative metabolism of other pharmaceutical agents.
| |
Footnotes |
|---|
Received July 27, 1999; accepted November 11, 1999.
1 This work was presented in partial fulfillment of the requirements for a doctoral degree (M.R.W.) in Toxicology at the University of New Mexico.
2 Present address: The Scripps Research Institute, Dept. of Molecular and Experimental Medicine, La Jolla, CA 92037.
Supported by National Institutes of Health Grants GM49511 (J.L.R., J.M.L.), AA07842 (J.M.L.), GM31001 (E.F.J.), and DK62274 (Liver Transplant, Procurement and Distribution System).
Send reprint requests to: Dr. Judy L. Raucy, The La Jolla Institute for Experimental Medicine, 505 Coast Blvd. S., La Jolla, CA 92037. E-mail: jraucy{at}agi.org
| |
Abbreviations |
|---|
Abbreviations used are:
CYP, cytochrome
P-450;
b5, cytochrome
b5;
P-450 reductase, NADPH:P-450
oxidoreductase;
anti-CYP2C9-P, polyspecific CYP2C9 antibodies;
anti-CYP2C9-M, monospecific CYP2C9 antibodies;
DLPC, L--dilauroylphosphatidylcholine;
DTT, dithiothreitol;
OHT, hydroxytolbutamide;
CYP2C19+, microsomes containing CYP2C19, CYP2C19, CYP2C19-deficient microsomes.
| |
References |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Purification, expression, mechanism, and relevance to drugs.
Drug Metab Dispos
26:
1175-1178This article has been cited by other articles:
|
|
 |
|
  R. A. Stringer, C. Strain-Damerell, P. Nicklin, and J. B. Houston Evaluation of Recombinant Cytochrome P450 Enzymes as an in Vitro System for Metabolic Clearance Predictions Drug Metab. Dispos., May 1, 2009; 37(5): 1025 - 1034. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Kumar, D. A. Rock, C. J. Warren, T. S. Tracy, and J. L. Wahlstrom Enzyme Source Effects on CYP2C9 Kinetics and Inhibition Drug Metab. Dispos., November 1, 2006; 34(11): 1903 - 1908. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Wester, J. K. Yano, G. A. Schoch, C. Yang, K. J. Griffin, C. D. Stout, and E. F. Johnson The Structure of Human Cytochrome P450 2C9 Complexed with Flurbiprofen at 2.0-A Resolution J. Biol. Chem., August 20, 2004; 279(34): 35630 - 35637. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Cai, R. W. Wang, R. W. Edom, D. C. Evans, M. Shou, A. D. Rodrigues, W. Liu, D. C. Dean, and T. A. Baillie VALIDATION OF (-)-N-3-BENZYL-PHENOBARBITAL AS A SELECTIVE INHIBITOR OF CYP2C19 IN HUMAN LIVER MICROSOMES Drug Metab. Dispos., June 1, 2004; 32(6): 584 - 586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Walsky and R. S. Obach VALIDATED ASSAYS FOR HUMAN CYTOCHROME P450 ACTIVITIES Drug Metab. Dispos., June 1, 2004; 32(6): 647 - 660. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. B. Koukouritaki, J. R. Manro, S. A. Marsh, J. C. Stevens, A. E. Rettie, D. G. McCarver, and R. N. Hines Developmental Expression of Human Hepatic CYP2C9 and CYP2C19 J. Pharmacol. Exp. Ther., March 1, 2004; 308(3): 965 - 974. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. Lee, J. A. Pieper, R. F. Frye, A. L. Hinderliter, J. A. Blaisdell, and J. A. Goldstein Tolbutamide, Flurbiprofen, and Losartan as Probes of CYP2C9 Activity in Humans J. Clin. Pharmacol., January 1, 2003; 43(1): 84 - 91. [Abstract] [Full Text]
|
|
|
|
|
|
R. Yuan, S. Madani, X.-X. Wei, K. Reynolds, and S.-M. Huang Evaluation of Cytochrome P450 Probe Substrates Commonly Used by the Pharmaceutical Industry to Study in Vitro Drug Interactions Drug Metab. Dispos., December 1, 2002; 30(12): 1311 - 1319. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Raucy, L. Mueller, K. Duan, S. W. Allen, S. Strom, and J. M. Lasker Expression and Induction of CYP2C P450 Enzymes in Primary Cultures of Human Hepatocytes J. Pharmacol. Exp. Ther., August 1, 2002; 302(2): 475 - 482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-S. Wang, X. Wen, J. T. Backman, and P. J. Neuvonen Effect of Albumin and Cytosol on Enzyme Kinetics of Tolbutamide Hydroxylation and on Inhibition of CYP2C9 by Gemfibrozil in Human Liver Microsomes J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 43 - 49. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Ando, E. Fuse, and W. D. Figg Thalidomide Metabolism by the CYP2C Subfamily Clin. Cancer Res., June 1, 2002; 8(6): 1964 - 1973. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Ginsberg, D. Hattis, B. Sonawane, A. Russ, P. Banati, M. Kozlak, S. Smolenski, and R. Goble Evaluation of Child/Adult Pharmacokinetic Differences from a Database Derived from the Therapeutic Drug Literature Toxicol. Sci., April 1, 2002; 66(2): 185 - 200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. W. Krausz, I. Goldfarb, J. T. M. Buters, T. J. Yang, F. J. Gonzalez, and H. V. Gelboin Monoclonal Antibodies Specific and Inhibitory to Human Cytochromes P450 2C8, 2C9, and 2C19 Drug Metab. Dispos., November 1, 2001; 29(11): 1410 - 1423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Dierks, K. R. Stams, H.-K. Lim, G. Cornelius, H. Zhang, and S. E. Ball A Method for the Simultaneous Evaluation of the Activities of Seven Major Human Drug-Metabolizing Cytochrome P450s Using an in Vitro Cocktail of Probe Substrates and Fast Gradient Liquid Chromatography Tandem Mass Spectrometry Drug Metab. Dispos., January 1, 2001; 29(1): 23 - 29. [Abstract] [Full Text]
|
|
|
|
|
|
D. F. McGinnity, A. J. Parker, M. Soars, and R. J. Riley Automated Definition of the Enzymology of Drug Oxidation by the Major Human Drug Metabolizing Cytochrome P450s Drug Metab. Dispos., November 1, 2000; 28(11): 1327 - 1334. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
), or anti-CYP2C9-M (
) IgG.
The total amount of IgG added (anti-CYP2C9 plus preimmune IgG) was
maintained at 200 µg. B, incubation mixtures contained liver
microsomes from subject H (Table 
