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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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). CYP2C9 is
primarily responsible for the metabolism of glyburide
(Miners and Birkett, 1998;
Brian, 2000).
An extensive literature review shows that most of the published drug
interactions for glyburide describe the effect of a concomitant drug on the
hypoglycemic activity of glyburide, leading to hypoglycemia. For example,
cotrimoxazole (Asplund et al.,
1983), cimetidine (Kubacka et
al., 1987), miconazole (Loupi
et al., 1982), fluconazole
(Albengres et al., 1998), and
gemfibrozil (Ahmad, 1991),
which are all CYP2C9 inhibitors, inhibit the pharmacokinetics and clinical
effect of glyburide.
Conversely, some authors have observed that glyburide inhibited the
metabolism of coadministered drugs with a consequent risk of toxic phenomena
(Jassel, 1991;
Islam et al., 1996). However,
there are no available data on the inhibitory effect of glyburide on
drug-metabolizing enzymes, especially
P4501 isoforms.
This study assessed the potential of glyburide to inhibit different P450 isoforms in vitro using human liver microsomes and to examine the mechanism of the interaction of glyburide with coadministered drugs.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-hydroxypaclitaxel, and 4'-hydroxymephenytoin were obtained from
Ultrafine Chemical Co. (Manchester, UK). Acetonitrile and methanol were
acquired from Fisher Scientific Co. (Pittsburgh, PA), and Bukwang
Pharmaceutical Co. (Seoul, Korea) kindly provided midazolam. All other
reagents and chemicals were of analytical or high-performance liquid
chromatography (HPLC) grade. Pooled human liver microsomes were obtained from
BD Gentest Corp. (Woburn, MA).
Incubation Studies. All incubations were performed in duplicate, and
the mean values were used for analysis. Briefly, each incubation was performed
with 1 mg/ml human liver microsomes in a final incubation volume of 0.25 ml.
The incubation medium contained 100 mM phosphate buffer (pH 7.4) containing an
NADPH-regenerating system (including 1.3 mM NADP, 3.3 mM glucose 6-phosphate,
3.3 mM MgCl2, and 1.0 U/ml glucose-6-phosphate dehydrogenase). The
incubation mixture containing glyburide (final concentration 1–100
µM) was preincubated for 5 min. To determine whether the inhibition of P450
isoforms by glyburide is mechanism-based, glyburide was preincubated with the
incubation medium at 37°C for 5 to 15 min, either in the presence or
absence of the NADPH-regenerating system. After preincubation, probe
substrates were added either with or without the NADPH-regenerating system.
The incubation conditions including incubation times, microsome
concentrations, and probe substrate concentrations have been reported
elsewhere (Bourrie et al.,
1996; Shin et al.,
1999,
2002). After incubation at
37°C for a specific period of time, the reaction was stopped by placing
the incubation tubes on ice and adding 70 µl of ice-cold acetonitrile or
10% perchloric acid depending on the reactions, as described previously
(Bourrie et al., 1996; Shin et
al., 1999,
2002). The incubation mixtures
were then centrifuged at 10,000g for 5 min at 4°C. Aliquots of
the supernatant were injected onto an HPLC system.
HPLC Analysis. HPLC assays for the respective products of the P450
marker reactions were performed. The reactions investigated were phenacetin
O-deethylation for CYP1A2
(Tassaneeyakul et al., 1993),
S-warfarin 7-hydroxylation for CYP2C9
(Rettie et al., 1992),
S-mephenytoin 4'-hydroxylation for CYP2C19
(Wrighton et al., 1993),
dextromethorphan O-demethylation for CYP2D6
(Broly et al., 1989),
paclitaxel 6-hydroxylation for CYP2C8
(Harris et al., 1994), and
midazolam 1-hydroxylation for CYP3A4
(Thummel et al., 1994). The
incubation conditions and analytical assays for the activities of these
isoforms were similar to the method previously described
(Bourrie et al., 1996; Shin et
al., 1999,
2002). The HPLC system
consisted of a Shiseido SI-1 system (Shiseido Co., Tokyo, Japan) and a Jasco
FP-2020 plus fluorescence detector (Jasco Co., Tokyo, Japan).
Data Analysis. The IC50 values (concentration of the inhibitor causing 50% inhibition of the original enzyme activity) were determined graphically. The apparent inhibitory constants (Ki) were calculated using graphical analysis of secondary plots of the slopes of Lineweaver-Burk plots of glyburide versus probe drug-catalyzed metabolite formation using GraphPad Prism Software (GraphPad Software Inc., San Diego, CA).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Kubacka et al., 1987;
Self et al., 1989;
Ahmad, 1991). Conversely,
several studies have shown that glyburide inhibited coadministered drugs
(Jassel, 1991;
Islam et al., 1996).
Of the P450-catalyzed reactions tested here, glyburide inhibited
CYP2C9-catalyzed S-warfarin 7-hydroxylation most significantly, with
a Ki of 2.4 µM. Although this effect is weaker than
that of sulfaphenazole (Ki < 1 µM)
(Rettie et al., 1992), a known
CYP2C9 inhibitor, it is more potent than any other CYP2C9 inhibitor, including
tolbutamide (Ki 
100 µM)
(Yamazaki and Shimada, 1997),
gemfibrozil (Ki = 5.8 µM)
(Wen et al., 2001), and
fluconazole (Ki = 8 µM)
(Kunze et al., 1996).
Following the standard daily dose of glyburide (1.75–7 mg), the peak
plasma concentrations of glyburide at a steady state, which show substantial
interindividual variation, range from 88 to 680 ng/ml (approximately
0.2–1.4 µM) (Jonsson et al.,
2000). Therefore, the Ki of glyburide is
approximately 1.5 to 6.5 times higher than its maximal concentration after
administration of a typical dose.
Theoretically, drug interactions based on the inhibition of hepatic
metabolism (FDV: fractional decrement in reaction velocity) can be calculated
from the Ki and the appropriate concentration of the
inhibitor ([I]) of the metabolic enzyme in the liver using the
following predictive model: FDV = [I]/([I] +
Ki), assuming that the inhibitory pattern is competitive
and that the substrate concentration is much less than the
Km (von Moltke et al.,
1998). However, the in vivo concentration of the inhibitor near
the metabolic enzyme is unknown, although glyburide is very highly lipophilic
(Siluk et al., 2002).
Therefore, it is possible that, with long-term administration, glyburide
accumulates in tissues, such as the liver, kidney, and brain. Although the
exact liver/plasma partition coefficient is unknown, we expect glyburide to
inhibit approximately 7 to 37% of the in vivo clearance of warfarin, a CYP2C9
substrate, if the peak plasma concentrations of glyburide are used in the
scaling model described above. Although this predicted inhibitory effect of
glyburide is not so potent, the above extrapolation formula does not consider
hepatic drug uptake, protein binding, rates of elimination, or intrahepatic
glyburide concentrations (von Moltke et
al., 1998). Considering these factors would increase the
possibility of drug interactions.
There are no in vivo studies of the effect of glyburide on CYP2C9-catalyzed
metabolism, but indirect clinical data support our in vitro findings, since
glyburide has been shown to enhance the anticoagulant effect of warfarin,
resulting in hypothrombinemia leading to death
(Jassel, 1991). Warfarin is a
mixture of S- and R-forms. The metabolism of
S-warfarin, the pharmacologically more active enantiomer, is
catalyzed by CYP2C9, whereas the metabolism of R-warfarin is
catalyzed by CYP1A2 and CYP3A4 (Kaminsky
and Zhang, 1997; Yamazaki and
Shimada, 1997). Therefore, it is reasonable to postulate that the
clinical interaction of glyburide and warfarin can be explained by inhibition
of CYP2C9-mediated S-warfarin metabolism leading to elevated warfarin
concentrations. Due to the lack of data, however, we could not confirm that
glyburide also inhibits other CYP2C9 substrates. When we evaluated the effect
of glyburide on the metabolism of phenytoin as a different CYP2C9 substrate
(Bajpai et al., 1996;
Shin et al., 2002), we found
its potent inhibition (Ki = 3.1 µM) and similar
inhibitory potency compared with that of warfarin (Ki =
2.4 µM). Glyburide also inhibited CYP3A4-catalyzed midazolam
1-hydroxylation, but showed weak inhibition (Ki =
42.5 µM); therefore, only minor drug interactions may be predicted. Islam
et al. (1996) reported that
the coadministration of cyclosporine, which is primarily metabolized by
CYP3A4, with glyburide resulted in a 57% increase in the AUC of cyclosporine
in kidney transplant patients. The authors suggested that this drug
interaction was caused by the inhibition of CYP3A4-catalyzed cyclosporine
metabolism by glyburide. Since the inhibitory potential of glyburide on CYP3A4
is weak compared with that of other CYP3A4 inhibitors [e.g., ketoconazole
(Ki = 0.015 µM), itraconazole (Ki =
0.27 µM), fluconazole (Ki = 1.27 µM)]
(von Moltke et al., 1996;
Gibbs et al., 1999), the
mechanism that causes drug interactions might not be related to direct
inhibition by glyburide. Further evaluation of the exact mechanism of the
interaction between these two drugs is needed.
In conclusion, this study demonstrated that glyburide is a potent inhibitor of CYP2C9 and a weak inhibitor of CYP3A4. However, the other P450s tested (CYP1A2, 2C8, 2C19, 2E1, and 2D6) were not affected by glyburide. The inhibition of CYP2C9 seems to explain the observed drug interaction of glyburide with warfarin. It is anticipated that clinically significant drug-drug interactions will likely ensue when glyburide is coadministered with agents having narrow therapeutic ranges that are cleared primarily by CYP2C9-mediated pathways.
Kyoung-Ah Kim
Ji-Young Park
East-West Medical Research Institute, Kyunghee University, Seoul, Korea (K.-A.K); and Department of Pharmacology, Gachon Medical School, Incheon, Korea (J.-Y.P.)
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Footnotes |
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Address correspondence to: Dr. Ji-Young Park, Department of Pharmacology, Gachon Medical School, 1198 Kuwol-dong, Namdong-gu, Incheon 405-760, Korea. E-mail: jypark{at}gachon.ac.kr
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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), CYP2C19-catalyzed
S-mephenytoin 4-hydroxylation (
), CYP2D6-catalyzed
dextromethorphan O-demethylation (
), CYP2E1-catalyzed
chlorzoxazone 6-hydroxylation (
), CYP3A4-catalyzed midazolam
1-hydroxylation (
), CYP2C8-catalyzed paclitaxel 6
), CYP2C9-catalyzed S-warfarin 7-hydroxylation (
), and
phenytoin p-hydroxylation (
). Each data point represents the
average of duplicate experiments.