Relationship between phenytoin and tolbutamide hydroxylations in human liver microsomes.

Doecke, Christopher J; Veronese, Maurice E.; Pond, S. M.; Miners, John O.; Birkett, D. J.; Sansom, LN; McManus, M E

doi:10.1111/j.1365-2125.1991.tb05499.x

Cited by 91 publications

(39 citation statements)

References 22 publications

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“…In the current study, a single enzyme Michaelis-Menten model described well the formation of hydroxytolbutamide in all experiments, consistent with previous findings about the kinetics of tolbutamide metabolism in human liver microsomes (Miners et al, 1988;Doecke et al, 1991;Hickman et al, 1998;Hemeryck et al, 1999;Shin et al, 1999;Komatsu et al, 2000a). The observed apparent K m (123 M) and V max (282 pmol/mg/min) values in pooled human liver microsomes were comparable with the mean values of published reports (K m ϭ 135 M and V max ϭ 257 pmol/mg/min) ( Table 1).…”

Section: Discussionsupporting

confidence: 79%

“…The Michaelis-Menten and Doecke et al, 1991;2, Hemeryck et al, 1999;3, Shin et al, 1999;4, Komatsu et al, 2000a;5, Hickman et al, 1998;6, Miners et al, 1988. f In vivo CL met of tolbutamide hydroxylation observed in human subjects (Data from Gross et al, 1999). g P Ͻ 0.0001 compared with the observed in vivo CL met of tolbutamide using a t test.…”

Section: Comparison Of Observed Values Of In Vivo Metabolicmentioning

confidence: 99%

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Effect of Albumin and Cytosol on Enzyme Kinetics of Tolbutamide Hydroxylation and on Inhibition of CYP2C9 by Gemfibrozil in Human Liver Microsomes

Wang

Xia²,

Backman³

et al. 2002

J Pharmacol Exp Ther

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The effect of human serum albumin (Hsa) and human liver cytosol (Hlc) on the in vitro enzyme kinetics of the formation of hydroxytolbutamide (CYP2C9 marker reaction) and the inhibitory effect of gemfibrozil on tolbutamide hydroxylation were examined using human liver microsomes. The addition of Hsa greatly decreased the unbound concentrations of tolbutamide and gemfibrozil in the incubation medium, whereas Hlc only slightly decreased them. The unbound K m value for tolbutamide hydroxylation was 123 M without Hsa and Hlc, and 73, 88, and 64 M in the presence of Hsa (5 mg/ml), Hlc (0.5 mg/ml), and Hsa plus Hlc, respectively. The predicted in vivo hepatic clearance (CL h ) of tolbutamide based on enzyme kinetics without Hsa and Hlc (0.06 ml/min/kg) was 40% of its in vivo clearance (0.15 ml/min/kg) based on published data. Addition of 5 mg/ml Hsa and 0.5 mg/ml Hlc to the incubation medium distinctly improved the prediction, with the coaddition of Hsa and Hlc yielding the most accurate value (0.14 ml/min/kg). The K i (6 M) of gemfibrozil for CYP2C9, calculated using total drug concentrations, was increased by Hlc (8 M), Hsa (40 M), or both (72 M). However, when the unbound substrate and inhibitor concentrations were considered, the K i (6 M without Hsa and Hlc) was not markedly altered by Hsa (4 M), Hlc (8 M), or both Hsa and Hlc (9 M). The present findings suggest that the addition of Hsa and Hlc to microsomal incubation media may yield enzyme kinetic estimates more comparable with in vivo results.Numerous attempts have been made to predict in vivo pharmacokinetics and drug-drug interactions on the basis of in vitro hepatic microsomal data (for reviews, see Bertz and Granneman, 1997;von Moltke et al., 1998). However, the utilization of human microsomal data for predicting the in vivo situation has remained uncertain and controversial. Factors such as the in vitro incubation conditions, extrahepatic drug metabolism, and transporters related to drug absorption and disposition can confound the in vitro-in vivo extrapolation.The addition of bovine serum albumin to the microsomal incubation medium has decreased the K m estimates and increased the intrinsic clearance (CL int ) of phenytoin p-hydroxylation, a reaction mainly catalyzed by cytochrome P-450 CYP2C9, yielding predicted clearance values more comparable with the in vivo values (Ludden et al., 1997;Carlile et al., 1999). However, because the albumin concentrations used in microsomal incubations have been unphysiologically high (comparable with serum levels), a recent consensus on the conduct of studies of metabolic and transport interactions warranted further studies to resolve whether albumin should be added to microsomal incubation mixtures (Tucker et al., 2001). In another study, phenytoin oxidation in human liver microsomes was substantially promoted by the addition of rat liver cytosol (Komatsu et al., 2000b). Both albumin and cytosolic components are probably present at the metabolic enzyme site in vivo (Shroyer and Nakane, 1987;Komatsu et al., 2000b...

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Section: Discussionsupporting

confidence: 79%

Section: Comparison Of Observed Values Of In Vivo Metabolicmentioning

confidence: 99%

Effect of Albumin and Cytosol on Enzyme Kinetics of Tolbutamide Hydroxylation and on Inhibition of CYP2C9 by Gemfibrozil in Human Liver Microsomes

Wang

Xia²,

Backman³

et al. 2002

J Pharmacol Exp Ther

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“…K m values for tolbutamide in pooled liver microsomes and rCYP2C9 were 147 Ϯ 4 and 82.0 Ϯ 2.5 M, respectively (Table 7; Fig. 2), as compared with previously cited values ranging from 60 to 580 M (Darby and Price-Evans, 1971; Purba et al, 1987;Back et al, 1988;Miners et al, 1988;Doecke et al, 1991;Chen et al, 1993;Sharer et al, 1995;Bourrie et al, 1996;Inoue et al, 1997;Hickman et al, 1998;Lasker et al, 1998;Carlile et al, 1999;Hemeryck et al, 1999;Palamanda et al, 2000;Yin et al, 2000;Tang et al, 2002;Wang et al, 2002) (Fig. 4).…”

Section: Validated Assays For Human Cytochrome P450 Enzymessupporting

confidence: 50%

Validated Assays for Human Cytochrome P450 Activities

Walsky

Obach²

2004

Drug Metab Dispos

465

380

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ABSTRACT:The measurement of the effect of new chemical entities on human cytochrome P450 marker activities using in vitro experimentation represents an important experimental approach in drug development. In vitro drug interaction data can be used in guiding the design of clinical drug interaction studies, or, when no effect is observed in vitro, the data can be used in place of an in vivo study to claim that no interaction will occur in vivo. To make such a claim, it must be assured that the in vitro experiments are performed with absolute confidence in the methods used and data obtained. To meet this need, 12 semiautomated assays for human P450 marker substrate activities have been developed and validated using approaches described in the GLP (good laboratory practices) as per the code of U.S. Federal Regulations. The assays that were validated are: phenacetin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), bupropion hydroxylase (CYP2B6), amodiaquine N-deethylase (CYP2C8), diclofenac 4-hydroxylase and tolbutamide methylhydroxylase (CYP2C9), (S)-mephenytoin 4-hydroxylase (CYP2C19), dextromethorphan O-demethylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1), felodipine dehydrogenase, testosterone 6␤-hydroxylase, and midazolam 1-hydroxylase (CYP3A4 and CYP3A5). High-pressure liquid chromatography-tandem mass spectrometry, using stable isotope-labeled internal standards, has been applied as the analytical method. This analytical approach, through its high sensitivity and selectivity, has permitted the use of very low incubation concentrations of microsomal protein (0.01-0.2 mg/ml). Analytical assay accuracy and precision values were excellent. Enzyme kinetic and inhibition parameters obtained using these methods demonstrated high precision and were within the range of values previously reported in the scientific literature. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of cytochrome P450 activities.Drug-drug interactions are of great interest to scientists involved in drug research, regulatory authorities who are responsible for public safety, physicians, and their patients. Since "polypharmacy," or the practice of simultaneous prescription of more than one drug to treat one or more conditions in a single patient, has become a more common practice, drug interactions have been cited as one of the major reasons for hospitalization and even death (Lazarou et al., 1998). Thus, a great deal of effort is expended by researchers engaged in new drug research in avoiding the development of compounds that will cause drug-drug interactions.The most common mechanism underlying drug-drug interactions is the inhibition of cytochrome P450 activities. Several drugs in common use cause large increases in exposure to other drugs. Examples include ketoconazole, itraconazole, erythromycin, clarithromycin, diltiazem, and nefazodone (CYP3A); quinidine, paroxetine, and terbinafine (CYP2D6); ticlopidine (CYP2C19); ...

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“…The remaining probe substrate activities used were as described previously in the literature. Coumarin 7-hydroxylase activity was determined at a substrate concentration of 100 M (Maurice et al, 1991), (S)-warfarin 7-hydroxylase activity was determined at a substrate concentration of 500 M (Doeke et al, 1991), (S)-mephenytoin 4-hydroxylase activity was determined at a substrate concentration of 500 M (Meier et al, 1985), bufuralol 1Ј-hydroxylase was determined at a substrate concentration of 10 M (Kronbach et al, 1987), 4-nitrophenol 3-hydroxylase activity was determined at a substrate concentration of 1000 M (Tassaneeyakul et al, 1993), and testosterone 6␤-hydroxylase activity was determined at a substrate concentration of 250 M (Funae and Imaoka, 1987).…”

Section: Methodsmentioning

confidence: 99%

Identification of the Cytochrome P450 Enzymes Involved in theN-Oxidation of Voriconazole

Hyland

Jones²,

Smith³

2003

Drug Metab Dispos

356

335

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Relationship between phenytoin and tolbutamide hydroxylations in human liver microsomes.

Cited by 91 publications

References 22 publications

Effect of Albumin and Cytosol on Enzyme Kinetics of Tolbutamide Hydroxylation and on Inhibition of CYP2C9 by Gemfibrozil in Human Liver Microsomes

Effect of Albumin and Cytosol on Enzyme Kinetics of Tolbutamide Hydroxylation and on Inhibition of CYP2C9 by Gemfibrozil in Human Liver Microsomes

Validated Assays for Human Cytochrome P450 Activities

Identification of the Cytochrome P450 Enzymes Involved in theN-Oxidation of Voriconazole

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