[30] Deuterium and tritium kinetic isotope effects on initial rates

Northrop, Dexter B.

doi:10.1016/s0076-6879(82)87032-8

Cited by 120 publications

(121 citation statements)

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“…At low values of f (Ͻ5%), such as those observed in the present experiments, this expression reduces to (Northrop, 1982) T…”

Section: Marco Et Almentioning

confidence: 45%

“…Formation of tritiated water from [6␤-3 H]testosterone was NADPH-dependent and inhibited by a series of CYP3A/5 inhibitors with IC 50 values that were not significantly different from those obtained with [1,2,6,7ated and unlabeled substrate makes it impossible to measure their separate reaction velocities. When trace concentrations of a tritiated substrate are used to measure reaction velocities in the presence of competing unlabeled substrate, it is possible to measure the kinetic isotope effect on the V/K ratio (but not on the individual values of V max and K m ) (Northrop, 1982). By measuring formation of tritiated water and 6␤-hydroxytestosterone in the same reaction mixture, the specific activity of the tritiated water product can be calculated and compared with the initial specific radioactivity of the substrate, and these values can be used to derive T V/K.…”

Section: Discussionmentioning

confidence: 99%

“…T V/K, the kinetic isotope effect on the V/K ratio, was determined according to the formula (Northrop, 1982) …”

Section: Marco Et Almentioning

confidence: 99%

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Development and Validation of a High-Throughput Radiometric Cyp3a4/5 Inhibition Assay Using Tritiated Testosterone

Marco¹,

Marcucci²,

Verdirame³

et al. 2004

Drug Metab Dispos

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ABSTRACT:A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 3A4/5 in human liver microsomes is described. In contrast to the conventional testosterone 6␤-hydroxylation assay, the new method does not require high-performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP3A4/5-mediated 6␤-hydroxylation of testosterone labeled with tritium in the 6␤ position. The radiolabeled product is separated from the substrate using 96-well solid-phase extraction plates. The pharmacokinetic and toxicokinetic properties of pharmaceuticals depend in great part on their biotransformation by drug-metabolizing enzymes. The main drug-metabolizing system in mammals is cytochrome P450 (P450), a family of microsomal enzymes present predominantly in the liver. Multiple P450 enzymes catalyze the oxidation of chemicals of endogenous and exogenous origin, including drugs, steroids, prostanoids, eicosanoids, fatty acids, and environmental toxins (Ioannides, 1996). When a drug that is metabolized by a particular P450 enzyme is coadministered with an inhibitor of that same enzyme, changes in its pharmacokinetics can occur, which can give rise to adverse effects (Bertz and Granneman, 1997;Lin and Lu, 1998;Thummel and Wilkinson, 1998). It is therefore important to predict and prevent the occurrence of clearance changes due to metabolic inhibition. During the drug discovery process, it is routine practice in the pharmaceutical industry to assess the P450 inhibition potential of drug candidates to exclude potent inhibitors from further development (Lin and Lu, 1998;Crespi and Stresser, 2000;Riley, 2001). CYP3A4/5 is the most abundant P450 in human liver and is involved in the metabolism of about 50% of drugs used in human therapy (Guengerich, 1999). Inhibition of CYP3A4/5 activity can give rise to clinically significant and potentially life-threatening drug interactions (Thummel and Wilkinson, 1998). Several assay methods are currently used for determining the potential of drug candidates to inhibit CYP3A4/5 activity, and each of these methods presents distinct advantages and disadvantages. The most widely used method is the testosterone 6␤-hydroxylation assay, which is specific for enzymes of the CYP3A family (CYP3A4/5) (Waxman et al., 1988;Maenpaa et al., 1993;Wang et al., 1997;Yamazaki and Shimada, 1997). According to recent surveys conducted by reviewers in the Center for Drug Evaluation and Research of the United States Food and Drug Administration, the testosterone 6␤-hydroxylation assay represents the most commonly used probe reaction in support of new drug applications (Yuan et al., 1999(Yuan et al., , 2002. The practical challenge posed by this assay is that it requires HPLC separation of the reaction product from the substrate, followed by UV or mass spectrometric detection. This renders the assay relatively laborious, time-consumThis work was supported in part by a grant from t...

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“…At low values of f (Ͻ5%), such as those observed in the present experiments, this expression reduces to (Northrop, 1982) T…”

Section: Marco Et Almentioning

confidence: 45%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Development and Validation of a High-Throughput Radiometric Cyp3a4/5 Inhibition Assay Using Tritiated Testosterone

Marco¹,

Marcucci²,

Verdirame³

et al. 2004

Drug Metab Dispos

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“…where T (V/K) is the kinetic isotope effect on the V max /K m ratio (Northrop, 1982). A NIH shift of the radiolabel during hydroxylation, with retention of tritium in 4Ј-hydroxymephenytoin, would further increase the SA 0 /SA P ratio.…”

Section: Marco Et Almentioning

confidence: 99%

High-Throughput Radiometric CYP2C19 Inhibition Assay Using Tritiated (S)-Mephenytoin

Marco

Cellucci²,

Chaudhary³

et al. 2007

Drug Metab Dispos

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ABSTRACT:A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C19 in human liver microsomes is described. The new assay, which does not require high-performance liquid chromatography (HPLC) separation or mass spectrometric detection, is based on the release of tritium as tritiated water that occurs upon CYP2C19-mediated 4-hydroxylation of (S)-mephenytoin labeled with tritium in the 4 position. Because this reaction is subject to an NIH shift, tritium was also introduced into the 3-and 5-positions of the tracer to enhance formation of a tritiated water product. Tritiated water was separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent and sensitive to CYP2C19 inhibitors. IC 50 values for 15 diverse drugs differed less than 2.5-fold from those determined by quantification of the unlabeled 4-hydroxy-(S)-mephenytoin product, using HPLC coupled to mass spectrometric detection. All of the steps of the new assay, namely incubation, product separation, and radioactivity counting, are performed in a 96-well format and can be automated. This assay represents a non-HPLC, high-throughput version of the classic (S)-mephenytoin 4-hydroxylation assay, which is the most widely used method to assess the potential for CYP2C19 inhibition of new chemical entities.

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“…9), with its estimated rate constants, yields values that may reflect the balance in a qualitatively similar way (as in Fig. 7) but be more appropriate, in that k cat values can be misleading due to the contribution of multiple steps (67,68). The ratio of the estimated rate of conversion of 17␣-OH progesterone (to androstenedione) to its k off rate was ϳ0.1, whereas the value for conversion of 17␣-OH pregnenolone to its k off rate was ϳ2.…”

Section: Discussionmentioning

confidence: 92%

Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2

Pallan

Nagy

Lei

et al. 2015

Journal of Biological Chemistry

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Background: Fish (and human) P450 17A1 catalyze both steroid 17␣-hydroxylation and 17␣,20-lyase reactions. A second fish P450, 17A2 (51% identical), catalyzes only 17␣-hydroxylation. Results: Crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1 are very similar. Conclusion: In kinetic analysis, the two-step oxidation of progesterone is more distributive than for pregnenolone. Significance: Small structural differences are associated with activities of the two fish P450s.

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[30] Deuterium and tritium kinetic isotope effects on initial rates

Cited by 120 publications

References 16 publications

Development and Validation of a High-Throughput Radiometric Cyp3a4/5 Inhibition Assay Using Tritiated Testosterone

Development and Validation of a High-Throughput Radiometric Cyp3a4/5 Inhibition Assay Using Tritiated Testosterone

High-Throughput Radiometric CYP2C19 Inhibition Assay Using Tritiated (S)-Mephenytoin

Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2

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