William F. Trager

Genetic association between sensitivity to warfarin and expression of CYP2C9*3.

Cytochrome P4502C9 (CYP2C9) is largely responsible for terminating the anticoagulant effect of racemic warfarin via hydroxylation of the pharmacologically more potent S-enantiomer to inactive metabolites. Mutations in the CYP2C9 gene result in the expression of three allelic variants, CYP2C9*1, CYP2C9*2 and CYP2C9*3. Both CYP2C9*2 and CYP2C9*3 exhibit altered catalytic properties in vitro relative to the wild-type enzyme. In the present study, a patient was genotyped who had proven unusually sensitive to warfarin therapy and could tolerate no more than 0.5 mg of the racemic drug/day. PCR-amplification of exons 3 and 7 of the CYP2C9 gene, followed by restriction digest or sequence analysis, showed that this individual was homozygous for CYP2C9*3. In addition, patient plasma warfarin enantiomer ratios and urinary 7-hydroxywarfarin enantiomer ratios were determined by chiral-phase high performance liquid chromotography in order to investigate whether either parameter might be of diagnostic value in place of a genotypic test. Control patients receiving 4-8 mg warfarin/day exhibited plasma S:R ratios of 0.50 +/- 0.25:1, whereas the patient on very low-dose warfarin exhibited an S:R ratio of 3.9:1. In contrast, the urinary 7-hydroxywarfarin S:R ratio of 4:1 showed the same stereoselectivity as that reported for control patients. Therefore, expression of CYP2C9*3 is associated with diminished clearance of S-warfarin and a dangerously exacerbated therapeutic response to normal doses of the racemic drug. Analysis of the plasma S:R warfarin ratio may serve as a useful alternative test to genotyping for this genetic defect.

Evaluation of distribution-free confidence limits for enzyme kinetic parameters

Abstract Monte Carlo experiments have been used to test the robustness of distribution-free confidence limits for the parameters of the Michaelis-Menten equation (Porter & Trager, 1977). When used in conjunction with the modified form of the direct linear plot (Cornish-Bowden & Eisenthal, 1978), they prove to be more robust than least-squares confidence limits. In circumstances where the least-squares assumptions are correct, the distribution-free confidence limits define the parameters somewhat less precisely than the corresponding least-squares confidence limits, but this effect is negligible unless there are eight or fewer observations.

Configurational and conformational analysis : Axial-axial and axial-equatorial coupling constants in six-membered ring compounds

Abstract Axial-axial and axial-equatorial coupling constants (J aa and J ae ) have been obtained directly from the NMR spectra of simple AB systems in a number of cis - and trans -l-(substituted)-2-arylcyclohexanes-3,3,6,6-d 4 measured in several solvents. Variations in J aa and J ae have been observed with various substituents. No simple correlation is found between electronegativity of substituents and substituent effects on coupling constants. A review of J aa and J ae in six-membered ring compounds is given.

Fluvoxamine-theophylline interaction: Gap between in vitro and in vivo inhibition constants toward cytochrome P4501A2

Several reports indicate that fluvoxamine decreases the clearance of cytochrome P4501A2 (CYP1A2) substrates. This study compared in vitro and in vivo inhibition potencies of fluvoxamine toward CYP1A2 with an approach based on inhibition constants (Ki) determined in vitro and in vivo.

The lipophilicity of deuterium atoms. A comparison of shake-flask and HPLC methods

Abstract Isotopic effects are demonstrated in the lipophilicity, measured by shake-flask and HPLC methods, of a series of deuterated aromatic compounds. The results indicate that deuterated compounds are less lipophilic than their protium isomers by about −0.006 per deuterium atom on the log P oct scale. This isotopic effect is satisfactorily accounted for by differences in molar volumes of isotopomers. The partition coefficient of benzene and toluene is critically evaluated in view of the volatility of these compounds.

Carbon-13 nuclear magnetic resonance studies of coumarin and related compounds

Fourier-transform ^(13)C NMR spectra of nine coumarinoid compounds of medicinal interest are reported. All of the carbon resonances are assigned with the aid of various spectral techniques and stable isotopic labeling. The substituent effects on the chemical shifts in several systems are also discussed.

Changes in the metabolic profiles of R- and S-warfarin and R- and S-phenprocoumon as a probe to categorize the effect of inducing agents on microsomal hydroxylases.

Abstract The biotransformation of R- and S-warfarin was examined using liver microsomes prepared from both noninduced rats and rats pretreated with Arochlor 1254, β-napthoflavone (BNF), pregnenolone-16α-carbonitrile (PCN), phenobarbital (PB), and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). For comparison, the metabolism of a closely related coumarin anticoagulant, R- and S-phenprocoumon, was determined using the same microsomal preparations. In noninduced microsomes the overall metabolism of warfarin was about five times that of phenprocoumon, with 7-hydroxywarfarin as the principle metabolite followed by the 6-, 4′- and 8-hydroxy derivatives (benzylic hydroxylation was not examined). For phenprocoumon a different regioselectivity was observed with 4′ hydroxylation being the greatest followed by 6-, and 7 and 8 hydroxylation. In the case of warfarin, hydroxylation with noninduced microsomes was either nonstereoselective (4′ hydroxylation) or selective for the R-enantiomer. The metabolic pattern observed for phenprocoumon showed hydroxylation to be either nonstereoselective (7 and 8 hydroxylation) or, in contrast to warfarin, selective for the S-enantiomer. Induction of cytochrome P-450 by Arochlor, BNF, and TCDD produced a similar metabolic pattern for both substrates in which 6 and 8 hydroxylation were greatly increased over control levels. In keeping with the pattern obtained from noninduced microsomes, a reversed stereoselectivity was again observed after induction with these three agents, i.e. warfarin metabolism was selective for the R-enantiomer and phenprocoumon metabolism was selective for the S-enantiomer. Based on cytochrome P-450 levels PCN decreased the metabolism of both substrates while PB had no effect. However, the induction with PB was readily apparent when calculations were performed on a per mg protein basis.

Interaction of secobarbital with warfarin pseudoracemates

To evaluate the interaction of secobarbital with racemic warfarin or R,S(±)‐warfarin, S(−)‐warfarin was synthesized with 13C‐label in the 2‐position of the coumarin nucleus and added to 12C‐R(+)‐warfarin to form a 12C‐/13C‐warfarin pseudoracemate. Six normal subjects received 1.5 mg/kg of this “cold‐labeled” pseudoracemate. It was given with and without a daily oral dose of secobarbital, 100 mg, beginning 7 days before the warfarin and continuing throughout the hypoprothrombinemia. Plasma samples were obtained daily and analyzed for warfarin and for one‐stage prothrombin activity. Unchanged warfarin in plasma was fractionated by forward‐phase high‐pressure liquid chromatography, and enantiomorphic ratios were determined by chemical ionization–mass spectrometry with pentadeuterio‐warfarin as the internal standard. There was a reduction of the hypoprothrombinemia of the pseudoracemate during the secobarbital regimen over that on warfarin alone (p < 0.001). There was an increase in plasma clearance of R‐warfarin (p < 0.05) and an increase in plasma clearance of S‐warfarin (p < 0.003) during the secobarbital regimen over that on warfarin alone. It was concluded that secobarbital diminished the hypoprothrombinemia of pseudoracemic warfarin by increasing plasma clearance of the more hypoprothrombinemic S‐warfarin and by increasing plasma clearance of the less hypoprothrombinemic R‐warfarin.

Studies on the mechanism of aromatase and other cytochrome P450 mediated deformylation reactions

Aromatase is a microsomal cytochrome P450 that converts androgens to estrogens by three sequential oxidations. The isolation of the 19-hydroxy and 19-oxo androgens suggests that the first two oxidations occur at the C19 carbon. However, the mechanism of the third oxidation, which results in C10--C19 bond cleavage, has not been determined. Two proposed mechanisms which remain viable involve either initial 1 beta-hydrogen atom abstraction or addition of the ferric peroxy anion from aromatase to the C19 aldehyde. Semiempirical molecular orbital calculations (AM1) were used to study potential reaction mechanisms initiated by initial 1 beta-hydrogen atom abstraction. Initially, the energetics of carbon--carbon bond cleavage of the keto and enol forms of C1-radicals were studied and were found to be energetically similar. A mechanism was proposed in which the 19-oxo intermediate is subject to initial nucleophilic attack by the protein. The geometry of the A-ring in the androgens is between that for the 1-radicals and estrogen, suggesting that some transition state stabilization for the homolytic cleavage reaction can occur. More recently, studies on liver microsomal cytochrome P450 mediated deformylation of xenobiotic aldehydes supports mechanisms involving an alkyl peroxy intermediate formed by addition of the ferric peroxy anion from aromatase to the C19 aldehyde. Although this intermediate could proceed through several different concerted or non-concerted pathways, one non-concerted pathway involves the heterolytic cleavage of the dioxygen bond resulting in an active oxygenating species (iron-oxene) and a diol. The diol could then undergo hydrogen atom abstraction followed by homolytic carbon--carbon bond cleavage as in the mechanisms modeled previously. When this cleavage was modeled for seven aldehydes, a good correlation with reported experimental aldehyde turnover numbers was obtained. However, when dialkoxy derivatives of the aldehydes are subject to microsomal metabolism, the rates of carbon-carbon cleavage products do not approach the rates of deformylation of the aldehyde analog.

Stereochemistry of Cytochrome P-450 Reactions

AbstractGenerally, most enzymes have a high degree of substrate specificity. In contrast, one of the primary characteristics of the cytochrome P-450s is a lack of substrate specificity. Specific isozymes will not only oxidize a number of different substrates but will often transform a single substrate into a number of different products. Indeed, it appears that almost any organic molecule that can reach the active site of the enzyme will be oxidized. This high degree of chemical reactivity demands that there must be some structural feature that controls access to the active site in order to prevent excess turnover of endogenous substances critical to the normal functioning of the organism. The structural feature that serves this role is the lipoidal character of the enzyme complex which results from the fact that it is membrane bound. Thus, the known endogenous substrates for the cytochrome P-450s, the steroids, prostaglandins, and fat-soluble vitamins, are all highly lipophilic substances.