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Biomedical subjects

W F Trager

Publications and source records attributed to W F Trager.

At least 73 records · Page 4Linked to original sources
PubMed1986

Prochiral selectivity and intramolecular isotope effects in the cytochrome P-450 catalyzed omega-hydroxylation of cumene.

A kinetic model is presented from which steady-state equations are derived that describe the intramolecular competition for the enzymatically mediated hydroxylation of two like groupings of a prochiral substrate. The observed isotope effect in such a system if one of the groupings is isotopically labeled is shown to be a function of three parameters: the equilibrium constant for the catalytically sensitive orientations of the two prochiral groupings at the active site, the intrinsic isotope effect associated with the bond-breaking step, and the relative rates of bond breaking vs. enzyme-substrate dissociation. The expected isotope effects associated with the omega-hydroxylation of racemic, (R)-, and (S)-2-phenylpropane-1,1,1-d3 and the product stereoselectivity associated with the omega-hydroxylation of (R)- and (S)-[1-13C]-2-phenylpropane were determined with microsomal preparations (cytochrome P-450) from untreated and phenobarbital- and beta-naphthoflavone-pretreated male Sprague-Dawley rats. The data from these experiments allow the observed isotope effect to be evaluated in terms of its component parts, i.e., expected isotope effects, product stereoselectivity, and equilibrium constant. These data further suggest that the intramolecular isotope effect is consistent with a hydrogen abstraction recombination mechanism and is largely dependent upon the chemical nature of the porphyrin-Fe-oxene complex but independent of specific apoprotein structure, product stereoselectivity is primarily dependent upon apoprotein structure, and product stereoselectivity is a good measure of the equilibrium constant and both parameters are dependent upon the chirality of the active site.

Animals↗
PubMed1986

The warfarin-sulfinpyrazone interaction: stereochemical considerations.

To allow the simultaneous evaluation of the interaction between sulfinpyrazone and each enantiomer of racemic warfarin, pseudoracemic warfarin (1:1 12C-R(+) and 13C-S(-)warfarin) was given to six normal subjects both before and during oral sulfinpyrazone dosing. Serial blood and urine samples were analyzed for unchanged warfarin and its metabolic products by GC/MS. A mass balance of an oral dose of pseudoracemic warfarin, containing a tracer quantity of 14C-warfarin, was carried out in one of the subjects by monitoring 14C levels in urine and feces for 15 days. Concomitant sulfinpyrazone dosing markedly increased hypoprothrombinemia, decreased clearance of (S)-warfarin, and increased clearance of (R)-warfarin. Sulfinpyrazone also decreased the urinary excretion of warfarin-related products but increased their fecal excretion by an equivalent amount. Virtually all of the administered warfarin dose could be accounted for either as unchanged drug or known metabolites. Pharmacokinetic analysis of the data suggests the following: At least four distinct enzymes (two oxidases and two reductases) are involved in the metabolism of warfarin. Sulfinpyrazone increases the hypoprothrombinemia caused by warfarin primarily by inhibition of the cytochrome P-450-mediated oxidation of (S)-warfarin, the biologically more potent enantiomer. The increased clearance of (R)-warfarin results not from induction, but from its selective displacement from plasma protein binding sites.

Administration, Oral↗
PubMed1985

Stereoselective and regioselective hydroxylation of warfarin and selective O-dealkylation of phenoxazone ethers in human placenta.

The oxidative metabolism of warfarin and a series of phenoxazone ethers was studied in two groups of human placentas which exhibited high or low levels of aryl hydrocarbon hydroxylase (AHH). Warfarin metabolism was stereoselective (mean R/S = 2.48) for the R-enantiomer and regioselective for the 6- and 8- positions in the high AHH group whereas warfarin metabolism in the low AHH group displayed no significant overall stereoselectivity (mean R/S = 1.24) and was regioselective for the 7- position. The high AHH group metabolized the methyl, ethyl, propyl and butyl ethers of phenoxazone rapidly, while the low AHH group catalyzed their biotransformation at very low or negligible rates. Neither group detectably metabolized phenoxazone or pentyloxyphenoxazone whereas both groups metabolized benzyloxyphenoxazone at low but similar rates. Rates of warfarin R-6 and R-8 hydroxylation were highly correlated with metabolism of benzo(alpha)pyrene (r = 0.99) and the C1-C4 phenoxazone ethers (r greater than 0.87), but poorly correlated with metabolism of benzyloxyphenoxazone (r less than 0.50). These data support the use of warfarin and the phenoxazone ethers as sensitive biochemical probes for P-450 isozymes in human extrahepatic tissues. They indicate the presence of a multiplicity of xenobiotic metabolizing P-450's in placental tissue which has not been exposed to inducing agents that elevate AHH.

Aryl Hydrocarbon Hydroxylases↗
PubMed1985

A stable isotope assay for phenprocoumon and its metabolites.

A gas chromatographic/mass spectrometric assay for quantifying phenprocoumon and its 4'-, 6-, 7- and 8-hydroxy metabolites in microsomal preparations is described. This assay which uses deuterium-labeled analogs of the phenprocoumon metabolites as internal standards has a lower limit of quantitation of 20 ng ml-1. Diazomethane is used to derivatize both metabolites and parent compound yielding along with the expected 4-methoxy derivative a minor amount of the 2-methoxychromone. Resolution of the methylated metabolites is accomplished by capillary gas chromatography.

4-Hydroxycoumarins↗
PubMed1985

Metabolic fate of phenprocoumon in humans.

Samples of urine and feces were collected daily from a normal human volunteer who had received a dose of pseudoracemic phenprocoumon [an equimolar mixture of (R)-[12C]- and (S)-[2-13C]phenprocoumon] containing a tracer dose of 10 microCi of [14C]phenprocoumon and analyzed by TLC, HPLC, and GC-MS. After 25 days, 96% of the radiolabeled material was recovered (62.8% in urine and 33.3% in feces). By isotopic dilution and comparison to the Rf values, retention times, and mass fragmentograms of synthetic standards, the metabolites of the drug were identified as the 4'-, 6-, and 7-hydroxy analogues of phenprocoumon. Virtually all of the recovered radioactivity could be accounted for by the parent drug (approximately 40%) and the three metabolites (approximately 60%). The formation of both 4'-(8.1% of administered dose) and 7- (33.4% of administered dose) hydroxyphenprocoumon was highly stereoselective, giving S/R ratios of 2.86 and 1.69, respectively. The formation of 6- (15.5% of administered dose) hydroxyphenprocoumon showed little stereoselectivity (S/R ratio equal to 0.85). The urinary excretion pattern was also confirmed in four additional healthy male subjects who received a single oral dose of pseudoracemic phenprocoumon and whose urine was analyzed by GC-MS. All the drug-related materials (both hydroxylated metabolites and parent compound) that were excreted into the urine were extensively conjugated.

4-Hydroxycoumarins↗
PubMed1985

Substrate probes for the mechanism of aromatic hydroxylation catalyzed by cytochrome P-450: selectively deuterated analogues of warfarin.

Optically pure analogues of (R)- and (S)-warfarin selectively deuterated in either the 6-, 7-, or 8-position were prepared and incubated with microsomal preparations from either nontreated, phenobarbital-pretreated, or beta-naphthoflavone-pretreated male Sprague-Dawley rats. The amount of deuterium retained and the relative amount of hydroxylated product formed (6-, 7-, 8-, or 4'-hydroxywarfarin) from each of the six substrates for each of the treatments were determined by capillary gas chromatography-mass spectrometry. The degree of deuterium retention in all products from all substrates was largely independent of both absolute configuration and induction state. Conversely, the relative amounts of product formed were highly dependent upon both absolute configuration and induction state. These results suggest that all the hydroxylation reactions proceed through an addition rearrangement step prior to or in the absence of epoxide formation, which appears to be dictated by the nature of the heme-Fe3+-oxene complex. In contrast, the position of hydroxylation or regioselectivity appears to be primarily dependent upon the nature of the apoprotein.

Animals↗
PubMed1985

Stereoselective metabolism of conformational analogues of warfarin by beta-naphthoflavone-inducible cytochrome P-450.

Previous studies have shown that the structurally related oral anticoagulants warfarin and phenprocoumon are regioselectively hydroxylated in the 6- and 8-positions by hepatic microsomes obtained from 3-methylcholanthrene (3-MC) or beta-naphthoflavone (BNF) pretreated rats. Stereoselectivity for hydroxylation is also observed and favors (R)-warfarin but (S)-phenprocoumon. The possibility that the stereoselectivity of warfarin hydroxylation is a function of the solution conformation of the drug was tested with conformationally restricted analogues. In these experiments the analogues were incubated with microsomes obtained from BNF-pretreated rats and any stereoselectivity associated with 6- and 8-hydroxylation was determined. The R enantiomer of cyclocoumarol, the cyclic ketal analogue of warfarin, was found to be selectively hydroxylated, in contrast to the S enantiomer of warfarin 4-methyl ether, the ring-opened analogue. The latter compound is known to have a preferred solution conformation similar to that of phenprocoumon. The results suggest that at the active site of BNF-induced cytochrome P-450 (R)-warfarin is metabolized in its cyclic hemiketal tautomer, a form which spatially mimics the preferred solution conformation of (S)-phenprocoumon.

Animals↗
PubMed1984

Intramolecular isotope effects associated with meta-hydroxylation of biphenyl catalyzed by cytochrome P-450.

Intramolecular isotope effects and the degree of deuterium retention were determined for the meta-hydroxylation of biphenyl as catalyzed by microsomal cytochrome P-450 obtained from rats pretreated with phenobarbital. The percent deuterium retention after meta-hydroxylation of 3,5,3',5'-[2H4]-biphenyl was found to be 77.3% +/- 1.9. The intramolecular isotope effects associated with 3,3'-[2H2]-biphenyl and 3,5-[2H2]-biphenyl were found to be 0.90 +/- .05 and 1.05 +/- .06, respectively. These data demonstrate conclusively that a direct insertion or abstraction mechanism is not operable in the meta-hydroxylation of biphenyl and suggest the possibility of an addition-rearrangement mechanism as opposed to initial and direct arene oxide formation.

Animals↗
PubMed1984

The preferred solution conformation of warfarin at the active site of cytochrome P-450 based on the CD spectra in octanol/water model system.

An octanol/water model system and circular dichroism (CD) spectroscopy have been used to study the solution conformation of warfarin in aqueous and lipid environments. Upon partitioning of (S)-warfarin from buffer pH 7.4 into octanol, the position of the absorption band due to the alpha, beta-unsaturated carbonyl chromophore shifts from 210 nm in the aqueous phase to 220 nm in the octanol phase. The shift is coupled to an increase in the molecular ellipticity of the band, suggesting the formation of a dissymmetric chromophore. Comparison of CD spectra of conformationally fixed analogues of warfarin to that of warfarin in solution suggests that the compound shifts from the open side chain keto form in the aqueous phase at pH 7.4 to the cyclic hemiketal form after partitioning into the lipid octanol phase. On the basis of these results, the hemiketal form is proposed as the preferred solution conformation of warfarin in the lipid environment of the active site of cytochrome P-450 and the relationship between solution conformation and stereoselectivity of warfarin metabolism by beta-naphthoflavone inducible cytochrome P-450 is discussed.

4-Hydroxycoumarins↗
PubMed1983

A sensitive and specific stable isotope assay for warfarin and its metabolites.

A capillary gas chromatographic mass spectrometric method for the quantification of warfarin and its known metabolites from microsomal incubations is described. Deuterium labelled 4', 6-, 7- and 8-hydroxy warfarins are used as internal standards and the method has detection limits of 1 ng ml-1 with 20 ng ml-1 being the lower limit for accurate quantification.

Animals↗
PubMed1980

A stable isotope assay for pseudoracemic warfarin from human plasma samples.

A pseudoracemic technique utilizing a seable isotope in one enantiomer was employed for the simultaneous determination of (R) and (S)-warfarin from plasma of human subjects. The assay includes high performance liquid chromatographic clean-up prior to mass spectral analysis to eliminate ion interferences from either co-administered drugs or contamination of the source. The assay is reliable, accurate and precise to within 5% at the submicrogram level.

Carbon Isotopes↗