Activity levels of tamoxifen metabolites at the estrogen receptor and the impact of genetic polymorphisms of phase I and II enzymes on their concentration levels in plasma.

The therapeutic effect of tamoxifen depends on active metabolites, e.g., cytochrome P450 2D6 (CYP2D6) mediated formation of endoxifen. To test for additional relationships, 236 breast cancer patients were genotyped for CYP2D6, CYP2C9, CYP2B6, CYP2C19, CYP3A5, UGT1A4, UGT2B7, and UGT2B15; also, plasma concentrations of tamoxifen and 22 of its metabolites, including the (E)-, (Z)-, 3-, and 4'-hydroxymetabolites as well as their glucuronides, were quantified using liquid chromatography-tandem mass spectrometry (MS). The activity levels of the metabolites were measured using an estrogen response element reporter assay; the strongest estrogen receptor inhibition was found for (Z)-endoxifen and (Z)-4-hydroxytamoxifen (inhibitory concentration 50 (IC50) 3 and 7 nmol/l, respectively). CYP2D6 genotypes explained 39 and 9% of the variability of steady-state concentrations of (Z)-endoxifen and (Z)-4-hydroxytamoxifen, respectively. Among the poor metabolizers, 93% had (Z)-endoxifen levels below IC90 values, underscoring the role of CYP2D6 deficiency in compromised tamoxifen bioactivation. For other enzymes tested, carriers of reduced-function CYP2C9 (*2, *3) alleles had lower plasma concentrations of active metabolites (P < 0.004), pointing to the role of additional pathways.

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry.

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C18 cartridges. Thereafter compounds were separated on a short narrow bore RP C18 column (30 x 2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C18 column (70 x 2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380-->316 and m/z 366-->302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25-250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 microl) was validated over a concentration range of 0.5-20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.

Determination of propafenone and its phase I and phase II metabolites in plasma and urine by high-performance liquid chromatography-electrospray ionization mass spectrometry.

A sensitive method was developed to determine propafenone, 5-hydroxypropafenone, N-despropylpropafenone and propafenone glucuronides in human plasma and urine by HPLC-electrospray ionization mass spectrometry with the respective deuterated analogues as internal standards. The analytes were extracted by a single solid-phase extraction, collecting two fractions, one containing the glucuronides and the other propafenone and the phase I metabolites 5-hydroxypropafenone and N-despropylpropafenone. The mobile phases used for HPLC were: (A) 5 mM ammonium acetate in water and (B) 5 mM ammonium acetate in methanol-tetrahydrofuran (50:50, v/v). Separation of the diastereoisomeric propafenone glucuronides was achieved on a Spherisorb ODS 2 column (150 x 2.0 mm I.D., particle size 5 microm) at a flow-rate of 0.3 ml/min using a linear gradient from 20% B to 50% B in 15 min. For separation of propafenone, 5-hydroxypropafenone and N-desalkylpropafenone a linear gradient from 50% B to 80% B in 10 min was employed. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH+ ions for quantification. The limits of quantification achieved with this method were 10 pmol/ml for propafenone, 5-hydroxypropafenone, R- and S-propafenone glucuronide and 20 pmol/ml for N-desalkylpropafenone using 0.5 ml of plasma. Reproducibility and accuracy was below 12% for each analyte over the whole concentration range measured. The method was applied to a pharmacokinetic study assessing the influence of rifampicin on propafenone disposition.

Influence of renal function on the steady-state pharmacokinetics of the antiarrhythmic propafenone and its phase I and phase II metabolites.

The aim of this study was to investigate the disposition of propafenone and its Phase I and II metabolites in relation to kidney function under steady-state conditions. The mechanism of the renal handling of propafenone glucuronides (filtration, secretion) was also examined. Racemic (R/S) propafenone was administered to 7 young volunteers, to 5 older patients with a normal glomerular filtration rate and to 4 patients with chronic renal failure. No difference was found in the plasma concentrations of propafenone and 5-hydroxypropafenone between the three groups. The propafenone glucuronide (PPFG) concentration was elevated in the older compared to the younger subjects (S-PPFG: 544 vs. 222 nmol.ml-1.mol-1; R-PPFG: 576 vs. 304 nmol.ml-1.mol-1). Although Glomerular filtration rate did not differ, the renal clearance of propafenone glucuronides was reduced in the former group, which could be attributed to their impaired renal secretion. A dramatic increase in propafenone glucuronide concentration was observed in the patients with renal failure (S-PPFG: 2783 nmol.ml-1.mol-1; R-PPFG: 7340 nmol.ml-1.mol-1). In summary, the disposition of propafenone and of its active metabolite 5-hydroxypropafenone was not affected by kidney dysfunction, indicating that no dose adjustment is necessary in patients with renal failure. The accumulation of drug glucuronides in older patients with apparently normal kidney function should be taken into account as a possible factor modifying drug therapy.

Rapid determination of CYP2D6 phenotype during propafenone therapy by analysing urinary excretion of propafenone glucuronides.

Metabolism of the antiarrhythmic, propafenone, cosegregates with the sparteine/debrisoquine polymorphism. Patients devoid of CYP2D6 activity have a higher incidence of adverse effects than those with normal enzyme function. In this paper we present a method for rapid assignment of CYP2D6 phenotype using urinary excretion of intact glucuronides of propafenone (PPFG). After establishing an HPLC assay, urinary excretion of PPFG was quantified during one dosage interval and related to individual CYP2D6 activity as determined by phenotyping. We observed a close correlation of urinary excretion of PPFG with individual CYP2D6 activity (r = 0.84, P < 0.01) and conclude that this method is suitable for rapid assignment of phenotype during propafenone therapy.