Drug discovery increasingly relies on the ability to rapidly identify "quality" molecules that possess the desired attributes of bioavailability, chemical tractability, selectivity, and potency. Traditional methods used to determine the pharmacokinetics (oral bioavailability, clearance, volume of distribution, and half-life) of molecules have presented a bottleneck in some drug discovery programs. We have increased throughput in in vivo pharmacokinetic screening of structurally related compounds by dosing mixtures of compounds intravenously to a single animal and using atmospheric pressure ionization (API) tandem liquid chromatography/ mass spectrometry (LC/MS/MS) for analysis. We have referred to this as N-in-one dosing where N is the number of compounds coadministered. The method was used to simultaneously determine the clearance (CL), steady-state volume of distribution (V ss ), and elimination half-life (t 1/2 ) of five R 1a receptor antagonists (compounds possessing selective R 1a antagonist properties may have potential therapeutic importance in the treatment of benign prostatic hyperplasia). 1-3 The mixture approach provides an opportunity to study the pharmacokinetics of several compounds under identical conditions while minimizing sample processing time and the number of animals required. To the best of our abilities, we found limited literature that capitialized on the advantages gained through simultaneously dosing compounds to determine pharmacokinetics and bioavailability after intravenous and intraduodenal administration, 4,5 and one of these utilized LC/MS. 6 Five R 1a receptor antagonists ( Figure 1) that had been previously studied by individual dosing (known range of CL, V ss , and t 1/2 ) were dosed intravenously as a mixture to a single dog. In the traditional individual dosing studies, plasma samples were analyzed for R 1a antagonist by reverse-phase HPLC with fluorescence detection. Plasma sample analysis of the R 1a antagonists in the mixture study relied heavily on the application of atmospheric pressure ionization (API) LC/MS methodology using a triple quadrupole instrument. LC/ MS with its inherent detection specificity, selectivity, and sensitivity enabled rapid analytical method development prior to dosing the animal as well as high throughput sample analysis.The compounds studied exhibited good mass spectrometric response in the positive ion mode using the API technique. To increase analyte specificity from the biological matrix, a reverse-phase LC/MS/MS method was developed. For this series of compounds, a characteristic neutral loss of CF 3 CH 2 OH (100 amu) was observed in the product ion mass spectra. This transition was optimized for sensitivity in the selected reaction monitoring (SRM) mode. Among the compounds studied, a pair of isobars (compounds 2 and 3 of molecular weight 638) produced the same abundant fragment ion at m/z 539. Consequently, the HPLC mobile phase conditions were optimized to resolve compounds 2 and 3, extending the analysis time to 6 min. An internal standar...
The purpose of this work was (1) to determine if useful in vivo pharmacokinetic data could be obtained after simultaneous administration of 5-22 compounds of a chemically congeneric series to dogs and (2) to determine if structure-pharmacokinetic relationships could be derived from such studies. Mixtures of structurally related alpha-1 antagonist compounds (5-22) were administered intravenously to conscious dogs. Blood samples were taken over the next 24 h and analyzed by LC/MS to determine plasma levels and pharmacokinetics of each compound. The pharmacokinetics of 17 of these compounds were also determined after individual administration. Results obtained in the N-in-One format for 17 compounds correlated well with results obtained when these same compounds were administered individually. The N-in-One method is a useful method for obtaining pharmacokinetic data on 5-20 molecules in a single animal at one time. The increased throughput in obtaining important pharmacokinetic information should enhance the drug discovery process. In addition, it was possible to determine the extent to which various chemical substitutions did or did not affect pharmacokinetic parameters.
An HPLC method for the determination of S-phenyl-N-acetylcysteine in urine is described. The sensitivity is 6 mumol/L (CV = 9%) urine. Exposure of rats to six different concentrations of benzene, ranging from 0-30 ppm, was highly associated with urinary excretion of S-phenyl-N-acetylcysteine (r = 0.86) and with total phenol (r = 0.81). A background level of phenol was found in urine of both non-exposed rats and of non-exposed referents. However, no background excretion of S-phenyl-N-acetylcysteine was found, either in rats or in humans. In urine of exposed rats, the level of S-phenyl-N-acetylcysteine was approximately five times lower than the phenol level. Workers occupationally exposed to benzene, showing high levels of urinary phenol, revealed low concentrations of urinary S-phenyl-N-acetylcysteine. The biological monitoring of industrial exposure to benzene by determination of S-phenyl-N-acetylcysteine in urine is not better than the determination of phenol in urine.
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