Simultaneous Pharmacokinetic Screening of a Mixture of Compounds in the Dog Using API LC/MS/MS Analysis for Increased Throughput

Abstract

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...

Molecular and Pharmacokinetic Properties of 222 Commercially Available Oral Drugs in Humans.

“…10,12) However, problems with Caco-2 cells have been reported with regard to their lower paracellular permeability than normal cells, 16,17) essential characteristics of tumor cells 18) and lower metabolic capacity, 19) and further refinements of this model are still needed for accurate prediction. 4,10) Cassette dosing with LC/MS/MS has been proposed to assess overall absorption in vivo in high throughput mode, [13][14][15] but this method has the major drawback of drugdrug interaction. Cassette dosing has also been applied for the Caco-2 system, 21) but whether this will be beneficial is still unclear.…”

“…[9][10][11][12] The development of the LC/MS/MS assay system has enabled us to evaluate overall bioavailability in vivo with clinical dose, and cassette dosing, combining many test compounds into one dose, has been proposed. [13][14][15] This is also promising, but the predictive capability of these methods has sometimes been questioned from the viewpoints of in vitro-in vivo correlations [16][17][18][19] and drug-drug interactions, 13) respectively. More recently, technological advances in HTS including smaller sample volume and higher density of the wells have facilitated rapid throughput of drug testing, discovery pipeline, and the assessment of oral absorption has again become the rate-limiting step, even when the Caco-2 assay system or cassette dosing is appropriately applied.…”

The application of cassette dosing for pharmacokinetic screening in small-molecule cancer drug discovery

“…37). This approach involves the simultaneous administration of a mixture of several compounds (typically belonging to the same structural class) at relatively low doses to a single animal (38,39).…”

“…Despite these potential issues, cassette dosing was widely adopted by the pharmaceutical industry following the first publication on this technique in 1997 (37). The results of two surveys that were conducted to assess the popularity of cassette dosing were published several years later in 2004 (43,44).…”

Results from a bench marking survey on cassette dosing practices in the pharmaceutical industry

“…This practice, made possible by the extraordinary specificity of tandem MS detection, is seen as a way to maximize the utilization of expensive LC/MS/MS instrumentation and to minimize the number of animals used. Since the initial reporting of N-in-one dosing by Berman and co-workers in 1997 [1], several reports have appeared in the literature [2][3][4][5]. Interested readers are also referred to a seminal article by White and Manitpisitkul on the pharmacokinetic implications of CD [6].…”