ABSTRACT:Thirty-one structurally diverse marketed central nervous system (CNS)-active drugs, one active metabolite, and seven non-CNSactive compounds were tested in three P-glycoprotein (P-gp) in vitro assays: transwell assays using MDCK, human MDR1-MDCK, and mouse Mdr1a-MDCK cells, ATPase, and calcein AM inhibition. Additionally, the permeability for these compounds was measured in two in vitro models: parallel artificial membrane permeation assay and apical-to-basolateral apparent permeability in MDCK. The exposure of the same set of compounds in brain and plasma was measured in P-gp knockout (KO) and wild-type (WT) mice after subcutaneous administration. One drug and its metabolite, risperidone and 9-hydroxyrisperidone, of the 32 CNS compounds, and 6 of the 7 non-CNS drugs were determined to have positive efflux using ratio of ratios in MDR1-MDCK versus MDCK transwell assays. Data from transwell studies correlated well with the brainto-plasma area under the curve ratios between P-gp KO and WT mice for the 32 CNS compounds. In addition, 3300 Pfizer compounds were tested in MDR1-MDCK and Mdr1a-MDCK transwell assays, with a good correlation (R 2 ‫؍‬ 0.92) between the efflux ratios in human MDR1-MDCK and mouse Mdr1a-MDCK cells. Permeability data showed that the majority of the 32 CNS compounds have moderate to high passive permeability. This work has demonstrated that in vitro transporter assays help in understanding the role of P-gp-mediated efflux activity in determining the disposition of CNS drugs in vivo, and the transwell assay is a valuable in vitro assay to evaluate human P-gp interaction with compounds for assessing brain penetration of new chemical entities to treat CNS disorders.Human P-glycoprotein (P-gp, MDR1) is known to be a determinant of drug absorption, distribution, and excretion of a number of clinically important drugs (Ambudkar et al., 1999;Fromm, 2000). P-gp is widely expressed in major organs, and, more specifically, P-gp is highly expressed in the capillaries of the blood brain barrier (BBB) and poses a barrier to brain penetration of its substrates (Schinkel, 1999). Given that P-gp efflux liability can be a major hurdle for CNS therapeutic drugs to cross the BBB and reach the target, the interactions of CNS compounds with P-gp may lead to the lack of CNS activity as a result of the decreased brain penetration. Thus, the prediction and understanding of the relevance of P-gp-mediated efflux transport have become important activities in the discovery and development of CNS drugs. In attempts to predict the effects of P-gp in vivo, a variety of in vitro P-gp assays have been developed to classify compounds as P-gp substrates. For instance, transwell-based assays using polarized cell lines such as the Madin-Darby canine kidney (MDCK) cell line. The MDCK cell line can be stably transfected with human MDR1 or mouse Mdr1a (MDR1-MDCK or Mdr1a-MDCK, respectively). Comparison of the efflux ratios between MDR1-MDCK and MDCK transwell assays can provide a measure of the specific human P-gp-mediated e...

The usefulness of MALDI for small-molecule work has been limited by matrix chemical interference in the mass range of interest, tedious sample preparation, and various crystallization and sample deposition issues. We report instrument characterization and small-molecule quantification performance data from a high repetition rate laser MALDI ion source coupled to a triple quadrupole mass spectrometer. The high repetition rate laser improves sensitivity and precision and allows a proportional increase in sample throughput. Tandem mass spectrometry is used to discriminate the signal from the high chemical background caused by the MALDI matrix. Successful quantification requires use of an internal standard and a means of sample cleanup for typical in vitro sample compositions. This instrument combination and analysis technique is relatively insensitive to sample crystal quality and spot homogeneity. Quantitative performance results are characterized for 53 small-molecule pharmaceutical compounds and compared to those obtained by ESI-MS/MS. Further comparison between MALDI and ESI is examined, and the potential for high-throughput MALDI-MS/MS quantification is demonstrated.

Understanding the mechanisms and energetics of ion solvation is critical in many scientific areas. Here, we present a methodlogy for studying ion solvation using differential mobility spectrometry (DMS) coupled to mass spectrometry. While in the DMS cell, ions experience electric fields established by a high frequency asymmetric waveform in the presence of a desired pressure of water vapor. By observing how a specific ion's behavior changes between the high- and low-field parts of the waveform, we gain knowledge about the aqueous microsolvation of that ion. In this study, we applied DMS to investigate the aqueous microsolvation of protonated quinoline-based drug candidates. Owing to their low binding energies with water, the clustering propensity of 8-substituted quinolinium ions was less than that of the 6- or 7-substituted analogues. We attribute these differences to the steric hinderance presented by subtituents in the 8-position. In addition, these experimental DMS results were complemented by extensive computational studies that determined cluster structures and relative thermodynamic stabilities.