Linking solubility and permeability assays for maximum throughput and reproducibility.

Solubility and permeability are intimately linked in drug absorption processes. They have, however, been traditionally assayed separately. To support this linkage, a combined solubility/permeability assay was developed for determining absorption properties of chemical entities. First, solubility is determined at 4 pH values by comparing the concentration of a saturated compound solution to its dilute, known concentration. The filtered, saturated solution from the solubility assay is then used as input material for the membrane permeability determination. The permeability assay is a parallel artificial membrane technique whereby a membrane is created on a solid support parallel artificial membrane permeation assay (PAMPA). The 2 artificial membranes presented here model the gastrointestinal tract and the blood-brain barrier (BBB). Data are presented for control compounds, which are well documented in the literature and exemplify a range of solubility and membrane permeability. The advantages of the combination method are 1) reduction of sample usage and preparation time, 2) elimination of interference from compound precipitation in membrane permeability determination, 3) maximization of input concentration to permeability assay for improved reproducibility, and 4) optimization of sample tracking by streamlining data entry and calculations. BBB permeability ranking of compounds correlates well with literature CNS activity.

A generic high-throughput screening assay for kinases: protein kinase a as an example.

A generic high-throughput screening assay based on the scintillation proximity assay technology has been developed for protein kinases. In this assay, the biotinylated (33)P-peptide product is captured onto polylysine Ysi bead via avidin. The scintillation signal measuring the product formation increases linearly with avidin concentration due to effective capture of the product on the bead surface via strong coulombic interactions. This novel assay has been optimized and validated in 384-well microplates. In a pilot screen, a signal-to-noise ratio of 5- to 9-fold and a Z' factor ranging from 0.6 to 0.8 were observed, demonstrating the suitability of this assay for high-throughput screening of random chemical libraries for kinase inhibitors.