A polychromatic turbidity microplate assay to distinguish discovery stage drug molecules with beneficial precipitation properties

Abstract

A material sparing microplate screening assay was developed to evaluate and compare the precipitation of discovery stage drug molecules as a function of time, concentration and media composition. Polychromatic turbidity time course profiles were collected for cinnarizine, probucol, dipyridamole as well as BMS-932481, and compared with turbidity profiles of monodisperse particle size standards. Precipitation for select sample conditions were further characterized at several time points by size, morphology, amount and form via laser diffraction, microscopy, size based particle counting and X-ray diffraction respectively. Wavelength dependent turbidity was found indicative of nanoprecipitate, while wavelength independent turbidity was consistent with larger microprecipitate formation. A transition from wavelength dependent to wavelength independent turbidity occurred for nanoparticle to microparticle growth, and a decrease in wavelength independent turbidity correlated with continued growth in size of microparticles. Other sudden changes in turbidity signal over time such as rapid fluctuation, a decrease in slope or a sharp inversion were correlated with very large or aggregated macro-precipitates exceeding 100μm in diameter, a change in the rate of precipitate formation or an amorphous to crystalline form conversion respectively. The assay provides an effective method to efficiently monitor and screen the precipitation fates of drug molecules, even during the early stages of discovery with limited amounts of available material. This capability highlights molecules with beneficial precipitation properties that are able to generate and maintain solubility enabling amorphous or nanoparticle precipitates.

Keywords: Crystallization; Form conversion; Particle size; Precipitation; Turbidity; Wavelength dependence.