Highthroughput solubility assays

PURPOSE AND RATIONALE In vitro testing of drug candidates and combinatorial chemistry lead to an increase of lipophilic compounds with poor solubility (Lipinsky). Poor solubility itself may lead to poor oral availability of a potential drug. The growing demand for solubility data in lead phase of drug discovery is answered by a variety of simple solubility assays, which allow the classification of a compound without real quantification (see previous section). One of the easiest way to detect saturation in a solvent is the turbidity of the solution if precipitation occurs. The turbidity caused by precipitation of a poorly soluble compound can be detected by a couple of detection methods (Van de Hulst, Hongve). Lipinsky describes the first methodology, which use UV as detection method and is able to screen hundreds of compounds a day with one instrument. 

Lipinsky dissolved compounds in DMSO at a concentration of 10 xg/ml. Complete dissolution is controlled by eye. One micro liter of this solution is added into a cuvette containing 2.5 ml pH 7 phosphate buffer. Mixing of the system is controlled via an integrated mixing device. The temperature is kept constant between 22 °C-25 °C. Stepwise one micro liter portions are added to the mixing chamber. After each step a equilibrium time of 5 minute is allowed before turbidity is analysed. These steps are repeated up to 14 times covering a range of 5 xg/ml to 65 xg/ml. If precipitation occurs the addition of compound is stopped after further two consecutive additions. The volume percent aqueous DMSO does not exceed 0.67%. 

Precipitated particles lead to an increase in UV absorbance due to light scattering. Lipinsky used a diode array UV (Hewlett Packard HP8452) at 600-820 nm for their experiment. UV absorbance (y-axis) vs. p,L DMSO plots (x-axis) is used to detect the precipitation point. A strong increase in the slope of the curve indicates precipitation. Precipitation defines the maximum solubility level in this experiment. The method allows a classification between poor, moderate and good 

The method underestimates compounds with slow dissolution rate. To minimize the risk of a wrong assessment control measurements with other methods (preferably HPLC-methods) are recommended. This could be done with one or two members of a compound class. 

Bevan introduced a high-throughput alteration of the method. He uses 10 mM DMSO stock solutions for a precipitation assay. In many biological assays 10 mM DMSO stock solutions are the standard stock solution, from which aliquots are added to the various assays. 10 mM DMSO stock solutions are readily available and the sample preparation can be minimized. 

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