|
Please provide an overview of the Cloe Screen Turbidimetric Solubility assay.
Aqueous solubility is measured using a high throughput turbidimetric assay. Initially, a stock DMSO solution is diluted in DMSO to produce a range of concentrations. These are then added to buffer, typically phosphate buffered saline at pH 7.4 (final test compound concentrations = 1µM, 3µM, 10µM, 30µM, 100µM, final DMSO concentration = 1%, 7 replicates per concentration) and incubated for 2hr at 37°C. At the end of the incubation period, the absorbance at 620nm is read for each concentration and each replicate.
Figure 1
Typical Solubility Profile showing the Effect of Increasing the Test Compound Concentration on Absorbance (at 620nm) for the Control Compound Pyrene.
Estimated precipitation range = 3 – 10µM
A program has been developed in-house to calculate the estimated solubility, with the onset of precipitation based on an increase in absorbance. As the calculation is to some extent based on inflection point, the maximum can vary from the set of concentrations tested. The solubility can also cover a wide range if the program does not feel it can accurately calculate the value.
Why is solubility important?
Poor solubility can limit the absorption of compounds from the gastrointestinal tract which reduces oral bioavailability. The quality of the data generated from the in vitro assays can also be affected by poor solubility.
How do I interpret the data from the solubility assay?
A precise measure of solubility can not be provided using the turbidimetric assay. The data are reported as an estimated precipitate range (i.e. lower bound, upper bound and calculated mid-range). It can be assumed that at some point within this range the compound precipitates. For ranking purposes clients typically use lower bound value. For drug discovery compounds, most clients would consider a compound which has a solubility value of less 1µM to be highly insoluble, between 1 and 100µM to be partially soluble and >100µM to be soluble. Although poor solubility is obviously an undesirable situation, it does not necessary preclude the development of an otherwise promising compound. If the compound has very high permeability, and is highly potent in vivo, then solubility may become less limiting. Formulation can also be used to improve the solubility.
The data can be used for a variety of purposes. Firstly, it can provide useful information for chemists to assess the effect of functional groups on the solubility of a series of compounds. The data can also be used to direct formulation strategy. Secondly, the solubility data can be useful for interpreting the results from the in vitro assays. If a compound has a solubility value lower than the test compound concentration in the in vitro screen then the results achieved for that screen may be inaccurate. Thirdly, solubility can be used in conjunction with other in vitro parameters to predict the oral pharmacokinetics of a compound using the simulation software, Cloe PK.
Inaccurate solubility readings may be caused by compounds which are insoluble in the original DMSO solution, or if the compound is a blue or green colour, which causes interference at the wavelength of 620nm. In drug discovery, most coloured compounds tend to be red or yellow, rather than blue or green. Therefore, the problem of interference is relatively rare.
Can the solubility assay be performed at different pH values?
Yes we are able to perform the assay at different pH values. Often clients will request pH 2 and pH 6 to mimic the conditions present in the stomach and small intestine respectively. For ionisable compounds, aqueous solubility is dependent on pH. If a compound has no ionisable groups then its solubility will be unaffected by the pH of the buffer.
What control compounds are used in the turbidimetric solubility screen?
Pyrene and nicardipine are used as the positive control compounds. The solubility of pyrene is pH-independent whereas nicardipine displays pH-dependent solubility.
back to top
|
