Understand if poor solubility is likely to be an issue in the development of your compound.
Turbidimetric solubility is one of Cyprotex's in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.
While thermodynamic solubility is useful when preparing a data package for a proposed development candidate, kinetic solubility seems most appropriate for high throughput in vitro assays in discovery.
1Kerns EH and Di L. (2005) Journal of the Association for Laboratory Automation 10(2); 114-123
|Final Test Article Concentration||1 μM, 3 μM, 10 μM, 30 μM and 100 μM|
|Buffer||0.01 M phosphate buffered saline pH7.4
(alternatives available on request)
|Final DMSO Concentration||1%|
|Number of Replicates||n = 7 per concentration|
|Incubation Time||2 hours|
|Test Article Requirements||150 μL of a 10 mM solution|
|Analysis Method||Absorbance at 620 nm|
|Data Delivery||Estimated solubility range (lower and upper bound and calculated mid-range in μM).|
|Prediction of Human Intestinal Absorption|
|Cyprotex's Turbidimetric Solubility data can be used in conjuction with Caco-2 Permeability data to predict dose dependent human intestinal absorption. Please refer to our Cloe Predict Human Intestinal Absorption Model section for further details.|
Data generated in Cyprotex's assay compare well with third party data.
|Compound||Cyprotex's Turbidimetric Solubility (μg/ml)||Blind Trial Solubility by|
Please provide an overview of Cyprotex's 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 = 1 µM, 3 µM, 10 µM, 30 µM, 100 µM, 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.
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 1 µM to be highly insoluble, between 1 and 100 µM to be partially soluble and > 100 µM 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 color, which causes interference at the wavelength of 620 nm. In drug discovery, most colored 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 ionizable compounds, aqueous solubility is dependent on pH. If a compound has no ionizable 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.
1 Kerns EH and Di L. (2005) Journal of the Association for Laboratory Automation 10(2); 114-123
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