SOLUBILITY (a physicochemical property) – 2

As discussed earlier, compounds with low solubility sometimes might end-up providing inaccurate in vitro biological data. 

·         For example, insufficient solubility can lead to inconsistencies between bioassay, underestimated activity, toxicity, reduced hit rates and inaccurate structure activity relationship.

·         Poor solubility can limit the absorption of compounds from the gastrointestinal tract which in turn reduces oral bioavailability.

·         Therefore, poor solubility can influence both pharmacokinetic and pharmacodynamic properties of the compound.

Therefore, solubility measurement is of utmost importance in drug discovery programs.

MEASUREMENT OF SOLUBILITY: 

During ADME profiling we come across two types of solubility data.  They are KINETIC SOLUBILITY data & THERMODYNAMIC SOLUBILITY data.

What do they mean and how do we measure these!

These two terms are related to TIME.  We do understand that KINETICS is time-dependent event and THERMODYNAMICS is time independent phenomenon.

KINETIC SOLUBILITY measurement is done something like this,

1.      In general stock solutions of desired compounds are prepared in DMSO (at a particular concentration depending on the final concentration in the assay).

2.      The above compound DMSO solution is then diluted in the buffer of interest and dispensed in microtiter plate. [When compound as DMSO solution is added to aqueous buffer – precipitation of the compound occurs – and this precipitation ofo the compound is measured at a range of concentrations]

3.      These plates are incubated at a particular temperature and time while shaking.

4.      After the above process, microplate’s content is filtered under vacuum and collected. 

5.      The concentration of compound in the filtrate is quantified by spectrophotometer.   

THERMODYNAMIC SOLUBILITY investigates the solubility of a compound as a saturated solution in equilibrium.  This measured solubility is dependent on the pH of the solution at the equilibrium and the pK_a of the compound.

Measurement is done something like this,

1.      Aqueous solvent is added to solid compound.  Here excess solid should be used and relatively long mixing times are performed to ensure equilibrium is achieved (16-72 hours – this is the incubation time)

2.      After this time, the saturated solution is filtered and quantified against a DMSO stock solution using spectrophotometer.

___________

Since the majority of known drugs contain ionizable groups, the aqueous solubility is assessed over a range of pH values.  The compound is dissolved in buffer solutions at the indicated pH values. The compound is allowed to reach thermodynamic equilibrium by incubating for 18 hours. Compound UV absorption is compared to fully saturated solution in 1-propanol.

Solubility report is given in mM units

___________

WHAT DID WE LEARN?  WHICH SOLUBILITY MEASUREMENT IS SUPERIOR!

Ø  When we perform in vitro discovery screens (activity testing) – compounds pre-dissolved in DMSO are used.  Therefore, kinetic solubility measurement (which is performed by using DMSO stocks) is preferred method at the early stage discovery programs. [kinetic solubility assay conditions mimic many early in vitro discovery screens – in that compounds are pre-dissolved in DMSO]

Ø  While determining Kinetic solubility – we are basically measuring precipitation rate rather than solubility.  Therefore, a stock solution of compound of interest in DMSO is gradually added to aqueous solvent (buffer solution) until the anti-solvent properties of the water drive the compound out of the solution.

Ø  Not surprisingly, thermodynamic solubility is less useful in early drug discovery.  This becomes important in late discovery and early development stages.  During these stages we must make sure about the previously obtained kinetic solubility results (to rule out potential artifacts and to generate high quality solubility data using crystalline material). This may be helpful to design formulation strategies for in vivo studies.