Thermodynamic solubility assay

Thermodynamic solubility assay protocol

Compound Requirements 2.5 mg solid (for solubility assessment) 1 mg solid (for standard preparation) Number of Replicates n = 2 (aliquots from filtrate) Incubation Time Overnight Incubation Temperature Ambient temperature Stirring Vial roller system Analysis method HPLC- UV (photodiode array detector acquiring between 220 nm and 300 nm wavelengths) Data Delivery Solubility (mg/mL)

In early development thermodynamic assays are performed to confirm earlier kinetic solubility results, to rule out potential artifacts, and to generate quality solubility data with crystalline material1.

Data from the Cloe Screen Thermodynamic Solubility assay

4 compounds were screened through the Cloe Screen Thermodynamic Solubility assay at 3 different pH values. The results clearly demonstrate the ability of the assay to identify compounds which undergo pH-dependent solubility. As expected, both nicardipine and ketoconazole are more soluble at acidic pH whereas metoprolol and pyrene do not exhibit pH dependency over the range of pH values investigated.

Figure 1

Mean solubility data for 4 compounds generated in the Cloe Screen Thermodynamic Solubility assay at 3 different pH values (error bars represent the standard deviation of 3 separate experiments).

 

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Questions and answers on thermodynamic solubility

Please provide an overview of the Cloe Screen Thermodynamic Solubility assay.

Thermodynamic (or equilibrium) solubility investigates the solubility of a compound as a saturated solution in equilibrium.

For this method, aqueous solvent is added to solid compound. Excess solid should be used and relatively long mixing times are performed to ensure equilibrium is achieved (typically 16-72hrs). At the end of the incubation period, the saturated solution is filtered and quantified against a DMSO stock solution using LC-UV.

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. Compounds with poor solubility can pose a development challenge and result in prolonged time frames and increased cost. For this reason, it is recommended that ideally, any potential issues are resolved with structural modifications at an early stage in drug discovery.

What is the difference between kinetic solubility and thermodynamic solubility and at what stage should I perform each assay?

Kinetic assessment evaluates the solubility of a compound already fully dissolved in an organic solvent (typically DMSO). A small percentage of the organic solvent is added to aqueous solvent and precipitation of the compound is measured at a range of concentrations. It does not allow for equilibrium to be reached between dissolved compound and solid compound. The conditions of the kinetic solubility assay mimic many early in vitro discovery screens in that compounds are pre-dissolved in DMSO. Therefore, kinetic solubility is a common choice at this early stage in discovery. Thermodynamic assessment evaluates the solubility of solid crystalline material in aqueous solvent as a saturated solution in equilibrium. The relevance of thermodynamic solubility is less useful in drug discovery as it is not common for early in vitro screens to start with solid material (DMSO stock solutions are typically used in discovery). The importance of thermodynamic assessment is greater in late discovery /early development where it is used to confirm earlier kinetic solubility results, rule out potential artifacts and to generate high quality solubility data using crystalline material.  

How do I interpret the data from the solubility assay?

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 or potentially misleading. Thirdly, solubility can be used in conjunction with other in vitro parameters to predict the oral pharmacokinetics or intestinal absorption of a compound using physiologically based modelling (e.g. Cloe PK or Cloe HIA)

Can the solubility assay be performed at different pH values or using different buffers?

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. Therefore for ionisable compounds, it is common for solubility to be assessed at different pH values to mimic the pH gradient observed in the gastrointestinal tract. Different buffers can also be assessed to mimic the effect of food effects e.g., simulated intestinal fluid conditions in the fasted and fed state.

What control compounds are used in the thermodynamic 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.

What approaches can be used to increase solubility?

In terms of structural modification, one of the most effective and commonly used approaches to increase solubility is the addition of an ionisable group. Other approaches include decreasing lipophilicity (LogP), reducing molecular weight, the introduction of hydrogen bond donors and acceptors, the design of a prodrug, the addition of a polar group or including an out of plane substitution. To improve dissolution rate, reduction in particle size, formulation with surfactants or preparation of salt forms are common approaches.

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