Small Molecules – Developing Better Drugs Through The Unseen

HORIZON: An Alternative Method for Solubility Measurement

Aqueous solubility of small molecule compounds is an essential parameter in drug discovery and development. Low solubility can impact biological assays, leading to underestimated potency and toxicity, inaccurate SAR, and difficult to interpret ADME results. Despite its critical importance at early stages, common detection methods for solubility measurement are inefficient, lack sensitivity, or are sensitive to sample processing.

The HORIZON® system with Backgrounded Membrane Imaging (BMI) is a subvisible particle analysis system that can measure compound solubility in high throughput via a simple and flexible workflow. In addition, highly resolved particle images provide valuable information on morphology of precipitated solids for more comprehensive solubility characterization.

Low Volume, High Throughput Subvisible Aggregate Analysis

BMI is a novel automated microscopy technology that images and analyzes insoluble aggregates captured on a membrane in low-volume, high throughput format. It offers the ease of use and throughput advantages homogeneous solution assays but with higher sensitivity and more comprehensive particle data. Since aggregates are measured on a membrane surface and not in solution, presence of solvent, matrix effects, or compound stickiness are not a concern with BMI. Every particle ≥2 μm is counted and analyzed, regardless of particle number or size distribution, so that changes in solubility can be detected even when only a few aggregates are present in the sample.

More Sensitive Kinetic Solubility Measurement

To demonstrate correlation of subvisible particle counts measured on the HORIZON® system to solubility changes, four control compounds with different aqueous solubilities were selected for analysis with BMI. A concentration series for each compound was prepared directly from DMSO stocks, then diluted into PBS, pH 7.4 to 1% DMSO final concentration.

Compound Solubility Profiles on the HORIZON. Relationship between increasing concentration in PBS 7.4 and surface coverage of the membrane well with insoluble particles. Differing compound profiles support emergence of particle aggregates as an indicator of solubility of the different compounds. Of note is that the inflection of the particle coverage curves and rate of increased precipitate formation vary for each compound.

Kinetic solubility range for each compound was reported as the concentration above and below where membrane area coverage by particles exceeded a baseline threshold. The midpoint of this range was assigned as the compound’s estimated solubility.

Kinetic solubility measurement of TIPT on the HORIZON system. Threshold for solubility was set at 0.5% of well area coverage signal saturation.

Kinetic solubility measurement of Dipyridamole on the HORIZON system. Threshold for solubility was set at 0.5% of well area coverage signal saturation

Kinetic solubility measurement of Griseofluvin on the HORIZON system. Threshold for solubility was set at 0.5% of well area coverage signal saturation

Kinetic solubility measurement of Diclofenac on the HORIZON system. Threshold for solubility was set at 0.5% of well area coverage signal saturation

Ranking of the four test compounds for solubility with the HORIZON system was identical to ranking by turbidimetry. However, with the HORIZON particle aggregates were detected at 5–10 times lower compound concentration.

Comparison of solubility measurements by the HORIZON versus turbidimetry. 150 μL (N=3) sample used for the turbidimetric analysis at A620 on a Spectra Max M2 Molecular Devices system. Turbidimetry threshold for solubility range determination was set at 0.05 absorbance units above background.

The Value of the Unseen

Small scale synthesis of compounds during selection stage can lead to variation of physical form, which can have a dramatic effect on the measured aqueous solubility of compounds. BMI provides high resolution images and digital image analysis of aggregate particles to aid in evaluation of solid-state form.

Brightfield image acquired on the HORIZON of crystalline particles

Brightfield image acquired on the HORIZON of needle-shaped particles

Brightfield image acquired on the HORIZON of amorphous solid particles.

Why use the HORIZON System for Small Molecule ADME Research?

  • Commonly used methods to measure solubility at early stages have limitations
    • Inefficient (filtration + LC-MS / HPLC) and not flexible for smaller projects
    • Lack sensitivity (light scattering / turbidimetry)
    • Subject to interference from solvent and matrix impurities (LC-MS, UV spectroscopy)
  • BMI is an easy-to-use, flexible solution to support early ADMET workflows
    • More sensitive than turbidimetry
    • Faster, easier, and more flexible than Mass Spec or HPLC detection
    • Dry measurement means no matrix interference
    • Particle images provide valuable information on solid-state form (crystalline vs amorphous)