Linking Solubility and Permeability Assays for Maximum Throughput and Reproducibility

Abstract

Solubility and permeability are intimately linked in drug absorption processes. They have, however, been traditionally assayed separately. To support this linkage, a combined solubility/permeability assay was developed for determining absorption properties of chemical entities. First, solubility is determined at 4 pH values by comparing the concentration of a saturated compound solution to its dilute, known concentration. The filtered, saturated solution from the solubility assay is then used as input material for the membrane permeability determination. The permeability assay is a parallel artificial membrane technique whereby a membrane is created on a solid support parallel artificial membrane permeation assay (PAMPA). The 2 artificial membranes presented here model the gastrointestinal tract and the blood-brain barrier (BBB). Data are presented for control compounds, which are well documented in the literature and exemplify a range of solubility and membrane permeability. The advantages of the combination method are 1) reduction of sample usage and preparation time, 2) elimination of interference from compound precipitation in membrane permeability determination, 3) maximization of input concentration to permeability assay for improved reproducibility, and 4) optimization of sample tracking by streamlining data entry and calculations. BBB permeability ranking of compounds correlates well with literature CNS activity. Key words: PAMPA, ADME-Tox, blood-brain-barrier, high throughput W ITH AN EVER INCREASING PRESSURE to push compounds through the drug discovery pipeline, there is a steady demand for reliable, high-throughput assay technologies in bottleneck areas. One bottleneck area that has received significant attention lately is ADME-Tox (Absorption, Distribution, Metabolism, Excretion, and Toxicology). The pressure is then compounded by the fact that 40% of late-stage failures are associated with poor pharmacokinetic properties, 1 such as low oral absorption. In the present article, we focus on the 2 major factors of absorption: solubility and membrane permeability.2-6 The focus of this report is to develop a high-throughput process that will integrate both solubility and permeability assays to increase throughput and decrease cost.Several different techniques are available for determining aqueous solubility.7-12 After reviewing the available technologies for solubility testing, the direct ultraviolet (UV) method 13 was chosen based on its adaptability into a high-throughput format, low cost, and reliability. One potential problem for the direct UV method is compounds with weak UV absorption; however, these compounds are in the minority. If required, other detection methods such as high-performance liquid chromatography or liquid chromatography/mass spectrometry can be implemented. The direct UV method requires a small amount (~0.25 mg) of compound and can be conducted over a wide range of pH values in the presence of many different excipients including DMSO. Importantly, the resulting filtrate from this t...

In vivo, in vitro and in silico methods for small molecule transfer across the BBB

“…Carrara et al179 showed that PAMPA BBB permeability is mostly dependent on the percentage of dodecane and the effect of phospholipids is relevant only for medium permeable compounds. Wexler et al180 developed a combined solubility/permeability assay for determining permeability properties (GI and BBB) of chemical entities. Firstly, solubility was determined at different pH values by comparing the concentration of a saturated compound solution to its dilute, known concentration.…”

“…An additional set of descriptors related, although nontrivially, to the physicochemical principles involved in passive diffusion described above, have been utilized to build log BB models yielding good correlation coefficients for training set, internal validation by leave‐one‐out (LOO) cross validation and external test set: r 2 , q 2 and predicted ‐ r 2 , respectively, from 0.70. These descriptors are, (i) COSMO σ‐moments obtained from quantum mechanical calculations using the continuum solvation model COSMO and a subsequent statistical decomposition of the resulting polarization charge densities, these descriptors have been already introduced as a general descriptor set for partition coefficients;222 (ii) properties like the solvent‐accessible surface area (SASA), and amphiphilic components of the SASA as determined from Monte Carlo simulations of the compounds in water;223 (iii) solvation free energies, G solv ;224, 225 (iv) Abraham or Linear Free Energy Relationship descriptors (LFER) 180. These five LFER descriptors ( E , S , A , B , and V ) are based on empirically determined values for simple molecules and “fragments” and can be regarded as a point in a five‐dimensional space.…”

Phytochemicals in olive‐leaf extracts and their antiproliferative activity against cancer and endothelial cells

“…The antioxidant potency per se is a well established biomarker, which indicates beneficial biological effects of plant derived phenolic substances [37 -39]. We selected the aqueous OLE, AOLE, for subsequent analysis on the criteria of its antioxidant potency (half maximal effective concentration (EC 50 ) = 49 lg/mL), easiness of preparation and water solubility, which indicates water solubility for its active compounds, a very desirable physical property for potential drugs [40,41]. MOLE was found to scavenge DPPH9 at a concentration of 71 lg/mL and was also further studied.…”

Peak Seeker: An Algorithm for Rapid Determination of Solubility

“…Given the ease of processing large numbers of samples using automated liquid handling and automated UV-visible spectra analysis using Peak Seeker, 128 compounds may be assayed at a single pH within 5 h. The data presented is in agreement with a literature report 5 that indicated more accurate solubility determinations are achieved with calibration curves than with flux ratios. 6…”