Combination of solubility and membrane permeability measurement methods for profiling of chemical compounds
Compound solubility and permeability are coordinately determined. High throughput solubility assays are set up, and the filtrate from the solubility assay is used as an input for PAMPA. In this way, the compound concentration during PAMPA is known exactly, and can be used as input to calculate Pe.
1 . A method for the combined determination of solubility and permeability of a compound, the method comprising:
performing a solubility assay and a permeability assay, wherein said permeability assay is performed using a filtrate from aqueous or cosolvent saturated solution obtained from said solubility assay, thereby providing each compound at a saturated, known concentration.
2 . The method according to claim 1 , wherein said solubility assay measures a spectrophotometric property of a saturated solution of said compound.
3 . The method according to claim 2 , wherein said solubility assay comprises:
preparing a sample solution of said compound in an aqueous buffer of known pH, and separating said sample solution from any precipitate to provide a filtrate,
preparing a reference solution that has a known concentration of said compound in said buffer, under conditions avoiding or suppressing precipitation,
preparing a blank solution free of said compound, but otherwise of the same composition as said reference solution,
measuring a spectrophotometric property of said sample filtrate, reference, and blank solutions;
determining the concentration of said sample solution by comparing the measured spectrophotometric property of said sample, reference and blank solutions, and calculating from said determination the aqueous solubility of said compound.
4 . The method according to claim 3 , wherein said plates are read with a UV/visible spectrophotometer.
5 . The method according to claim 4 , wherein the concentration of the compound in the solvent is determined by heuristically matching, scaling and background correcting the spectra and determining appropriate OD values to compare reference solutions to solutions containing an analyte of unknown concentration.
6 . The method according to claim 1 , wherein said permeability assay is a parallel artificial membrane permeation assay (PAMPA).
7 . The method according to claim 6 , wherein said permeability assay comprises:
placing an aliquot of said filtrate in a donor compartment;
covering said donor compartment with an artificial membrane barrier;
placing an initial acceptor solution in an acceptor compartment on the opposite side of said artificial membrane barrier;
preparing an acceptor-blank solution of the same composition as said initial acceptor solution
measuring an initial spectrophotometric property of said reference, donor-blank, and acceptor-blank solutions and measuring a spectrophotometric property of the final donor and final acceptor solutions after the start of the assay; and
determining the relative concentration of said final donor and acceptor solutions by comparing the measured spectrophotometric property of said final acceptor, final donor, reference, acceptor-blank and donor-blank solutions.
8 . The method according to claim 7 , wherein said artificial membrane barrier provides a gastrointestinal tract (GIT) model of phospholipid dissolved in organic solvent.
9 . The method according to claim 7 , wherein said artificial membrane barrier provides a blood brain barrier (BBB) model.
10 . The method according to claim 9 , wherein said artificial membrane comprises brain lipid extract dissolved in n-dodecane.
11 . The method according to claim 1 , wherein said assay is performed in a plate having 384 wells or greater than 384 wells.
12 . A method for high-throughput spectrophotometric measurement of aqueous solubility and permeability of a test compound, the method comprising:
preparing a sample solution of said compound in an aqueous buffer of known pH, and separating said sample solution from any precipitate to provide a filtrate,
preparing a reference solution that has a known concentration of said compound in said buffer, under conditions avoiding or suppressing precipitation,
preparing a blank solution free of said compound, but otherwise of the same composition as said reference solution,
measuring a spectrophotometric property of said sample filtrate, reference, and blank solutions;
determining the concentration of said sample solution by comparing the measured spectrophotometric property of said sample, reference and blank solutions, and calculating from said determination the aqueous solubility of said compound
placing an aliquot of said filtrate in a donor compartment;
covering said donor compartment with an artificial membrane barrier;
placing an initial acceptor solution in an acceptor compartment on the opposite side of said artificial membrane barrier;
preparing an acceptor-blank solution of the same composition as said initial acceptor solution
measuring an initial spectrophotometric property of said reference, donor-blank, and acceptor-blank solutions and measuring a spectrophotometric property of the final donor and final acceptor solutions after the start of the assay; and
determining the relative concentration of said final donor and acceptor solutions by comparing the measured spectrophotometric property of said final acceptor, final donor, reference, acceptor-blank and donor-blank solutions.