Plasma stability plays an important role in drug discovery and development. In addition to hepatic metabolism, compounds may be subject to degradation / modification by enzymes in plasma, particularly hydrolases and esterases. Unstable compounds tend to have rapid clearance and short half-life, resulting in poor in vivo performance.¹
The plasma stability assay can be used to alert teams to labile structural motifs, to prioritize compounds for in vivo studies, and to screen prodrugs.
Investigation of plasma stability should be performed early in the discovery process in order to assess potential degradation and/or protein binding issues.²
Compounds may exhibit interspecies differences in their stability in plasma.
Readout: t1/2or % of parent compound remaining
Controls: Tetracaine3, Propanolol
Assay Description:
Plasma and test compound or control compound (100 µM in DMSO) are added to the individual wells of a 96-well microtiter plate. The plate is incubated at 37 °C with gentle agitation. During the incubation, aliquots are withdrawn at 0, 15, 30, 60, and 120 minute time points and quenching solution (25/50 ng/mL terfenadine/tolbutamide in ACN/MeOH (1:1, v/v)) is added. After mixing, the quenched aliquots are centrifuged and supernatant is withdrawn for analysis by LC-MS/MS.
The MS detection is performed by using a SCIEX API 4000 Q trap instrument. Each compound is analyzed by reversed phase HPLC using a Kinetex 2.6μ C18 100Å column (3.0 mm X 30 mm, Phenomenex). Mobile phase – solvent A: water with 0.1% formic acid, solvent B: ACN with 0.1% formic acid.
Data Analysis:
The slope of the ln(%remaining) vs. time point line is used to calculate t1/2 according to the following formula:
Half life (t1/2) = – ln(2) / Slope
Abbreviations:
ACN Acetonitrile
DMSO Dimethylsulfoxide
HPLC High-performance liquid chromatography
LC Liquid chromatography
MS Mass spectrometry
ln Natural logarithm
Literature:
Di, L.; Kerns, E.H.; Hong, Y; Chen, H., “Development and application of high throughput plasma stability assay for drug discovery”; Int. J. of Pharmaceutics 297, 110, (2005).
Chung, T. D. Y.; Terry, D. B.; Smith, L. H.; “In Vitro and In Vivo Assessment of ADME and PK Properties During Lead Selection and Lead Optimization – Guidelines, Benchmarks and Rules of Thumb”, Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004. Available from: https://www.ncbi.nlm.nih.gov/books/NBK53196/.
“Tetracaine – hydrolyzed by cholinesterases in the plasma”: Gilman AG, Rall TW, Nies AS, Taylor P, editors. Goodman and Gilman’s the pharmacological basis of therapeutics. 8th ed. New York: Pergamon Press, 312-8, (1990).
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