Plasma Stability (mouse, rat, dog, monkey, or human plasma)
- 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: t1/2 or % of parent compound remaining
Controls: Tetracaine3, Propanolol
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 During the incubation, aliquots are withdrawn at 0, 15, 30, 60, and 120 minute time points and quenching solution (25/50 ng/mL terfenadine/tolbutamide in ACN/MeOH (1:1, v/v)) is added. After mixing, the quenched aliquots are centrifuged and supernatant is withdrawn for analysis by LC-MS/MS.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.
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
HPLC High-performance liquid chromatography
LC Liquid chromatography
MS Mass spectrometry
ln Natural logarithm
- 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).