Single use red plate

The free fraction of PT55 and PT119 in plasma was determined by rapid equilibrium dialysis using a Thermo Scientific™ Single-Use RED Plate. A solution of each compound was prepared in mouse plasma to match the C_max determined in the PK study (1.82 μg mL^–1 for PT55 and 0.79 μg mL^–1 for PT119). Then 200 μL of each solution was transferred to the plasma chamber of the Thermo Scientific™ Single-Use RED Plate and PBS was placed in the corresponding buffer chamber. The plate was sealed and incubated at 37 °C on an orbital shaker at ∼250 rpm for 4 h. Subsequently, the amount of compound in the plasma and buffer samples was determined by LC/MS/MS and the free fraction was calculated by taking the ratio of compound concentration in the buffer and plasma chambers.

The University of Michigan Pharmacokinetics (PK) Core (Ann Arbor MI, USA) determined plasma protein binding affinities to RAGER (Fig. S2). Three doses of RAGER were prepared in DMSO (1 mM, 100 μM, and 10 μM) and verapamil in DMSO (100 μM) was used as a positive control owing to its high level of plasma protein binding (∼90%) [25 ]. RAGER was added to mouse plasma (final RAGER concentrations of 10 μM, 1 μM, and 0.1 μM) in duplicate. 200 μL of sample was added into the sample chamber, and 350 μL of phosphate buffered saline (PBS) was added to the neighboring buffer chamber. Solutions were incubated at 37 °C for 5 h in the sample chamber (Thermo Single-use RED plate). Post incubation, corresponding plasma and buffer samples were mixed in equal volume and the internal standard was added in cold acetonitrile to precipitate proteins. The solution was vortexed and incubated on ice for 30 min. The entire solution was centrifuged for 10 min at 3500 rpm; the supernatant was analyzed by LC/MS/MS (Column: Waters XBridge C₁₈).