Analysis of KCN1 stability by high-performance liquid chromatography (HPLC). HPLC

grade methanol and triethylamine were purchased from Fisher Chemicals and from Sigma. Using

an Agilent 1120 instrument, KCN1 was separated on a Zorbax SB-C18 (5 μm, 150×4.6 mm)

analytical column with a Zorbax Reliance Cartridge Guard Column (SB-C18), and the eluate was

monitored by UV at 254 nm. The mobile phase was composed of “A” (methanol+0.1% Et3N)-

“B” (ddH2O+0.1% Et3N) (80:20, v/v). Prior to application, the mobile phase was filtered and

degassed using a Millipore glass filter system with a nylon membrane (0.2 μm).

Measurement of Permeability coefficients (PC) of KCN1. Human brain microvascular

endothelial cells (HH8) and brain endothelial cells (bEnd5) were gifts from Lonza and Dr. Britta

Engelhardt (Theodor Kocher Institute, University of Bern, Switzerland), respectively, and mouse

brain endothelial cells (bEnd3) were purchased from ATCC. bEnd3 and bEnd5 cells were seeded

at a density of 20,000 cells/cm2 on 12-well transwell plates (0.4-μm pore size) and grown to

confluence in 4 days. HH8 cell were seeded at a density of 5,000 cells/cm2 on transwell plates

that were pretreated with collagen and grown for 7 days. Passage 16 was used for HH8 and

bEnd5 cells and passage 29 was used for bEnd3.

HH8 cells were grown in endothelial cell basal media with growth supplements (Lonza),

incubated at 33oC and bEnd5, bEnd3 were grown in media described previously (1, 2). The

experiment was performed at the end of the cell confluence. [3H]-mannitol and [14C]-KCN1 (3)

were added to the apical side of each well. All the transport studies were performed in the apical

to basolateral direction and plates were rotated at 50 rpm using a shaker. Throughout the

experiments, aliquots (100µl) were taken from the basolateral chambers at different time

intervals (0, 30, 60, 90 and 120 min). Mannitol and KCN1 transported across cell monolayer was

determined by counting the samples in a Beckman LS 6500 liquid scintillation counter

(Beckman Instruments, Inc., Fullerton, CA). Apical-to-basolateral permeability across

monolayer was expressed as a PC (centimeters per minute), calculated from the following

equation:

PC= dQ/dt X 1/A X Co, where dQ/dt is the flux of tracer across the cell monolayer (dpm/min), A

is the surface area available for transport (1.2 cm2 for 12-well Transwell), and C0 is the original

donor concentration (dpm/ml).

References:

1. Yang T, Roder KE, Abbruscato TJ. Evaluation of bEnd5 cell line as an in vitro model for the blood-brain barrier under normal and hypoxic/aglycemic conditions. Journal of Pharmaceutical Sciences. 2007;96:3196-213. 2. Omidi Y, Campbell L, Barar J, Connell D, Akhtar S, Gumbleton M. Evaluation of the immortalised mouse brain capillary endothelial cell line, b.End3, as an in vitro blood-brain barrier model for drug uptake and transport studies. Brain Research. 2003;990:95-112. 3. Shi Q, Yin S, Kaluz S, Ni N, Devi NS, Mun J, Wang D, Damera K, Chen W, Burroughs S, Mooring SR, Goodman MM, Van Meir EG, Wang B, Snyder JP. Binding Model for the Interaction of Anticancer Arylsulfonamides with the p300 Transcription Cofactor. ACS Medicinal Chemistry Letters. 2012;in press.