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bph2143-sup-0001-fs1.JPG1079K

Figure S1 NS6180 and TRAM-34 inhibit ConA-stimulated proliferation of splenocytes from wild-type mice but not from KCa3.1−/− mice. Cells were stimulated for 48 h with 5 μg·mL−1 ConA in the presence and absence of TRAM-34 or NS6180. Control counts were normalized to 1, and fractional [3H]-thymidine incorporations from triplicate wells from one representative experiment are shown as mean ± SD. Splenocytes (frozen MNC) from KCa3.1−/− mice (Si et al., 2006) and C57Bl6 littermates were a generous gift from Dr Ralf Köhler, University of Southern Denmark, Odense. The cells were thawed, allowed to recover for 3 h and then seeded at 1 × 105 cells into flat-bottom 96-well plates and stimulated with 5 μg·mL−1 ConA.

bph2143-sup-0002-fs2.JPG1023K

Figure S2 Pharmacokinetics of NS6180 in rats. Total NS6180 plasma concentrations following i.v., i.p. and oral administration at 10 mg·kg−1 (n = 3 per route of application). Following i.v. administration, plasma concentrations fell from a peak of 9 ± 2 μM after 5 min to 90 nM at 24 h (n = 3). Similar to TRAM-34 (Chen et al., 2011), the i.v. data were best fitted triexponentially reflecting a three-compartment model with rapid distribution from blood into tissue (t1/2 distribution = 0.15 h) followed by elimination (t1/2 = 3.8 h), and slow repartitioning from body fat acting as a deep compartment back into plasma (t1/2 = 18 h). Bioavailability by i.p. and p.o. routes were low and resulted in Cmax concentrations of 186 ± 14 nM and 33 ± 4 nM only. Inset: Same data with rescaled X– and Y-axes to better visualize the i.p. and oral data. All values are means ± SD.

bph2143-sup-0003-fs3.JPG2501K

Figure S3 Effect of NS6180 and TRAM-34 on CRAC-, TRPV1-, TRPV2- and TRPV4-mediated currents expressed in HEK293 cells. (A) CRAC currents from the co-expression of human GFP-Myc-Orail1 and human mCherry-STIM1. Left: IV relation during voltage ramps (−120 to +40 mV) following application of 1 and 10 μM NS6180 (not leak-subtracted). Right: Time course of currents measured at −110 mV during application of NS6180 (1 and 10 μM) and 2-APB (30 μM). Ca2+ currents were elicited by passive store depletion with 2 mM external Ca2+. Pipette solution contained (in mM): CsAsp 133, CsCl 2, MgCl2 8, HEPES 15 and BAPTA 11 (pH 7.2 adjusted with CsOH; 309 mOsm). (B) Human TRPV1 currents: Left: Normalized IV plots under control conditions, after application of 250 nM capsaicin, 10 μM NS6180 plus 250 nM capsaicin or 10 μM TRAM-34 plus 250 nM capsaicin. Currents at each voltage were generated by 200 ms depolarizing steps (−120 to +70 mV in 10 mV increments; 1 s delay between steps). Right: Voltage ramps (−120 to +80 mV) under control conditions, after application of 250 nM capsaicin, and 10 μM NS6180 plus 250 nM capsaicin or 10 μM TRAM-34 plus 250 nM capsaicin. (C) Human TRPV2 currents. Voltage ramps (−120 to +80 mV) under control conditions, after application of 200 μM 2-aminoethoxydiphenylborane (2-APB), 10 μM NS6180 plus 200 μM 2-APB or 10 μM TRAM-34 plus 200 μM 2-APB. (D) Human TRPV4 currents. Voltage ramps (−120 to +80 mV) under control conditions, after application of 500 nM 4α-phorbol 12,13-didecanoate (4α-PDD), and 10 μM NS6180 plus 500 nM 4α-PDD (top) or 10 μM TRAM-34 plus 500 nM 4α-PDD (bottom). For all recordings from TRP channels pipette solutions contained (in mM): NaF 160, MgCl2 2, HEPES 10, EGTA 10 (pH 7.2 adjusted with NaOH; 301 mOsm). Na+-Ringer was used as an external solution for all recordings (in mM); NaCl 160, KCl 4.5, MgCl2 1, CaCl2 2, HEPES 10 (pH 7.4 adjusted with NaOH; 301 mOsm).

bph2143-sup-0004-fs4.jpg1017K

Figure S4 Additional data from the rat colitis experiment illustrated in Figure 8. (A) Colonic macroscopic damage score after 8 days of treatment. The individual colons were scored for adhesions (0–2), strictures (0–2), ulcers/inflammation (0–5) and wall thickness (1–2) and summarized. The bar graph depicts the averaged values from all animals (n = 8). Sulfasalazine reduced the total score significantly (one-way anova followed by Dunnett's post hoc test), whereas the NS6180 trends did not reach statistical significance. (B) Cumulative daily scorings for stool appearance comprising rectal bleeding (left column) and stool consistency (right column). Both parameters were scored (0, 1, 2), and the results from all animals are averaged daily. Both doses of NS6126 as well as sulfasalazine significantly (one-way anova followed by Dunnett's post hoc test) reduced the scores on days 6, 7 and 8.

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