Peter van Mourik

52 Chapter 3 0 3.125 6.25 12.5 25 50 100 200 0 500 1000 1500 [Genistein] ( µ M) 5 µ M fsk Organoidswelling (AUC t = 60 min) 0 0.00003 0.0003 0.003 0.03 0.3 3 30 0 500 1000 1500 [VX-770] ( µ M) 5 µ M fsk Organoidswelling (AUC t = 60 min) 0 3.125 6.25 12.5 25 50 100 200 0 500 1000 1500 [Curcumin] ( µ M) 5 µ M fsk Organoidswelling (AUC t = 60 min) 0.008 0.02 0.05 0.128 0.32 0.8 2 5 -200 0 200 400 600 800 [Forskolin] ( µ M) DMSO Genistein (50 µ M ) VX-770 (3 µ M ) VX-770+genistein Organoidswelling (AUC t = 60 min) 0.05 0.128 0.32 0.8 2 5 0 400 800 1200 [Forskolin] ( µ M) DMSO Genistein (10 µ M ) VX-770 (0.1 µ M ) VX-770+genistein Organoidswelling (AUC t = 60 min) 0.02 0.05 0.128 0.32 0.8 2 5 0 1000 2000 3000 [Forskolin] ( µ M) DMSO Gen (10 µ M ) VX-770 (0.1 µ M ) VX-809 (0.1 µ M ) VX-809+VX-770 VX-809+VX-770+genistein Organoidswelling (AUC t = 60 min) Suboptimal [potentiator] (0.32 µ M forskolin) a b c d e f g h + VX-809 (0.1 µ M ) Near-saturating [potentiator] (0.05 µ M forskolin) Fig. 4h VX-770 (3 µ M) Genistein (50 µ M) Curcumin (50 µ M) VX-770+gen VX-770+cur Gen+cur VX-770+gen+cur 0 500 1000 1500 Organoidswelling (AUC t=60min) VX-770 (0.1 µ M) Genistein (10 µ M) Curcumin (10 µ M) VX-770+gen VX-770+cur Gen+cur VX-770+gen+cur 0 500 1000 1500 2000 Organoidswelling (AUC t=60min) p=0.0125 ns p=0.0073 ns ns p=0.0197 ns ns ns ns ns ns Calculated additivevalues Measured values Calculated additivevalues Measured values + VX-809 (0.1 µ M ) Figure 4. Incubation of VX-770, genistein and curcumin in rectal organoids derived from F508del / F508del CF subjects. (a-c) Forskolin(fsk)-induced swelling (FIS) expressed as the absolute area under the curve (AUC) calculated from time tracings shown in Fig. 2b (baseline = 100%, t = 60 min) of organoids stimulated with multiple dosages of VX-770 (a), genistein (b) or curcumin (c) at the indicated forskolin concentrations. (d,e) FIS of organoids stimulated with near-saturating (d) or suboptimal (e) concentrations of VX-770, genistein or both using a dose range of forskolin. (f) The effect of VX-809 preincubation (24 h) on FIS of organoids stimulated with VX-770 or genistein using a dose range of forskolin. (g,h) FIS of organoids pre-incubated with VX-809 for 24h, and stimulated with near-saturating (g) or suboptimal (h) concentra- tions of VX-770, genistein (gen), curcumin (cur) or their combinations at the indicated concentrations of forskolin, corrected for the VX-809-repaired FIS without addition of a potentiator. (All figures represent data averaged from 3 F508del / F508del subjects. Each subject was measured at 2 to 5 independent culture time points in duplicate. Mean ± SD. The SD indicates the inter-subject variation). DISCUSSION We characterized interactions between VX-770 (ivacaftor, KALYDECO TM ) and the natural food components genistein and curcumin using rectal CF biopsies and primary intestinal CF organoids as ex vivo models. While CFTR repair by corrector / corrector or corrector / potentiator combinations has been abundantly examined 15,29,33,39-41 , only a few studies have reported interactions between potentiators 28,39 , and similar studies in primary cells including VX-770 are completely lacking. In this study, we