Preclinical in vitro Evaluation: Combination FDL169/FDL176 is Superior to Tezacaftor/Ivacaftor

Transcription

1 Mike MZ Sports Shots Preclinical in vitro Evaluation: Combination /FDL176 is Superior to Tezacaftor/Ivacaftor Flatley Discovery Lab

2 Preclinical in vitro Evaluation of CFTR Modulator Combinations Flatley Discovery Lab is developing CFTR modulator combination -FDL176 for the treatment of cystic fibrosis. is a corrector of CFTR FDL176 is a potentiator of CFTR Objective: To characterize combinations -FDL176 and Tezacaftor-Ivacaftor in vitro to determine the effect of each treatment on F58del-CFTR chloride transport and expression. 2

3 Ieq NAUC (% of positive control) % Positive Control Corrector Restores F58del-CFTR Chloride Transport with Greater in vitro Efficacy and Potency than Tezacaftor Chloride Transport Dose Response Maximum Response 1 EC 5 = 77 nm +/- 27 nm 1 8 TEZ EC 5 = 182 nm +/- 1 nm Log Conc M TEZ Data normalized to positive control Lumacaftor (3 µm) treated cells acutely stimulated with forskolin + Ivacaftor (1 µm) Equivalent current assay (Ieq; TECC-24 robot) in primary human homozygous F58del-CFTR HBE cells Treatment with corrector test compounds for 24 hours followed by acute stimulation with forskolin + Ivacaftor (1 µm) 3

4 Potency is Less Sensitive to Human Serum than Tezacaftor : 3.5-fold EC 5 Shift TEZ: 2-fold EC 5 Shift Normalized Ieq Fold Increase over vehicle control 4 + 2% Human Serum Vehicle only Emax: 3.4-fold Normalized Ieq Fold Increase over vehicle control 4 TEZ TEZ + 2% Human Serum Vehicle only Emax: 2.5-fold Conc ( M) Tezacaftor Conc ( M) Data reported as increase of chloride current over vehicle treatment control cells Robot equivalent current assay (Ieq; TECC-24 system) in primary human homozygous F58del-CFTR cells Treated with corrector test compounds ± 2% human serum for 24 hours and stimulated with forskolin + ivacaftor (1 µm) 4

5 Ieq AUC ( A*s/cm 2 ) Chronic Treatment with /FDL176 Demonstrates 2-Fold Higher in vitro Efficacy than Tezacaftor/Ivacaftor Chloride Transport 3 2 Corrected 2x TEZ Corrected Chronic treatment with + FDL176 is more efficacious than TEZ + IVA Chronic treatment with + FDL176 is less sensitive to reduction of chloride current compared to acute potentiation conditions 1 (3 µm) 24 hr + + FDL176 (3 µm) 24 hr + Acute FDL176 + forskolin + + FDL176 + IVA + TEZ TEZ TEZ (3 µm) 24 hr + + IVA (.2 µm) 24 hr + Acute IVA + forskolin + + Chronic treatment with TEZ + IVA results in a substantial reduction of chloride current compared to acute potentiation conditions Robot equivalent current assay (Ieq: TECC-24 system) Primary human homozygous F58del-CFTR cells Incubation with test compound(s) for 24 hours Cells stimulated with 1 µm forskolin + potentiator 5

6 RLU Chronic Treatment with /FDL176 Demonstrates Approximately 2-Fold Higher F58del-CFTR Expression than Tezacaftor/Ivacaftor F58del-CFTR Plasma Membrane Expression Corrected TEZ Corrected CFTR-HRP Cell Surface Assay N x F58 NBD1 R NBD2 C TMD2 TMD1 HRP CFTR DMSO vehicle + FDL176 (1µM) + (3 µm) + + IVA (1µM) + TEZ (3 µm) + + RLU = relative luminescence unit F58del-CFTR CFBE41o- cells labeled with horseradish peroxidase (HRP) on the fourth extracellular loop Incubation with test compound(s) for 24 hours HRP exposed on the cell surface is measured by chemiluminescence, representing the plasma membrane expression of F58del-CFTR 6

7 Ieq NAUC Ieq NAUC and FDL176 Demonstrate a Dose-Responsive Increase of F58del-CFTR Chloride Transport Maximum Response: 3 µm + 3 µm FDL176 (3 M) Chloride transport evaluated in a concentration matrix experiment Correctors and TEZ give a dose-responsive increase of chloride transport [FDL176] M FDL176 demonstrates a dose responsive increase of chloride current in corrected cells Maximum Response: 3 µm TEZ +.1 µm IVA Tezacaftor (3 M) IVA demonstrates a doseresponsive reduction of chloride current in TEZ corrected cells.75.5 Equivalent current assay (Ieq: TECC-24) Emax [Ivacaftor] M Primary human F58del-CFTR cells Treatment with test compounds for 24 hr Cells stimulated with forskolin Data normalized to positive control: cells treated with corrector for 24 hr and acutely stimulated with forskolin + potentiator (1 µm) 7

8 Isc (µa/cm 2 ) RLU Triple Combination -FDL176-FD25216 Further Increases Chloride Transport and CFTR Expression FD25216 is F58del-CFTR corrector that is additive with -FDL176 Chloride Transport Ussing Chamber Assay (Primary F58del-CFTR cells, 24hr) Fold Increase FDL176 (1 µm) (3 µm) FD25216 (1 µm) Data reported as increase of chloride current over vehicle treated control cells ~Response of chronic IVA/TEZ (~4X) Cell Surface Expression CFTR-HRP Assay (CFBE 41o- cells, 24 hr) DMSO Vehicle FDL176 1µM µm DMSO (3 M) + FDL176 (1 M) 2.2-Fold Increase FD25216 (1 M) + FDL176 + FD25216 FD µM RLU = relative luminescence unit Band C Expression Western Blot (Primary F58del-CFTR cells, 48 hr) Band C Band B Na/K ATPase C/B DMSO vehicle FDL176 1µM µm FD µM Band C = mature, fully-glycosylated CFTR Band B = immature, core-glycosylated isoform 8

9 Acknowledgements Special thanks to FDL Biology Mauri Krouse and the EPY team Priyanka Bhatt Violaine Bailey Justin Chin Castera Bresilla Vy Mai Weiling An Afia Dasgupta Iris Kwok (Molecular Biology) FDL Research and Development Mike Zawistoski Karen Handley Abhijeet Kanawade Eric Lui Jinliang Sui Chris Oalmann Andrew Kolodziej John Ferkany Jingwen Chai Jianmin Mao Brett Truitt Timothy O'Toole Kanwen Wang Mary Hoffee Claudia Ordonez Richard Fitzpatrick John Flatley