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INO-4995 Therapeutic Efficacy Is Enhanced with Repeat Dosing in Cystic Fibrosis Knockout Mice and Human Epithelia

¹ Case Western Reserve University, Cleveland, Ohio; ² ISM Therapeutics, Seattle, Washington; ³ Washington University, St. Louis, Missouri; ⁴ University of North Carolina, Chapel Hill, North Carolina; ⁵ Department of Medicine, University of Washington, Seattle, Washington; and ⁶ Cancer Targeted Technology, Seattle, Washington

Correspondence and requests for reprints should be addressed to Alexis Traynor-Kaplan, Ph.D., ISM Therapeutics, 454 North 34th Street, Seattle, WA 98103. E-mail: alexis{at}ismtherapeutics.com

Progressive lung damage in cystic fibrosis (CF) has been linked to inadequate airway mucosal hydration. We previously demonstrated that an inositol tetrakisphosphate analog, 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl)ester (INO-4995), regulates airway secretory and absorptive processes, affecting mucosal hydration by prolonged (24 h) inhibition of Na⁺ and fluid absorption in CF human nasal epithelia (CFHNE). The objectives of this study were to further assess clinical potential of INO-4995 in CF through ascertaining in vivo activity in mice with CF, determining the effects of repeated administration on potency and determining cytoplasmic half-life. Uptake and metabolism of [³H]INO-4995 was monitored with HPLC to calculate intracellular half-life. INO-4995 was administered in vitro repeatedly over 4 to 8 days to CFHNE. Fluid absorption was assessed by blue dextran exclusion, and basal short-circuit current was measured in Ussing chambers. INO-4995 (1–100 µg/kg) was dosed intranasally either as a single dose or once per day over 4 days to gut-corrected CF mice. [³H]INO-4995 was rapidly taken up by epithelial cultures and converted to the active drug, which had a half-life of 40 hours. Repeated daily application of INO-4995 to CFHNE lowered the effective concentration for inhibition of fluid absorption and amiloride-sensitive short-circuit current in cultured CFHNE, and reduced nasal potential difference to nearly control levels in gut-corrected CF mice. Ca²⁺-activated Cl^– channel activity was also boosted in cultures. Mouse nasal levels fell from abnormal levels to within 2 µA of normal with repeated exposure to 0.8 µg/kg over 4 days. These data support further development of INO-4995 for the treatment of CF.

Key Words: cystic fibrosis • nasal potential difference • inositol polyphosphate

CLINICAL RELEVANCE This study demonstrates the clinical potential for a compound for the treatment of cystic fibrosis. Its effectiveness serves to validate the use of inositol-based therapeutics in drug development, and demonstrates the role of inositol tetrakisphosphates in airway physiology.