Development of modified human thioredoxin as an inhaled biologic therapeutic for cystic fibrosis

SUMMARY Effective management of obstructive pulmonary diseases characterized by thickenedhighly viscoelastic mucus represents a significant unmet medical needespecially in cystic fibrosisCFan inherited disease affecting approximatelyindividuals in the USIn CFgenetic dysfunction of the CFTRCystic Fibrosis Transmembrane Regulatortrans membrane channel results in impaired mucociliary transport and defective mucus clearance that drives a cycle of chronic infection and inflammation leading to impaired lung function and deathNew CFTR modulator therapies can restore a degree of CFTR functionalitybut these are limited to certain CF genotypesare prone to side effectsand provide only marginal disease modificationHencelarge treatment gaps remainOur approach is designed to target all CF patients via an inhaled enzyme based treatmentORPthat restores a native airway mechanism for maintenance of normal mucus protein viscosity and stiffness that is lost when airways become acidified due to loss of CFTR mediated bicarbonate effluxORPis an engineered variant of human thioredoxina selective and specific protein disulfide reductase that is a highly potent normalizer of mucinmucus proteindisulfide bondsIt is hypothesized that low pH inactivates the thioredoxin substitute cofactor glutathionethus interfering with regeneration of active thioredoxin in the CF airwayORPis delivered in a pre activated form that does not depend on glutathione and has been engineered to bind covalently to mucin disulfide bondsThis both increases potency through prevention of disulfide re formation and enhances safety via sequestration of the drug in the mucus layerOur prior work has demonstrated that ORPhas a superior ability to normalize CF patient mucus sputum viscoelasticity vsnative human thioredoxinas well as the approved mucolytic drugs N acetyl cysteineNACand inhaled human DNaseDornase alfaPulmozymeMost significantlyORPis twice as active as DNase in restoring mucociliary transport to CF sputum applied in situ to rat tracheaea state of the art model for evaluating efficacy of mucus drugsWe have established a scalable and efficient ORPmanufacturing process in Ecoli and initial storage and delivery formulations that confer oxidative stabilityAcute aerosol inhalation of ORPin rats showed no toxicity atmg kgmany times greater than the anticipated human dosewith potential anti inflammatory effects observed in rodents in vivo as well as in vitro in primary human bronchial epithelia from CF donorsIn Phaseof this Fast Track project we will focus on establishment of sensitive bioanalytical assays for quantification of ORPin matrices from rat and monkeyand development of aerosols to facilitate inhalation toxicology studiesA successful outcome will enable in Phasethe analysis of formulation stabilityevaluation of ORPcompatibility with the eFlow nebulizer deviceand repeat dose inhalation studies in rat and non human primate species to provide dose proportionality and toxicology data as essential prerequisites to IND enabling GLP toxicology and subsequent human clinical trials