Effective management of obstructive pulmonary diseases characterized by thickened, highly viscoelastic mucus represents a significant unmet medical need, especially in cystic fibrosis (CF) an inherited disease affecting approximately 30,000 individuals in the US. In CF, genetic dysfunction of the CFTR (Cystic Fibrosis Transmembrane Regulator) trans-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 death. New CFTR modulator therapies can restore a degree of CFTR functionality, but these are limited to certain CF genotypes, are prone to side-effects, and provide only marginal disease modification. Hence, large treatment gaps remain. Our approach is designed to target all CF patients via an inhaled enzyme-based treatment (ORP-100) that 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 efflux. ORP100 is an engineered variant of human thioredoxin, a selective and specific protein disulfide reductase that is a highly potent normalizer of mucin (mucus protein) disulfide bonds. It is hypothesized that low pH inactivates the thioredoxin substitute cofactor glutathione, thus interfering with regeneration of active thioredoxin in the CF airway. ORP-100 is delivered in a pre-activated form that does not depend on glutathione and has been engineered to bind covalently to mucin disulfide bonds. This both increases potency through prevention of disulfide re-formation and enhances safety via sequestration of the drug in the mucus layer. Our prior work has demonstrated that ORP-100 has a superior ability to normalize CF patient mucus/sputum viscoelasticity vs. native human thioredoxin, as well as the approved mucolytic drugs N-acetyl cysteine (NAC) and inhaled human DNase (Dornase alfa, Pulmozyme®). Most significantly, ORP-100 is twice as active as DNase in restoring mucociliary transport to CF sputum applied in situ to rat tracheae, a state of the art model for evaluating efficacy of mucus drugs. We have established a scalable and efficient ORP-100 manufacturing process in E. coli and initial storage and delivery formulations that confer oxidative stability. Acute aerosol inhalation of ORP-100 in rats showed no toxicity at 40 mg/kg, many times greater than the anticipated human dose, with potential anti-inflammatory effects observed in rodents in vivo as well as in vitro in primary human bronchial epithelia from CF donors. In Phase 1 of this Fast-Track project we will focus on establishment of sensitive bioanalytical assays for quantification of ORP-100 in matrices from rat and monkey, and development of aerosols to facilitate inhalation toxicology studies. A successful outcome will enable in Phase 2 the analysis of formulation stability, evaluation of ORP-100 compatibility with the eFlow nebulizer device, and 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.
Public Health Relevance Statement: PROJECT NARRATIVE The goal of the project is to develop a novel and potentially disease-modifying treatment for cystic fibrosis (CF), a chronic life-threatening inherited disease with great unmet therapeutic need that affects approximately 30,000 people in the US. We have shown that ORP-100, an improved version of a secreted human enzyme (thioredoxin), restores airway mucus fluidity and normal rates of transport/clearance to CF patient mucus secretions in situ. We are developing ORP-100 as an inhaled drug that can potentially treat all CF patients, and in the proposed research will be investigating repeated-dose toxicology in two species as a follow up to earlier acute aerosol studies that demonstrated safety in rats at a large multiple of the anticipated effective human clinical dose.
Project Terms: Active Sites; Acute; Adverse effects; Aerosols; Affect; analytical method; Animal Model; Anions; Antiinflammatory Effect; assay development; Autopsy; Bacterial Infections; base; Bicarbonates; Binding; Biological; Biological Assay; Blood; Breathing; bronchial epithelium; Cells; Cessation of life; Characteristics; Chronic; Clinical; Clinical Trials; clinically relevant; cofactor; Complement; cost effective; Coughing; Cysteine; Cystic Fibrosis; cystic fibrosis airway; cystic fibrosis patients; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Dehydration; Deoxyribonucleases; Deposition; design; Detection; Development; Devices; Discrimination; Disease; disulfide bond; Disulfides; Dose; Drug Kinetics; Engineering; Enzymes; Escherichia coli; Evaluation; extracellular; Fermentation; fluidity; follow-up; Formulation; Functional disorder; Genetic; Genotype; Glutathione; Goals; Human; Impairment; improved; In Situ; In Vitro; in vivo; Individual; Infection; Inflammation; Inhalation Toxicology; Inherited; Investigational New Drug Application; Life; Liquid Chromatography; Lung; Lung diseases; Maintenance; manufacturing process; Maximum Tolerated Dose; Mediating; Medical; Methodology; Methods; Modeling; Modification; Monkeys; Mucins; Mucociliary Clearance; Mucolytics; Mucous body substance; Natural regeneration; Nebulizer; nonhuman primate; novel; Outcome; Oxidoreductase; Particulate; Pathologic; Peptides; Pharmaceutical Preparations; Pharmacology; Phase; Prevention; Process; product development; Production; Proteins; Rattus; recombinant human DNase; Recovery; Reducing Agents; Research; respiratory; Respiratory physiology; Risk; Rodent; Safety; small molecule; Sputum; stability testing; standard of care; System; tandem mass spectrometry; Testing; Therapeutic; Time; Tissues; Toxic effect; Toxicology; Trachea; Tracheal Epithelium; TXN gene; Variant; viscoelasticity; Viscosity; Work
