(389) The toxicology of ELD607, a novel immunomodulator of inflammatory responses in CF lungs

Background: Management of airway inflammation represents a vital aspect of CF treatment. However, beyond ibuprofen, there are no currently approved anti-inflammatory drugs to treat CF patients. Orai1 is a plasma membrane Ca²⁺ channel that regulates inflammation by controlling gene expression and cytokine secretion. We have generated ELD607, a fully optimized Orai1 inhibitory peptide. ELD607 is CFTR-mutation agnostic, proteolytically stable and has an IC₅₀ of 9 nM. ELD607 inhibits Orai1 in the lungs of neutrophilic mouse models to reduce pulmonary inflammatory responses. While we have shown efficacy of ELD607, the toxicological effects are also needed for advancement as a therapeutic. Here, we assess the safety and toxicology of ELD607.

Methods: To determine potential cytotoxicity, HEK293T and human primary bronchial epithelial cells were incubated with 100 µM ELD607 (approximately 10,000 times the IC₅₀) for 24 h and cell viability was measured with calcein-AM. The human Ether-à-go-go-Related gene (hERG) K⁺ channel plays a vital role in cardiac repolarization and inhibition of hERG is fatal. hERG activity in response to 100 µM ELD607 was measured using a patch clamp assay. 100 µM ELD607 was also screened against the Eurofins Safety 44 Panel. We next switched to an in vivo model to evaluate the safety of ELD607. C57Bl/6J mice were dosed with 10 mg/kg ELD607 (20 times the predicted therapeutic dose) intratracheally for 7 days. To determine the location of inhaled ELD607, mice were dosed with 1.0 mg/kg ELD607-labeled Tamra intratracheally and the lung was excised 1 h later. Whole lungs were fixed and imaged using light sheet microscopy. To analyze the pharmacokinetics of ELD607, mice were exposed to 0.5 mg/kg ELD607 intratracheally. Bronchoalveolar lavage (BAL) and blood were collected at timed intervals post-inhalation and samples were measured for ELD607 levels by HPLC. We are currently dosing nonhuman primates with ELD607 as nonhuman primates’ airways better mimic human airways.

Results: There was no change in cell viability when exposed to ELD607. Additionally, there was no change in the transepithelial electrical resistance, indicating that ELD607 does not change epithelial integrity and permeability. ELD607 did not have any effect on hERG activity and did not bind to any of the targets. After a 7-day dosing, mouse weight did not change nor were there histopathological changes in the lungs, heart, liver or kidneys. Light sheet microscopy revealed that ELD607-Tamra was found in the airway lumen (from the main bronchi to the alveoli), but was not detected outside of the airways. HPLC analysis revealed low levels of ELD607 were detected in the BAL with 20% of the initial dose found at 10 minutes and declining to 10% 2 h after dosing. Extremely low levels of ELD607 were found in the blood (0.2 % of initial dose) throughout the timed-interval collections. Results from the nonhuman primate studies will be discussed at the time of presentation.

Conclusions: ELD607 has no significant toxic effects in vitro and in vivo in small animal models, and may serve as a safe novel therapeutic for anti-inflammatory effects in CF patients.

Acknowledgements: Funded by the NIH/NIAID and the CFF.

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