(M1430-07-40) Developing Cyclodextrin-Complexed Edaravone – An Inhaled Repurposed Therapy for Idiopathic Pulmonary Fibrosis (IPF)

Purpose: Idiopathic pulmonary fibrosis (IPF) is a rare chronic devastating lung disorder leading to lung destruction and scarring. According to various sources, it has a prevalence of 13 to 20 per 100,000 people worldwide¹. In the United States (US), 30,000 to 40,000 new cases are diagnosed each year. In line with the recent scientific evidence, oxidative stress plays a crucial role in IPF progression. Lungs are exposed to more oxygen levels compared with any other organ in the human body. Exogenous oxidants and pollutants generate reactive oxygen species (ROS) and trigger oxidative stress and inflammatory pathways which eventually activate several pathological pathways to form fibrotic tissue in the lungs. Historically, it is also supported by the abnormal presence of ROS and oxidative stress-related markers in the serum of IPF patients. Although currently approved therapies slow the disease progression, there is a need for developing the new drugs to further improve the IPF patients’ quality of life^2,3,4. It is also to be noted that the pulmonary/inhalation route of drug delivery has more advantages including direct drug accumulation in the lungs, and minimal systemic side effects compared to other routes; particularly for the local treatment of lung disorders⁵. Based on the oxidative stress’ critical role in IPF pathophysiology, and the advantages of the inhalation route of administration, we were motivated to investigate an antioxidant via pulmonary route as IPF therapy. By utilizing drug repurposing strategy and scientific literature review, we have shortlisted Edaravone (Eda), an FDA-approved drug for amyloid lateral sclerosis (ALS) treatment. Since this drug (BCS-Class IV) has poor solubility and permeability, we are utilizing cyclodextrin (CD) complexation as our formulation strategy.

Methods: Edaravone cyclodextrin (Eda-CD) complex was formulated by applying the phase solubility studies as reported previously⁶. Sulfobutylether-β-cyclodextrin (SBECD) (0-200mM) and Eda (10 mg) were used for this study. The amount of Eda solubilized was quantified using the UPLC method. Further, the Eda-CD complex was characterized by Differential Scanning Calorimetry (DSC), X-ray diffraction (XRD), Nuclear Magnetic Resonance (NMR), and Fourier transform infrared (FTIR). Aerosolization ability of the complex was studied using Next-Generation Impactor (NGI). Release of the drug from the complex in the simulated lung fluid (SLF) and stability of the complex using accelerated stability conditions (75% RH (relative humidity), 40°C temperature) were also investigated. ROS scavenging activity of the plain drug and the complex was investigated using DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical scavenging assay. Further, the efficacy of the Eda-CD complex was evaluated by checking the protective activity of the Eda-Cd complex, and plain drug against hydrogen peroxide (H₂O₂) induced cell toxicity by cytotoxicity assay, caspase assay, and Superoxide dismutase (SOD) enzyme activity assay in A549 lung epithelial cell line.

Results: Based on phase solubility studies, an optimum CD concentration was found to be 100 mM which was able to complex 8 mg of Eda (Fig. 1). At this CD concentration, approximately a 3.2-fold solubility increase was observed compared with Eda in plain water without CD (which is 2.5 mg). Complexation of Eda inside the CD cavity was confirmed by DSC (disappearance of Eda melting point endotherm), XRD (disappearance of Eda crystalline peaks), NMR (chemical shift changes), and FTIR spectrum studies. From the in-vitro release study, >90% of Eda is released from the complex within the first 4 hours, whereas only 20% of the plain drug is recovered in the release medium at all time points (Fig 1). In addition, the complex also demonstrated good aerosolization behavior with Mass Median Aerodynamic Diameter (MMAD), Fine Particle Fraction (FPF), and Geometric standard deviation (GSD) of 3.4±0.2 μm, 83.3±3.2%, and 2.5±0.0 μm respectively (Fig 2). The prepared complex was also found to be stable in accelerated stability studies with 75%RH, 40°C. Both plain Eda and Eda-CD complex demonstrated concentration-dependent ROS scavenging activity from 6.25 to 50 μM concentration. Concentration-dependent protective activity is observed with both plain drug and complex from 5 μM to 30 μM concentration followed by no effect/ mild cytotoxic effect in H₂O₂ (400 μM) induced cytotoxicity in A549 cells (Fig. 3). Based on this observation, 30 μM (with the highest protective activity) concentration was used for further studies including caspase assay, and SOD assay (Fig. 3), and as expected, caspase enzyme levels (apoptotic enzymes) were reduced while SOD enzyme levels were increased in H₂O₂ + Eda and / H₂O₂ + Eda-CD treated groups compared to only H₂O₂ (400 μM) treated group.

Conclusion: From the results, it can be concluded that the optimized Eda-CD exhibited appropriate aerosolization, release, and stability. Eda-CD enhanced the in vitro anti-fibrotic efficacy of Eda. These findings suggest that an inhalable Eda-CD complex delivery system could be a potential and beneficial strategy for IPF treatment. While the results obtained from this study are promising, further in-vivo studies (for example, in Bleomycin induced rat IPF model) are required to comprehend the full spectrum of this approach’s feasibility including the molecular mechanisms.

References: 1. Idiopathic pulmonary fibrosis: MedlinePlus Genetics [Internet]. [[updated 2020 Jul 01, cited 2022 Apr 14]. Available from:
https://medlineplus.gov/genetics/condition/idiopathic-pulmonary-fibrosis/
2. Oldham JM, Vancheri C. Rethinking Idiopathic Pulmonary Fibrosis. Clinics in Chest Medicine. 2021;42(2):263–73.
3. Yue L, Shi Y, Su X, Ouyang L, Wang G, Ye T. Matrix metalloproteinases inhibitors in idiopathic pulmonary fibrosis: Medicinal chemistry
perspectives. European Journal of Medicinal Chemistry. 2021 Nov;224:113714.
4. Kliment CR, Oury TD. Oxidative stress, extracellular matrix targets, and idiopathic pulmonary fibrosis. Free Radical Biology and
Medicine. 2010 Sep 1;49(5):707–17
5. Borghardt JM, Kloft C, Sharma A. Inhaled Therapy in Respiratory Disease: The Complex Interplay of Pulmonary Kinetic Processes. Can
Respir J. 2018;2732017.
6. Wang, X., Parvathaneni, V., Shukla, S.K. et al. Cyclodextrin Complexation for Enhanced Stability and Non-invasive Pulmonary Delivery
of Resveratrol—Applications in Non-small Cell Lung Cancer Treatment. AAPS PharmSciTech 21, 183 (2020).

Acknowledgements: This work was supported by start-up funds to VG from the College of Pharmacy and Health Sciences (CPHS), St. John’s University. SS was supported by an assistantship from St. John’s University. All authors disclose no conflict of interest.


Phase solubility studies of Eda with CD. Improvement of Eda aqueous solubility with increasing CD concentrations until 100mM (Left side). In-vitro release profile of Eda-CD compared with plain Eda over a period of 8 hrs (>90% of Eda is released from the complex in the first 4 hrs.) (Right side). Data represent mean ± SD (n=3).


In-vitro aerodynamic performance of Eda-CD. % Cumulative deposition of Eda in Next-Generation Impactor (NGI) (Left side). In vitro aerodynamic deposition of Eda-CD at various stages in NGI (right side). Tabulated summary of aerosolization parameters obtained and calculated using NGI. Data represent mean ± SD (n=3).


Protective effect of Eda and Eda-CD on A549 cells, after treatments with Hydrogen peroxide (H2O2) at 400 μM concentration for 24 hr. followed by Eda and Eda-CD treatments for 24 hr. Eda and Eda-CD demonstrated protective activity against H2O2-induced cytotoxicity (Left side). Antioxidant enzyme SOD activity is reduced after treatment with H2O2 at 400 μM concentration. Upon treatments with Eda, Eda-CD at 30 μM concentrations, SOD levels were found to be restored to a good extent (Right side). Data represent mean ± SD (n=3)