Development of an aerosol intervention for COVID-19 disease: Tolerability of soluble ACE2 (APN01) administered via nebulizer

Abstract

As ACE2 is the critical SARS-CoV-2 receptor, we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung, and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here, after demonstrating in vitro neutralization of SARS-CoV-2 by APN01, and after obtaining preliminary evidence of its tolerability and preventive efficacy in a mouse model, we pursued development of an aerosol formulation. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers has now been initiated (NCT05065645), with subsequent Phase II testing planned for individuals with SARS-CoV-2 infection.

Conflict of interest statement

Gerald Wirnsberger and Sonja Holler and Romana Gugensberger were employed by Apeiron Biologics A.G. Apeiron supplied the APN01 for study. Josef M. Pettinger was a founder of Apeiron, is a current shareholder and inventor of APN01. David L. McCormick is a Section Editor for PLOS One. Other authors declare no competing interests.

Figures

Fig 1. APN01 neutralization of SARS-CoV-2 in the absence of cytotoxicity.

Serial dilutions of APN01 were prepared in assay medium (MEM supplemented with 2% fetal bovine serum and 50 μg/mL gentamicin) and a suspension of SARS-CoV-2 (USA-WA1/2020) was added to assess neutralization. For assessment of APN01 on viability, assay medium without virus was added. After one-hour incubation at 37°C, the dilutions were transferred to wells containing Vero E6 target cells (Multiplicity Of Infection, 0.001). Incubation was continued for four days and cell numbers were assessed with a neutral red endpoint. Cytopathic Effect (CPE) was calculated as the average optical density (OD) for replicate infected and treated wells divided by average control OD X 100 (expressed as a percentage). Viability was calculated as the average optical density for replicate uninfected and treated wells divided by average control OD X 100 (expressed as a percentage).

Fig 2. Intranasal APN01 protects from disease in a mouse-adapted SARS-CoV-2 respiratory infection model.

(A) Experimental outline for infection of male BALB/c mice (n = 5 for both groups) with SARS-CoV-2 (strain maVie16) and daily intranasal treatment with APN01 for five days. (B) Body weight, temperature and survival curves for infected BALB/c mice treated with vehicle control or APN01. Body weights and temperature were compared using mixed-effect analyses. Survival differences were analyzed using a Mantel-Cox test. (C) Lung tissue weight as assessed 5 days after infection of mice; data were analyzed with the Mann-Whitney test. Statistical significances are indicated by asterisks (p-value < 0.05: *; p-value < 0.001: ***).

Fig 3. APN01 binding to the SARS-CoV-2 RBD is not altered by nebulization.

Binding of increasing doses of APN01 to plate-immobilized RBD domain was assessed by ELISA. Curves depict pre-nebulization samples of APN01 (material remaining as liquid in the nebulizer cup), APN01 collected after nebulization (post-nebulization), and non-treated control APN01. For this experiment, APN01 was aerosolized at 2.5 mg/ml.

Fig 4. APN01 enzymatic activity is not altered by nebulization.

Three samples were tested as defined in the legend to Fig 3. Samples were diluted to APN01 concentrations of 25 ng/ml, 50 ng/ml, and 100 ng/ml and assayed for enzymatic function by cleavage of quenched fluorogenic MAPL-DNP substrate and subsequent measurement of fluorescence activity. Data are plotted as ΔRFU (change in relative fluorescence units) vs. time (min). One representative experiment out of 4 biological replicates is shown.