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

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.

Development of a novel, pan-variant aerosol intervention for COVID-19.

To develop a universal strategy to block SARS-CoV-2 cellular entry and infection represents a central aim for effective COVID-19 therapy. The growing impact of emerging variants of concern increases the urgency for development of effective interventions. Since ACE2 is the critical SARS-CoV-2 receptor and all tested variants bind to ACE2, some even at much increased affinity (see accompanying paper), 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 we show that intranasal administration of APN01 in a mouse model of SARS-CoV-2 infection dramatically reduced weight loss and prevented animal death. 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 can now be initiated, with subsequent Phase II testing in individuals with SARS-CoV-2 infection. This strategy could be used to develop a viable and rapidly actionable therapy to prevent and treat COVID-19, against all current and future SARS-CoV-2 variants.

A robust protocol for regional evaluation of methacholine challenge in mouse models of allergic asthma using hyperpolarized 3He MRI.

Hyperpolarized (HP) (3)He magnetic resonance imaging has been recently used to produce high-resolution images of pulmonary ventilation after methacholine (MCh) challenge in mouse models of allergic inflammation. This capability presents an opportunity to gain new insights about these models and to more sensitively evaluate new drug treatments in the pre-clinical setting. In the current study, we present our initial experience using two-dimensional (2D), time-resolved (3)He MRI of MCh challenge-induced airways hyperreactivity (AHR) to compare ovalbumin-sensitized and challenged (N = 8) mice to controls (N = 8). Imaging demonstrated that ovalbumin-sensitized and challenged animals exhibited many large ventilation defects even prior to MCh challenge (four out of eight) compared to no defects in the control animals. Additionally, the ovalbumin-sensitized and challenged animals experienced a greater number of ventilation defects (4.5 +/- 0.4) following MCh infusion than did controls (3.3 +/- 0.6). However, due to variability in MCh delivery that was specific to the small animal MRI environment, the difference in mean defect number was not statistically significant. These findings are reviewed in detail and a comprehensive solution to the variability problem is presented that has greatly enhanced the magnitude and reproducibility of the MCh response. This has permitted us to develop a new imaging protocol consisting of a baseline 3D image, a time-resolved 2D series during MCh challenge, and a post-MCh 3D image that reveals persistent ventilation defects.

Ventilation-synchronous magnetic resonance microscopy of pulmonary structure and ventilation in mice.

Increasing use of transgenic animal models for pulmonary disease has raised the need for methods to assess pulmonary structure and function in a physiologically stable mouse. We report here an integrated protocol using magnetic resonance microscopy with gadolinium (Gd)-labeled starburst dendrimer (G6-1B4M-Gd, MW = 192 +/- 1 kDa, R(h) = 5.50 +/- 0.04 nm) and hyperpolarized (3)helium ((3)He) gas to acquire images that demonstrate pulmonary vasculature and ventilated airways in live mice (n = 9). Registered three-dimensional images of (1)H and (3)He were acquired during breath-hold at 2.0 T using radial acquisition (total acquisition time of 38 and 25 min, respectively). The macromolecular Gd-labeled dendrimer (a half-life of approximately 80 min) increased the signal-to-noise by 81 +/- 30% in the left ventricle, 43 +/- 22% in the lung periphery, and -4 +/- 5% in the chest wall, thus increasing the contrast of these structures relative to the less vascular surrounding tissues. A constant-flow ventilator was developed for the mouse to deliver varied gas mixtures of O(2) and N(2) (or (3)He) during imaging. To avoid hypoxemia, instrumental dead space was minimized and corrections were made to tidal volume lost due to gas compression. The stability of the physiologic support was assessed by the lack of spontaneous breathing and maintenance of a constant heart rate. We were able to stabilize the mouse for >8 hr using ventilation of 105 breath/min and approximately 0.2 mL/breath. The feasibility of acquiring both pulmonary vasculature and ventilated airways was demonstrated in the mouse lung with in-plane spatial resolution of 70 x 70 microm(2) and slice thickness of 800 microm.

Dynamic lung morphology of methacholine-induced heterogeneous bronchoconstriction.

Hyperpolarized (HP) 3helium (3He) dynamic MRI was used to investigate airway response in rats following intravenous (i.v.) bolus administration of a contractile agent, methacholine (MCh). The method provides direct visualization of the ventilated regions within the lung. Heterogeneous bronchoconstriction following the i.v. MCh injection was evident using this technique. These 3He dynamic lung images revealed that the inspired fresh air was shunted to the less-constricted regions after the MCh challenge in a similar manner as described by Laplace's relationship for the stability between adjacent alveoli. The airways in the more-constricted regions became nearly closed, resulting in air trapping, while the airways in the less-constricted regions remained effectively open, leading to overinflation. These data suggest a lung model of airway constriction partitioned into ventilated and nonventilated regions. These nonventilated regions are heterogeneously distributed in the lung and this distribution cannot be deduced from spirometric measurement of the whole lung. We demonstrate that a combination of functional 3He images and anatomical 1H images provide an effective method to diagnose regional lung abnormalities in rats.

Measurement of regional lung function in rats using hyperpolarized 3helium dynamic MRI.

Dynamic regional lung function was investigated in rats using a radial acquisition cine (RA-CINE) pulse sequence together with hyperpolarized (HP) (3)He gas delivered by a constant flow ventilator. Based on regional differences in the behavior of inspired air, the lung was conceptually divided into two regions (the major airways and the peripheral airspace) for purposes of functional analysis. To measure regional function in the major airways, a large RF flip angle (24 degrees) was applied to reduce (3)He magnetization in the peripheral airspace, and signal intensity (SI) was normalized with the projected airway diameter to estimate local airflow. Higher normalized signal intensity was observed in the left branch airway as compared to the right branch airway. To determine regional function in the peripheral airspace, a small RF flip angle (6 degrees) was used. Incremental increases of peripheral SI in successive lung images were consistent with the increase in lung volume. A new "skipping" scanning strategy using dummy frames allows a trade-off between the number of frames acquired for dynamic information, the RF flip angle, and the penetration depth of (3)He magnetization into the lung. This work provides a novel approach to simultaneously assess dynamic regional function and morphology.