A Treatment to Eliminate SARS-CoV-2 Replication in Human Airway Epithelial Cells Is Safe for Inhalation as an Aerosol in Healthy Human Subjects

Abstract

Background: Low airway surface pH is associated with many airway diseases, impairs antimicrobial host defense, and worsens airway inflammation. Inhaled Optate is designed to safely raise airway surface pH and is well tolerated in humans. Raising intracellular pH partially prevents activation of SARS-CoV-2 in primary normal human airway epithelial (NHAE) cells, decreasing viral replication by several mechanisms.

Methods: We grew primary NHAE cells from healthy subjects, infected them with SARS-CoV-2 (isolate USA-WA1/2020), and used clinical Optate at concentrations used in humans in vivo to determine whether Optate would prevent viral infection and replication. Cells were pretreated with Optate or placebo prior to infection (multiplicity of infection = 1), and viral replication was determined with plaque assay and nucleocapsid (N) protein levels. Healthy human subjects also inhaled Optate as part of a Phase 2a safety trial.

Results: Optate almost completely prevented viral replication at each time point between 24 h and 120 h, relative to placebo, on both plaque assay and N protein expression (P < .001). Mechanistically, Optate inhibited expression of major endosomal trafficking genes and raised NHAE intracellular pH. Optate had no effect on NHAE cell viability at any time point. Inhaled Optate was well tolerated in 10 normal subjects, with no change in lung function, vital signs, or oxygenation.

Conclusions: Inhaled Optate may be well suited for a clinical trial in patients with pulmonary SARS-CoV-2 infection. However, it is vitally important for patient safety that formulations designed for inhalation with regard to pH, isotonicity, and osmolality be used. An inhalational treatment that safely prevents SARS-CoV-2 viral replication could be helpful for treating patients with pulmonary SARS-CoV-2 infection.

Keywords: COVID-19; SARS-CoV-2; airway pH.

Fig. 1.

Optate raises intracellular pH of NHAE cells. (A) Optate raised intracellular pH. NHAE cells were loaded with BCECF-AM, which increases in fluorescence intensity as pH increases across the physiological range (top), or pHrodo Red, which decreases in fluorescence intensity as pH increases (bottom). Cells were imaged before or after treatment. (B) Fluorescence intensity increased in cells loaded with BCECF-AM and decreased in cells loaded with pHrodo Red (4 each, P = .039 and P = .001, respectively). As a control, we treated cells with ZnCl², which acidifies the intracellular space by inhibiting voltage-gated proton channel Hv121. Zinc had an effect opposite Optate (3 and 5, P = .036 and P = .001, respectively). Center lines denote means and whiskers show standard error. NHAE = normal human airway epithelial; BCECF-AM = 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester.

Fig. 2.

Optate decreases SARS-CoV-2 viral replication in Vero E6 and NHAE cells. (A) PFUs were measured in culture media from control and Optate-treated Vero E6 cells infected with SARS-CoV-2. Optate ablated viral infection in the Vero E6 cells (3 experiments, 2 replicates each, P < .001). (B) Control and Optate-treated primary NHAE cell cultures were then infected with SARS-CoV-2, and PFUs in culture media were analyzed under similar conditions for 120 h, starting from 24 h after infection. After infection was established (24 h), viral infection was ablated in the Optate-treated cells (3 experiments using cells from 2 subjects, 2 replicates each, P < .001). (C, D) N protein expression was then studied in cell lysates from NHAE cells described in (B). SARS-CoV-2 N protein expression, normalized to β-actin, was almost completely suppressed at time points after 24 h (P < .001). Center lines or points denote means, and whiskers show standard error. *P < .01, **P < .001. NHAE = normal human airway epithelial; PFU = plaque-forming units.

Fig. 3.

Optate alters endocytosis and endosomal trafficking genes in NHAE cells. RNA sequencing results were compared between control and Optate-treated NHAE cells. Genes with significant differences between the groups are presented in a volcano plot. Genes decreased in the Optate-treated group are negative (to the left), and those that increase are positive (to the right). Downregulated genes for endocytosis and endosomal trafficking are labeled in green. Genes in blue have a Bonferroni-corrected P < .001 and the absolute log_(FC) < 1.5; genes in magenta have a Bonferroni-corrected P < .001 and the absolute log_(FC) > 1.5. Genes in black are below the significance threshold of .001. NHAE = normal human airway epithelial.

Fig. 4.

Optate safely is well tolerated in the human airway in vitro and in vivo. (A) Human subjects had no significant change in FEV₁ (P = .84), FVC (P = .99), heart rate (P = .42), or ${\text{S}}_{{\text{pO}}_2}$ (P = .99) after Optate inhalation (n = 10 normal volunteers). (B) NHAE cells exposed to Optate for 5 d did not differ from the control group in cytotoxicity evaluated with lactate dehydrogenase (LDH) levels (top) or percentage viability determined with Trypan Blue exclusion (bottom). Center lines or points denote means, and whiskers show standard error. NHAE = normal human airway epithelial.