Evaluation of Biologics ACE2/Ang(1–7) Encapsulated in Plant Cells for FDA Approval: Safety and Toxicology Studies

<b>Background/Objectives:</b> For several decades, protein drugs (biologics) made in cell cultures have been delivered as sterile injections, decreasing their affordability and patient preference. Angiotensin Converting Enzyme 2 (ACE2) gum is the first engineered human blood protein expressed in plant cells approved by the FDA without the need for purification and is a cold-chain and noninvasive drug delivery. This biologic is currently being evaluated in human clinical studies to debulk SARS-CoV-2 in the oral cavity to reduce coronavirus infection/transmission (NCT00543318). <b>Methods:</b> Chemistry, manufacturing, and control (CMC) studies for the ACE2/Ang(1-7) drug substances (DSs) and ACE2 gum drug product (DP) were conducted following USP guidelines. GLP-compliant toxicology studies were conducted on Sprague Dawley rats (<i>n</i> = 120; 15/sex/group) in four groups-placebo, low (1.6/1.0 mg), medium (3.2/2.0 mg), and high (8.3/5.0 mg) doses IP/kg/day. Oral gavage was performed twice daily for 14 days (the dosing phase) followed by the recovery phase (35 days). Plasma samples (<i>n</i> = 216) were analyzed for the product Ang(1-7) by ELISA. <b>Results:</b> The ACE2 protein was stable in the gum for at least up to 78 weeks. The toxicology study revealed the dose-related drug delivery to the plasma and increases in the AUC (56.6%) and Cmax (52.9%) after 28 high-dose gavages (95% C.I.), although this quantitation excludes exogenously delivered membrane-associated ACE2/Ang(1-7). Vital biomarkers and organs were not adversely affected despite the 10-fold higher absorption in the tissues, demonstrating the safety for the first in-human clinical trials of ACE2/Ang(1-7). The NOAEL observed in the rats was 2.5-7.5-fold higher than that of the anticipated efficacious therapeutic dose in humans for the treatment of cardiopulmonary disorders, and it was 314-fold higher than the NOAEL for topical delivery via chewing gum. <b>Conclusions:</b> This report lays the foundation for the regulatory process approval for noninvasive and affordable human biologic drugs bioencapsulated in plant cells.