Title: Novel Formulations Containing EC16 for Future Antiviral and Virucidal Products

Abstract:

Background: Norovirus is the world-leading cause of acute gastroenteritis associated with severe symptoms and deaths. However, vaccines against norovirus are currently not available, and medications that specifically target human norovirus infection are still under development. Surface disinfectants with activity against norovirus often contain toxic chemicals such as chlorine-based compounds (e.g., hypochlorite/bleach, ammonium chloride), acids, hydrogen peroxide, or a combination of these chemicals. However, these chemicals are not suitable for hand hygiene products to help prevent norovirus outbreaks or certain healthcare associated infections. The current studies evaluated the virucidal and antiviral activities of epigallocatechin-3-gallate-palmitate (EC16), a compound derived from green tea polyphenols, against murine norovirus (MNV S99, a surrogate for human norovirus) and other microorganisms.

Method: Initially, formulation suitability tests were conducted to compare EGCG (epigallocatechin-3-gallate), EC16 and tea polyphenol-palmitate in alcohol solution and hand hygiene formulations. The virucidal activity of EC16 was then tested in hand sanitizer gel and hand sanitizer foam formulations using a TCID50 time-kill suspension assay. The hand sanitizer gel prototype was also tested according to the EN13727:2012 (S. aureus and P. aeruginosa), EN13624 (Candida albicans), and EN 14476:2019 (norovirus) protocols. The surface disinfectant spray prototype was tested against S. aureus, P. aeruginosa, and S. enterica according to the AOAC Official Method 961.02, and against feline calicivirus according to ASTM test method E1053-20. The prototypes were also tested against spores of Bacillus cereus and Clostridium sporogenes using time-kill suspension tests. The In vitro treatment and prevention tests were performed using a 1-hour incubation of EC16 or EGCG with RAW264.7 cells, either pre-infection or post-infection with MNV.

Results: Unlike EC16, both EGCG and tea polyphenol-palmitate showed auto-oxidation (color change) and precipitation in alcohol solution and hand hygiene formulations, and were thus less suitable for potential hand hygiene products or new drug development. The time-kill suspension test results demonstrated that EC16 in both sanitizer gel and foam formulations reduced MNV by >99.99% (>log10 4) after 60 sec direct contact. The hand sanitizer gel showed activities against S. aureus, P. aeruginosa, C. albicans, and murine norovirus with a >log₁₀ 7.4 reduction for bacteria, >log₁₀ 6.4 for the yeast, and >log₁₀ 5.5 for murine norovirus. The surface disinfectant spray tests passed EPA required standards with all species tested. The sporicidal activities of similar formulations showed >log₁₀ 5 reductions against two bacterial spores. Without alcohol, one-hour incubation of EC16 with RAW264.7 cells either before or after MNV infection (i.e., without direct contact with MNV), resulted in >99% (>log10 2) reduction of MNV infectivity.

Conclusion: EC16 is a candidate for use as a virucidal and antiviral compound to prevent and treat norovirus infection, with potential to be developed as a new drug against norovirus, pending in vivo and clinical tests.The hand sanitizer and surface disinfectant formulations containing EC16 have the potential to be further developed into novel hand hygiene and surface disinfectant products with bactericidal, fungicidal, virucidal, and sporicidal activities, pending additional studies and tests.

Biography:

Dr. Stephen Hsu earned a bachelor’s degree from Wuhan University, China, a Master of Arts degree from Montclair State University and a PhD. degree from the University of Cincinnati College of Medicine. He spent four years at Memorial Sloan-Kettering Cancer Center before a career change.  During the four years as a sports anchor for ESPN International, Dr. Hsu taught at the National University of Singapore. Dr. Hsu joined Augusta University in 1999 and serves as Course Director for Nutrition and for Biochemistry.  His research has been supported by the NIH and other agencies, with more than 80 research articles published