Nanoemulsion Formulation with BZK: The nanoemulsion formulation was prepared with 0.13% benzalkonium chloride (BZK) as the active ingredient.12 BZK, a quaternary ammonium compound, was chosen due to its inherent antimicrobial activity and is currently use in numerous nasal spray and skin antiseptic products. In NE-BZK, the BZK resides at the interface between the oil and water phases of the nanodroplets with the hydrophobic tail distributed in the oil core and the polar cationic head group residing at the oil-water interface.
Virus Strains Used for Demonstration of Antiviral Activity with NE-BZK: Broad spectrum antiviral activity of NE-BZK was tested using the following viral isolates: SARS-CoV-2 Victoria/1/2020 strain (Public Health England (PHE), Porton Down, Salisbury, UK); Human coronavirus 229E (ATCC: VR-740); Influenza B (VR-1931); and Respiratory Syncytial Virus (BlueWillow Biologics in-house strain: NBL-14-001-2UC). The virus growth media was either Minimum Essential Medium – Eagle with Earle's BSS (MEM Eagle EBSS) from Lonza (Rochester, NY) or Dulbecco’s Modified Eagle’s Medium (DMEM) from Corning Inc (Corning, NY). The cells used in the virus studies were obtained from ATTC (Manassas, VA): Vero E6 cells (ATCC# CRL 1586) for SARS-CoV2, MRC-5 cells for HCoV229E (CCL-171 ATCC), MDCK cells for Influenza B (CCL-34 ATCC) and Vero cells for RSV (CCL-81 ATCC).
In Vitro Determination of Antiviral Activity
Using the time kill procedures described in the Standard Guide ASTM E1052-11, the antiviral activity of the NE-BZK was assessed by inoculating the formulation with a suspension of viral particles (final concentration of 1.5-3.1 x106 PFU/ml).13 At a predetermined exposure time, an aliquot was taken and neutralized to remove residual effect of NE-BZK by diluting at 1:100 dilution in cell growth media containing 1-2% FBS. A comparative inactivation of HCoV 229E by NE-BZK and AQ-BZK was also evaluated by using the test samples at full strength, 1/10 dilution, and1/20 dilution.Virus control with virus alone and toxicity control with test sample alone was also included. Concentration of active virus particles was determined quantitively by plaque or TCID50 assay. TCID50 was calculated by the Karber method.14 Number of PFU recovered from the test sample was converted into log10 format and compared to an initial starting concentration to determine a log reduction.
Ex Vivo Persistence of Antiviral Activity on Human Cadaver Skin Following Application of NE-BZK
Cryopreserved, dermatomed human cadaver abdominal skin from Caucasian donors was obtained from Science Care organ donor bank (Phoenix, AZ). The permeation and retention of each antiseptic preparation in human skin was determined using the ex vivo permeation technique described by Franz.15 In brief, human skin was placed onto a Franz diffusion cell chamber and secured. The skin was maintained at a temperature and humidity that match typical in vivo conditions. Human skin was dosed with either the nanoemulsion antiseptic (NE-BZK) or aqueous BZK solution (AQ-BZK) applied at a single dose of 100 mL/cm2. At either 4 or 8 hours post topical application, 10µL of viral particles in suspension (final concentration of 1-3 x105 PFU/ml) was applied to the skin surface for a contact time of 20 minutes. The skin surface was washed with growth media, pooled and neutralized to remove residual effect of the test formulation by diluting at 1:100 in growth media. Concentration of virus particles was determined quantitively by plaque or TCID50 assay.
In Vivo Rabbit Safety and Toxicology Study
The safety of NE-BZK was evaluated in New Zealand white rabbits following bi-dose nasal swab application for two weeks. The exploratory study was performed at IITRI in accordance with protocols approved by the animal care and use committee. Following acclimation, the animals were randomly assigned to either naïve control or NE-BZK treatment groups with 2/sex in control group and 4/sex in NE-BZK group. The NE-BZK was applied to the inner nostril and nasal mucosa using a puritan foam tip. Two hours post last dose application and two days later, blood was collected by bleeding the central ear artery. Serum was analyzed by HPLC for BZK. The animals were sacrificed 2 days post last administration by an overdose of sodium pentobarbital (150-200 mg/kg by intravenous administration) and nasal cavity was examined macroscopically and scored for erythema and edema by Draize method of scoring for dermal irritation. 16
Analysis of Serum Samples for BZK
Rabbit serum samples collected at two hours and two days post NE-BZK application was analyzed using qualified reverse phase HPLC method. The samples were extracted with acetonitrile and filtered before injecting into column. The samples were run on Phenomenex Luna 5µ column, using 0.04M sodium acetate in acetonitrile as mobile phase. The samples were run at a flow rate of 2mL/ minute with 100µL injection volume. The peaks were detected using 254nm wavelength. The analytical assay detection limit was 0.094µg/mL.