A Non-Prescription Nitric Oxide Releasing Gel Demonstrates In Vitro Anti-fungal Activity and Supports Nail Clearance in Patients with Onychomycosis
Authors: Regev Gilly , Martins James , Miller Chris 
Published: November 2024

Background: Onychomycosis is a common fungal nail infection. Nitric oxide has broad-spectrum antifungal activity; NO releasing formulations are in development to support nail wellness.  
Objective: to examine antifungal properties of NO releasing formulations liquid (NORS) and gel (NORS-gel) to support nail clearance in onychomycosis. 
Methods:
NORS In Vitro antifungal testing: Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were used to compare NORS with efinaconazole. Trichophyton tonsurans, Trichophyton mentagrophytes, and Epidermophyton floccosum were selected and MFC was performed. Cidal activity: ≥ 99.9% reduction in CFUs/mL from baseline; Static activity: <99.9% reduction from baseline CFU/mL. (CFU: colony forming units).
NORS keratin binding: Six tubes were prepared with 1 mL + 107 conidia/mL of T. mentagrophytes strain (37035) + sterilized cadaver nail; six treatment tubes included: NORS, NORS+Keratin (50mg/mL), Terbinafine (0.002 µg/mL)+keratin (50 mg/mL), Terbinafine (0.002 µg/mL), Growth control (T. mentagrophytes+saline). After 15-min, tubes were centrifuged, supernatant decanted, and pellets resuspended in sterile saline; 10-fold serial dilutions were plated (triplicate).
Ex-Vivo in Onychomycosis: Toenail samples from patients with suspected onychomycosis were shredded and either placed onto agar plates incubated at 30°C for 1-month, or, treated with 1.5mL NORS-gel to 50mL tube, placing the clippings onto the gel and then overlaying another 1.5mL NORS-gel, samples were stored in the dark at room temperature for 8-hours, washed, dried, plated and incubated at 25°Cx7days
Nail penetration: Underside of cadaver toenails were inoculated with T. mentagrophytes 107 conidia/mL and placed on inert gel agar. A bead of NORS-gel (approx. 0.125mL), control gel and Terbinafine 0.002 µg/mL were spread to cover the topside of the nail. Covered petri dishes were placed for 15-minutes and 8-hours; the nail was removed, washed 3x in sterile saline, and pulverized; serial dilutions with plated samples incubated at 25°Cx7days. Statistics: Log CFU ± standard deviation, analyzed by t-test, p ≤ 0.05.
Human Volunteers: Three individuals with confirmed onychomycosis applied daily NORS-gel to infected nails (covered overnight), for one month. Photographs were taken at baseline, day 1 after application, and 4-5 months post-application.  Volunteers self-reported nail appearance.  
 Results:
NORS In Vitro antifungal testing: NORS showed potent antifungal activity against dermatophytes, including those less susceptible to efinaconazole.
NORS keratin binding: NORS with and without keratin demonstrated the lowest fungal burdens with average log CFUs/mL ± SD of 0 ± 0, and 2.45 ± 0.5, respectively, demonstrating significant activity vs control, and terbinafine (P-values < 0.0001). 
Ex Vivo: NORS-gel demonstrated significant fungicidal activity as indicated by the absence of fungal growth on petri dishes. 
Nail penetration: NORS-gel treated nail showed no fungal growth (average log CFUs/g ± SD of 0 ± 0) vs.  terbinafine–burden of 3.58 ± 0.2 average log CFU/g, at 8 hrs. NORS-gel significantly reduced fungal burden vs. terbinafine and control (P-value < 0.05).  (preliminary results; methodology undergoing refinement.) 
Human Volunteers: Daily application of NORS-gel for one-month improved nail clarity in volunteers with confirmed onychomycosis.  Temporary discoloration was observed during treatment.
Conclusions:  NORS and NORS-gel demonstrated antifungal activity; NORS-gel supported nail clearance and was well tolerated in individuals with onychomycosis.