Nitric Oxide-Releasing Macromolecule Exhibits Broad-Spectrum Antifungal Activity and Utility as a Topical Treatment for Superficial Fungal Infections.

Cutaneous and superficial fungal infections affecting the skin, nails, and hair of humans are caused primarily by dermatophytes of the genera Trichophyton and Epidermophyton or by yeasts of the genera Candida and Malassezia. Onychomycosis is a common fungal infection of the nail that frequently coexists with tinea pedis, the most prevalent mycotic skin infection. Efficacy rates for current topical onychomycosis therapies are hampered by low drug penetration across the nail plate, which is theoretically obviated with nitric oxide (NO)-based topical therapies. The Nitricil technology platform is comprised of polysiloxane-based macromolecules that stably release therapeutic levels of NO. In the reported studies, NVN1000, the lead candidate of the platform, was assessed for its spectrum of in vitro activity against a broad range of filamentous fungi and yeast species commonly associated with cutaneous fungal infections. Time-kill assays demonstrated that NVN1000 exhibited fungicidal activity as early as 4 h. Additionally, the penetration of several unique NVN1000 NO-releasing drug product formulations (gel, cream, and lacquer) was evaluated following a single topical application in an in vitro infected human nail assay, with all formulations showing similar inhibition of fungal growth. Repeated topical application in this model demonstrated that a lower-strength dose of NO could achieve the same efficacy as a higher-strength dose after 7 days. Together, these in vitro results demonstrate that NO-releasing treatments rapidly penetrate the nail plate and eradicate the fungal infection, representing promising novel topical therapies for the treatment of onychomycosis and other cutaneous fungal infections.

A Phase 2 Controlled Study of SB206, a Topical Nitric Oxide-Releasing Drug for Extragenital Wart Treatment.

BACKGROUND: Nitric oxide (NO), a free radical gas, is endogenously produced in human cells. In high concentration, NO neutralizes many disease-causing microbes. The topical investigational drug SB206 releases NO and has the potential to treat skin diseases caused by viruses. Genital warts (condyloma acuminata) are primarily caused by human papillomavirus (HPV) types 6 and 11. Available treatments have low tolerability and efficacy rates and are inconvenient for the patient. Genital warts can recur if HPV is incompletely eradicated during treatment. OBJECTIVE: Topical SB206 (berdazimer sodium plus carboxymethyl cellulose hydrogel) was assessed for tolerability, safety, and efficacy for up to12 weeks in patients with external genital and/or perianal warts (EGW/PAW) in a phase 2, double-blind, randomized, dose-escalation study. METHODS: Patients (N=108) were randomly assigned to SB206 or vehicle in a 3:1 ratio: SB206 4% once (QD) or twice daily (BID), 8% QD, 12% QD, or corresponding vehicle. Treatment duration was up to 84 days. The primary efficacy endpoint was complete clearance of baseline EGW/PAW at or before week 12. Pearson's Chi Square tests compared the efficacy of active vs vehicle treatments. Safety was assessed through adverse event and tolerability reports, physical examination findings, and clinical laboratory test results. RESULTS: In the Intent-to-Treat population, the percentage of patients with complete clearance of baseline EGW/PAW at or before week 12 was higher for SB206 groups than for vehicle groups, with the greatest difference between SB206 12% QD (33.3%; P=0.010) and vehicle QD (4.3%). CONCLUSION: Berdazimer sodium (SB206) plus hydrogel was efficacious and well tolerated in the treatment of EGW/PAW. J Drugs Dermatol. 2018;17(10):1100-1105.

A Phase 2, Controlled, Dose-Ranging Study of SB208, an Investigational Topical Nitric Oxide-Releasing Drug, for the Treatment of Tinea Pedis.

BACKGROUND: Tinea pedis, or athlete's foot, is a superficial, skin infection caused by dermatophytes. It is usually topically treated. Nitric oxide is endogenously produced in humans and has a variety of physiologic and antimicrobial properties. SB208 is a novel topical treatment comprising berdazimer sodium (a nitric oxide-storing macromolecule) and a hydrogel. Admixing these two components releases nitric oxide to the application site. METHODS: A phase 2, double-blind, randomized trial evaluated the safety and efficacy of 3 doses of SB208 (2%, 4%, and 16%) vs matching vehicle, administered once daily for 14 days, in subjects with culture-confirmed interdigital tinea pedis. The primary efficacy outcome was the proportion of subjects with negative fungal cultures at end of treatment (day 14). Secondary outcomes at days 14 and 42 were the proportion of subjects with mycological cure (negative potassium hydroxide wet mount skin test and culture), clinical cure (reduced signs and symptoms from baseline graded on a 4-point scale). Safety was monitored through physical examinations, adverse events, and hemoglobin and methemoglobin levels. Efficacy outcomes were analyzed using a two-sided Cochran-Mantel-Haenszel test for general association, stratified by site. RESULTS: At day 14, a higher proportion of patients had negative fungal cultures in the pooled SB208-treated group (62%; P=0.04) than the vehicle-treated group (43%). Of SB208 groups, the 4% group had higher incidence of negative fungal cultures vs the vehicle group (67.6% vs 42.9%; P=0.03). At day 42, pooled SB208-treated groups had significantly more mycological cure vs vehicle group (47% vs 31%, respectively; P=0.08), and clinical cure was maintained in 23% of pooled SB208-treated patients vs 14% of vehicle-treated patients. No safety concerns were reported. Adverse events were mild, not serious, and considered unrelated to study medications. CONCLUSIONS: Topical SB208 was effective and well tolerated in the treatment of tinea pedis. J Drugs Dermatol. 2018;17(8):888-893.

Efficacy and hazards of 425 nm oral cavity light dosing to inactivate SARS-CoV-2.

OBJECTIVE: Using a battery of preclinical tests to support development of a light-based treatment for COVID-19, establish a range of 425 nm light doses that are non-hazardous to the tissues of the oral cavity and assess whether a 425 nm light dose in this non-hazardous range can inactivate SARS-CoV-2 in artificial saliva. METHODS: The potential hazards to oral tissues associated with a range of acute 425 nm light doses were assessed using a battery of four preclinical tests: (1) cytotoxicity, using well-differentiated human large airway and buccal epithelial models; (2) toxicity to commensal oral bacteria, using a panel of model organisms; (3) light-induced histopathological changes, using ex vivo porcine esophageal tissue, and (4) thermal damage, by dosing the oropharynx of intact porcine head specimens. Then, 425 nm light doses established as non-hazardous using these tests were evaluated for their potential to inactivate SARS-CoV-2 in artificial saliva. RESULTS: A dose range was established at which 425 nm light is not cytotoxic in well-differentiated human large airway or buccal epithelial models, is not cytotoxic to a panel of commensal oral bacteria, does not induce histopathological damage in ex vivo porcine esophageal tissue, and does not induce thermal damage to the oropharynx of intact porcine head specimens. Using these tests, no hazards were observed for 425 nm light doses less than 63 J/cm2 delivered at irradiance less than 200 mW/cm2. A non-hazardous 425 nm light dose in this range (30 J/cm2 at 50 mW/cm2) was shown to inactivate SARS-CoV-2 in vitro in artificial saliva. CONCLUSION: Preclinical hazard assessments and SARS-CoV-2 inactivation efficacy testing were combined to guide the development of a 425 nm light-based treatment for COVID-19. CLINICAL SIGNIFICANCE: The process used here to evaluate the potential hazards associated with 425 nm acute light dosing of the oral cavity to treat COVID-19 can be extended to other wavelengths, anatomical targets, and therapeutic applications to accelerate the development of novel photomedicine treatments.

Visible blue light inactivates SARS-CoV-2 variants and inhibits Delta replication in differentiated human airway epithelia.

The emergence of SARS-CoV-2 variants that evade host immune responses has prolonged the COVID-19 pandemic. Thus, the development of an efficacious, variant-agnostic therapeutic for the treatment of early SARS-CoV-2 infection would help reduce global health and economic burdens. Visible light therapy has the potential to fill these gaps. In this study, visible blue light centered around 425 nm efficiently inactivated SARS-CoV-2 variants in cell-free suspensions and in a translationally relevant well-differentiated tissue model of the human large airway. Specifically, 425 nm light inactivated cell-free SARS-CoV-2 variants Alpha, Beta, Delta, Gamma, Lambda, and Omicron by up to 99.99% in a dose-dependent manner, while the monoclonal antibody bamlanivimab did not neutralize the Beta, Delta, and Gamma variants. Further, we observed that 425 nm light reduced virus binding to host ACE-2 receptor and limited viral entry to host cells in vitro . Further, the twice daily administration of 32 J/cm 2 of 425 nm light for three days reduced infectious SARS-CoV-2 Beta and Delta variants by >99.99% in human airway models when dosing began during the early stages of infection. In more established infections, logarithmic reductions of infectious Beta and Delta titers were observed using the same dosing regimen. Finally, we demonstrated that the 425 nm dosing regimen was well-tolerated by the large airway tissue model. Our results indicate that blue light therapy has the potential to lead to a well-tolerated and variant-agnostic countermeasure against COVID-19.

A randomized, controlled, feasibility study of RD-X19 in subjects with mild-to-moderate COVID-19 in the outpatient setting.

The RD-X19 is an investigational, handheld medical device precisely engineered to emit blue light through the oral cavity to target the oropharynx and surrounding tissues. At doses shown to be noncytotoxic in an in vitro three-dimensional human epithelial tissue model, the monochromatic visible light delivered by RD-X19 results in light-initiated expression of immune stimulating cytokines IL-1α and IL-1ß, with corresponding inhibition of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) replication. A single exposure of 425 nm blue light at 60 J/cm2 led to greater than 99% reductions against all SARS-CoV-2 strains tested in vitro, including the more transmissible (Alpha) and immune evasive (Beta) variants. These preclinical findings along with other studies led to a randomized, double-blind, sham-controlled early feasibility study using the investigational device as a treatment for outpatients with mild to moderate coronavirus disease 2019 (COVID-19). The study enrolled 31 subjects with a positive SARS-CoV-2 antigen test and at least two moderate COVID-19 signs and symptoms at baseline. Subjects were randomized 2:1 (RD-X19: sham) and treated twice daily for 4 days. Efficacy outcome measures included assessments of SARS-CoV-2 saliva viral load and clinical assessments of COVID-19. There were no local application site reactions and no device-related adverse events. At the end of the study (day 8), the mean change in log10 viral load was -3.29 for RD-X19 and -1.81 for sham, demonstrating a treatment benefit of -1.48 logs (95% confidence internal, -2.88 to -0.071, nominal p = 0.040). Among the clinical outcome measures, differences between RD-X19 and sham were also observed, with a 57-h reduction of median time to sustained resolution of COVID-19 signs and symptoms (log rank test, nominal p = 0.044).

Economic and clinical impact of a novel, light-based, at-home antiviral treatment on mild-to-moderate COVID-19.

OBJECTIVES: Antiviral treatments for early intervention in patients with mild-to-moderate COVID-19 are needed as a complement to vaccination. We sought to estimate the impact on COVID-19 cases, deaths, and direct healthcare costs over 12 months following introduction of a novel, antiviral treatment, RD-X19, a light-based, at-home intervention designed for the treatment of mild-to-moderate COVID-19 infection. METHODS: A time-dependent, state transition (semi-Markov) cohort model was developed to simulate infection progression in individuals with COVID-19 in 3 US states with varying levels of vaccine uptake (Alabama, North Carolina, and Massachusetts) and at the national level between 1 June 2020 and 31 May 2021. The hypothetical cohort of patients entering the model progressed through subsequent health states after infection. Costs were assigned to each health state. Number of infections/vaccinations per day were incorporated into the model. Simulations were run to estimate outcomes (cases by severity, deaths, and direct healthcare costs) at various levels of adoption of RD-X19 (5%, 10%, 25%) in eligible infected individuals at the state and national levels and across three levels of clinical benefit based on the results from an early feasibility study of RD-X19. The clinical benefit reflects a decline in the duration of symptomatic disease by 1.2, 2.4 (base case), and 3.6 days. RESULTS: In the base case analysis with 10% adoption, simulated infections/deaths/direct healthcare costs were reduced by 10,059/275/$69 million in Alabama, 21,092/545/$135 million in North Carolina, and 16,670/415/$102 million in Massachusetts over 12 months. At the national level, 10% adoption reduced total infections/deaths/direct healthcare costs by 686,722/17,748/$4.41 billion. CONCLUSION: At-home, antiviral treatment with RD-X19 or other interventions with similar efficacy that decrease both symptomatic days and transmission probabilities can be used in concert with vaccines to reduce COVID-19 cases, deaths, and direct healthcare costs.

Visible blue light inhibits infection and replication of SARS-CoV-2 at doses that are well-tolerated by human respiratory tissue.

The delivery of safe, visible wavelengths of light can be an effective, pathogen-agnostic, countermeasure that would expand the current portfolio of SARS-CoV-2 intervention strategies beyond the conventional approaches of vaccine, antibody, and antiviral therapeutics. Employing custom biological light units, that incorporate optically engineered light-emitting diode (LED) arrays, we harnessed monochromatic wavelengths of light for uniform delivery across biological surfaces. We demonstrated that primary 3D human tracheal/bronchial-derived epithelial tissues tolerated high doses of a narrow spectral band of visible light centered at a peak wavelength of 425 nm. We extended these studies to Vero E6 cells to understand how light may influence the viability of a mammalian cell line conventionally used for assaying SARS-CoV-2. The exposure of single-cell monolayers of Vero E6 cells to similar doses of 425 nm blue light resulted in viabilities that were dependent on dose and cell density. Doses of 425 nm blue light that are well-tolerated by Vero E6 cells also inhibited infection and replication of cell-associated SARS-CoV-2 by > 99% 24 h post-infection after a single five-minute light exposure. Moreover, the 425 nm blue light inactivated cell-free betacoronaviruses including SARS-CoV-1, MERS-CoV, and SARS-CoV-2 up to 99.99% in a dose-dependent manner. Importantly, clinically applicable doses of 425 nm blue light dramatically inhibited SARS-CoV-2 infection and replication in primary human 3D tracheal/bronchial tissue. Safe doses of visible light should be considered part of the strategic portfolio for the development of SARS-CoV-2 therapeutic countermeasures to mitigate coronavirus disease 2019 (COVID-19).

Reduced ischemia/reperfusion injury via glutathione-initiated nitric oxide-releasing dendrimers.

We report the therapeutic potential of S-nitroso-N-acetylpenicillamine-derivatized generation-4 polyamidoamine dendrimers (G4-SNAP) for reducing ischemia/reperfusion (I/R) injury in an isolated, perfused rat heart. The use of this dendrimer scaffold to deliver the nitrosothiol therapeutic did not inhibit NO donor activity as the required dose of G4-SNAP to minimize I/R injury (31nM corresponding to 2microM SNAP) was consistent with the optimum concentration of small molecule SNAP alone. An exploration of G4-SNAP NO release kinetics in the presence of physiologically relevant concentrations of glutathione (GSH) indicated enhanced NO release (t[NO]=1.28microM NO/mg) at 500microM GSH. Reperfusion experiments conducted with 500microM GSH further lowered the optimal therapeutic G4-SNAP dose to 230pM (i.e., 15nM SNAP). The unique combination of G4-SNAP dendrimer and glutathione trigger represents a novel strategy with possible clinical relevance toward salvaging ischemic tissue.

S-nitrosothiol-modified dendrimers as nitric oxide delivery vehicles.

The synthesis and characterization of two generation-4 polyamidoamine (PAMAM) dendrimers with S-nitrosothiol exteriors are reported. The hyperbranched macromolecules were modified with either N-acetyl-D, L-penicillamine (NAP) or N-acetyl-L-cysteine (NACys) and analyzed via 1H and 13C NMR, UV absorption spectroscopy, MALDI-TOF mass spectrometry, and size exclusion chromatography. Treatment of the dendritic thiols with nitrite solutions yielded the corresponding S-nitrosothiol nitric oxide (NO) donors (G4-SNAP, G4-NACysNO). Chemiluminescent NO detection demonstrated that the dendrimers were capable of storing approximately 2 micromol NO x mg (-1) when exposed to triggers of S-nitrosothiol decomposition (e.g., light and copper). The kinetics of NO release were found to be highly dependent on the structure of the nitrosothiol (i.e., tertiary vs primary) and exhibited similar NO release characteristics to classical small molecule nitrosothiols reported in the literature. As a demonstration of utility, the ability of G4-SNAP to inhibit thrombin-mediated platelet aggregation was assayed. At equivalent nitrosothiol concentrations (25 microM), the G4-SNAP dendrimer resulted in a 62% inhibition of platelet aggregation, compared to only 17% for the small molecule NO donor. The multivalent NO storage, the dendritic effects exerted on nitrosothiol stability and reactivity, and the utility of dendrimers as drug delivery vehicles highlight the potential of these constructs as clinically useful S-nitrosothiol-based therapeutics.

Influence of glutathione and its derivatives on fibrin polymerization.

A complex relationship exists between reduced, oxidized, and nitrosated glutathione (GSH, GSSG, and GSNO, respectively). Although previous studies have demonstrated S-nitrosoglutathione (GSNO) has potent antiplatelet efficacy, little work has examined the role of GSNO and related species on subsequent aspects of coagulation (e.g., fibrin polymerization). Herein, the effects of GSH, GSSG, and GSNO on the entire process of fibrin polymerization are described. Relative to normal fibrinogen, the addition of GSH, GSSG, or GSNO leads to prolonged lag times, slower rates of protofibril lateral aggregation and the formation of clots with lower final turbidities. Dose-dependent studies indicate the influence of GSH on fibrin formation is a function of both GSH and fibrinogen concentration. Studies with Aalpha251 recombinant fibrinogen (lacking alphaC regions) showed GSH had no influence on its polymerization, suggesting the glutathione species interact within the alphaC region of fibrinogen.

Cytotoxicity of polypropylenimine dendrimer conjugates on cultured endothelial cells.

The cytotoxicity and time-dependent membrane disruption by polypropylenimine dendrimer conjugates on cultured human umbilical vein endothelial cells (HUVEC) is reported. Fluorescently labeled derivatives of generation 5 polypropylenimine dendrimers were prepared via conversion of amines to acetamides or through the covalent attachment of high molecular weight poly(ethylene glycol) (PEG) chains. Direct interactions between the fluorescent dendrimer conjugates and HUVEC were monitored using confocal fluorescence microscopy to track dendrimer movement across the plasma membrane and the fluorescent staining of cell nuclei. Propidium iodide and lactate dehydrogenase cytotoxicity assays confirmed that chemical modification of the surface amines of the parental dendrimer to neutral acetamide or PEG functionalities eliminated their acute cytotoxicity. Cationic primary-amine-containing dendrimers demonstrated drastic time-dependent changes in the plasma membrane permeability and prominent cytotoxicity. However, complete removal of the primary amines or masking of the cationic surface via PEGylation decreased dendrimer cytotoxicity. Thus, preventing electrostatic interactions of dendrimers with cellular membranes apparently is a necessary step toward minimizing the toxicity of delivery vehicles to the endothelium.

Results of a Phase 2 Efficacy and Safety Study with SB204, an Investigational Topical Nitric Oxide-releasing Drug for the Treatment of Acne Vulgaris.

OBJECTIVE: To compare efficacy, tolerability, and safety of two concentrations of topical SB204 and vehicle twice daily for 12 weeks in the treatment of acne vulgaris. DESIGN: Randomized, double-blind, placebo-controlled, three-arm, Phase 2 study. SETTING: Dominican Republic, Panama, and Honduras. PARTICIPANTS: Subjects with acne, age 12 to 40, with 25 to 70 noninflammatory lesions, 20 to 40 inflammatory lesions, and a baseline Investigator's Global Assessment score of mild, moderate, or severe. MEASUREMENTS: The primary efficacy assessment was the absolute change in noninflammatory lesion counts. Other assessments included inflammatory lesion counts, success on dichotomized Investigator's Global Assessment, reported adverse events, physical examinations, laboratory testing, and tolerability. RESULTS: One hundred fifty-three subjects were randomized to vehicle (n=52), SB204 1% (n=51), or SB204 4% (n=50). When compared to vehicle, subjects treated with SB204 1% and SB204 4% had significantly greater mean percent reduction in noninflammatory lesions from baseline and subjects treated with SB204 4% had a significantly greater mean percent reduction in inflammatory lesion count from baseline at Week 12. There were no significant differences in the IGA success rates between groups. Both concentrations of SB204 were safe and well-tolerated. CONCLUSIONS: When compared to vehicle, both SB204 1% and SB204 4% significantly decreased the percentage of noninflammatory lesions and SB204 4% also significantly decreased the percentage of inflammatory lesions in subjects with acne vulgaris treated for 12 weeks. Treatment with SB204 1% and SB204 4% was safe and well-tolerated. Registry: clinicaltrials.gov (NCT01844752).

Bactericidal efficacy of nitric oxide-releasing silica nanoparticles.

The utility of nitric oxide (NO)-releasing silica nanoparticles as novel antibacterial agents is demonstrated against Pseudomonas aeruginosa. Nitric oxide-releasing nanoparticles were prepared via co-condensation of tetraalkoxysilane with aminoalkoxysilane modified with diazeniumdiolate NO donors, allowing for the storage of large NO payloads. Comparison of the bactericidal efficacy of the NO-releasing nanoparticles to 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO), a small molecule NO donor, demonstrated enhanced bactericidal efficacy of nanoparticle-derived NO and reduced cytotoxicity to healthy cells (mammalian fibroblasts). Confocal microscopy revealed that fluorescently labeled NO-releasing nanoparticles associated with the bacterial cells, providing rationale for the enhanced bactericidal efficacy of the nanoparticles. Intracellular NO concentrations were measurable when the NO was delivered from nanoparticles as opposed to PROLI/NO. Collectively, these results demonstrate the advantage of delivering NO via nanoparticles for antimicrobial applications.

Water-soluble nitric oxide-releasing gold nanoparticles.

The synthesis and characterization of water-soluble nitric oxide (NO)-releasing monolayer-protected gold clusters (MPCs) are reported. Tiopronin-protected MPCs ( approximately 3 nm) were functionalized with amine ligands and subsequently exposed to 5 atm of NO to form diazeniumdiolate NO donors covalently bound to the gold MPC. Diazeniumdiolate formation conditions, NO-release, and nanoparticle stability were examined as a function of the structure of the protecting ligand, pH, and storage time. Despite their aqueous solubility, proton-initiated decomposition of the diazeniumdiolate-modified Tio-MPCs resulted in only modest NO-release (<0.023 micromol/mg) for short durations (<1.5 h). To increase the NO storage capacity of gold nanoparticles, polyamine-stabilized MPCs ( approximately 5 nm) were synthesized with significantly enhanced NO-release properties (0.386 micromol/mg) and durations (up to 16 h). Transmission electron microscopy, thermogravimetric analysis, nuclear magnetic resonance spectroscopy, elemental analysis, and UV-vis spectroscopy were used to characterize both nanoparticle systems before and after NO exposure. The MPCs represent the smallest water-soluble NO-release nanoparticles to date (3-5 nm).

Dendrimers as a scaffold for nitric oxide release.

The preparation and characterization of nitric oxide (NO)-releasing dendrimer conjugates are reported. Generation 3 and 5 polypropylenimine dendrimers (DAB-Am-16 and DAB-Am-64) were modified at the exterior to impart different amine functionalities. The ability to store NO on a dendritic scaffold using N-diazeniumdiolate NO donors was examined via the reaction of primary amine, secondary amine, and amide functionalities with high pressures of NO (5 atm). The secondary amine dendrimer conjugates exhibited a high storage capacity for NO (up to 5.6 micromol NO/mg), greatly increasing the "payload" of released NO over existing macromolecular NO donors. The mechanism of diazeniumdiolate decomposition was proton initiated, generating NO spontaneously under physiological conditions (pH 7.4, 37 degrees C). The NO release durations (>16 h) observed for the secondary amine dendrimers were significantly longer compared to small molecule alkyl secondary amine diazeniumdiolates, thus illustrating a dendritic effect on NO release kinetics. The multivalent exterior of dendrimers allows for the future combination of NO donors and other functionalities on a single molecular scaffold, enabling diverse utility as NO storage/delivery systems.