Updates on Nitric Oxide As a Topical Anti-infective
NO continues to show promise as a safe and effective topical treatment candidate for acne and other infectious dermatoses.
Adam Friedman, MD is Associate Professor of Dermatology, Residency Program Director, and Director of Translational Research in the Department of Dermatology at George Washington School of Medicine and Health Sciences in Washington, DC. Among his multiple research initiatives, he has investigated nanotechnology and nitric oxide. Co-chair of the Scientific Advisory Board at Nano Biomed, Inc., Dr. Friedman discusses the status of NO research to date.
What does the latest research regarding NO and acne show?
Adam Friedman, MD: Nitric oxide (NO) is a potent bioactive diatomic gaseous molecule derived from diet as well as from endogenous enzymatic sources that exhibits broad spectrum antimicrobial activity and immunomodulatory properties as well as numerous signaling functions. Why is this so important? Antibiotic agents that exert multiple mechanisms of antimicrobial action limit pathogens’ ability to develop resistance; such drugs are advantageous for this reason. In thinking of acne vulgaris, a disease for which dermatologists frequently use antibacterial dosing of antibiotics for extended time periods even in the wake of emerging and established multi-drug resistant organisms, identifying active ingredients to which bacteria cannot develop resistance is paramount. The risk of bacterial resistance to both innate production and exogenous delivery of NO is minimized because NO exhibits multiple mechanisms of antimicrobial action both by inhibiting cell growth and by directly killing the pathogen.
It has been well known for some time that the bacterium P. acnes that is associated with acne vulgaris is highly sensitive to nitric oxide. However, this is only a small piece of the puzzle. We now know that there is an inappropriate immune response to this organism, and possibly other bystander stimuli, that contribute to the onset of this exceedingly common skin disease. It is actually through this inflammatory response that the tetracycline class of antibiotics is imparting its clinical benefit. Initiation of the first line of defense against microbes by the innate immune system is often through recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Although these pathways combat infection and prevent foreign invasion, they also result in inflammation and tissue injury. The inflammatory response to PAMPs and DAMPs is mediated by the inflammasome, a caspase 1–activating cytoplasmic complex that induces the secretion of crucial proinflammatory cytokines.
The exact mechanism by which P. acnes exerts its proinflammatory activity has been somewhat unclear, though P. acnes–induced inflammation has been shown to be mediated by proinflammatory cytokines tumor necrosis factor α, IL-1, IL-6, IL-8, and IL-12. Recently it was found that P. acnes is a potent trigger of IL-1β generation via activation of a specific inflammasome, the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome (J Invest Dermatol 134:381–8). We therefore sought to better understand if and how nitric oxide can influence this inflammasome, and found that nitric oxide inhibits multiple elements of this inflammasome resulting in inhibition of multiple pro-inflammatory cytokines of P. acnes stimulated keratinocytes and monocytes (J Invest Dermatol. 2015; 135: 2723–2731).
What is still unclear is through what mechanism NO exerts this robust anti-inflammatory activity. It is likely both NO itself as well as NO-derived products, such as those generated through S-nitrosylation. S-nitrosylation is the covalent modification of a protein or peptide cysteine thiol by an NO group to generate an S-nitrosothiol (SNO) (e.g. nitrosoglutathione). In fact, both NO and S-nitrosothiols impact the inflammatory cascade. We found that NO generated from our nanoparticle platform did not suppress gene expression of the receptor of this inflammasome, rather it impacted its activity which supports the nitrosation theory. Therefore, a technology that can both generate and release NO as well as facilitate S-nitrosylation is ideal. This nanoparticle platform does both most likely through the production of a dinitrogen trioxide intermediate that is a potent S-nitrosating agent even in the absence of oxygen.
Table 1. Disease Targets for NO
Multi-drug resistant bacterial and fungal skin and soft tissue infections:
Martinez, L, Han, G, Chacko M, Mihu, MR, Jacobson, M, Gialanella, P, Friedman, A, Nosanchuk, J, Friedman, JM. Antimicrobial and Healing Efficacy of Sustained Release Nitric Oxide Nanoparticles against Staphylococcus aureus Skin Infections. J Invest. Dermatol. 2009: 129; 2463–2469.
Han, G, Martinez, LM, Mihu, MR, Friedman, A, Friedman, JM, Nosanchuk, J. Nitric Oxide Releasing Nanoparticles are Therapeutic for Staphylococcus aureus Abscesses in Murine Model of Infection. PLoS ONE 2009; 4(11): e7804.
Mihu MR, Sandkovsky U, Han G, Friedman JM, Nosanchuk JD, Martinez LR. The use of nitric oxide releasing nanoparticles as a treatment against Acinetobacter baumannii in wound infections. Virulence. 2010;1(2):62-7.
Friedman, A, Blecher, K, Sanchez, D, Tuckman-Vernon, C, Gialanella, P, Friedman, JM, Martinez, L, Nosanchuk, J. Susceptibility of Gram Positive and Negative Bacteria to Novel Nitric Oxide-Releasing Nanoparticle Technology. Virulence. 2011; 2(3). 217-21.
Friedman, A, Blecher, K, Schairer, D, Tuckman-Vernon, C, Nacharaju, P, Sanchez, D, Gialanelll, P, Martinez, L, Friedman, J, and Nosanchuk, J. Improved Antimicrobial Efficacy with Nitric Oxide Releasing Nanoparticle Generated S-Nitrosoglutathione. Nitric Oxide. 2011; 25: 381-386.
Schairer, D, Martinez, L, Blecher, K, Chouake, J, Nacharaju, P Gialanella, P, Friedman J, Nosanchuk, J, Friedman, A. Nitric Oxide Nanoparticles: Pre-clinical Utility as a Therapeutic for Intramuscular Abscesses. Virulence 2012; 3(1):1-6.
Chouake, J, Schairer, D, Kutner, A, Sanchez, D, Makdisi, J, Blecher-Paz, K, Nacharaju, P, Tuckman-vernon, C, Gialanella, P, Friedman, JM, Nosanchuk, JD, Friedman, A. Nitrosoglutathione Generating Nitric Oxide Nanoparticles as an Improved Strategy for Combating Pseudomonas aeruginosa – Infected Wounds. J Drugs Dermatol. 2012; 11(12):1471-1477.
Marcherla, C, Sanchez, DA, Ahmadi, M, Vellozi, EM, Friedman, AJ, Nosanchuk, JD, Martinez, LR. Nitric Oxide Releasing Nanoparticles for the Treatment of Candida albicans Burn Infections. Front. Microbio. 2012;3:193. [Epub 2012 Jun 8.]
Mordorski B, Pelgrift R, Krausz A, Adler B, Batista A, Liang H, Gunther L, Clendaniel A, Harper S, Friedman JM, Nosanchuk JD, Nacharaju P, Friedman AJ. S-nitrosocaptopril Nanoparticles as Nitric Oxide-Liberating and Transnitrosylating Anti-infective Technology. Nanomedicine. 2015; 11(2): 283-291.
Landriscina A, Rosen J, Blecher-Paz K, Long L, Ghannoum M, Nosanchuk J, Friedman AJ. Nitric oxide-releasing nanoparticles as a treatment for cutaneous dermatophyte infections. Sci Lett 2015, 4: 193
Han G, Nguyen LN, Macherla C, Chi Y, Friedman JM, Nosanchuk JD, Martinez LR. Nitric oxide-releasing nanoparticles accelerate wound healing by promoting fibroblast migration and collagen deposition. Am J Pathol. 2012 Apr;180(4):1465-73.
Blecher, K Martinez, LR, Tuckman-Vernon, C, Nacharaju, P, Schairer, D, Chouake, J, Friedman, JM, Alfieri, A, Guha, C Nosanchuk, JD, Friedman, A. Nitric oxide-releasing nanoparticles accelerate wound healing in NOD-SCID mice. Nanomedicine. 2012;8(8):1364-71.
Han, G, Tar, M, Dwarakka, K, Friedman, A, Melman, A, Friedman, JM, Davies, KP. Nanoparticles as a Novel Delivery Vehicle for Therapeutics Targeting Erectile Dysfunction. J Sexu Med. 2010; 7(1 Pt 1):224-33.
Tar M, Cabrales P, Navati M, Adler B, Nacharaju P, Friedman A, Friedman J, Davies K. Topically applied NO-releasing nanoparticles can increase intracorporal pressure and elicit spontaneous erections in a rat model of radical prostatectomy. J Sex Med. 2014;11(12):2903-14
Cabrales, P, Han, G, Roche C, Nacharaju, P, Friedman, A, Friedman, J. Sustained Release Nitric Oxide from Long Lived Circulation Nanoparticles. Free Rad Med. 2010; 15;49(4):530-8.
Cabrales, P, Han, G, Nacharaju, P, Friedman, AJ, Friedman, JM. Reversal of Hemoglobin-Induced Vasoconstriction with Sustained Release of Nitric Oxide. Am J Physiol Heart Circ Physiol. 2011; 300(1):H49-56.
Nacharaju, P, Friedman, AJ, Friedman, JM, Cabrales, P. Exogenous Nitric Oxide Prevents Collapse during Hemorrhagic Shock. Resuscitation. 2011; 82(5):607-13.
What about applications beyond acne?
Dr. Friedman: The limitation to date with respect to the clinical use of nitric oxide has not been the number of potential applications, as these are practically limitless. Rather, it has been the development of a practical, safe, and translatable delivery system for NO, as this extraordinary biomolecule is reactive and short lived under physiological conditions. My collaborative group has explored many applications for our nitric oxide generating nanoplatform well beyond the scope of even dermatology ranging from acne to bacterial and fungal skin and soft tissue infections to the topical treatment of erectile dysfunction and other diseases involving vascular inflammation. Systemic administration of these nanoparticles show considerable promise in treating the inflammatory cascades associated with hemorrhagic shock and hemorrhagic fevers such as seen in Ebola. Table 1 provides a short list of disease targets we have and are currently exploring with associated relevant publications.
Can you describe NO delivery? Are there different approaches to delivery?
Dr. Friedman: The utilization of nanomaterials in dermatology has enumerable benefits but for the sake of brevity I will list a few: Nanoscopic carriers can be specifically designed to
1. have longer resident time on the skin’s surface, releasing their payload in a controlled manner to overcome potential adverse events associated with higher concentrations or even difficult dosing regimens,
2. target specific structures such as the follicular unit or specific cell types or receptors as most biological processes occur at the nanoscale (think tiny dart going after a big bullseye), and
3. even more importantly, serve as a vehicle through which unstable and elusive therapeutically relevant agents or molecules can be harnessed.
Nitric oxide (NO) represents one such molecule. While there are several NO donating compounds available, some even commercially such as the organic nitrates, they all suffer from various set backs ranging from stability to toxicity. The main limitation of organic nitrates for example, such as nitroglycerin, is decreased efficacy with prolonged continuous use; a so-called ‘nitrate tolerance.’ Nanotechnology has been utilized to deliver some of the newer NO donors, such as diazeniumdiolates, as a means of stabilizing the donor chemical entity and making said donor less toxic. Therefore, nanotechnology here is more allowing for the delivery of an NO donor that would otherwise not be clinically translatable. The nanoparticle platform on which I work (designated NO-nps) is unique in that it actually generates nitric oxide from it’s precursor, sodium nitrite, and this process is dependent on a unique chemistry afforded by the makeup of the nanoparticle in which it is encapsulated. The rich hydrogen bonded matrix and anhydrous environment that comprises these nanoparticles facilitates the formation of N2O3, a potent S-nitrosating agent and NO donor which when exposed to water, will spontaneously generate NO. Therefore, the NO remains trapped in the matrix when dry, permitting easy storage, but upon exposure to an aqueous environment, the NO-nps swell and release the encapsulated NO. The quantity and rate of NO release is controlled by modifying various steps in the nanoparticle synthesis, making it a highly malleable and versatile system for numerous clinical applications.
What should dermatologists watch for in acne therapeutics in 2016?
Dr. Friedman: I think dermatologists overall are tired of the acne armament redundancy. While unique vehicles now allow for the topical delivery of previously incompatible drugs such as benzoyl peroxide and adapalene, or even simpler, enable the topical delivery of a drug for which systemic administration is not warranted, such as dapsone, we crave new active ingredients entirely resulting from cutting edge investigations into the biological underpinnings of acne vulgaris. Case in point, the history of the biologics in psoriasis and now atopic dermatitis underscore this point. There are several new AIs coming down the pipeline with new mechanisms of action and biological targets. I may be biased but no question NO will a much larger role to play in dermatology and medicine overall then acne vulgaris, and this potential will be realized through nanotechnology.
M. Joyce Rico, MD, MBA is Chief Medical Officer at Novan Therapeutics, headquartered in Durham, NC. Here she gives her take on NO research and provides updates on Novan’s topical drug candidates.
What is the rationale for nitric oxide as an anti-inflammatory or anti-infective agent and specifically an anti-acne agent?
Dr. Rico: We know nitric oxide to be a powerful anti-microbial agent that has a low propensity for the development of resistance. Novan has demonstrated in in vitro and clinical studies that the drug candidate SB204 can kill P. acnes. As a dermatologist, I’m excited about opportunity to have a new topical anti-acne drug that targets P. acnes with a low likelihood of developing resistance.
Nitric oxide’s anti-inflammatory activity is a component of the innate immune response. For example, Drs. Adam Friedman and Jenny Kim recently published that nitric oxide inhibits activation of the NRLP-3 inflammasome, a key component of the inflammatory cascade in acne and many other disorders (J Invest Dermatol, 2015).
Could you briefly summarize the data for SB204 and highlight what strikes you in the data so far?
Dr. Rico: In Novan’s Phase 2b study, SB204 demonstrated statistically significant reductions in the percent change of non-inflammatory and inflammatory lesions at Week 12 with all doses of SB204 compared to Vehicle. SB204 demonstrated excellent cutaneous tolerability with no treatment-related serious adverse events in over 400 dosed subjects to date.
What should clinicians look for in the next round of trials? What is important to demonstrate or learn at this point?
Dr. Rico: Novan is starting Phase 3 clinical trials with once daily SB204 in acne subjects ages nine through 40 during the first quarter of 2016. These studies aim to confirm the significant and rapid reduction of acne lesions and excellent cutaneous tolerability profile seen in Phase 2. This will reinforce Novan’s ability to deliver topical nitric oxide to targeted locations.
Are there any emerging misconceptions?
Dr. Rico: Up until now, the challenge faced with a nitric oxide product is the ability to stably store and safely deliver therapeutic quantities of nitric oxide. The Novan technology can store and release more nitric oxide than any other topical nitric oxide delivery system. The Phase 2 results from the acne program validate our platform and prove our ability to deliver therapeutic levels of nitric oxide for additional indications.
What's the status of Nitricil and what can we expect to see next in terms of its development?
Dr. Rico: In addition to our acne program, Novan is conducting a Phase 2 study with SB206 in subjects with external genital warts/perianal warts and expects top line results in the third quarter of 2016. We also plan to initiate a Phase 2 clinical program in onychomycosis with SB208 in the first half of 2016. n