Topical treatment of COVID-19 in early symptomatic disease using Plasma produced Nitric Oxide
Background
COVID-19, a Corona virus (CoV), has the caused an epidemic respiratory illness in China with an associated mortality rate of about 2% [1]. People who were exposed to the illness in China have conveyed the illness in relatively small numbers of people, to date, in other countries [2].
CoV are relatively large (120–160 nm in diameter), enveloped viruses with a single-strand of positive-sense RNA genome [3]. We know of some 7- human pathogenic CoVs. Susceptible persons normally develop a common cold-like infection, which is self-limiting and often free of any serious sequelae. In contrast, the SARS, MERS, and COVID-19 can cause serious and potentially fatal forms of pneumonia and gastroenteritis. The risk of environmental contamination with those three viruses is thus much higher.
The evidence from healthcare settings strongly suggests droplet transmission of CoV and to a much-limited degree by aerosols. We do know whether pathogenic CoV can enter the body via the mouth and conjunctivae as well; nor do we know if the inhaled virus trapped in the throat can translocate to the gut. Virus shedding occurs for about 6 days post-infection with a peak around day 4 post-infection. The minimal infective dose for human CoV is unknown.
Work on potential treatments for COVID-19 are underway. A clinical trial of the combination tablet of Lopinavir and ritonavir [4], a successful therapy for HIV, has begun and a Remdesivir study is due to begin this month [5]. It has been tried, but failed, in Ebola.
Work is advancing on several promising vaccines. One such programme is using a similar platform as has shown preliminary success for MERS [6].
Nitric Oxide and Viral infections
Endogenous NO is considered an important defence to a range of viral infections. The evidence that supports this view of NO has been gained from in vivo and ex-vivo studies. Of interest there have been studies over the last 30 years, for example ex vivo cell-based studies that have shown exogenous NO delivered by NO donor molecules inhibit the intracellular replication of viruses. Such virus infections include Influenza virus [7,8], Rhinovirus [9], together with some of the corona viruses [10,11,12,13].
The question of use of NO donors or gaseous NO is important to the quantitative and applicability of the therapeutic. Carbon Monoxide (CO) has a similar ability to NO to increase cGMP. Similarly, the gas CO has been shown to inhibit replication of Rhinovirus in cultured cells at low concentrations of 100ppm [14]. This study argues in favour of sufficient uptake of the CO into the cell culture fluid and cells over period of 10 minutes once a day. Similar results have been seen with gaseous NO and influenza with 10 to 160ppm [7]. We would attempt in a clinical trial the same duration and exposure with NO which has been used for the treatment of chronic skin ulcers [15].
Topical application of gaseous Nitric Oxide (NO) has been shown to be successful in treating chronic skin ulceration with diabetics and patients with chronic vascular disease [15]. The putative mechanisms for hastening healing are believed to be long-lasting enhanced peripheral blood flow [16] and dispersal of bacterial biofilms [17] over the surface of the ulcer. This dispersal can greatly enhance the effectiveness of antibiotics.
In order to deliver gaseous NO to the skin, several electric arc plasma devices have been developed. One of which is call Plason [18]. This device is portable and operates using an electric battery to sustain the direct electrical current arc. This generates NO from flowing air through a reaction chamber. The air enriched with NO is cooled so that the gas flow is at body temperature when directed at the skin surface. Concentration of NO ranges from 400 to 1100 ppm and the gas flow is applied for minutes at a time to affect the healing of the chronic ulcers.
Therapeutic topical application of NO to lung epithelium is well established treatment of neonatal pulmonary arterial hypertension [19]. Indeed, plasma devices have been developed for this precision therapeutic need [20].
Use of exogenous NO for the treatment of COVID-19
There are two main therapeutic goals for new treatments for this virus infection once symptoms have begun:
1. To reduce the viral “load” within the initial site of contact. Presently the consideration is given to the treatment of the epithelial surfaces of the nose, eyes and throat. The expectation is the by so reducing the viral load the consequences of the illness will be reduced — provided, that patients are diagnosed early.
2. To reduce the replication rate of the virus and therefore limit the subsequent viral sheading reducing the risk of infection to other people.
Our intention is to study the effect topical NO to conjunctiva, nose and throat (plus standards of care for COVID-19 infection compared with a sham treatment (plus standard of care for the illness).
The outcome measures would focus on the novel nucleic acid in nose, conjunctival and throat swabs over a period of 12 days together with clinical indices of respiratory and general ill health.
Study synopsis — “BANISH”
Primary outcome measurements
1) Novel COVID-19 unique nucleic genomic sequence in swabs from nose, eyes and throat measured at 0, 2, 3, 4, 7, 10, 14, 21 days
We intend to user recombinase aided amplification (RAA) assay is a novel isothermal nucleic acid amplification technique in recent years, which has a variety of the advantages including high specificity and sensitivity, rapid detection (30 min), low cost, low equipment requirements and simple operation. This RAA assay for COVID-19 has the advantages of high speed, simple operation and low cost, and overcomes the shortcomings of the existing molecular detection methods. A real time reverse-transcription RAA (RT-RAA) assay for detection of 2019-nCoV. This assay was performed at 42°C within 30min using a portable real-time fluorescence detector, Recombinant plasmids containing conserved ORF1ab genes was used to analyse the specificity and sensitivity. In parallel, we also used the commercial RT-qPCR assay kit for 2019-nCoV as a reference.
2) Physical measurements:
a. Temperature daily for 21 days
b. Disease progress daily for 21 days
c. O2 Sat% daily for 21 days
d. BP Daily
e. Chest Xray (CT) 0, 2, 4, 7, 10, 14, 21
3) Daily Lab tests:
a. LFT, AST, ALT TBIL
b. Renal eGFR and Create
c. Myocardial enzymes
d. Chemo and Cytokine measurement
Undertake study in one site
Analyses
Primary outcome variable:
Testing for presence of the virus gained on the three site swabs daily comparison between active plus conventional care versus sham plus conventional care ANOVA with adjustment for multiple comparisons
Secondary outcome variables, physical and lab test:
Similar quantitative comparisons between treatment groups
Requirements:
1. 50 Plason devices
2. Costs for the tests
3. Ethics approval
4. Identify a Principal Investigator in China
Tim Higenbottam PFPM
References:
1. Na Zhu, Dingyu Zhang, Wenling Wang, et. al. for the China Novel Coronavirus Investigating and Research Team. A Novel Coronavirus from Patients with Pneumonia in China, 2019. This article was published on January 24, 2020, and updated on January 29, 2020, at NEJM.org. https: DOI: 10.1056/NEJMoa2001017.
2. Lan T. Phan, Thuong V. Nguyen, Quang C. Luong, et.al. Importation and Human-to-Human Transmission of a Novel Coronavirus in Vietnam. This letter was published on January 28, 2020, at NEJM.org. https: DOI: 10.1056/NEJMc2001272.
3. de Groot RJ, Baker SC, Baric R, Enjuanes L, Gorbalenya AE, Holmes KV, Perlman S, Poon L, Rottier PJ, Talbot PJ, Woo PC, Ziebuhr J (2011). “Family Coronaviridae”. In AMQ King, E Lefkowitz, MJ Adams, EB Carstens (eds.). Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier, Oxford. pp. 806–828.
4. https://clinicaltrials.gov/ct2/show/NCT04252885?cond=Corona+Virus+Infection&draw=2&rank=1.
5. https://clinicaltrials.gov/ct2/show/NCT04261907?cond=Corona+Virus+Infection&draw=2&rank=10
6. http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(19)30397-4/fulltext
7. Gilly Regev-Shoshani, Selvarani Vimalanathan, Bevin McMullin, Jeremy Road, Yossef Av-Gay, Chris Miller. Gaseous nitric oxide reduces influenza infectivity in vitro. Nitric Oxide 2013; 31: 48–53.
8. Guus F. Rimmelzwaan, Marianne M. J. W. Baars, Pim DE Lijster, Ron A. M. Fouchier, AND Albert D. M. E. Osterhaus. Inhibition of Influenza Virus Replication by Nitric Oxide Journal of Virology. 1999; 33: 8880–8883.
9. Scherer P. Sander S, Edward S. Siekierski, Jacqueline D. Porter, Stephen M. Richards, David Proud. Nitric Oxide Inhibits Rhinovirus-Induced Cytokine Production and Viral Replication in a Human Respiratory Epithelial Cell Line Journal OF Virology. 1998; 72: 934–942
10. Sara Åkerstrom, Mehrdad Mousavi-Jazi, Jonas Klingstrom, Mikael Leijon, Åke Lundkvist, Ali Mirazimi. Nitric Oxide Inhibits the Replication Cycle of Severe Acute Respiratory Syndrome Coronavirus Journal of Virology. 2005; 79: 1966–1969.
11. Sara Åkerström, Vithiagaran Gunalan, Choong Tat Keng, Yee-Joo Tan, Ali Mirazimi. Dual effect of nitric oxide on SARS-CoV replication: Viral RNA production and palmitoylation of the S protein are affected. Virology 2009; 395: 1–9.
12. Chuanmin Liu, Libin Wen, Qi Xiao, Kongwang He. Nitric oxide-generating compound GSNO suppresses porcine circovirus type 2 infection in vitro and in vivo. BMC Veterinary Research 2017; 13:59 68 (DOI 10.1186/s12917–017–0976–9).
13. Murine Coronavirus Thomas E. Lane, Alyssa D. Paoletti, Michael J. Buchmeier Disassociation between the In Vitro and In Vivo Effects of Nitric Oxide on a Neurotropic. Virology 2009, 395: 1–9.
14. Xue Deng, Hiroyasu Yasuda, Takahiko Sasaki, Mutsuo Yamaya. Low-Dose Carbon Monoxide Inhibits Rhinovirus Replication in Human Alveolar and Airway Epithelial Cells. Tohoku J. Exp. Med., 2019, 247, 215–222.
15. I.V. Suzdaltsev I.V., I.A. Polapin. Clinical-laboratory evaluation of application of air-plasma in South Africa in the treatment of necrotizing diabetic foot ulcer complications. Medical Sciences (Russian) 2013; 5: 1–4.
16. Martin Feelisch, Takaaki Akaike, Kayleigh Griffiths et. al., Long-lasting blood pressure lowering effects of nitrite are NO-independent and mediated by hydrogen peroxide, persulfides, and oxidation of protein kinase G1a redox signalling. Cardiovascular Research published November 2019 https: doi:10.1093/cvr/cvz202.
17. Lisa-Marie Nisbett, Lucas Binnenkade, Bezalel Bacon, et al. Multicomponent c‑Di-GMP Network and Biofilm Formation in Shewanella oneidensis Biochemistry November 2019 https. DOI: 10.1021/acs.biochem.9b00706.
18. Anatoly B. Shekhter, Alexander V. Pekshev, Andrey B. Vagapov, Physicochemical parameters of NO-containing gas flow affect wound healing therapy. An experimental study. European Journal of Pharmaceutical Sciences 128 (2019) 193–201.
19. Mei-Yin Lai, Shih-Ming Chu, Satyan Lakshminrusimha, Hung-Chih Lin. Beyond the inhaled nitric oxide in persistent pulmonary hypertension of the newborn. Paediatrics and Neonatology 2018; 59: 15–23.
20. Fernanda Blasina, Lucía Vaamonde, Fernando Silvera, Efficacy and safety of a novel nitric oxide generator for the treatment of neonatal pulmonary hypertension: Experimental and clinical studies. Pulmonary Pharmacology & Therapeutics 54 (2019) 68–76.
