The University of Alabama at Birmingham has been selected to begin enrolling patients in an international study assessing the use of inhaled nitric oxide (iNO) to improve outcomes for COVID-19 patients with severely damaged lungs.
Acute respiratory distress syndrome, a severe form of lung failure, is the leading cause of death in COVID-19.
iNO has been used for the treatment of failing lungs, but it was also found to have antiviral properties against coronaviruses.
When lungs are failing, some parts of the lungs receive air while some do not.
iNO is a gas that improves the blood flow to those areas of the lung that are receiving air, boosting the amount of oxygen circulating in the blood stream.
It also reduces the work of the right side of the heart, which is under extreme stress during conditions of lung failure, such as severe COVID-19 infection.
With the start of this trial, any COVID-19 patient who is admitted to UAB’s ICU and is breathing with the assistance of a ventilator may potentially qualify for the study.
“This trial will allow the sickest COVID-19 patients at UAB access to a rescue therapy that may have antiviral benefits in addition to improving the status of lungs,” said Vibhu Parcha, M.D., a research fellow with UAB’s Division of Cardiovascular Disease.
Pankaj Arora, M.D., assistant professor in the division, is spearheading UAB’s efforts in providing this treatment option to eligible COVID-19 patients.
The mechanism of benefit of iNO could be the direct antiviral effect as shown in the SARS 2003 pandemic, modulation of oxidative stress, or improvement of the ventilation perfusion matching in the lungs, Arora says. His group plans to study the cardiovascular effects of high-dose inhaled NO in an ancillary effort to the primary clinical trial.
“In humans, nitric oxide is generated within the blood vessels and regulates blood pressure, and prevents formation of clots and also destroys potential toxins,” Arora said.
The UAB team says this pandemic has led to an extraordinary unifying response by the medical community, including ICU physicians, nurses, respiratory therapists, clinical trial specialists, reviewers and medical administrators, allowing for faster than normal approvals for potentially lifesaving research studies.
“The fact that we are able to get this trial started quickly was due to collaborations across specialties and fields of expertise at UAB with the common goal of providing the highest quality of scientifically proven care for our COVID-19 patients,” Arora said.
“We are all trying to fight this together, and I hope, with our resilience, we shall overcome these difficult times.”
Nitric Oxide (inhaled):
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Severe acute respiratory syndrome (SARS) has re- cently emerged as a new severe human disease, re- sulting globally in 774 deaths from 8098 reported probable cases (as of the 26th of September 2003).
A novel member of the Coronaviridae family has been identiﬁed as the causative agent of this pul- monary disease.1
Thus far, treatment of SARS cases has been largely empirical and has usually included
an antiviral agent such as ribavirin or a combination of lopinavir/ritonavir and steroids. It is however un- clear whether any of these treatments were able to alter the ultimate outcome of the disease.2,3
During the SARS epidemic, Chen and colleagues included inhalation of NO gas in the treatment of a number of SARS patients. Medicinal NO gas, a gaseous blend of nitric oxide (0.8%) and nitrogen (99.2%), was given for three days or longer, initially at 30 ppm and then at 20 and 10 ppm on the second and third day (unpublished data).
Their ﬁndings suggest not only an immediate improvement of oxygenation but also a lasting effect on the disease itself after termination of inhalation of NO.
NO is a key molecule in the pathogenesis of infectious diseases.
In a variety of microbial infections, NO biosynthesis occurs through the expression of an inducible nitric oxide synthase (iNOS). This molecule has been reported to have antiviral effects against a variety of DNA and RNA viruses, including mouse hepatitis virus (MHV), a murine coronavirus.4
In a recent study, replication of two SARS-CoV isolates (FFM-1 and FFM-2) was shown to be greatly inhibited by glycyrrhizin, an active compound of liquorice roots.5
Glycyrrhizin upregulates the expression of iNOS and production of NO in macrophages.6
Although the initial global outbreak of SARS appears to have been successfully contained, SARS will remain a serious concern while there continues to be no suitable vaccine or effective drug treatment.
Materials and methods
In this study we examined the antiviral activ- ity of nitric oxide (NO) against SARS coronavirus (SARS-CoV) isolate Frankfurt-1 (FFM-1).
Two NO donor compounds, S-nitroso-N-acetylpenicillamine (SNAP, Sigma, Belgium) and sodium nitroprusside (SNP, Sigma, Belgium), were added to conﬂuent African Green monkey (Vero E6) cells. SNAP releases NO in aqueous solutions with a half-life of approximately 4 hours.7 The non-nitrosylated form of SNAP, N-acetylpenicillamine (NAP, Sigma, Bel- gium) was included as a control compound in the assay.
Antiviral activity and cytotoxicity measure- ments were based on the viability of cells that had been infected or not infected with 100 CCID50 (50% cell culture infective doses) of the SARS-CoV in the presence of various concentrations of the test compounds.
Three days after infection, the number of viable cells was quantiﬁed by a tetrazolium-based colorimetric method, in which the reduction of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) dye (CellTiter 96 AQueous One Solution kit, Promega, The Netherlands) by cellular dehydro- genases to an insoluble coloured formazan was measured in a spectrophotometer (Multiskan EX, Thermo Labsystems, Belgium) at 492 nm.8,9
The selectivity index was determined as the ratio of the concentration of the compound that reduced cell viability to 50% (CC50 or 50% cytotoxic concentration) to the concentration of the compound needed to inhibit the viral cytopathic effect to 50% of the control value (IC50 or 50% inhibitory concentration).
The amount of NO produced by SNAP in cul- ture medium was determined by assaying its stable end-product, NO2− (nitrite) in a cell culture environment.
Freeze-thawed cell culture samples were centrifuged at 300 g for 10 min; equal volumes (100 µl) of the sample supernatants and Griess reagent (1% sulphanilamide, 0.1% N-1-naphthylethylenediamine, 5% H3PO4) (Sigma, Belgium) were mixed and incubated for 10 min at 37 ◦C.
The optical density at 540 nm was measured with an automated multiscan spectrophotometer. A range of sodium nitrite dilutions served to generate a standard curve for each assay.
Results and discussion
SNAP inhibited SARS-CoV replication at non-toxic concentrations (222 µM) with a selectivity index of 2.6 (Table 1).
The NO concentration released by 222 µM SNAP is between 30—55 µM NO.
No protective effect below the CC50 could be demonstrated for SNP. The difference in activity between these two NO donor compounds might be explained by a different mechanism of releasing NO. SNAP is a direct donor of NO and generates NO in aqueous solutions through hydrolysis, while SNP only releases NO after reaction with a reducing agent.10—12
No protective effect could be obtained with N-acetylpenicillamine (NAP), which is the non-nitro- sylated form of SNAP and does not release NO in solution (Figure 1).
These results illustrate that the protective effect of SNAP is a consequence of NO release and not of a potential solitary antiviral effect of the N-acetyl-penicillamine moiety.
In this study, we provide additional evidence that NO and NO-donors may have an antiviral ef- fect against the SARS-CoV and we speculate that the prolonged effect of inhalation of NO gas observed earlier could be an antiviral effect of NO against SARS-CoV. Based on our results we encourage the inclusion of inhalation of NO in the treatment of SARS. NO-donors, including SNAP, have been described as potential therapeutics in the treatment of cardiovascular disease.13
To conﬁrm the anti-SARS-CoV effect of NO gas and NO donors and before SNAP can be used in SARS treatment, additional in vivo experiments are required.
As resurgence of the SARS outbreak is a dis- tinct possibility, the search for antivirals effective against the SARS-CoV remains an important endeavour.
This work was supported by a fellowship of the Flemish Fonds voor Wetenschappelijk Onderzoek (FWO) to Leen Vijgen, and by FWO-grant G.0288.01.
- Drosten C, Gunther S, Preiser W, et al. Identiﬁcation of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 2003;348:1967—76.
- Zhaori G. Antiviral treatment of SARS: can we draw any conclusions? CMAJ 2003;169:1165—6.
- Chu CM, Cheng VC, Hung IF, Wong MM, Chan KH, Chan KS, et al. Role of lopinavir/ritonavir in the treatment of SARS: initial virological and clinical ﬁndings. Thorax 2004;59:252— 6.
- Lane TE, Paoletti AD, Buchmeier MJ. Disassociation be- tween the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus. J Virol 1997;71:2202—10.
- Cinatl J, Morgenstern B, Bauer G, Chandra P, Rabenau H, Doerr HW. Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. Lancet 2003;361:2045—6.
- Jeong HG, Kim JY. Induction of inducible nitric oxide syn- thase expression by 18[3-glycyrrherinic acid in macrophages. FEBS Lett 2002;513:208—12.
- Ignarro LJ, Lippton H, Edwards JC, Baricos WH, Hyman AL, Kadowitz PJ, et al. Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and ni- tric oxide: evidence for the involvement of S-nitrosothiols as active intermediates. Pharmacol Exp Ther 1981;218:739— 49.
- Pauwels R, Balzarini J, Baba M, Snoeck R, Schols D, Herdewijn P, et al. Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. J Virol Methods 1988;20:309—21.
- Goodwin CJ, Holt SJ, Downes S, Marshall NJ. Micro- culture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS. J Immunol Methods 1995;179:95—103.
- Bates JN, Baker MT, Guerra Jr R, Harrison DG. Nitric oxide generation from nitroprusside by vascular tissue. Evidence that reduction of the nitroprusside anion and cyanide loss are required. Biochem Pharmacol 1991;42:157—65.
- Marks GS, McLaughlin BE, Brown LB, Beaton DE, Booth BP, Nakatsu K, et al. Interaction of glyceryl trinitrate and sodium nitroprusside with bovine pulmonary vein ho- mogenate and 10,000 x g supernatant: biotransforma- tion and nitric oxide formation. Can J Physiol Pharmacol 1991;69:889—92.
- Kowaluk EA, Seth P, Fung HL. Metabolic activation of sodium nitroprusside to nitric oxide in vascular smooth muscle. J Pharmacol Exp Ther 1992;262:916— 22.
- Megson IL. Nitric oxide donor drugs. Drugs of the Future 2000;25:701—15.