WO2021240568A1 - Adenosine for the prevention and treatment of acute respiratory distress syndrome (ards) - Google Patents

Abstract

The present invention concerns adenosine for the prevention and treatment of acute respiratory distress syndrome (ARDS), wherein adenosine is administered by inhalation.

Description

Adenosine for the prevention and treatment of acute respiratory distress syndrome (ARDS)

The present invention concerns adenosine for the prevention and treatment of acute respiratory distress syndrome (ARDS). In particular, the present invention concerns adenosine for the prevention and treatment of acute respiratory distress syndrome (ARDS), wherein adenosine is administered by inhalation.

Acute respiratory distress syndrome (ARDS) represents a severe inflammatory mediated damage of the alveolar tissue that becomes unable to allow O2/CO2 exchanges with microvassels needed for blood oxygenation. ARDS is clinically defined by the occurrence of severe dyspnea, radiological picture of interstitial pneumonitis, a Horowitz index (Pa02/Fi02 ratio) <250 (that describes the ratio of partial pressure of oxygen in blood (Pa02, mm/hg) and the fraction of oxygen in the inhaled air (Fi02)). It is caused by endogenous and exogenous multifactorial events and different triggers, including: bacterial toxins, microbial pathogens (including human syncytial virus, H1 N1 virus, SARS, MERS, SARS-C0V2, etc), inappropriate immuneresponse, immunomodulating drugs, chemical and physical insults and ischemy. These factors ignite an orchestrated cascade of events culminating in sustained overwhelming inflammation and severe lung injury.

At the presents, there is no recommended treatment for ARDS that presents a high morbidity rate and often a lethal outcome. These patients require active oxygenation to counteract the occurrence of hypoxia; however, there is clear evidence that this hyper-oxygenation of inflamed lung greatly exaggerates the inflammatory mediated damage and the spectrum of lung injury by dampening the natural anti-inflammatory defence of the tissue. Hence, by the time ARDS is recognized, significant intra-pulmonary inflammation and injury has already occurred.

Anti-inflammatory therapy initiated in patients with established ARDS provides disappointing results.

Therefore, ARDS frequently requires active ventilation and may have fatal outcome.

As mentioned above, ARDS comprises also Covid-19-related lung injury.

Covid-19 outbreak has been declared as pandemic by the WHO, reporting more than four million new cases worldwide with 300.000 related deaths (1). Almost 20% of these patients develops interstitial pneumonitis that may evolve in ARDS requiring hyperoxic active ventilation, with mostly fatal outcomes (2-5). The pathogenesis of the Covid-19-related lung injury is still controversial; however, a massive and uncoordinated release of inflammatory cytokines and a post-ischemic reaction to micro-vascular damage and micro-embolization seem to be involved (5-10). The eagerly need of hindering the escalation of these events sustained the rationale for testing biological drugs in a number of ongoing trials aimed to hamper the effects of the most relevant cytokines involved the first phases of the inflammatory process (11-14). In this context, mAbs to IL1 b (Kanakinumab), IL6-Receptor (Tocilizumab) and inhibitors of Janus kinases (JAK)-1/2 (Baricitinib and Ruxolitinib) are being tested on patients with Covid19-related interstitial pneumonitis with unsatisfactory results (14- 16). In this light, it has been reported that more than 65-80% of patients with Covid19-related lung injury requiring active oxygen ventilation ultimately die with the suspect that some iatrogenic complication other than a mechanical lung damage does occur (17-18).

Therefore, no effective therapy is available for patients with COVID- 19 and severe interstitial pneumonitis, until now.

In the light of the above, it is therefore apparent the need to provide therapies for the treatment and prevention of ARDS, which overcome the disadvantages of known treatments.

It is known that oxygenation inhibits the physiological tissue- protecting mechanism and thereby exacerbates acute inflammatory lung injury (19).

Based on the results of those preclinical studies in mice, the inventors of the present invention hypothesized that mechanical ventilation and hyper-oxygenation produce unacceptable inflammatory side effects also in patients with Covid19-related severe pulmonary complications. In fact, the life-saving oxygenation weakens the local tissue hypoxia-driven and adenosine A2A receptor (A2AR)-mediated anti-inflammatory mechanism (20-22) that unleashes not only the neutrophils, but also lung macrophages and pulmonary natural killer T cells (23-25). Without this major physiological tissue-protecting mechanism, neutrophils and other immune cells are no longer inhibited and unleashed to destroy the still healthy alveolar tissue, leading to the strongly exacerbated inflammatory lung injury (23-25). These studies gave a strong impulse to the research of drugs aimed to target A2A or A2B adenosine receptors as an important field in inflammation research and rheumatology (24,25).

It is known that the deadly immunological side effects of the otherwise life-saving oxygenation is prevented by the airways administration of selective A2AR agonists to compensate for the oxygenation-related loss of the lung tissue-protecting endogenous nucleoside (26-29). Preclinical studies in mice are also known that show that intra-bronchial administration of chemical agents with A2 receptor agonist activity could restore the natural anti-inflammatory and tissue protective activity of the endogenous pathway (19, 31-34).

According to the present invention, it has been now found that the administration of adenosine by inhalation is able to re-establish the physiological tissue-protecting mechanism and reduce acute inflammatory lung injury in ASDR. Therefore, adenosine can be advantageously used as a lung specific anti-inflammatory drug to compensate the oxygen- related distress on the naturally generated nucleoside in inflamed lungs.

Specifically, according to the present invention, fourteen patients with Covid-19 and severe lung inflammation received inhaled adenosine. The results showed that the treatment therapeutically compensates for oxygen-related distress in the endogenous adenosine->A2AR signalling that is no longer able to physiologically mitigate the otherwise destructive inflammatory cascade.

Due to the acute medical need and shortage of time related to the escalating Covid19 outbreak, it was impossible to gain access to a synthetic A2A receptor agonists while adenosine was already available for clinical use (Adenoscan® and Krenosin®) as it already has a number of different applications in human pathologies (30). Additionally, adenosine has already been tested as aereosol formulation in a range of 6-40 mg dosage, presenting an acceptable safety profile with no hemodynamic or other side-effects in normal subjects. On these bases adenosine is currently used in diagnosis to discriminate patients with small respiratory tract asthmatic disease from chronic inflammatory lung disease (31-34).

As mentioned above, fourteen patients with Covid19-related interstitial pneumonitis and Pa02/Fi02 ratio<300 received off-label- treatment with inhaled adenosine 9mg every 12 hours in the first 24 hours and subsequently, every 24 days for the next 4 days. Monitoring included hemodynamic/hematochemical study, CT scans, and SARS-CoV2-tests.

The treatment resulted safe with the report of one case of moderate bronchospasm. An early clinical benefit was reported in 13 patients with a significant mean Pa02/Fi02-ratio increase (from 215 ± 45 to 464 ± 136, P= 0.0002). A radiological response was demonstrated in 7 patients, while SARS-CoV-2 load rapidly decreased in 13 cases.

Therefore, the treatment was well tolerated and was associated with a early improvement in Pa02/Fi02 ratio, respiratory symptoms and performance status, so that ten patients were dismissed from the hospital within one week since the beginning of the treatment with adenosine. These results were also confirmed by the radiological imaging study showing the significant improvement in the signs of interstitial lung pneumonitis in six out ten studied patients.

As an additional and unexpected finding a significant decrease in SARS-Cov-2 viral load was also detected.

Presently, enough information to explain the effect of inhaled adenosine therapy on SARS-Cov-2 load are not available; however, it can be hypothesized that adenosine given in hypoxic conditions is able to induce the following effects: i) to restore an appropriate virus specific immune-response previously attenuated by the inflammatory storm; ii) to exert a direct anti-viral host effects mediated throughout the A2R pathway; iii) to convert the intracellular adenosine in pro-apoptotic metabolites (like deoxy-adenosine/deoxy-ATP) in some infected cells. In this light, there is large concordance on the fact that mechanical damage to the lung associated to active ventilation can add to the SARS-CoV-2 -induced lung damage (5). On the other hand, it is now widely known that abuse of hyperoxic breathing itself can inhibit the major physiological tissue- protecting hypoxia-A2-adenosinergic mechanism leading to massive tissue damage consequences. The treatment according to the present invention is aimed to restore the A2 adenosine receptors signaling and thereby ensuring that healthy lung tissue are again protected -even in the presence of continuing oxygenation.

Additionally, the experimental results reported in the Examples show that the use of inhaled adenosine in COVID19 patients allows a reduction of hospitalizations length, on average 6 days. This result is strengthened by the decrease in SARS-CoV-2 positive days. In treated patients compared to control, a clear improvement in Pa02/Fi02 was observed together with a reduction in inflammation parameters, such as the decrease of CRP level. Furthermore, the efficacy of inhaled exogenous adenosine led to an improvement of the prognosis indices, NLR and PLR. The treatment seems to be safe and modulates the immune system, allowing an effective response against the viral infection progression, reducing length of stay and inflammation parameters.

Therefore, the present experimental data show that the treatment strategy according to the present invention results in an accelerated increase in Pa02/Fi02 ratio and performance status and an antiviral specific effect. In this view, inhaled adenosine is the first treatment for Covid-19 aimed to exert rapidly both clinical benefit and antiviral activity in critical patients greatly improving the way how these patients are treated and cured.

It is therefore object of the present invention adenosine or a pharmaceutical composition comprising or consisting of adenosine in association with one or more pharmaceutically acceptable excipients and/or adjuvants, for use in the prevention and treatment of acute respiratory distress syndrome, wherein the adenosine or the pharmaceutical composition is administered by inhalation, such as for example by inhalation to lung alveoli.

According to the present invention, acute respiratory distress syndrome is chosen from the group consisting of acute respiratory distress syndrome related to pneumonia, such as viral, bacterial or fungal pneumonia or pneumonia not associated with infections or associated to lung cancer. In particular, acute respiratory distress syndrome can be ARDS related to SARS-CoV-2 infection.

The pharmaceutical composition according to the present invention can be in a form suitable for inhalation. The administration of adenosine by inhalation advantageously allows the use of this nucleoside without incurring serious adverse events related to the ubiquitous distribution of its receptors in all the tissues, organs and systems of the body, thus obtaining minimal toxicity.

According to an embodiment of the present invention, the pharmaceutical composition can further comprise hyaluronic acid and or liposomes.

According to the present invention, adenosine or the pharmaceutical composition can be administered by inhalation in combination with oxygen, wherein the percentage of oxygen is the air oxygen percentage, i.e. about 21% or in hypoxia conditions. The administration of adenosine should not occur in hyperoxia conditions. For example adenosine or the receptor agonist can be administered by inhalation via nebulization system (Aerogen USB Controller) connected to a High Flow Nasal Cannula (HFNC) high flow generator with 21% Fi02 regulation and 60 I/m flow setting in non-intubated patients or via nebulization system connected to the inhalation route of the respiratory circuit in the intubated patient.

Further object of the present invention is a combination of adenosine with one or more compounds chosen from the group consisting of oxygen in air oxygen percentage, an anti-inflammatory drug, a (beta2) adrenergic receptor agonist or an anticancer agent, such as PD-1/PDL-1 immunecheckpoint inhibitors, for separate or sequential use in the prevention and treatment of acute respiratory distress syndrome, wherein the adenosine is administered by inhalation.

The administration of adenosine can be also combined with radiotherapy.

According to the present invention, “separate use” is understood as meaning the administration, at the same time, of the compounds (adenosine and the other compounds) of the combination according to the invention in distinct pharmaceutical forms. “Sequential use” is understood as meaning the successive administration of the compounds (adenosine and the other compounds) of the combination according to the invention, each in a distinct pharmaceutical form.

As mentioned above, acute respiratory distress syndrome is chosen from the group consisting of acute respiratory distress syndrome related to pneumonia, such as viral, bacterial or fungal pneumonia or pneumonia not associated with infections or associated to lung cancer. In particular, acute respiratory distress syndrome can be an ARDS related to SARS-CoV-2 infection.

As mentioned above, adenosine is administered by inhalation, therefore, it is in a form suitable for inhalation.

The present invention concern also adenosine or a pharmaceutical composition comprising or consisting of adenosine together with one or more pharmaceutically acceptable excipients and/or adjuvants, for use in antiviral therapy. In particular, adenosine or the pharmaceutical composition according to the present invention can be advantageously used for anti-SARS-CoV-2 therapy.

The present invention now will be described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to the enclosed drawings, which show:

Figure 1 - Respiratory and inflammatory marker monitoring before and after adenosine treatment

Panel A)- Adenosine treatment shows a significant improvement in the mean Pa02/Fi02 ratio in fourteen patients who received adenosine treatment;

Panel B)- The plot shows a post-treatment decline in IL-6 serum level. However, the differences did not achieve statistical significance (P=0.07). There was no significant treatment-related changes in blood cell counts as well as serum C-reactive protein and Lactate Dehydrogenase (LDH) levels (data not shown)

Figure 2 - The High Resolution Computerized Scan (HRCT) monitoring before and after adenosine treatment

Panel. 2.1 -Patient #2- (A-B) baseline HRCT shows signs of interstitial pneumonitis with focal area of ground-glass in the RSL; (C) ^§Pre-treatment volume rendering. (D-E) Post-treatment HRCT shows widespread reduction in the interstitial pneumonitis. (F) ^§Post-treatment volume rendering.

Panel 2.2 -Patient #4- (A-B) baseline HRCT shows signs of interstitial pneumonitis with areas of ground-glass in the RSL and LIL with pleura-parenchimal branches in the periphery; (C) ^§Pre-treatment Volume rendering. (D-E) Post-treatment HRCT shows wide-spread reduction in the interstitial engagement. (F) ^§Post-treatment volume rendering shows reduction in amorphous increase in lung density

Panel 2.3 -Patient #5- (A-B) baseline HRCT shows widespread signs of interstitial pneumonitis with areas of ground-glass and pleura- parenchymal branches, present in both lungs with spread to the periphery in the LIL and RIL. (C) ^§Pretreatment volume rendering. (D-E) post treatment HRCT shows widespread reduction in the interstitial engagement. (F) ^§Post-treatment volume rendering showis significant reduction of parenchymal thickenings.

Panel 2.4 -Patient #6- (A-B) baseline HRCT shows widespread signs of interstitial pneumonitis with areas of ground glass and crazy paving, present in both lung fields with spreading to the periphery. (C) ^§Pre-treatment volume rendering. (D-E) Post-treatment HRCT shows widespread reduction in interstitial engagement and crazy paving. (F) ^§Post-treatment volume rendering.

Panel 2.5 -Patient #10- (A-B) baseline HRCT shows widespread signs of interstitial pneumonitis with areas of ground glass in both lungs with spreading to the periphery. (C) ^§Pretreatment volume rendering. (D-E) Post-treatment HRCT shows widespread reduction in interstitial engagement. (F) ^§Post-treatment volume rendering.

Panel 2.6 -Patient #13- (A-B) Baseline HRCT shows widespread signs of interstitial pneumonia, fibrous septa and pulmonary thickening, with areas of ground glass in both lungs spreading to the periphery. (C) ^§Pretreatment volume rendering. (D-E) Post-treatment HRCT shows widespread reduction in interstitial pneumonia and septal thickening. (F) ^§Post-treatment volume rendering showing reduction thickenings. ^§ In the volume rendering study, green area represent the normal lung parenchyma while red areas indicate the inflammatory involvement.

Figure 3 - The monitoring of threeSARS-CoV-2 RNA target genes before and after adenosine treatment - Evaluation of SARS- COV-2 RNA N gene Ct value in respiratory specimens over time. N gene Ct value peaked at the baseline and decreased in responder patients. Above 40 Ct RNA-N genes not detectable. From the left to the right: 1^st column, baseline; 2^nd column, 48 hours; 3^rd column, 120 h; up to 15 days (8^th column) from the beginning of the treatment.

Example 1: Study on the effects of inhaled adenosine on the performance status and the mean Pa02/Fi02 ratio in patients with severe Covidl 9-associated inflammatory lung disease and SARS-Cov-2 load

Methods

Fourteen hospitalized patients positive for the expression of SARS- Cov-2 and without clear improvement with hydroxychloroquine (HC), azitromycin (AZM) and low molecular weight heparin (LMWH), signed an informed consent and received an off-label treatment with inhalatory adenosine at the dosage of 9 mg every 12 hours in the first 24 hours and subsequently, every 24 days for four consecutive days. Adenosine was nebulized and dispensed by an Aerogen USB Controller linked to a high flux device with 21% F1O2, a flow of 60 I/m in five minutes.

Inhaled adenosine dose was extrapolated from the preclinical studies in mice (24,30-32) as well as from the clinical studies using adenosine as aereosol formulation showing dose limiting efficacy over 10 mg and no adverse events in normal individuals and patients with non asthmatic disease (30-34).

The off label treatment and patient monitoring was approved for each single individual by the Hospital Safety Team and by the Ethical Committee of South Calabria. Patients’ privacy and sensitive data were appropriately protected.

Patients were daily monitored for vital parameters, arterial pressure (AP) and heart-rate (HR), hemogas-analysis, blood cell counts, biochemistry, ECG, inflammatory markers (CRP, LDH and ESR), coagulation asset and D-Dimer. Interleukin-6 was also measured in the serum of these patients at baseline and 120 hours after the beginning of the treatment by Electroluminescent immune Assay (kit-ECLIA, Roche) in the laboratory of Clinical Pathology of the Grand Metropolitan hospital (GOM), RC, Italy.

The detections of SARS-Cov-2 in upper or lower upper or lower respiratory specimens was performed at baseline, 48, 120 hours and 15 days after the beginning of treatment. It was evaluated by laboratory of Microbiology& Virology of the GOM, RC, Italy. Upper (nasopharyngeal swabs) and lower (bronchoalveolar lavages, bronchoaspirates and tracheal aspirates) respiratory tract specimens, were collected using Copan Universal Transport Medium (UTM-RT®) System or sterile container at 4°C and processed within 24 hours. Real-time reverse transcription-PCR is currently the most reliable diagnostic method for COVID-19 around the world. RNA-COVID 19 was evaluated by using an Allplex 2019-nCoV Assay that identifies three different target genes: E (envelope), RdRp (RNA-dependent RNA polymerase, and N (nucleoprotein gene) according to the international recommended guidelines by the World Health Organization. This test has also received CE-IVD mark and KFDA approval. The test assay was performed following the manufacturer’s instructions. According to the interpretation criteria, detection of only one of multiple genes has been interpreted as COVID-19 positive.

High resolution CT scan were performed at baseline and after treatment and results were analysed by the same dedicated radiologists.

Results

Patients population -Fourteen patients, 10 males and 4 females with a mean age of 57± 19 year were approved to receive life-saving off- label treatment with inhaled adenosine, dispensed by a high flow device and 21% O2. All patients positive for the expression of SARS-Cov-2 had been previously hospitalized and 13 of them had received empiric treatments commonly used for Covid19 with no clinical or biological improvement for more than three weeks. Before receiving adenosine they had presented a Pa02/Fi02 ratio <250 and a CT scan picture suggestive of severe interstitial pneumonitis. Eleven patients had received previous treatment with HC, AZM, LMWH. Four of them had also received previous off-label treatments with Tolicizumab more than three weeks prior adenosine administration. Ten patients, hosted in the Unit of Infectious Disease required high flux oxygenation, while further four patients requiring active ventilation, were hosted in the Resuscitation unit of the Grand Metropolitan Hospital.

Table 1 shows patient features, treatments, and outcome.

Table 1

Patient performance status at baseline was evaluated according to the Eastern Cooperative Oncology Group (ECOG) scale (1-4); adverse events (AE) were evaluated according to the World Health Organization (WHO) scale grade (g)

Legend: HC= hydroxychloroquine, AZR= Azitromycin, IOT= intubation oro-tracheal, VM= Mechanical ventilation; CPAP= Continuous positive airway pressure; HNFC = High Flow oxygenation; COPD Chronic Obstructive Pulmonary disease. Low molecular weight heparins = LMWH; Bidaily = bd; CT score finding:-1 =worse; -2=no change; -3=slight improvement (reduction focal or diffuse pneumonia <50%); -4= improvement > 50%; -5=no evidence; -ND=not done.

Adverse events - Adenosine treatment was well tolerated and there was no effect on either HR or AP during and after the treatment procedure. There was a case of reversible brochospasm during the third adenosine dose administration in a mechanically ventilated patient that consequently discontinued the treatment. A temporary flushing was also recorded in another case six hours after the first adenosine dose (Table 1).

Treatment response - Inhalatory adenosine administration resulted in a fast and significant rise in the Pa02/Fi02 ratio within 120 hours since the beginning of the treatment (215 ± 45 vs. 464 ± 136, P= 0.0002) (Figure 1A). Thirteen patients presented a clinical benefit from the treatment with decrease in symptoms and improvement in performance status within few days. Eight of them did not require further oxygen administration and could be released by the hospital within one week from the beginning of the treatment. Two patients in active ventilation could be extubated 72 hours after the beginning of the treatment and addressed to high flux ventilation out of the resuscitation facility. A high resolution CT scan monitoring could be performed in ten patients only as four patients refused the post treatment scan. A complete resolution of the lung disease was recorded in two patients and a significant improvement of the picture was observed in further four cases (Figure 2). Two patients showed a minimal radiological benefit, while two patients showed the presence of new lung consolidative areas and pleural effusion suggestive of a new bacterial complication.

Laboratory response - No treatment-related changes were recorded in peripheral blood cell counts, neutrophil to lymphocyte ratio, biochemistry, inflammatory markers as well as coagulation assets ( data not shown). The analysis revealed a trend to decline in serum IL-6 levels 120 hours after the end of the treatment that did not achieve statistical significance (P=0.075) (Figure 1b). The detection of SARS-Cov-2 was performed at baseline and 48 and 120 hours and 15 days after the beginning of adenosine treatment. Eight patients showed complete disappearance of viral load while five of them showed the persistence of a very low virus load where only SARS-Cov-2 N gene could be detected, at higher Ct respect to baseline value which disappeared above the 40 Ct. As an additional finding early decrease in the virus load was observed with no detection in RdRp/E genes since the first post-treatment analysis performed after 49 hours after the beginning of the treatment (Figure 3 and Table 1). All of the thirteen responsive patients, however resulted SARS-Cov-2 free after 15 days (Figure 3).

Example 2: Efficacy and effect of inhaled adenosine treatment in hospitalized COVID-19 patients.

Introduction

Lack of specific antiviral treatment for COVID-19 has resulted in long hospitalizations and high mortality rate. By harnessing the regulatory effects of adenosine on inflammatory mediators, according to the present invention a new therapeutic treatment with inhaled adenosine in COVID-19 patients have been instituted, with the aim of reducing inflammation, the onset of cytokine storm, and therefore to improve prognosis.

Methods

The study population was represented by hospitalized native COVID-19 patients meeting eligibility criteria. This single-center case-control study involved off-label treatment with adenosine, administered to Sars-Cov2 positive patients, who arrived at the Emergency Department of the "Bianchi Melacrino Morelli" Grande Ospedale Metropolitano (GOM) of Reggio Calabria, Italy, between March 19th and April 13 th, 2020.

After Sars-Cov2 positivity confirmation, patients were transferred to the Infectious Diseases Unit, underwent chest CT, drug therapy with low molecular weight heparin, azithromycin, HCQ, and Lopinavir/ritonavir.

At baseline, after interrupting Lopinavir/ritonavir administration, biochemical-clinical analyses were performed to all eligible patients and off-label treatment was started in the treated group (TG), represented by the patients who accepted compassionate treatment. The control group (CG) included eligible patients, with characteristics homogenous to TG, who did not accept the off-label compassionate use of Adenosine.

The following additional information was collected (35): myocardial infarction (Ml); atrial fibrillation; type 2 diabetes mellitus; chronic liver disease; chronic obstructive pulmonary disease (COPD); chronic kidney failure (CKF); stroke; cancer in the last 5 years.

At 10 days from baseline, patients underwent follow-up of blood chemistry and clinical parameters.

On the 21st day after admission to the Infectious Diseases Unit, all patients underwent a chest CT-scan.

The off-label treatment was administered after obtaining informed consent and approval for each patient from the Hospital Safety Team.

All patients’ history and clinical status data were collected at baseline and follow up. All chest CT scans, SarS-Cov2 research, and blood chemistry analyses were performed at the GOM.

Days of SarS-Cov2 positivity are evaluated from the first positive test to the second negative test in 48 hours. In addition, hospitalization days were examined from transfer to the Infectious Diseases Unit until discharge. The discharge was allowed with two negative SarS-Cov2 tests within 48 hours and remission of clinical symptoms.

Inclusion criteria: - biologically confirmed by SARS-CoV-2 PCR test;

- COVID-19 diagnosis clinically and radiologically confirmed by chest CT scan.

Exclusion criteria :

- patients with a history of neutropenia;

- patients with acquired immunodeficiency;

- patients with cancer history in the last 5 years;

- patients who underwent transplants;

- patients who received previous immunosuppressive therapies or corticosteroids;

- women who are pregnant or breastfeeding.

Withdrawal Criteria

Participants were free to withdraw from participation in the study at any time upon request or at the request of their legally acceptable representative.

An investigator may discontinue or withdraw a participant from the study for the following reasons:

- pregnancy;

- non-compliance to study intervention;

- occurrence of any clinical adverse event, laboratory abnormality, or medical condition compromising continued participation in the study and not in the best interest of the participant;

- severe side effects clearly related to the study device;

- disease progression requiring other treatments;

- if the participant meets an exclusion criterion (either newly developed or not previously recognized) that precludes further study participation;

- participant unable to receive study intervention for > 2 days/week;

- subject noncompliant to investigational procedures; subject noncompliant to visits.

Therapy - All patients received standard therapeutic protocol including low molecular weight heparin 4000 units two times a day, azithromycin 1 tablet a day when needed, HCQ (Plaquenil®) 200 mg, 2 tablets two times a day, Lopinavir/Ritonavir (Kaletra®) 100/25 mg, 2 tablets two times a day for 7 days (36).

Respiratory support was used when needed, according to current guidelines.

The off-label treatment involved the use of inhaled adenosine (Krenosin®), 9 mg every 12 hours for the first 24 hours and subsequently, every 24 hours for four days (37-38). Nebulized adenosine was delivered by an Aerogen USB® controller connected to a high flow 60 L for 5 minutes device, with 21% Fi02. The safe posology has been established from preclinical studies (39-40).

The administration of aerosolized adenosine has a dose-limiting efficacy greater than 10 mg. Its use did not show adverse effects in non asthmatic subjects (41-42).

Adverse effects were monitored throughout the study and patients underwent continuous monitoring of cardiocirculatory and respiratory function.

Sars-CoV2 Swab Test - SARS-CoV-2 was detected by RNA sequencing assay (Seegene ‘AllplexTM 2019-CoV Assay, catalogue number #RP10243X 100 rxn), targeting SARS-CoV-2 RdRp, E and N genes. Data were analysed by CFX96 Manager software and Seegene viewer software. The responses were “2019-nCoV detected”, “negative” or “invalid” (Allplex 2019-nCoV assay IFU) (43).

Anthropometry - The Body Mass Index (BMI) was calculated by weight and height measured, according to Romano et al. (44) or referred when unable to carry out the assessment.

Biological specimen collection and laboratory evaluations - The following blood analysis were performed at each evaluation time: blood gas analysis parameters, as arterial oxygen partial pressure (Pa02) and arterial carbon dioxide partial pressure (PaC02); fractional inspired oxygen (Fi02) in patients with respiratory support; arterial oxygen partial pressure and fractional inspired oxygen ratio (Pa02/Fi02) (51); platelets (103/pL); white blood cells (WBC) (103/pL); red blood cells (RBC) (106/mI_); hemoglobin (g/dL); neutrophils (103/pL); lymphocytes (103/pL); monocyte (109/L); fibrinogen (mg/dl_); D-dimer (ng/mL); CRP(mg/L); glycemia (mg/dl_); albumin (g/dL); AST (U/L); HALT (U/L); gamma- glutamyl transpeptidase (GGT); indirect bilirubin (mg/dL); direct bilirubin (mg/dL); amylase (U/L); lipase (U/L); potassium (mEq/L); sodium (mEq/L). According to Qin, neutrophils to lymphocytes (NLR) and platelet to lymphocyte (PLR) were calculated (45).

Chest CT and Findings - CT examination was performed with high-resolution acquisitions for the study of lung interstitium, with CT GE Medical System Optima CT 660, followed by multi-parametric reconstructions according to coronal and sagittal and 3D plans.

Two radiologists independently and blindly examined the Chest CT scans and reported the radiographic findings, according to Chung (46-47).

Statistical Analysis - All statistical analyses were conducted with SPSS 23 software (version 23.0, IBM, Armonk, NY, USA). Data collected before statistical evaluations were analyzed for the presence of outliners and for normally distribution with the Shapiro-Wilk test. The data presented are expressed as mean, standard deviation, percentage in contingency tables and as D%, to evaluate differences between the times. Before, the differences between CG and TG patients were assessed by Independent samples T test and Mann Whitney test. Subsequently, the two-tailed Student’s paired t-test or Wilcoxon rank test were used to assess the presence of differences in the variables examined between baseline and follow-up. Conclusively, for each study variable, to compare the trend over time, D% were calculated equal to the percentage variation of each parameter calculated as an absolute margin of variation from the baseline value. The differences in A% between baseline and follow-up among groups were assessed with the Anova one-way test. The presence of difference in contingency tables were analyzed with the Chi square test. Statistical significance was set to a value of p < 0.05. All p values shown are two-tailed.

Results

Of the 30 Caucasian patients enrolled for prospective analytical case-control study, 6 subjects were excluded from the study for the following reasons: 2 subjects did not meet inclusion criteria (transfer in intensive care unit), 1 subject died before starting treatment; 3 subjects were excluded for incomplete data. Finally, 24 patients were included in the study. The age of subjects was 56.86 ± 15.65 years, 37.50% females and 62.50% males. The eligible patients were allocated into two groups: 12 patients in the control group (CG) (41.67% females and 58.33% males) and 12 patients in the treatment group (TG) (33.33% females and 66.67% males). It was observed that the days of hospitalization and test positivity were statically decreased in TG compared to CG (respectively p= 0.032; p= 0.002). No statistical differences between groups were observed in age, BMI, days of HDCL and AZT treatment. At baseline, only PLR was statistically increased in TG compared to CG (p=0.004). Lymphocytes and potassium were significantly reduced in TG compared to CG (respectively p=0.021 ; p= 0.022). No other statistical differences between groups were detected. At baseline no statistical differences were observed in the frequency of airway support, hypertension, diabetes mellitus and dementia. Furthermore, none of the enrolled patients suffered from COPD, CKF, Ml and cancer in the past 5 years. Between baseline and follow-up, it was observed that NLR, RBC, haemoglobin and neutrophils were statically reduced in CG (respectively p=0.028; p=0.032; p=0.032; p= 0.038), the Pa02/Fi02, platelets and lipase concentration were statically increased in CG (p=0.042; p= 0.038; p= 0.037). Between baseline and follow-up, NLR, PLR, F1O2, CRP, amylase, lipase and D-dimer concentration were statistically decreased in TG (respectively p= 0.008; p= 0.003; p=0.042; p=0.005 p= 0.025; p=0.042; p= 0.020); Pa02, Pa02/Fi02 and lymphocytes were significantly increased in TG (respectively p=0.045; p=0.014; p=0.013). Moreover, PLR and CRP showed a greater significant D% reduction in TG (respectively p=0.017; p=0.046). It was observed that D% Pa02/Fi02 was significantly raised in TG (p=0.046) and D% platelets in CG (p=0.013). Qualitative changes in CT findings are reported. It was observed that ground-glass opacities or consolidation and pleural effusion frequencies were overall, and in each group, statically reduced (respectively p=0.002; p=0.021 ; p=0.021 and p=0.007; p=0.021 ; p=0.048). Ground-glass opacities with consolidation, rounded morphology and pneumopathy >25% frequencies were significantly decreased in overall and TG (respectively p=0.012; p=0.045; p=0.023; p=0.012; p=0.041 and p=0.048). More than two lobes affected, and bilateral lung disease frequencies were statistically increased in CT (p= 0.035). Pneumopathy frequencies was observed statically reduced only in overall (p= 0.029). Conclusion

The use of inhaled adenosine in COVID19 patients has allowed reduction of length of stay, on average 6 days. This result is strengthened by the decrease in SARS-CoV-2 positive days. In treated patients compared to control, a clear improvement in Pa02/Fi02 was observed together with a reduction in inflammation parameters, such as the decrease of CRP level. Furthermore, the efficacy of inhaled exogenous adenosine led to an improvement of the prognosis indices, NLR and PLR. The treatment seems to be safe and modulates the immune system, allowing an effective response against the viral infection progression, reducing length of stay and inflammation parameters.

References

1. https://www.who.com

2. Yang X, Yu Y, Xu J, Shu H, Xia J, Liu H, Wu Y, Zhang L, Yu Z, Fang M, Yu T, Wang Y, Pan S Zou X, Yuan S, Shang Y. Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study. Lancet Respir Med. 2020 May;8(5):475-481. doi: 10.1016/S2213- 2600(20)30079-5.

3. Wang D, Hu B, Hu C, Zhu F, Liu X, Zhang J, Wang B, Xiang H, Cheng Z, Xiong Y, Zhao Y, Li Y, Wang X, Peng Z.CIinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-lnfected Pneumonia in Wuhan, China. JAMA. 2020 Feb 7. doi: 10.1001/jama.2020.1585

4. ARDS Definition Task Force, Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, Fan E, Camporota L, Slutsky AS. Acute respiratory distress syndrome: the Berlin Definition. AMA. 2012 Jun 20;307(23):2526-33. doi: 10.1001/jama.2012.5669.

5. Wu C, Chen X, Cai Y, Xia J, Zhou X, Xu , Huang H, Zhang L, Zhou X, Du C, Zhang Y, Song J, Wang S, Chao Y, Yang Z, Xu J, Zhou X, Chen D Xiong W, Xu L, Zhou F, Jiang J, Bai C, Zheng J, Song Y. Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China. JAMA Intern Med. 2020 Mar 13. doi:

10.1001 /jamainternmed.2020.0994.

6. Guan WJ, Ni ZY, Hu Y, Liang W, Ou CQ, He JX, Liu L, Shan H, Lei CL, Hui DSC, Du B, Li LJ, Zeng G, Yuen KY, Chen RC, Tang CL, Wang T, Chen PY, Xiang J, Li SY, Wang JL, Liang ZJ, Peng YX, Wei L, Liu Y, Hu YH, Peng P, Wang JM, Liu JY, Chen Z, Li G, Zheng ZJ, Qiu SQ, Luo J, Ye CJ, Zhu SY, Zhong NS; China Medical Treatment Expert Group for Covid-19. Clinical Characteristics of Coronavirus Disease 2019 in China. N Engl J Med. 2020 Apr 30;382(18):1708- 1720. doi: 10.1056/NEJMoa2002032.

7. Wu Z, McGoogan JM. Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72314 Cases From the Chinese Center for Disease Control and Prevention. JAMA. 2020 Feb 24. doi:

10.1001 /jama.2020.2648.

8. Shi S, Qin M4, Shen B, Cai Y, Liu T, Yang F, Gong W, Liu X, Liang J, Zhao Q, Huang H, Yang B, Huang C. Association of Cardiac Injury With Mortality in Hospitalized Patients With COVID-19 in Wuhan, China. JAMA Cardiol. 2020 Mar 25. doi:

10.1001 /jamacardio.2020.0950.

9. Guo T, Fan Y, Chen M, Wu X, Zhang L, He T, Wang H, Wan J, Wang X, Lu Z. Cardiovascular Implications of Fatal Outcomes of Patients With Coronavirus Disease 2019 (COVID-19). JAMA Cardiol. 2020 Mar 27. doi: 10.1001/jamacardio.2020.1017

10. Ruan Q, Yang K, Wang W, Jiang L, Song J. Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China. Intensive Care Med. 2020 May;46(5):846-848. doi: 10.1007/s00134-020-05991 -x.

11. Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, Qiu Y, Wang J, Liu

Y, Wei Y, Xia J, Yu T, Zhang X, Zhang L. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet. 2020 Feb

15;395(10223):507-513. doi: 10.1016/S0140-6736(20)30211 -7.

12. Cron RQ, Chatham WW. The Rheumatologist's Role in COVID-19. J Rheumatol. 2020 May 1 ;47(5):639-642. doi: 10.3899/jrheum.200334.

13. Mehta P, McAuley DF, Brown M, Sanchez E, Tattersall RS, Manson JJ; HLH Across Speciality Collaboration, UK. COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet. 2020 Mar 28;395(10229):1033-1034. doi: 10.1016/S0140-6736(20)30628- 0.

14. Seo SU, Kweon MN. Virome-host interactions in intestinal health and disease. Curr Opin Virol. 2019 Aug;37:63-71. doi:

10.1016/j.coviro.2019.06.003.

15. Zhang X, Song K, Tong F, Fei M, Guo H, Lu Z, Wang J, Zheng C. First case of COVID-19 in a patient with multiple myeloma successfully treated with tocilizumab. Blood Adv. 2020 Apr 14;4(7):1307-1310. doi: 10.1182/bloodadvances.2020001907.

16. Michot JM Albiges L, Chaput N, Saada V, Pommeret F, Griscelli F, Balleyguier C, Besse B, Marabelle A, Netzer F, Merad M, Robert C, Barlesi F, Gachot B, Stoclin A. Tocilizumab, an anti-IL6 receptor antibody, to treat Covid-19-related respiratory failure: a case report. Ann Oncol. 2020 Apr 2. pii: S0923-7534(20)36387-0. doi: 10.1016/j.annonc.2020.03.300.

17. Grasselli G, Zangrillo A, Zanella A, Antonelli M, Cabrini L, Castelli A, Cereda D, Coluccello A, Foti G, Fumagalli R, lotti G, Latronico N Lorini L, Merler S, Natalini G, Piatti a, Ranieri MV, Scandroglio AM, Storti E, Cecconi M, Pesenti A; COVID-19 Lombardy ICU Network, Nailescu A, Corona A, Zangrillo A, Protti A, Albertin A, Forastieri Molinari A, Lombardo A, Pezzi A, Benini A, Scandroglio AM, Malara A, Castelli A, Coluccello A, Micucci A, Pesenti A, Sala A, Alborghetti A, Antonini B, Capra C, Troiano C, Roscitano C, Radrizzani D, Chiumello D, Coppini D, Guzzon D, Costantini E, Malpetti E, Zoia E, Catena E, Agosteo E, Barbara E, Beretta E, Boselli E, Storti E, Harizay F, Della Mura F, Lorini FL, Donato Sigurta F, Marino F, Mojoli F, Rasulo F, Grasselli G, Casella G, De Filippi G, Castelli G, Aldegheri G, Gallioli G, Lotti G, Albano G, Landoni G, Marino G, Vitale G, Battista Perego G, Evasi G, Citerio G, Foti G, Natalini G, Merli G, Sforzini I, Bianciardi L, Carnevale L, Grazioli L, Cabrini L, Guatteri L, Salvi L, Dei Poli M, Galletti M, Gemma M, Ranucci M, Riccio M, Borelli M, Zambon M, Subert M, Cecconi M, Mazzoni MG, Raimondi M, Panigada M, Belliato M, Bronzini N, Latronico N, Petrucci N, Belgiorno N, Tagliabue P, Cortellazzi P, Gnesin P, Grosso P, Gritti P, Perazzo P, Severgnini P, Ruggeri P, Sebastiano P, Covello RD, Fernandez-Olmos R, Fumagalli R, Keim R, Rona R, Valsecchi R, Cattaneo S, Colombo, Cirri S, Bonazzi S, Greco S, Muttini S, Langer T, Alaimo V, Viola U. Baseline Characteristics and Outcomes of 1591 Patients Infected With SARS-CoV-2 Admitted to ICUs of the Lombardy Region, Italy. JAMA. 2020 Apr 6. doi: 10.1001 /jama.2020.5394.

18. Grasselli G, Pesenti A, Cecconi M Critical Care Utilization for the COVID-19 Outbreak in Lombardy, Italy: Early Experience and Forecast During an Emergency Response. JAMA. 2020 Mar 13. doi: 10.1001 /jama.2020.4031.

19. Manfred Thiel, Alexander Chouker, Akio Ohta, Edward Jackson Charles Caldwell, Patrick Smith, Dmitry Lukashev, Iris Bittmann, and Michail V Sitkovsky corresponding author Oxygenation Inhibits the Physiological Tissue-Protecting Mechanism and Thereby Exacerbates Acute Inflammatory Lung Injury. PLoS Biol. 2005 Jun; 3(6): e174. doi: 10.1371/journal.pbio.0030174

20. Fredholm BB, IJzerman AP, Jacobson KA, Klotz KN, Linden J. International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors Pharmacol Rev. 2001 Dec;53(4):527-52.

21. Sitkovsky MV, Lukashev D, Apasov S, Kojima H, Koshiba M, Caldwell C, Ohta A, Thiel M. Physiological control of immune response and inflammatory tissue damage by hypoxia-inducible factors and adenosine A2A receptors. Annu Rev Immunol. 2004;22:657-82.

22. Ralevic V1 , Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev. 1998 Sep;50(3):413-92

23. Nowak-Machen M, Schmelzle M, Hanidziar D, Junger W, Exley M, Otterbein L, Wu Y, Csizmadia E, Doherty G, Sitkovsky M, Robson SC. Pulmonary natural killer T cells play an essential role in mediating hyperoxic acute lung injury. Am J Respir Cell Mol Biol. 2013 May;48(5):601-9. doi: 10.1165/rcmb.2012-0180OC.

24. Aggarwal NR1 , D'Alessio FR, Eto Y, Chau E, Avalos C, Waickman AT, Garibaldi BT, Mock JR, Files DC, Sidhaye V, Polotsky VY, Powell J, Florton M, King LS. Macrophage A2A adenosinergic receptor modulates oxygen-induced augmentation of murine lung injury Am J Respir Cell Mol Biol. 2013 May;48(5):635-46. doi: 10.1165/rcmb.2012-0351 OC.

25. Cronstein BN, Sitkovsky M..Adenosine and adenosine receptors in the pathogenesis and treatment of rheumatic diseases. Nature Rev Rheumatology 2017 13(1 ):41 -51

26. Ohta A, Sitkovsky M. Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. 2001 Dec 20-27;414(6866):916-20.

27. Hatfield SM, Kjaergaard J Lukashev D, Schreiber TH, Belikoff B,

Abbott R, Sethumadhavan S, Philbrook P, Ko K, Cannici R, Thayer M, Rodig S, Kutok JL, Jackson EK, Karger B, Podack ER, Ohta A, Sitkovsky MV. Immunological mechanisms of the antitumor effects of supplemental oxygenation. SciTransl Med. 2015 Mar

4;7(277):277ra30. doi: 10.1126/scitranslmed.aaa1260.

28. Eltzschig HK1 , Sitkovsky MV, Robson SC. Purinergic signaling during inflammation. N Engl J Med. 2012 Dec 13;367(24):2322-33. doi: 10.1056/NEJMra1205750.

29. Power Coombs MR, Belderbos ME, Gallington LC, Bont L, Levy O. Adenosine modulates Toll-like receptor function: basic mechanisms and translational opportunities. Expert Rev Anti Infect Ther. 2011 Feb;9(2):261-9. doi: 10.1586/eri.10.158.

30. Jacobson KA, Tosh DK, Jain S, Gao ZG. Historical and Current Adenosine Receptor Agonists in Preclinical and Clinical Development Front Cell Neurosci. 2019 Mar 28;13:124. doi: 10.3389/fncel.2019.00124. eCollection 2019.

31. Lucia Spicuzza GiuseppeDiMaria Riccardo Polosa Adenosine in the airways: Implications and applications Author links open overlay panel European Journal of Pharmacology Volume 533, Issues 1-3, 8 March 2006, Pages 77-88 https://doi.Org/10.1016/j.ejphar.2005.12.056 Get rights and content

32. Holgate ST, Cushley MJ, Mann JS, Hughes P, Church MK (1986) The action of purines on human airways. Arch Int PharmacodynTher 280: [Suppl] 240-252

33. Cushley MJ, Tattersfield AE, Holgate ST (1983) Inhaled adenosine and guanosine on airwayresistance in normal and asthmaticsubjects. Br J ClinPharmacol 15: 161-165]

34. van der Wiel E Lexmond AJ, van den Berge M, Postma DS, Hagedoorn P, Frijlink HW, Farenhorst MP, de Boer AH, Ten Hacken NHT. Targeting the small airways with dry powder adenosine: a challenging concept. Eur Clin Respir J. 2017 Sep 6;4(1):1369328. doi: 10.1080/20018525.2017.1369328. eCollection 2017.

35.L’epidemiologia per la sanita pubblica. Istituto Superiore di Sanita. Gli Studi interventistici per I’emergenza COVID-19. https://www.epicentro.iss.it/coronavirus/sars-cov-2-analisi-studi- interventistici [Accessed September 4, 2020]. 36. Md Insiat Islam Rabby (2020). Current Drugs with Potential for

Treatment of COVID-19: A Literature Review. J. Pharm. Pharm. Sci. 23(1), 58-64. https://doi.org/10.18433/jpps31002. Correale, P., Caracciolo, M., Bilotta, F., Conte, M., Cuzzola, M., Falcone, C., Mangano, C., Falzea, A.C., luliano, E., Morabito, A., Foti, G., Armentano, A., Caraglia, M., De Lorenzo, A., Sitkovsky, M., Macheda, S. Therapeutic effects of Adenosine in high flow 21% oxygen aereosol in patients with Covidl 9-Pneumonia. (2020) Plos One. Falcone, C., Caracciolo, M., Correale, P., Macheda, S., Vadala, E. G., La Scala, S., Tescione, M., Danieli, R., Ferrarelli, A., Tarsitano, M. G., Romano, L., & De Lorenzo, A. (2020). Can Adenosine Fight COVID-19 Acute Respiratory Distress Syndrome?. J. Clin. Med. 9(9), E3045. https://doi.org/10.3390/jcm9093045 Aggarwal, N. R., D'Alessio, F. R., Eto, Y., Chau, E., Avalos, C., Waickman, A. T., Garibaldi, B. T., Mock, J. R., Files, D. C., Sidhaye, V., Polotsky, V. Y., Powell, J., Horton, M., & King, L. S. (2013). Macrophage A2A adenosinergic receptor modulates oxygen-induced augmentation of murine lung injury. Am. J. Respir. Cell. Mol. Biol. 48(5), 635-646. https://doi.org/10.1165/rcmb.2012-03510C Jacobson, K. A., Tosh, D. K., Jain, S., & Gao, Z. G. (2019). Historical and Current Adenosine Receptor Agonists in Preclinical and Clinical Development. Front. Cell. Neurosci.13, 124. https://doi.Org/10.3389/fncel.2019.00124 Cushley, M. J., Tattersfield, A. E., & Holgate, S. T. (1983). Inhaled adenosine and guanosine on airway resistance in normal and asthmatic subjects. Br. J. Clin. Pharmacol.15(2), 161-165. https://doi.Org/10.1111/j.1365-2125.1983.tb01481.x van der Wiel, E., Lexmond, A. J., van den Berge, M., Postma, D. S., Hagedoorn, P., Frijlink, H. W., Farenhorst, M. P., de Boer, A. H., & Ten Hacken, N. (2017). Targeting the small airways with dry powder adenosine: a challenging concept. Eur. Clin. Respir. J. 4(1), 1369328. https://doi.Org/10.1080/20018525.2017.1369328 Gualtieri, P., Falcone, C., Romano, L., Macheda, S., Correale, P., Arciello, P., Polimeni, N., & Lorenzo, A. (2020). Body Composition Findings by Computed Tomography in SARS-CoV-2 Patients: Increased Risk of Muscle Wasting in Obesity. Int. J. Mol. Sci. 21 (13), 4670. https://doi.org/10.3390/ijms21134670 44. Romano, L., Marchetti, M., Gualtieri, P., Di Renzo, L., Belcastro, M., De

Santis, G. L., Perrone, M. A., & De Lorenzo, A. (2019). Effects of a Personalized VLCKD on Body Composition and Resting Energy Expenditure in the Reversal of Diabetes to Prevent Complications. Nutrients. 11 (7), 1526. https://doi.org/10.3390/nu11071526 45. ARDS Definition Task Force, Ranieri, V. M., Rubenfeld, G. D.,

Thompson, B. T., Ferguson, N. D., Caldwell, E., Fan, E., Camporota, L., & Slutsky, A. S. (2012). Acute respiratory distress syndrome: the Berlin Definition. JAMA, 307(23), 2526-2533. https://doi.Org/10.1001 /jama.2012.5669 46.Qin, B., Ma, N., Tang, Q., Wei, T., Yang, M., Fu, H., Hu, Z., Liang, Y.,

Yang, Z., & Zhong, R. (2016). Neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) were useful markers in assessment of inflammatory response and disease activity in SLE patients. Mod Rheumatol. 26(3), 372-376. https://doi.Org/10.3109/14397595.2015.1091136

47. Chung, M., Bernheim, A., Mei, X., Zhang, N., Huang, M., Zeng, X., Cui, J., Xu, W., Yang, Y., Fayad, Z. A., Jacobi, A., Li, K., Li, S., & Shan, H. (2020). CT Imaging Features of 2019 Novel Coronavirus (2019-nCoV). Radiology, 295(1), 202-207. https://doi.org/10.1148/radiol.2020200230

Claims

1) Adenosine or a pharmaceutical composition comprising or consisting of adenosine in association with one or more excipients and/or adjuvants, for use in the prevention and treatment of acute respiratory distress syndrome, wherein the adenosine or the pharmaceutical composition is administered by inhalation.

2) Adenosine or pharmaceutical composition according to claim 1 , for use according to claim 1 , wherein acute respiratory distress syndrome is chosen from the group consisting of acute respiratory distress syndrome related to pneumonia, such as viral, bacterial or fungal pneumonia or pneumonia not associated with infections or associated to lung cancer.

3) Adenosine or pharmaceutical composition according to claim 1 , for use according to anyone of claims 1-2, wherein acute respiratory distress syndrome is related to SARS- CoV-2 infection.

4) Adenosine or pharmaceutical composition according to anyone of claim 1-3, for use according to anyone of claims 1-3, wherein the pharmaceutical composition is in a form suitable for inhalation.

5) Adenosine or pharmaceutical composition according to anyone of claims 1-4, for use according to anyone of claims 1-4, wherein the pharmaceutical composition further comprises hyaluronic acid, liposomes.

6) Adenosine or pharmaceutical composition according to anyone of claims 1-5, for use according to anyone of claims 1-5, wherein the inhalation is in combination with oxygen, wherein the percentage of oxygen is the air oxygen percentage.

7) Combination of adenosine with one or more compounds chosen from the group consisting of oxygen in air oxygen percentage, an anti inflammatory drug, a (beta2) adrenergic receptor agonist, an anticancer agent, such as PD-1/PDL-1 immunecheckpoint inhibitors, for separate or sequential use in the prevention and treatment of acute respiratory distress syndrome, wherein the adenosine is administered by inhalation.

8) Combination according to claim 7, for use according to claim 7, wherein acute respiratory distress syndrome is chosen from the group consisting of acute respiratory distress syndrome related to pneumonia, such as viral, bacterial or fungal pneumonia or pneumonia not associated with infections or associated to lung cancer.

9) Combination according to claim 7, for use according to anyone of claims 7-8, wherein acute respiratory distress syndrome is related to SARS- CoV-2 infection.

10) Adenosine or a pharmaceutical composition comprising or consisting of adenosine together with one or more excipients and/or adjuvants, for use in antiviral therapy.

11) Adenosine or a pharmaceutical composition according to claim 10, wherein the antiviral therapy is anti-SARS-CoV-2 therapy.