Intensive Care Unit outcomes of COVID-19: A National Cohort study from Malta

Introduction: The COVID-19 virus has caused a massive strain on medical services worldwide. Throughout 2020 and 2021, hospitals and their Intensive Care Units (ICUs) have been inundated with patients suffering from critical illness due to COVID-19, many of whom developed multiorgan failure and required prolonged ICU stays.1 Malta is a Mediterranean island with a population of 500,000 people, with one main acute general hospital (Mater Dei Hospital) with a single 20-bed ICU. This meant that the COVID-19 pandemic surge had to be managed locally by increasing ICU capacity without access to a referral network of other hospital ICUs at different phases of the pandemic or the possibility of referral to ECMO services abroad. Objectives: Establish the demographics and outcomes of all patients admitted to ICU with COVID-19 in Malta. Methods: A single centre prospective cohort study conducted in the Intensive Care Units caring for COVID-19 patients at Mater Dei Hospital, Malta. Data was collected on admission and then daily until death or discharge from ICU. Results: The COVID-19 pandemic resulted in 252 patients being admitted to ICU from March 2020 to May 2021. The peak of admissions occurred in March 2021 with a maximum of 21 admissions in a week resulting in a peak of 33 COVID-19 ICU beds being utilized. This represents 165% of the normal 20 bed ICU capacity. There were 9 readmissions, these were excluded from data analysis. All patients admitted to ICU were treated with Dexamethasone and Remdesivir, and Tocizulimab from January 2021, unless contraindicated. Overall ICU mortality was 34% and increased to 46% in those requiring mechanical ventilation. Males were responsible for 75% of admissions but gender was not associated with ICU mortality. Older patients and those with ischemic heart disease (IHD) and diabetes had a significantly increased mortality as were those patients with a higher Sequential Organ Failure Assessment (SOFA) and lower PaO2/FiO2 (P/F) on admission (Table 1). The total number of patients requiring intubation during their admission was 173 (69%) with a median time to intubation of two days [IQR 1-4]. Proning was used for 124 (69%) of the mechanically ventilated patients for a median of 2 days per patient [IQR: 1 -3], similarly muscle relaxant infusion was also used in 124 of mechanically ventilated patients (69%) for a median of three days per patient [IQR: 2 -5]. The median duration of mechanical ventilation was 11 days [IQR: 6 -22.2] with a maximum of 63 days. Tracheostomies were performed in 59 (34%) of mechanically ventilated patients with a median duration of 14.5 days intubated prior to tracheostomy [IQR: 13 -17]. The median length of stay was 11.5 days [7-23]. Conclusion: This observational study represents all COVID-19 ICU admissions that occurred in Malta from March 2020 to May 2021 in the single institution caring for these patients in the country. We have demonstrated a predominantly male, elderly admission population with an increased mortality associated with age, ischemic heart disease and diabetes. Overall ICU mortality was 34% and 46% in ventilated patients, which is comparable to that found in other national databases.2.

Self-Masked Aldehyde Inhibitors of Human Cathepsin L Are Potent Anti-CoV-2 Agents.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of compounds, self-masked aldehyde inhibitors (SMAIs) which are based on the dipeptide aldehyde inhibitor (Cbz-Phe-Phe-CHO, 1), for which the P1 Phe group contains a 1'-hydroxy group, effectively, an o-tyrosinyl aldehyde (Cbz-Phe-o-Tyr-CHO, 2; (Li et al. (2021) J. Med. Chem. 64, 11,267-11,287)). Compound 2 and other SMAIs exist in aqueous mixtures as stable δ-lactols, and apparent catalysis by the cysteine protease cruzain, the major cysteine protease of Trypanosoma cruzi, results in the opening of the lactol ring to afford the aldehydes which then form reversible thiohemiacetals with the enzyme. These SMAIs are also potent, time-dependent inhibitors of human cathepsin L (K i = 11-60 nM), an enzyme which shares 36% amino acid identity with cruzain. As inactivators of cathepsin L have recently been shown to be potent anti-SARS-CoV-2 agents in infected mammalian cells (Mellott et al. (2021) ACS Chem. Biol. 16, 642-650), we evaluated SMAIs in VeroE6 and A549/ACE2 cells infected with SARS-CoV-2. These SMAIs demonstrated potent anti-SARS-CoV-2 activity with values of EC50 = 2-8 µM. We also synthesized pro-drug forms of the SMAIs in which the hydroxyl groups of the lactols were O-acylated. Such pro-drug SMAIs resulted in significantly enhanced anti-SARS-CoV-2 activity (EC50 = 0.3-0.6 µM), demonstrating that the O-acylated-SMAIs afforded a level of stability within infected cells, and are likely converted to SMAIs by the action of cellular esterases. Lastly, we prepared and characterized an SMAI in which the sidechain adjacent to the terminal aldehyde is a 2-pyridonyl-alanine group, a mimic of both phenylalanine and glutamine. This compound (9) inhibited both cathepsin L and 3CL protease at low nanomolar concentrations, and also exerted anti-CoV-2 activity in an infected human cell line.

Self-Masked Aldehyde Inhibitors of Human Cathepsin L Are Potent Anti-CoV-2 Agents

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of compounds, self-masked aldehyde inhibitors (SMAIs) which are based on the dipeptide aldehyde inhibitor (Cbz-Phe-Phe-CHO, 1), for which the P1 Phe group contains a 1′-hydroxy group, effectively, an o-tyrosinyl aldehyde (Cbz-Phe-o-Tyr-CHO, 2;(Li et al. (2021) J. Med. Chem. 64, 11,267–11,287)). Compound 2 and other SMAIs exist in aqueous mixtures as stable δ-lactols, and apparent catalysis by the cysteine protease cruzain, the major cysteine protease of Trypanosoma cruzi, results in the opening of the lactol ring to afford the aldehydes which then form reversible thiohemiacetals with the enzyme. These SMAIs are also potent, time-dependent inhibitors of human cathepsin L (Ki = 11–60 nM), an enzyme which shares 36% amino acid identity with cruzain. As inactivators of cathepsin L have recently been shown to be potent anti-SARS-CoV-2 agents in infected mammalian cells (Mellott et al. (2021) ACS Chem. Biol. 16, 642–650), we evaluated SMAIs in VeroE6 and A549/ACE2 cells infected with SARS-CoV-2. These SMAIs demonstrated potent anti-SARS-CoV-2 activity with values of EC50 = 2–8 μM. We also synthesized pro-drug forms of the SMAIs in which the hydroxyl groups of the lactols were O-acylated. Such pro-drug SMAIs resulted in significantly enhanced anti-SARS-CoV-2 activity (EC50 = 0.3–0.6 μM), demonstrating that the O-acylated-SMAIs afforded a level of stability within infected cells, and are likely converted to SMAIs by the action of cellular esterases. Lastly, we prepared and characterized an SMAI in which the sidechain adjacent to the terminal aldehyde is a 2-pyridonyl-alanine group, a mimic of both phenylalanine and glutamine. This compound (9) inhibited both cathepsin L and 3CL protease at low nanomolar concentrations, and also exerted anti-CoV-2 activity in an infected human cell line.

High Flow Nasal Cannula Use: A Survey on Weaning Practices

Rationale: High flow nasal cannula (HFNC) is first line therapy for patients with acute hypoxic respiratory failure (AHRF). HFNC utilization among hospitalized patients with AHRF has increased substantially during the COVID-19 pandemic. While good evidence exists to guide initiation of HFNC, evidence-based strategies for HFNC weaning are lacking. Therefore, we sought to characterize HFNC weaning practices among respiratory therapists (RTs) to determine the degree of practice variability and assess general approaches to HFNC weaning. Methods: We conducted a cross-sectional survey evaluating HFNC weaning practices of adult inpatient RTs at an academic, tertiary care medical center in the United States. Survey participants were asked about their practice of HFNC weaning and whether use of an institutional protocol provided guidance for HFNC weaning. Survey questions also assessed RT knowledge of institutional policies regarding HFNC use in hospitalized patients, including location of HFNC use within the hospital, personnel involved in HFNC titration, and methods of weaning through clinical vignettes. Survey responses were analyzed using descriptive statistics. Results: Overall, 21 of 68 RTs surveyed completed the survey (response rate 31%). The majority of participants (95%) worked primarily in an ICU, general wards, or emergency department. Approximately one-third of participants were unaware of a HFNC weaning protocol at their institution. Among those who endorsed the existence of a HFNC weaning protocol at their institution, 79% reported using the protocol “always or often.” We found substantial variation in RT knowledge of institutional policies regarding which practitioners were permitted to wean HFNC, with approximately one-half of participants believing that any practitioner could wean FiO2 and 43% believing that only RTs could wean flow. Additionally, participants' approaches to weaning varied substantially in response to clinical vignettes. For example, in the vignette of a clinically stable patient on HFNC at 60L and 100%, 62% of participants chose to wean only FiO2, 14% to wean only flow, and 24% to wean both. Conclusion: Nearly one-third of respiratory therapists were unaware of the existing HFNC weaning protocol at their institution, potentially contributing to the substantial variability in HFNC weaning practices between surveyed RTs. More research is needed to identify and successfully implement optimal weaning strategies for HFNC among patients hospitalized with AHRF.

The impact of COVID-19 pandemic on intensive care workload at Mater Dei Hospital in Malta

Introduction: The aim of the study was to determine the impact of COVID-19 pandemic on intensive care workload [1,2] at our only acute main general hospital on the island. During the pandemic surge in March 2021, our intensive care was running at 200% capacity. Mater Dei Hospital has a 20-bedded adult intensive care catering for a population of 500,000. Methods: This is a prospective cohort study conducted in the COVID- 19 Intensive Care Unit at Mater Dei Hospital, Malta. Data analysed is from March 2020 to May 2021. Data collected daily from admission until death or discharge from ICU. Results: A total of 261 patients with severe acute respiratory distress syndrome coronavirus 2 (SARS-Cov-2) required admission to our intensive care. ICU facilities required expansion into a total of 5 Intensive Care Units, therefore reaching a capacity of 44 intensive care beds during the peak month of March 2021. A maximum of 21 patients were admitted per week culminating to a total of 33 COVID-19 Intensive Care beds during the month of March 2021. A total of 179 patients (68.6%) required mechanical ventilation for a median duration of 11 days per patient. Proning was required in 124 mechanically ventilated patients (70.5%). 50 patients (20%) required CRRT with a maximum number of 7 patients per day requiring CRRT. Conclusions: COVID-19 pandemic transformed the way how we provide critical care with improved bed capacity, ICU triage and ICU devices. This study highlighted the need for more clinical guidelines and their availability for online use. This will positively impact the care of non-COVID patients. It also highlighted the need for more training of non-ICU staff to allow for surges in ICU capacity. The COVID-19 pandemic has seen Mater Dei hospital already investing in ICU personnel and equipment as this cannot be reactive to large scale events but must be a proactive planned strategy to enhance resilience of our ITU.

High flow nasal oxygen in the intensive care setting during the COVID-19 pandemic at Mater Dei Hospital, Malta

Introduction: The aim of this study was to describe the use of high flow nasal oxygen (HFNO) in COVID-19 intensive care unit (ICU) patients [1] locally, and establish their demographics and outcomes. Mater Dei Hospital is the only main acute general hospital on the island. It houses a 20-bedded adult ICU catering for a population of 500,000. Methods: We conducted a single-centre prospective observational cohort study at the ICU at Mater Dei Hospital in Malta between March 2020 and May 2021. Data collected included use of HFNO, mechanical ventilation (MV), duration of MV, length of stay, and 28-day survival. Results: 240 COVID-19 ICU patients were included. 108 (45%) received HFNO for a median of 3 days, the rest received MV for a median of 12 days. No major differences in demographics were noted (age: 66.5 vs 68 years, p = 0.225;70% male, 30% female vs 79% male, 21% female, p = 0.191). Forty-two (38.2%) patients failed HFNO after a median of 2 days, needing MV for a median of 10 days (p < 0.001). Median length of stay was lower in HFNO patients (6 vs 13 days;p < 0.001). 28-day survival was highest in the HFNO-only group (94%), followed by the HFNO + MV group (61%), and finally the MV-only group (52%;p < 0.0001). This is not simply due to severity since FiO2 was higher for HFNO patients and PaO2 tended to be lower. Cox proportional hazards analysis showed that respiratory support was more significant than admission P/F ratios, PaO2s, or SOFA, with MV being linked to a hazards ratio of 8.4 (p < 0.001) when adjusted for the above criteria. Conclusions: HFNO offers considerable practical advantages over MV. Avoiding MV might be linked to a reduced incidence of ventilator-associated pneumonias, shorter ICU stay and lower mortality. It is also a safe tool to use and the risk of aerosolization should not deter from its use.

A Clinical-Stage Cysteine Protease Inhibitor blocks SARS-CoV-2 Infection of Human and Monkey Cells.

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 µM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 µM. There was no toxicity to any of the host cell lines at 10-100 µM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.

Dinitrosyl iron complexes (DNICs) as inhibitors of the SARS-CoV-2 main protease.

By repurposing DNICs designed for other medicinal purposes, the possibility of protease inhibition was investigated in silico using AutoDock 4.2.6 (AD4) and in vitro via a FRET protease assay. AD4 was validated as a predictive computational tool for coordinatively unsaturated DNIC binding using the only known crystal structure of a protein-bound DNIC, PDB- (calculation RMSD = 1.77). From the in silico data the dimeric DNICs TGTA-RRE, [(µ-S-TGTA)Fe(NO)2]2 (TGTA = 1-thio-ß-d-glucose tetraacetate) and TG-RRE, [(µ-S-TG)Fe(NO)2]2 (TG = 1-thio-ß-d-glucose) were identified as promising leads for inhibition via coordinative inhibition at Cys-145 of the SARS-CoV-2 Main Protease (SC2Mpro). In vitro studies indicate inhibition of protease activity upon DNIC treatment, with an IC50 of 38 ± 2 µM for TGTA-RRE and 33 ± 2 µM for TG-RRE. This study presents a simple computational method for predicting DNIC-protein interactions; the in vitro study is consistent with in silico leads.

Self-Masked Aldehyde Inhibitors: A Novel Strategy for Inhibiting Cysteine Proteases.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas disease-Trypanosoma cruzi. These SMAIs exerted potent, reversible inhibition of cruzain (Ki* = 18-350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.

Bepridil is potent against SARS-CoV-2 in vitro.

Guided by a computational docking analysis, about 30 Food and Drug Administration/European Medicines Agency (FDA/EMA)-approved small-molecule medicines were characterized on their inhibition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). Of these small molecules tested, six displayed a concentration that inhibits response by 50% (IC50) value below 100 µM in inhibiting Mpro, and, importantly, three, that is, pimozide, ebastine, and bepridil, are basic molecules that potentiate dual functions by both raising endosomal pH to interfere with SARS-CoV-2 entry into the human cell host and inhibiting Mpro in infected cells. A live virus-based modified microneutralization assay revealed that bepridil possesses significant anti-SARS-CoV-2 activity in both Vero E6 and A459/ACE2 cells in a dose-dependent manner with low micromolar effective concentration, 50% (EC50) values. Therefore, the current study urges serious considerations of using bepridil in COVID-19 clinical tests.

A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells.

K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 µM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 µM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2 , differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.

Targeting the SARS-CoV-2 Main Protease to Repurpose Drugs for COVID-19 (preprint)

Guided by a computational docking analysis, about 30 FDA/EMA-approved small molecule medicines were characterized on their inhibition of the SARS-CoV-2 main protease (MPro). Of these tested small molecule medicines, six displayed an IC50 value in inhibiting MPro below 100 M. Three medicines pimozide, ebastine, and bepridil are basic small molecules. Their uses in COVID-19 patients potentiate dual functions by both raising endosomal pH to slow SARS-CoV-2 entry into the human cell host and inhibiting MPro in infected cells. A live virus-based microneutralization assay showed that bepridil inhibited cytopathogenic effect induced by SARS-CoV-2 in Vero E6 cells completely at and dose-dependently below 5 M and in A549 cells completely at and dose-dependently below 6.25 M. Therefore, the current study urges serious considerations of using bepridil in COVID-19 clinical tests.