Senicapoc treatment in COVID‐19 patients with severe respiratory insufficiency—A randomized, open‐label, phase II trial

Abstract Background The aim of the current study was to determine if treatment with senicapoc, improves the PaO2/FiO2 ratio in patients with COVID‐19 and severe respiratory insufficiency. Methods Investigator‐initiated, randomized, open‐label, phase II trial in four intensive care units (ICU) in Denmark. We included patients aged ≥18 years and admitted to an ICU with severe respiratory insufficiency due to COVID‐19. The intervention consisted of 50 mg enteral senicapoc administered as soon as possible after randomization and again after 24 h. Patients in the control group received standard care only. The primary outcome was the PaO2/FiO2 ratio at 72 h. Results Twenty patients were randomized to senicapoc and 26 patients to standard care. Important differences existed in patient characteristics at baseline, including more patients being on non‐invasive/invasive ventilation in the control group (54% vs. 35%). The median senicapoc concentration at 72 h was 62.1 ng/ml (IQR 46.7–71.2). The primary outcome, PaO2/FiO2 ratio at 72 h, was significantly lower in the senicapoc group (mean 19.5 kPa, SD 6.6) than in the control group (mean 24.4 kPa, SD 9.2) (mean difference −5.1 kPa [95% CI −10.2, −0.04] p = .05). The 28‐day mortality in the senicapoc group was 2/20 (10%) compared with 6/26 (23%) in the control group (OR 0.36 95% CI 0.06–2.07, p = .26). Conclusions Treatment with senicapoc resulted in a significantly lower PaO2/FiO2 ratio at 72 h with no differences for other outcomes.


| INTRODUCTION
Coronavirus disease 2019  is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 1,2 COVID-19 is primarily characterized by upper or lower respiratory tract symptoms. 3 Although the majority of COVID-19 cases are asymptomatic or only have mild disease, some patients require respiratory support such as high flow oxygen therapy or mechanical ventilation. 4 In these patients, short-term mortality rates are reported around 30%. 5,6 Interleukin-6 receptor inhibitors and glucocorticoids, both targeting an excessive inflammatory response, have been reported to improve survival among severely ill patients in large randomized trials. [7][8][9] However, no therapies have to date demonstrated an effect of directly targeting the pulmonary tissue injury induced by SARS-CoV-2. Preclinical studies suggest that ion channels situated in the lung's endothelial and epithelial cell layers play a crucial role in activating an inflammatory response and fluid transport across the alveolar-capillary barrier. [10][11][12] The calcium-activated potassium channel of intermediate conductance (KCa3.1) is an ion channel highly expressed in the epithelium and white blood cells. It is a key regulator of fluid transport and inflammatory processes. [13][14][15][16] In a recent study in a mouse model of acute respiratory distress syndrome (ARDS), we found that a single dose of a KCa3.1 channel blocker, senicapoc, improved gas exchange measured as arterial-to-inspired oxygen (PaO 2 /FiO 2 ) ratio, attenuated reduction in lung compliance, and diminished the pulmonary inflammatory response e.g., reduced neutrophil recruitment and pro-inflammatory cytokine release. 17 Furtermore, senicapoc protected against changes in the alveolar-capillary barrier permeability and reduced neutrophil recruitment in a porcine models of ARDS. 17,18 Moreover, senicapoc has been reported to inhibit replication of the arenavirus. 19 In phase III clinical trials for sickle cell anemia, senicapoc was found to be safe and welltolerated in non-critically ill patients. 20 patients were enrolled based on an emergency basis (e.g., with consent from a doctor who was independent of the trial). Subsequently, written consent was obtained from a surrogate. Finally, written consent was obtained from the patient when able.

| Participants
Patients were included if they were aged ≥18 years and admitted to an ICU with severe respiratory insufficiency due to COVID-19. COVID-19 was defined as a positive polymerase chain reaction (PCR) test for SARS-CoV-2, within 14 days prior to ICU admission. Severe respiratory insufficiency was defined as requiring supplemental oxygen ≥10 L/min or mechanical ventilation with an FiO 2 ≥ 40%. Exclusion criteria were severe heart failure (ejection fraction <30%), severe renal insufficiency (eGFR <30 ml/min/1.73 m 2 ), severe hemodynamic instability (noradrenalin dose >0.3 μg/kg/min), prior enrollment in the trial, pregnancy, allergy to senicapoc, inability to take enteral medication, more than 24 h since ICU admission, limitations of care, and anticipated death within 24 h. During the study period, on Sept. 23, 2020 (6 patients included at that time), the exclusion criteria "more than 24 h since ICU admission" was changed from "more than 12 h since ICU admission" due to prolonged response times for confirmation of coronavirus in PCR-base tests.

| Randomization
Eligible patients were randomized in a 1:1 ratio to either enteral senicapoc in addition to standard of care or standard of care alone in blocks with random sizes of 2 or 4. The randomization was stratified according to the baseline PaO 2 /FiO 2 ratio (above or below 20 kPa

| Intervention
The intervention consisted of 50 mg enteral senicapoc (5 Â 10 mg tablets) administered as soon as possible after randomization and again after 24 h. Senicapoc was administered enterally as it is not available as an intravenous drug. Patients in the control group received standard care only. Physicians, patients, and individuals who assessed the outcomes were not blinded to the assigned treatment.
All clinical interventions were left at the discretion of the clinical team for both groups. The level of oxygen therapy and the oxygen level being targeting was determined by the treating ICU physician independent of the trial. Senicapoc is not labeled for the treatment of COVID-19 and the product is still investigational.

| Clinical and laboratory data
Data on demographic characteristics were collected at inclusion, while laboratory values and physiological variables were collected daily for the first 10 days. The use of mechanical ventilation and other oxygen supportive therapies, including neuromuscular blocking agents, prone positioning, and extracorporeal membrane oxygenation (ECMO), were collected daily through day 10. The radiographic assessment of lung edema (RALE) score was used to evaluate baseline chest X-rays. 22

| Outcomes
The primary outcome was the PaO 2 /FiO 2 ratio 72 h after randomiza-  Additional outcomes included vasopressor-free days, need for renal replacement therapy within 28 days, and health-related quality of life (EQ-5D-5L) at 28 days. 23 For patients unable to respond to the EQ-5D-5L questionnaire due to health reasons (e.g., respiratory insufficiency, incompetent, still on mechanical ventilation) we assigned worst values. For EQ-5D-5L, an index value based on Danish data was calculated using the "Crosswalk Index Value Calculator." 24 Blood samples for measurement of senicapoc plasma concentrations and SARS-CoV-2 were drawn at baseline and after 24, 48, 72, 120, and 168 h. Blood samples were collected in EDTA tubes, centrifuged for 10 min at 3000 rpm, and the plasma was stored at À80 C.
A rapid, sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to quantify senicapoc. 25 SARS-CoV2 was measured by PCR in plasma, see Supplement Material. 26

| Adverse events
To assess specific potential adverse events, we collected data on the following: cardiac arrhythmias, vasopressor refractory shock, allergic reaction, acute coronary syndrome, anemia, leucopenia, and hyperglycemia. For definitions, see Supplementary Material.

| Sample size
The sample size was based on the primary outcome of the PaO 2 /FiO 2 ratio at 72 h. Given the novelty of COVID-19 at the time of protocol writing, there were limited data to support a definitive sample size calculation.
Based on preliminary data, 3,27,28 we anticipated a PaO 2 /FiO 2 ratio of 120 mmHg (16 kPa) in the control group and 180 mmHg (24 kPa) in the senicapoc group. With a common standard deviation of 70 mmHg (9 kPa), an alpha of 5%, and based on a t-test, 46 patients were needed to have 80% power to detect a statistically significant difference.

| Statistical analysis
Continuous variables are presented as medians with interquartile range (IQR) and categorical variables as counts with frequencies. Renal disease 0 (0) 2 (7.7) Median Frailty score prior to acute illness (IQR)

(2-3) (3-4)
Median RALE score (IQR) a 10 (4-21) 14 (  F I G U R E 3 Mean PaO 2 /FiO 2 ratio from baseline to day 10 in the two groups. All patients with a missing PaO 2 /FiO 2 ratio from day 8 and forward were discharged home, except for one patient in the standard care group dying at day 8. Error bars SD 3 | RESULTS

| Trial flow and baseline characteristics of participants
From 28th of April 2020 to 28th of December 2020, 48 patients were randomized. Two patients were excluded from the trial. One patient withdrew consent and one patient received a limitation of care order immediately after randomization and before trial drug administration ( Figure 1). Of the included patients, 20 were randomized to senicapoc, while 26 were randomized to the control group. The trial groups had similar characteristics regarding age, body mass index, the interval between ICU admission and randomization, and the majority of coexisting illnesses (Table 1). More patients in the senicapoc group had diabetes, while more patients in the control group were men and had a higher Sequential Organ Failure Assessment (SOFA) Score and RALE score. Patients in the two groups were balanced with regards to COVID-19 disease characteristics, including symptoms, COVID-19 treatments, and enrollment into other trials (eTable 1). More patients in the control group were on non-invasive/invasive ventilation at baseline (54% vs. 35%); otherwise, groups were balanced at baseline for respiratory and arterial blood gas parameters (eTable 2).

| Senicapoc
In controls, the senicapoc concentration was below the detection limit in all samples. Senicapoc concentrations in the intervention group is displayed in Figure 2.

| Primary outcome
The PaO 2 /FiO 2 ratio was significantly lower in the senicapoc group at 72 h than in the control group ( lower in the senicapoc group from baseline to day 8 but higher from day 8 and forward ( Figure 3). All patients with a missing PaO 2 /FiO 2 ratio from day 8 and forward were discharged home, except for one patient in the standard care group dying at day 8 ( Figure 3).

| Secondary outcomes
Secondary outcomes are listed in

| Respiratory parameters and arterial blood gas values
Respiratory parameters and arterial blood gas values are shown in e- Ventilator settings and arterial blood gas values were in general comparable between the two groups.

| SARS-CoV2 was measured by PCR
The number of SARS-CoV2 viral copies are displayed in eFigure 9.
There was no change in number of copies over time or between groups.

| Adverse events
Specific adverse events are shown in eTable 4. Specific adverse events were low in both groups with no serious adverse events attributed to the study treatment.

| DISCUSSION
In this trial, senicapoc treatment in patients admitted to the ICU due to severe respiratory insufficiency resulted in a statistically significantly lower PaO 2 /FiO 2 ratio. There were no differences in the secondary outcomes.
The current clinical trial is the first investigating the effect of senicapoc administered to patients with severe respiratory insufficiency.
Previous clinical trials of senicapoc have only included outpatients with sickle cell disease or asthma with a limited number of treatment-related adverse effects. 21,30 In contrast, patients with severe respiratory insufficiency admitted to the ICU are at high risk of organ failure, and there is increased concern regarding the interaction with other drugs. In general, the reported number of specific adverse events was low, with no serious adverse events attributed to the study drug. These observations should, of course, be interpreted in light of the small sample size. The safety of the intervention is therefore still unknown.
Senicapoc was administered orally or administered through a gastric feeding tube. Based on previous dose-findings studies, a plasma concentration of 56.5 ng/ml and 115.5 ng/ml was anticipated at 24 and 48 h. Although slightly lower plasma concentrations were measured, the achieved concentrations were above the desired concentration of 3.23 ng/ml. 31 The study was therefore successful in obtaining the desired plasma concentrations.
Contrary to our hypothesis of an improvement in PaO 2 /FiO 2 ratio, we observed a significantly lower PaO 2 /FiO 2 ratio in the senicapoc group at 72 h. Although the study was randomized and strati- with the publication of studies demonstrating a protective effect of corticosteroids, dexamethasone was implemented as a standard of care. 8,42 Consequently, more than 90% of the included patients in both groups received dexamethasone, which could have dampened a potential protective anti-inflammatory effect of senicapoc, as dexamethasone was not included as a standard of care therapy in the original animal studies. 18,34 Although the primary endpoint favored standard care, several secondary endpoints pointed towards a protective effect of senicapoc, including 28-day mortality and the number of patients receiving renal replacement therapy. However, none of the differences were statistically significant, and in any case, the comparisons should be considered explorative, only. The higher ventilator-free hours and vasopressor-free hours in the senicapoc group should be interpreted in the light of a lower number of patients being on non-invasive/ invasive ventilation at baseline. A higher number of patients in the senicapoc group were therefore never exposed to non-invasive/ invasive ventilation and as consequence of this vasopressor therapy as vasopressor therapy is more frequently used in sedated mechanically ventilated patients.
The current trial has important strengths. The trial was completed within a short period of time, in a setting without a significant burden on the health care system and a constant mortality rate during the entire pandemic. 5,43 There was no loss to follow-up, and detailed data on respiratory parameters and laboratory data were included.
The trial also has some limitations. were randomized to standard care than the control group. Also, considerable imbalances existed at baseline which is a limitation. This is likely caused by the small study size, one site recruiting a limited number of patients, and 2 patients being excluded in the senicapoc group after randomization. Exclusion of patients after randomization may have introduced bias.

| CONCLUSION
Treatment with senicapoc resulted in a significantly lower PaO 2 /FiO 2 ratio at 72 h with no difference for other outcomes.

DATA AVAILABILITY STATEMENT
Six months after the publication of the results, all deidentified individual patient data will be made available for data sharing.