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Inhibition of Bruton tyrosine kinase in patients with severe COVID-19 - Science
Jun 05, 2020 9 mins, 54 secs
Acalabrutinib, a selective BTK inhibitor, was administered off-label to 19 patients hospitalized with severe COVID-19 (11 on supplemental oxygen; 8 on mechanical ventilation), 18 of whom had increasing oxygen requirements at baseline.

Over a 10-14 day treatment course, acalabrutinib improved oxygenation in a majority of patients, often within 1-3 days, and had no discernable toxicity.

At the end of acalabrutinib treatment, 8/11 (72.7%) patients in the supplemental oxygen cohort had been discharged on room air, and 4/8 (50%) patients in the mechanical ventilation cohort had been successfully extubated, with 2/8 (25%) discharged on room air.

Ex vivo analysis revealed significantly elevated BTK activity, as evidenced by autophosphorylation, and increased IL-6 production in blood monocytes from patients with severe COVID-19 compared with blood monocytes from healthy volunteers.

The spectrum of COVID-19 ranges from a mild respiratory illness to a severe disease requiring hospitalization in up to a third of patients, with frequent progression to acute respiratory distress syndrome (ARDS) and a high mortality (2).

During severe COVID-19, the heightened levels of IL-1β in several COVID-19 patients (11, 12) indicates the formation of an NLRP3 inflammasome that converts pro-IL-1β to mature IL-1β (54).

In an effort to reduce inflammation and improve clinical outcome of patients with severe COVID-19, we administered acalabrutinib, a highly specific covalent inhibitor of BTK approved in the United States for the treatment of lymphoid malignancies (28).

Herein, we present a prospective off-label clinical study of 19 hospitalized patients with COVID-19 and severe hypoxia who also had evidence of inflammation and/or severe lymphopenia.

This prospective off-label clinical study includes 19 hospitalized patients with severe COVID-19 who received off-label acalabrutinib between March 20, 2020 (date of treatment of the first patient) through April 10, 2020 with formal data collection completed on April 23, 2020 (Table S1).

Eleven (58%) patients were receiving supplemental oxygen for a median of 2 days (range: 1-12), 7/11 (64%) of whom were on high flow nasal cannula at the time they began acalabrutinib (“supplemental oxygen cohort”).

In addition, 8 (42%) patients were receiving invasive mechanical ventilation for a median of 1.5 (range: 1-22) days prior to acalabrutinib administration (“mechanical ventilation cohort”).

In the supplemental oxygen cohort, concomitant drugs for the treatment of COVID-19 included steroids and/or hydroxychloroquine in 5/11 (45%) patients each, and in the mechanical ventilation cohort, 6/8 (75%) and 3/8 (38%) patients, respectively, received these drugs.

Laboratory evidence of inflammation with elevated CRP and/or ferritin was present in 18/19 (95%) patients with significantly elevated baseline laboratory abnormalities prior to acalabrutinib dosing including elevated CRP (> 10 mg/dL) in 15/19 (79%) patients [median (range) of 18.7 (2-31.5)]; ferritin (> 500 ng/mL) in 16/19 (84%) patients [median (range) 1240 (155-4168)]; fibrinogen (> 400 mg/dl) in 10/10 (100%) patients [median (range) 605 (409- >1000)]; D-dimer (> 0.5 mcg/mL) in 15/17 (88%) patients [median (range) 1.65 (0.48- >20)]; IL-6 (≥ 15 pg/mL) in 9/9 (100%) patients [median (range) 44 (25-89.8)]; and severely decreased ALC (≤ 1000 cells/μL) in 15/18 (83%) patients [median (range) 675 (250-1700)] (Table 1).

Among 11 patients in the supplemental oxygen cohort, the median duration of follow-up from the initiation of acalabrutinib treatment was 12 (range 10-14) days.

At the time of formal data collection, 8 (73%) patients no longer required supplemental oxygen and had been discharged from the hospital.

Eight patients on invasive mechanical ventilation were followed for a median of 12 days (range 7-30) from the initiation of acalabrutinib treatment and received the anticipated treatment duration of 10 to 14 days, with the exception of 2 patients who died (Fig. 3).

In the supplementary oxygen cohort, 9 patients had been discharged on room air and remained clinically well, one was still hospitalized, and one died.

In the mechanical ventilation cohort, 3 patients were discharged on room air and remained well, one was discharged to rehabilitation, and 4 patients died.

In the 11 patients on supplemental oxygen, CRP returned to normal in 10 (91%) patients and was decreasing in one (9%) patient (Fig. 2, 3).

The 8 patients who began acalabrutinib while on mechanical ventilation showed a more variable and blunted change in laboratory values compared to those on supplemental oxygen.

Mixed-effect regression analysis showed that over time, patients in the supplemental oxygen cohort generally increased their oxygen uptake efficiency (p= 3.65E-6) and ALC (p= 0.0252) and decreased their CRP levels (p=1.15E-4) (Table S8), as illustrated by the trend lines in Fig.

CRP levels were inversely associated with oxygen uptake efficiency in both the supplemental oxygen cohort (p=1.82E-3) and in the mechanical ventilation cohort (p=1.46E-2) (Fig. 4B).

ALC was directly associated with oxygen uptake efficiency in the supplemental oxygen cohort (p=2.11E-4), but not in the mechanical ventilation cohort (Fig. 4B).

Plots of oxygen uptake efficiency (SpO2/FiO2), CRP and ALC levels versus days of acalabrutinib treatment for all patients at all time points.

Patients in the supplemental oxygen and mechanical ventilation cohorts are indicated in red and blue, respectively.

To examine whether the target of acalabrutinib, BTK, was activated in patients with COVID-19, we studied BTK autophosphorylation at residue Y223 in whole blood samples from 3 patients with severe COVID-19 (Table S10) and in 5 healthy volunteers (Fig. S1).

We observed a significantly increased mean fluorescence intensity of phosphorylated BTK in CD14+ monocytes from patients with severe COVID-19 relative to that observed in healthy volunteers, an increase that was not due to differential levels of total BTK (Fig. 5A).

Flow cytometric analysis of unstimulated whole blood samples revealed a significant increase in the percentage of IL-6+ CD14+ monocytes in patients with severe COVID-19 (n=4) compared with healthy volunteers (n=5; Fig. 5B).

Treatment of these whole blood samples with the small molecule R848, a mimic of TLR7 and TLR8 activation by single strand RNA, increased the percentage of IL-6+ blood monocytes, with significantly higher levels in samples from COVID-19 patients compared to healthy controls (Fig. 5B).

Of note, the percentage of IL-6+ monocytes in patients with severe COVID-19 without ex-vivo restimulation was comparable to that observed in monocytes from healthy volunteers following R848 stimulation (Fig. 5B).

In keeping with these findings, blood IL-6 levels in COVID-19 patients on our clinical study decreased during acalabrutinib treatment (p=6.5E-4) (Fig. 5C).

Left panels: histograms of BTK phosphorylation in CD14+ blood monocytes from 3 patients with severe COVID-19 (A, B, C; Table S10) and 4 healthy volunteers, as indicated.

Right panels: Summary data showing significant increase in mean fluorescence intensity of phosphorylated BTK (residue Y223) in CD14+ monocytes from 3 COVID-19 patients compared with 5 healthy volunteers, with no evident BTK phosphorylation in CD19+ B cells.

Left panels: Representative contour plots of intracellular IL-6 production in CD14+ monocytes from a patient with severe COVID-19 (Patient C; Table S10) and a healthy volunteer, either as unstimulated ex vivo cells or following R848 (10 μM) stimulation, as indicated.

Plot of blood IL-6 concentrations (pg/ml) on a log scale versus days of acalabrutinib treatment for patients in whom there were at least two IL-6 measurements during the plotted time course.

Patients in the supplemental oxygen (n=5) and mechanical ventilation (n=3) cohorts are indicated in red and blue, respectively.

In accordance with World Health Organizations guidance (29), we prospectively administered acalabrutinib off-label with therapeutic intent to 19 hospitalized patients with severe COVID-19, based on the known role of BTK in innate immune cells.

All but one patient had increasing oxygen requirements at the time of treatment initiation, and all but 4 patients were on high-flow oxygen or invasive mechanical ventilation, indicating the severity of the disease in this series.

The oxygenation and clinical status of most patients on supplemental oxygen improved relatively rapidly following acalabrutinib initiation, which was temporally associated with a normalization of inflammatory markers.

Although the patients on mechanical ventilation had a more variable clinical response to acalabrutinib, improved oxygenation in half of these patients allowed them to be extubated.

Our laboratory studies of ex vivo blood samples from patients hospitalized with COVID-19 revealed significantly elevated BTK phosphorylation in peripheral blood monocytes compared with healthy volunteers, demonstrating that the target of acalabrutinib is activated in these innate immune cells.

Many patients in this series had a severely depressed ALC, which has also been associated with severe COVID-19.

The apparent beneficial effect of acalabrutinib was clearly different between patients who were on supplemental oxygen and those who required mechanical ventilation.

The association between oxygen uptake efficiency and normalization of CRP was also evident in the mechanical ventilation cohort.

Since our study investigated the effect of a limited course of acalabrutinib in severe COVID-19, we were interested in whether the disease recurred after acalabrutinib cessation.

However, since we have only treated a small cohort of patients, the safety profile of acalabrutinib in patients with severe COVID-19 needs to be confirmed in a prospective clinical trial.

Ex vivo analysis of blood samples from patients with severe COVID-19 revealed BTK activation in monocytes in all cases, as evidenced by significantly increased BTK phosphorylation compared with monocytes from healthy volunteers.

While BTK inhibitors interfere with B cell activation and could potentially lower anti-viral antibody titers, this concern may be mitigated by the timing of administration to patients with severe COVID-19, who are typically hospitalized 7 or more days following initial infection.

A more complete understanding how BTK inhibitors modulate the immune pathophysiology of COVID-19 will require the use of preclinical model systems in concert with detailed immune profiling of patients with COVID-19, before and during treatment with a BTK inhibitor.

After we initiated our prospective off-label clinical study of acalabrutinib in COVID-19, investigators interested in the role of BTK in COVID-19 reported that among 6 patients with confirmed COVID-19 who were taking the BTK inhibitor ibrutinib chronically for their hematologic malignancy, only one patient was hospitalized (37).

In this regard, ibrutinib has a higher incidence of serious bleeding and pro-arrhythmic side effects than acalabrutinib, toxicities that may worsen the outcome of patients with severe COVID-19 (39).

BTK activation occurs in macrophages when TLRs bind single-stranded RNA, as may occur in SARS-CoV-2 infection, leading to NF-κB-dependent expression of multiple inflammatory cytokines and chemokines, including IL-6 which we observed was induced in COVID-19 monocytes and decreased in plasma following acalabrutinib treatment (Fig. 1).

Given the activation of BTK and production of IL-6 that we detected in COVID-19 monocytes, we propose that BTK inhibitors target pathological monocyte/macrophage activation and dampen the cytokine storm, which consequently may improve outcomes in these patients.

The selection criteria included hospitalized patients with confirmed COVID-19 and hypoxia (room air blood oxygen saturation (SpO2) of 94% or less) requiring supplemental oxygen and ferritin ≥ 500 ng/mL, C-reactive protein ≥ 10 mg/dL and/or an absolute lymphocyte count < 1000 cells/μL.

We communicated with physicians at five hospitals to identify hospitalized patients who met these criteria and had individual case-based discussions with the treating physicians regarding the use of acalabrutinib as an off-label treatment for patients who were either deteriorating or not improving on best supportive care.

Patients received the approved acalabrutinib dose of 100 mg orally or per enteric feeding tube twice daily for 10 days (patients on supplemental oxygen) and 14 days (patients on mechanical ventilation).

Patients who are receiving corticosteroids for COVID-19 at the time of acalabrutinib institution should be weaned off as appropriate.

The treating physician was included in these discussions to inform on other treatment options for severe COVID-19.

Four patients who were hospitalized with severe COVID-19 at the NIH Clinical Center (n = 2) or George Washington University Hospital (n = 2) enrolled in NIH IRB-approved protocols (NCT00001467; NCT01200953).

Comparison of the frequency of IL-6+ CD14+ monocytes or B cells under unstimulated or stimulated conditions and of the mean fluorescence intensity (MFI) of phosphorylated BTK in CD14+ monocytes or B cells between patients with severe COVID-19 and healthy volunteers were performed using an unpaired t test or Mann-Whitney test where appropriate, using GraphPad Prism 8.0 and were presented as means ± SEM.

Comparisons of the frequency of IL-6+ CD14+ monocytes or B cells under unstimulated or stimulated conditions and of the mean fluorescence intensity (MFI) of phosphorylated BTK in CD14++monocytes or B cells between patients with severe COVID-19 and healthy volunteers were performed using an unpaired t-test or Mann-Whitney test where appropriate, using GraphPad Prism 8.0 and were presented as means ± SEM.

Laboratory tests for inflammatory markers during acalabrutinib treatment in supplemental oxygen cohort.

Laboratory tests for inflammatory markers during acalabrutinib treatment in mechanical ventilation cohort.

Other laboratory tests during acalabrutinib treatment in supplemental oxygen cohort.

Other laboratory tests during acalabrutinib treatment in mechanical ventilation cohort.

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