key: cord-0910233-b1etd7sd authors: Frame, David; Scappaticci, Gianni B.; Braun, Thomas M.; Maliarik, Mary; Sisson, Thomas H.; Pipe, Steven W.; Lawrence, Daniel A.; Richardson, Paul G.; Holinstat, Michael; Hyzy, Robert C.; Kaul, Daniel R.; Gregg, Kevin S.; Lama, Vibha N.; Yanik, Gregory A. title: Defibrotide therapy for SARS-CoV2 Acute Respiratory Distress Syndrome (ARDS) date: 2022-04-09 journal: Chest DOI: 10.1016/j.chest.2022.03.046 sha: bb4677c67a312b6c46f7dd95146a22f0772bd526 doc_id: 910233 cord_uid: b1etd7sd Background SARS-CoV2 related acute respiratory distress syndrome (ARDS) is associated with endothelial dysfunction and profound dysregulation of the thrombotic/fibrinolytic pathway. Defibrotide (DF) is a polyanionic compound with fibrinolytic, anti-thrombotic and anti-inflammatory properties. Research Question What is the safety and tolerability of defibrotide in patients with severe SARS-CoV2 infections? Study Design and Methods We report a prospective, open label, single center safety trial of DF for the management of SARS-CoV2 related ARDS. Eligible subjects were ≥18 years in age, with clinical and radiographic signs of ARDS, no signs of active bleeding, a serum D-Dimer >2X ULN, and a positive PCR-based assay for SARS-CoV-2. Defibrotide (6.25 mg/kg/dose IV q.6hours) was administered for a planned 7-day course, with serum D-Dimer levels and respiratory function monitored daily during therapy. Results Twelve subjects (median 63 years) were treated, with 10 on mechanical ventilation and six on vasopressor support at study entry. The median D-Dimer was 3.25 mcg/ml (1.33-12.3) at study entry. The median duration of therapy was seven days. No hemorrhagic or thrombotic complications occurred during therapy. No other adverse events attributable to DF were noted. Four subjects met the day 7 pulmonary response parameter, all four having a decrease in serum D-Dimer levels within the initial 72-hours of DF therapy. Three subjects died from progressive pulmonary disease, 11, 17 and 34 days following study entry. Nine subjects (75%) remain alive, 64 to 174 days following initiation of DF. Day 30 all-cause mortality was 17%(95%CI:0-35%). All subjects with a baseline PaO2/FiO2 >125 mmHg survived, whereas the three subjects with a baseline PaO2/FiO2 <125 mmHg died. Interpretation The use of DF for management of SARS-CoV-2-related ARDS proved safe and tolerable. No hemorrhagic or thrombotic complications were reported during therapy, with promising outcomes in a patient population with a historically high mortality rate. The use of DF for management of SARS-CoV-2-related ARDS proved safe and tolerable. No hemorrhagic or thrombotic complications were reported during therapy, with promising outcomes in a patient population with a historically high mortality rate. The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), has affected over 300 million individuals, with 5.5 million deaths reported 1 . Comorbidities that increase disease severity include older age, diabetes, hypertension, and prior pulmonary or cardiovascular disease 2,3 . One commonality in these disorders is chronic endothelial cell (EC) dysfunction, , with EC dysfunction a major factor in the pathogenesis and outcomes related to SARS-CoV2 infections [4] [5] [6] . Ultimately, EC dysfunction leads to end organ damage in the setting of microvascular and macrovascular emboli, potentially resulting in acute pulmonary, hepato-renal, myocardial and neurologic sequelae 7, 8 . Autopsy data from patients with SARS-CoV2 pneumonitis have identified diffuse alveolar damage, widespread microscopic thrombi and associated organ infarcts including hepatic sinusoidal damage, renal thrombotic microangiopathy (TMA), and myocardial infarction, even in patients that had been anticoagulated [9] [10] [11] . Severe SARS-CoV2 infections have been associated with elevated D-dimers levels, with increasing mortality as the D-dimer value rises, and mortality rates >50% with values >6-fold upper limit of normal (ULN) 12 . Approximately 25% of patients requiring hospitalization for SARS-CoV2 related pneumonia may exhibit a D-dimer >0.5 mcg/L, with elevated levels often persisting for several months in patients 13 . Of note, hemorrhagic symptomatology are not common with SARS-CoV2 infections, with infrequent reports of pulmonary or gastro-intestinal bleeding 14, 15 . Defibrotide is a polydisperse mixture of porcine-derived single-stranded oligonucleotides that was FDA approved in March 2016 for the treatment of hepatic veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) with either pulmonary or renal dysfunction following hematopoietic cell transplantation (HCT) 16 . Defibrotide displays endothelial-stabilizing properties, with profibrinolytic, anti-inflammatory, and endothelial adhesion effects, leading to increases in tissue plasminogen activator (tPA), and reductions in plasminogen activator inhibitor-1 (PAI-1) levels [17] [18] [19] [20] [21] [22] [23] [24] . The agent has anti-inflammatory effects, potentially modulated through decreased activation of p38 mitogen-activated protein kinase (MAPK) signaling pathways, the down regulation of intercellular adhesion molecule-1 (ICAM-1) expression in ECs, as well as suppression of heparanase expression in a variety of experimental models [21] [22] [23] [24] . The agent may play a role in reducing oxidative stress, increasing nitric oxide generation and inhibiting the generation of reactive oxygen species, potential key mediators of the endothelial injury associated with SARS-CoV2 infections 25 . Based upon this rationale, we have completed a safety trial of defibrotide for the management of patients with SARS-COV2 related ARDS. A prospective, single center, open label trial to evaluate the safety of defibrotide for the treatment of patients with SARS-CoV2 pneumonitis was performed. The trial was approved by the Institutional Review Board (IRB) at Michigan Medicine with written informed consent required from patients or their legal authorized representative (LAR). In situations in which a subject was incapable of providing consent (i.e. the subject was sedated on mechanical ventilation), the subject's LAR provided consent. The trial was registered at ClinicalTrials.gov as NCT04530604. Eligible patients had evidence of an active COVID-19 infection confirmed via real-time reverse transcription polymerase chain reaction (RT-PCR) from naso-pharyngeal or lung lavage samples. Patients were 18-70 years in age with a serum D-Dimer ≥2.0 mcg/ml, and acute J o u r n a l P r e -p r o o f respiratory distress syndrome (ARDS), with radiographic evidence of bilateral lung disease and/or impaired oxygenation (PaO2/FiO2 ≤ 300 mmHg). Patients were ineligible if they had received thrombolytic therapy and/or anticoagulants within 12 hours of study entry, excluding heparin flushes for centrally placed catheters. Patients with clinical evidence of active bleeding or hemorrhage within the prior 72 hours, mechanical ventilation for >96 consecutive hours, thrombocytopenia (platelets <50,000/mm3), hypofibrinogenemia (<150 mg/dl), uncontrolled infection other than SARS-CoV2, hemodynamic instability defined as the use of ≥2 vasopressors, the use of extracorporeal membrane oxygenation (ECMO), or pregnancy were additionally excluded. There was no exclusion based upon hepatic or renal function, including the use of hemodialysis. Transfusions of platelets to attain a level >50,000/uL or infusions of fresh frozen plasma (or cryoprecipitate) to attain a serum fibrinogen > 150 mg/dl were not allowed to meet eligibility criteria. After the first 3 subjects had enrolled, the study was amended (amendment v1.0) to include subjects >70 years in age, to lower the D-Dimer threshold to >2X upper limit of normal (ULN), and allow subjects on mechanical ventilation for ≤7 consecutive days to enroll. After the 9 th subject had enrolled, the study was amended (amendment v2.0) to allow the use of nontherapeutic doses of heparin (≤7,500 units q.8 hours) or low molecular weight heparin (≤1 mg/kg/day) concurrent with defibrotide therapy. Thrombolytic treatment and/or anticoagulant treatment at therapeutic doses within 12 hours of study entry remained an exclusion criterion. Defibrotide was administered intravenously at a dose of 25 mg/kg/day divided in 4 doses daily for a planned duration of 7 days (28 doses). Individual doses were diluted in 0.9% sodium chloride or 5% dextrose in water, mixed to a concentration of 4-20 mg/ml and infused with a 0.2 micron in-line filter over a 2-hour period. No other medications were co-administered through the same intravenous line during the defibrotide infusion. Clinical ordinal scores for activity and respiratory support were assigned to subjects at baseline, then daily throughout the study period ( Table 1) . 26 Response to therapy was defined as a reduction in the WHO ordinal score ≥2 points for 48 consecutive hours, or a complete cessation of supplemental oxygen support by day 7 of therapy. Subjects who met the response parameter (or were discharged) prior to day 7 were allowed to discontinue defibrotide at that time, without completing the 7-day course. Subjects with a ≥ 20% reduction in supplemental oxygen requirement (%FiO2) by day 7 were allowed to receive an additional 7 days of therapy, for a maximum 14-day course. No outpatient dosing was allowed. Defibrotide therapy was held if a subject developed signs of bleeding as defined by International Thrombosis and Haemostasis (ISTH) criteria 27 . Blood-tinged endotracheal secretions, microscopic hematuria, or mild menorrhagia did not require withholding defibrotice, unless the treating medical team deemed the event required medical intervention. Subjects with central nervous system or alveolar hemorrhage were required to discontinue therapy immediately, without the option to resume. Defibrotide was held for surgical procedures or to accommodate central line placements, the agent held > 2 hours prior to the procedure and for 12-24 hours postprocedure, based upon the discretion of the medical team. No additional doses were given to account doses held during this period. Defibrotide therapy was discontinued if a subject initiated ECMO therapy. Hematologic parameters (complete blood counts, CBC), serum chemistries, D-Dimer, prothrombin time (PT) and serum fibrinogen levels were required daily while receiving defibrotide therapy. Platelet transfusions and or use of fresh frozen plasma / cryoprecipitate were allowed if clinically indicated during therapy. Pulmonary indices (level and type of supplemental oxygen support) were recorded daily till cessation of therapy and again at day 14 from study entry. Biomarkers of hemostasis, including PAI-1 and tPA were obtained at day 1 (baseline), and day 4 of study, provided the patients were still receiving defibrotide therapy at that time. The study was designed as a feasibility study to examine the use of defibrotide in patients with SARS-CoV2 ARDS. The primary study endpoint was the occurrence of major toxicity, including hemorrhagic complications during study therapy. Secondary endpoints included overall survival (OS), day 7 response, day 14 survival, and day 14 ventilator free survival, with ventilator free survival summarized by the proportion of patients alive and ventilator free on day 14 of study. Stopping rules for hemorrhagic complications and day 14 all-cause mortality were present, with the study suspended if three of the first six patients developed a hemorrhagic event, or five of the first six patients died by day 14. Subjects who received at least one dose of defibrotide were considered evaluable for the primary and secondary endpoints. Toxicity assessments using Common Terminology Criteria for Adverse Events version 5.0 (CTCAEv5.0) were used. Overall survival was determined using the Kaplan-Meier method, with survival defined from the time of study entry to the date of death or last contact. The study was designed to have a planned sample size of 12 patients, the sample size deemed reasonable to assess feasibility. A data safety monitoring committee (DSMC) reviewed all toxicity data and response assessments. Thirteen subjects were enrolled between October 2020 and March 2021, with 12 subjects (median 63 years, range 35-73 years) treated ( Table 2) . One patient developed a positive blood culture after consent was signed but prior to receipt of their first dose of defibrotide. The patient was deemed ineligible and replaced on study. The median time from diagnosis of SARS-Cov2 to study entry was 9 days (range 1 to 26 days), with supplemental oxygen given a median 7 days (range 3 to 26 days) prior to enrollment. Ten of the 12 subjects required mechanical ventilation and 6 subjects required vasopressor support (WHO ordinal score=7) at study entry ( Table 2 Therapy duration ranged from 1 to 14 days (median 7 days), with 6 subjects receiving < 7 days of therapy due to meeting the response criteria (n=3), ventilator associated infections (n=2) or progressive pulmonary disease (n=1) ( Table 3 ). The infusions were well tolerated with no infusion related reactions reported. All defibrotide doses were given as scheduled, with minor adjustments in the timing (±1-2 hours) required given the complexities of intensive care unit (ICU) care for SAR-CoV2 positive patients. The first nine subjects received defibrotide without any other anti-coagulation being given. Following approval of amendment 2.0, subjects 10-12 received defibrotide in conjunction with prophylactic doses of intravenous (n=1) or low molecular weight heparin (n=2). No hemorrhagic or bleeding episodes occurred during study therapy, including the three subjects (10, 11, 12) who received defibrotide with concurrent heparin prophylaxis ( Table 3) . Conversely, no thrombotic events developed while subjects were on therapy, including the nine Four subjects (1, 5, 6, 7) met the day 7 response parameter, with two subjects (5 and 6) having a complete cessation of oxygen support within this 7-day period. Subject 6, a 48-year old female with severe myasthenia gravis, was extubated following 4-days of defibrotide therapy and discontinued all supplemental oxygen by day 7 (Figure 1) . Overall, six subjects remain off supplemental oxygen support, 64 to 174 days from study entry. Seven of the 12 subjects had a ≥2 point decline in WHO ordinal scores within 30 days of completion of study therapy (Figure 2) . All four subjects who met the day 7 response parameter had a decrease in serum D-Dimer values within 72-hours of initiation of defibrotide therapy, including subject 5 whose D-Dimer decreased from 14.9 to 5.81 mcg/ml within the initial 24-hours of therapy ( Table 4) . Two subjects (4, 9) had a 3-fold increase in D-Dimer within 72-hours of starting therapy. Both patients died from progression of their SARS-CoV2 ARDS. One of two patients (3, 5) with a baseline D-Dimer > 10 mcg/ml responded, while both patients (11, 12) with a baseline D-Dimer < 2.0 mcg/ml failed to meet the Day 7 response parameter. Nine subjects (75%) remain alive, in an outpatient setting following their primary hospitalization for SARS-Cov2 ARDS, including seven of ten male and both female subjects Total PAI-1 and tPA levels were available in 7 of the 12 patients. At baseline, the median PAI-1 level was 167 ng/ml (105-264 ng/ml), decreasing to 104 ng/ml (55-166 ng/ml) by day 4 of therapy. All 7 subjects exhibited a decline in PAI-1 levels within the initial week of therapy. Total tPA levels increased from a median 3.02 ng/ml (0.72 -36.1 ng/ml) at baseline to 4.5 ng/ml (1.1-8.2 ng/ml) by day 4, increasing in 5 of 7 subjects. Given the small sample sizes, no definitive correlation of PAI-1 or tPA levels with outcome or response could be determined. We report the first safety study of defibrotide therapy in a trial of critically ill patients with SARS-CoV2 associated ARDS. The lack of both hemorrhagic and/or thrombotic complications, the response to therapy and overall survival were promising in this prospective, open label trial. Historically, the mortality for patients with SARS-CoV2 ARDS is high, with day 28 mortality rates 26%-61.5% in patients requiring mechanical ventilation [27] [28] [29] [30] [31] [32] . The median duration from ICU care to death is often short, within 7 days in several reports [28] [29] [30] . In a prospective cohort study of 4643 critically ill adults with COVID-19 infections in northern Europe, 63% were intubated within 24 hours of ICU admission, with 80% requiring mechanical ventilation during their ICU course 29 . Mortality rates paralleled the severity of the ARDS at the time of ICU admission, with mortality rates of 30%, 34% and 50% respectively for mild, moderate and severe ARDS. In comparison, day-30 mortality was 17% (95%CI: 0-35%) in our study with defibrotide. Though our study was not designed to assess efficacy, our day 30 and 60 survival are promising, considering the high acuity of our subjects at study entry. Defibrotide was safe in our patient population, with no major adverse events attributable to the agent noted. Defibrotide is currently FDA approved for the treatment of adult and pediatric patients with veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) with renal or pulmonary dysfunction following HCT. Common toxicities (any grade) reported in HCT recipients included hypotension (37%), diarrhea (24%), vomiting (18%), epistaxis (14%), pulmonary alveolar hemorrhage (9%), gastro-intestinal (GI) hemorrhage (9%), septicemia (7%), and cerebral hemorrhage (2%), with grade 4-5 pulmonary hemorrhage noted in 8% of cases 16 . The agent was well tolerated in our current study, with no hypotension, GI toxicity or pulmonary hemorrhage noted. No bleeding (pulmonary or non-pulmonary) occurred during study therapy in any patient, including the 3 patients who received concurrent heparin prophylaxis. The incidence of ventilator associated pneumonia in our patient population (33%) was similar to published reports in patients requiring ICU level care for SARS-CoV2 disease, in which secondary infection rates of 21% to 58% have been reported [29] [30] [31] [32] [33] . Considering that subjects in our study were all receiving concurrent systemic corticosteroids, the infection rate for our patients was promising, with secondary infections primarily due to gram negative pathogens. Furthermore, the use of defibrotide has not been associated with an increased risk of infections in other (non-SARS-CoV2) patient populations, particularly HCT recipients. 16 Though our current study followed dosing guidelines established in HCT recipients (6.25 mg/kg IV q.6 hours), we used a much shorter course of therapy (7-14 days) than typically given to HCT recipients, in which 21 days of therapy are routinely administered 16 . The use of a longer therapy course (>7 days) may not be required, as all responders did so within 7 days of study initiation. On-going trials in the critical care setting will evaluate the optimal duration of therapy in different patient subgroups 34, 35 . We are also unable to address whether defibrotide can be safely given with other fibrinolytics or anticoagulants, as only 3 of 12 patients received prophylactic heparin concurrent with defibrotide therapy. Importantly, none of the three had any bleeding From a mechanistic standpoint, defibrotide is an attractive agent for investigation into the management of the thrombo-inflammation associated with SARS-CoV2 infections, through its modulation of cytokine release and other markers of endothelial stress. Specifically, both preclinical and clinical data indicate the agent may augment fibrinolysis, decrease thrombin generation, reduce PAI-1 levels, reduce oxidative stress, and decrease the platelet activation seen in pro-thrombotic states. Another potential mechanism of action may involve inhibition of neutrophil extracellular trap (NET) formation in SARS-CoV2 affected patients, with the severity of SARS-CoV2 infections directly correlating with NET over-expression [39] [40] [41] [42] [43] . Histone generation during NET formation has already been shown to lead to endothelial cell injury in other clinical settings 44 . Pre-clinical models by investigators at our center have shown that defibrotide directly neutralizes NET derived cationic proteins (histones), inhibits the activation and permeability of cultured endothelial cells, and protects endothelial cells from histone induced cell death 45 . Given the small sample size, our pilot study had several potential limitations. The study was not designed to address the efficacy of defibrotide in this clinical setting. In addition, the study could not address the role of concurrent anticoagulation, with only 3 of 12 patients receiving heparinization during study therapy. Furthermore, defibrotide was only given in conjunction with prophylactic, but not therapeutic doses of heparin in our study. We do not know the impact of defibrotide when combined with therapeutic heparin dosing. For patients with SARS-CoV2 infections, defining both the optimal patient population, and the optimal timing for initiation of defibrotide remain under investigation, with larger phase II and phase III clinical trials required. Several trials are currently in progress, including a randomized, placebo-controlled trial (NCT04348383), and two trials targeting patients with high acuity disease (NCT04652115, NCT04335201). Despite the small sample size of our pilot study, it is reasonable to infer that defibrotide therapy could be considered in patients with less acute pulmonary disease (i.e. WHO ordinal scores <6). Our results support this assumption, in which all patients with severe pulmonary disease at baseline (PaO2/FiO2 <125 mmHg) died within 60 days of therapy, and all patients with a baseline PaO2/FiO2 > 125 mmHg survived. Preliminary results from an Italian trial likewise show significant promise for both safety and efficacy in less severely ill patients with SARS-CoV2 pneumonitis. 35 Given that SARS-CoV2 related thrombo-inflammation, complement activation and resultant endothelial damage may begin early in a patient's clinical course, one might consider initiating defibrotide therapy in any patient hospitalized for SARS-CoV2 in whom prophylactic heparinization is being considered. Combining an immunomodulatory and antithrombotic agent such as defibrotide with prophylactic doses of low-molecular weight heparin may be an attractive option to consider in this clinical situation 22, 25, 46 . Our findings should motivate additional studies examining earlier initiation of defibrotide therapy, specifically targeting those with lower WHO ordinal scores. COVID-19) Dashboard Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. 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