Efficacy and tolerability of bevacizumab in patients with severe Covid-19

On the basis of Covid-19-induced pulmonary pathological and vascular changes, we hypothesize that the anti-vascular endothelial growth factor (VEGF) drug bevacizumab might be beneficial for treating Covid-19 patients. From Feb 15 to April 5, 2020, we conducted a single-arm trial (NCT04275414) and recruited 26 patients from 2-centers (China and Italy) with severe Covid-19, with respiratory rate ≥30 times/min, oxygen saturation ≤93% with ambient air, or partial arterial oxygen pressure to fraction of inspiration O2 ratio (PaO2/FiO2) >100 mmHg and ≤300 mmHg, and diffuse pneumonia confirmed by chest imaging. Followed up for 28 days. Among these, bevacizumab plus standard care markedly improves the PaO2/FiO2 ratios at days 1 and 7. By day 28, 24 (92%) patients show improvement in oxygen-support status, 17 (65%) patients are discharged, and none show worsen oxygen-support status nor die. Significant reduction of lesion areas/ratios are shown in chest computed tomography (CT) or X-ray within 7 days. Of 14 patients with fever, body temperature normalizes within 72 h in 13 (93%) patients. Relative to comparable controls, bevacizumab shows clinical efficacy by improving oxygenation and shortening oxygen-support duration. Our findings suggest bevacizumab plus standard care is highly beneficial for patients with severe Covid-19. Randomized controlled trial is warranted.

c These data refer to rates of improvement of oxygen-support class.
d Discharge or mortality rates within the duration of 28 days.

Supplementary Figure 1. Changes in fever symptom of individual control patients.
Dynamic changes of fever status of 13 controls who had fever at day 0. Red, orange, and blue columns indicate the duration of fever or normal body temperature status.
Diamonds represent discharge, and solid square symbols represent death.

Supplementary Figure 2. CONSORT flow diagram of external controls.
PaO2/FiO2=partial arterial oxygen pressure to fraction of inspiration O2 ratio

BACKGROUND
Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a subfamily of growth factors. Hypoxia induces VEGF expression through activation of the Prolyl hydroxylases (PHD)-hypoxia inducible factor (HIF)-1 pathway, which upregulates VEGF expression through transcription activation. Patients with severe Covid-19 suffer from severe hypoxia, and VEGF levels in patients with severe Covid-19 are markedly elevated. VEGF is a potent vascular permeability factor that induces vascular leakiness in Covid-19-infected lung tissues, resulting in plasma extravasation and pulmonary edema, which further increases tissue hypoxia. Bevacizumab is a recombinant humanized monoclonal antibody. As an anti-VEGF medication, it has been prevalently utilized in oncotherapy since 2004, with considerable reliability and clinical safety.
Supportive clinical and nonclinical profile for the use of bevacizumab in severe patients with Covid-19 includes: l Patients with severe Covid-19 suffer from severe hypoxia. Hypoxia is known to significantly induce VEGF expression through activation of the HIF-1 pathway 1,2 .
l VEGF levels in patients with severe Covid-19 are markedly elevated 3 . VEGF contributes to increased vascular permeability and pulmonary edema 4,5 .
l Pulmonary edema frequently presents in Covid-19 patients. Autopsy of Covid-19 patients shows excessive extravasates in alveoli of the infected lungs 6 .
l Overreactive inflammatory response happens in Covid-19. VEGF is known to enhance inflammation in the lung 7 .
l In a mouse model of Acute Respiratory Distress Syndrome (ARDS), Watanabe M et al. used intratracheal adenovirus (Ad)-mediated overexpression of human vascular endothelial growth factor in mouse lung to induce alveolar permeability and consequent pulmonary edema, and demonstrated that bevacizumab suppressed vascular endothelial growth factor-induced high-permeability pulmonary edema.
l David R et al found plasma VEGF was significantly elevated in patients with ARDS compared with at-risk patients. In vitro study showed, the peripheral blood mononuclear cells from patients with ARDS produced remarkably more VEGF in vitro than at-risk patients, and VEGF inhibitors significantly ameliorated the permeability of human lung epithelial cells.
l Bevacizumab has been widely used in cancer treatment since 2004, with considerable reliability and clinical safety. It was reported that bevacizumab rapidly reduced macular edema and contributed to improve visual acuity in patients with wet age-related macular degeneration 8 .
2) Confirmed COVID-19 diagnosis. A confirmed case is based on epidemiological history (including cluster transmission), and results of SARS-CoV-2 nucleic acid detection.

Exclusion Criteria
1) Patients with severe hepatic dysfunction (Child-Pugh score ≥ C or aspartate aminotransferase level > 5 times the upper reference limit, URL); 2) Patients with severe renal dysfunction (estimated glomerular filtration rate ≤ 30 mL/min/1.73 m 2 ) or who required continuous renal replacement therapy, haemodialysis, or peritoneal dialysis; 3) Patients with uncontrolled hypertension (sitting systolic blood pressure > 160 mmHg or diastolic blood pressure >100 mmHg) or a history of hypertension crisis or hypertensive encephalopathy; 4) Patients with poorly controlled heart diseases, such as New York Heart Association class II or higher cardiac insufficiency, unstable angina pectoris, myocardial infarction within 1 year before enrollment, or supraventricular or ventricular arrhythmia needing treatment or intervention; 5) Patients with hereditary bleeding tendency or coagulopathy, and patients who received full-dose anticoagulant or thrombolytic therapy within 10 days before enrollment, or non-steroidal anti-inflammatory drugs with platelet suppression within 10 days before enrollment (except those who used small doses of aspirin [≤325 mg/day] for preventive use);

Dosage
Bevacizumab at a dose range of 5-15 mg/kg is routinely used in oncology. A single dose of 500mg (about 7.5 mg/kg) used in this study was within the lower range.

Route of Administration
A single dose of bevacizumab 500mg + saline 100ml is administered intravenously in no less than 90 min under electrocardiogram monitoring.

Pharmacokinetics of Bevacizumab
The pharmacokinetic profile of bevacizumab is provided according to the drug manuscript as follows: The pharmacokinetic profile of bevacizumab was assessed using an assay that measured total serum bevacizumab concentrations (i.e., the assay did not distinguish between free bevacizumab and bevacizumab bound to VEGF ligand). Based on a population pharmacokinetic analysis of 491 patients who received 1 to 20 mg/kg of bevacizumab every week, every 2 weeks, or every 3 weeks, Bevacizumab pharmacokinetics are linear and the predicted time to reach more than 90% of steady state concentration is 84 days. The accumulation ratio following a dose of 10 mg/kg once every 2 weeks is 2.

Informed Consent
The researcher informs the patient about the trial's detailed information. After signing the informed consents, the patients will be assessed if they are eligible for this study.

Prior to Drug Administration
Physical examination and vital signs assessment will be conducted. Arterial blood gas (ABG) assay will be performed within 24 hours prior to bevacizumab administration. Chest CT or chest X-ray will be performed within 48 hours prior to bevacizumab treatment. Patient's demographic information, medical history, diagnosis of Covid-19, current medication, oxygen-support status and laboratory tests will be collected.

ABG assay
ABG assay will be performed on day 1 and day 7 post-bevacizumab administration.

Chest Radiological imaging
Chest CT scanning will be performed on day 7 ( ± 24hrs) post-bevacizumab administration. Take consideration that the medical resources during the pandemic situation are limited, alternative Chest X-ray will be acceptable, which will be performed on day 3 and day 7 (±24hrs) post-bevacizumab administration.

Oxygen-support status
Oxygen-support status including extracorporeal membrane oxygenation, mechanical support, non-invasive support, an intermediate status of alternation of non-invasive support and high-flow oxygen, high-flow oxygen, and low-flow oxygen will be recorded daily from the day of bevacizumab administration to day 28. Venturi mask is regarded as low-flow oxygen. Reservoir with oxygen flow≥10 L/min is considered as high-flow oxygen. Oxygen inhalation through nasal catheter or mask regardless of the oxygen flow is identified as low-flow oxygen.

Others
It is recommended that use of corticosteroids be avoided, if possible.
Physical examination and vital signs will be performed daily.
Laboratory tests including blood routine, hepatic and renal function tests, CRP and coagulation factors) will be performed at baseline (within 48 hours prior to bevacizumab administration) and day 7 (±24hrs) post-bevacizumab administration.

BENEFIT-RISK ASSESSMENT
No investigational drugs with proven clinical efficacy for severe Covid-19 is available. Dyspnoea and inflammatory pulmonary edema present in almost all patients with severe Covid-19 and needs oxygen-support and long hospital stay. The levels of VEGF, a potent vascular permeability factor that induces vascular leakiness, are markedly elevated in Covid-19 patients. VEGF also significantly participates in lung inflammation. Blocking VEGF and the VEGFR-mediated signalling by would improve oxygen perfusion and anti-inflammatory response and alleviate clinical symptoms in patients with severe Covid-19. Bevacizumab would address the urgent need for developing effective drugs in this serious pandemic situation.
The pharmacokinetic profile of bevacizumab has been assessed using an assay that measured total serum bevacizumab concentrations (i.e., the assay did not distinguish between free bevacizumab and bevacizumab bound to VEGF ligand) as mentioned above. The rate of infusion is based on the patient's tolerance, and the first intravenous infusion needs to last 90 minutes. In the dose range of 1 to 10 mg/kg, the pharmacokinetics of bevacizumab show a linear relationship. The metabolism and elimination of bevacizumab is similar to endogenous IgG, which is mainly through proteolytic catabolism of human body including endothelial cells, instead of the kidney and the liver. The binding of IgG to FcRn protects it from being metabolized by cells and has a long terminal half-life. The clearance of Bevacizumab varies by body weight, and sex. The estimated elimination half-life of a typical female patient is 18 days, and a typical male patient is 20 days. The pharmacokinetics of bevacizumab doses not differ significantly between different ages in adults. Pharmacokinetic studies of bevacizumab in patients with kidney injury or liver damage have not been conducted because the kidney or the liver are not the main organ for bevacizumab metabolism or excretion.
Over the past years, several clinical trials of bevacizumab in the treatment of different malignant tumors have been carried out, most of which are combined with chemotherapy drugs. This paragraph describes the safety results obtained from the clinical trial population of approximately 5,500 patients. The most serious adverse reactions are: gastrointestinal perforation, bleeding (mostly pulmonary hemorrhage and hemoptysis in patients with non-small cell lung cancer) and arterial thromboembolism. The most frequent adverse reactions include hypertension, fatigue, diarrhea and abdominal pain. The analysis of clinical safety data suggests that the incidence of hypertension and proteinuria may be dose-dependent when receiving bevacizumab.
In pre-clinical animal experiments, the incidence and severity of bevacizumab's toxic and side effects are related to the dose, and can be partially recovered after drug withdrawal, which includes growth plate dysplasia, reduced wound healing ability, and possibly affecting fertility, the dose is 0.4-20 times the recommended dose for human use. In trials tested by human volunteers, the highest dose (20 mg/kg body weight, given every 2 weeks, intravenous infusion) may cause severe migraine in some patients.

SAFETY MONITORING
Possible adverse events related to this study include: hypertension, nausea, vomiting, diarrhea, abdominal pain, neutropenia, leukocytopenia, thrombocytopenia, lymphopenia, anemia, headache, increased tearing, epistaxis, stomatitis, exfoliative dermatitis, proteinuria, gastrointestinal perforation, bleeding and arterial thromboembolism, etc. Adverse events will be monitored and adjudicated by the Safety Monitoring Committee. All the adverse events will be handled timely with proper medical treatment to avoid further damage.

Chest CT (within 48h prior drug administration)
□Chest CT has been performed within 48h prior study drug administration □Chest CT can not be performed. Chest X ray has been performed within 48h prior study drug administration.