Ramatroban, an orally bioavailable immunomodulator and antithrombotic agent for treatment of COVID-19 disease: A Case Report

SARS-CoV-2 pneumonia and COVID-19 disease are characterized by a maladaptive immune response and prothrombotic state. The Spike protein of SARS-CoV-2 virus unleashes a storm of lipid mediators with very high levels of thromboxane B 2 and prostaglandin D 2 in the bronchoalveolar lavage uid. DPr2 receptors for prostaglandin D 2 and TPr receptors for thromboxane A 2 have been proposed as therapeutic targets in COVID-19 in order to decrease viral load and inhibit platelet dependent thrombosis, respectively. Ramatroban is an oral, dual receptor antagonist of the DPr2 and TPr receptors with established safety prole, having been used in Japan for the treatment of allergic rhinitis for over 20 years. We report two patients, an 88-year-old woman and a 31-year-old man, both with COVID-19 pneumonia who were successfully treated in ambulatory setting with oral ramatroban, leading to rapid improvement in oxygen saturation from about 80–82% to about 90–95% over 24–48 hours followed by gradual recovery from the disease. The mechanism of action of ramatroban and its ecacy reported here supports clinical trials on this drug as a potential treatment for COVID-19.

successfully treated in ambulatory setting with oral ramatroban, leading to rapid improvement in oxygen saturation from about 80-82% to about 90-95% over 24-48 hours followed by gradual recovery from the disease. The mechanism of action of ramatroban and its e cacy reported here supports clinical trials on this drug as a potential treatment for COVID-19.

Background
During the current pandemic over 145 million patients have been infected with SARS-CoV-2 virus and more than 3 million have been deceased, as of April 2021. Despite vaccine rollout many countries continue to experience a surge in cases including India, Brazil, Chile, Canada and many European countries. COVID-19 disease is caused by SARS-CoV-2 virus which enters host cells through the ACE2 receptors. Severe SARS-CoV-2 infection is characterized by rst, respiratory infection with the development of ARDS; and second, vascular disease manifesting as thrombotic microangiopathy, pulmonary thrombosis, pedal acro-ischemia ("COVID-toes"), arterial clots, strokes, cardiomyopathy, coronary and systemic vasculitis, bleeding, deep venous thrombosis, pulmonary embolism; and microvascular thrombosis in renal, cardiac and brain vasculature. [1][2][3][4][5][6] The underlying pathobiology of SARS-CoV-2 infection comprises a trilogy of a maladaptive in ammatory response, immunosuppression and thromboin ammation. First, emerging evidence suggests that in patients with severe COVID-19 the immune response is disproportionately shifted from a Th1 to a Th2 response characterized by an increase in plasma levels of type 2 cytokines produced by Th2 cells, including IL-4, IL-13 and IL-5. 7-9 Second, IL-13 is known to upregulate monocyte-macrophage derived suppressor cells (MDSC), which play a role in immune suppression and lymphopenia. [10][11][12] Lymphopenia is one of the characteristic features of COVID-19 disease in adults, and a predictor of disease severity. 13 Third, endothelial cell injury by the SARS-CoV-2 virus and direct association of virus with platelets leads to platelet activation, release of platelet microvesicles, platelet-neutrophil and platelet-monocyte interaction and release of neutrophil extracellular traps (NETs). 5,14−16 This leads to in ammatory microvascular thrombi in the pulmonary, hepatic, renal and cardiac microvasculature and multi-organ failure. 1,5 A proin ammatory lipid storm lies at the heart of the above trilogy. Archambault and colleagues measured eicosanoids in the bronchoalveolar lavage uid (BALF) in 33 severely ill patients with COVID-19 within 2 hours of initiation of mechanical ventilation, compared with 25 healthy controls. 17 Severe COVID-19 patients had marked increases in fatty acid levels as well as an accompanying in ammatory lipid storm with predominance of cyclooxygenase metabolites notably thromboxane B 2 > > PGE 2 > PGD 2 . 17 PGD 2 action on DPr2 receptor mediates the maladaptive type 2 immune response. 18, 19 Thromboxane A 2 action on thromboxane prostanoid receptors (TPr) mediates thromboin ammation. 11,12,20 DPr2 and TPr have been proposed as therapeutic targets in COVID-19, in order to decrease viral load and inhibit plateletdependent thrombosis, respectively. 21 Ramatroban is the only dual receptor antagonist of the DPr2 and TPr receptors available for medicinal use. Ramatroban has an established safety pro le having been used for over 20 years in Japan for the treatment of allergic rhinitis. 22,23 Here, we report the rst use of ramatroban, a DPr2 and TPr antagonist to treat COVID-19 disease.
Case Reports 1st case S.D., an 87-year old Indian lady (SD) experienced sudden onset of fever, cough, diarrhea, anorexia, profound weakness and slight shortness of breath, 10 days following a 2-hour ight from New Delhi to Indore, Madhya Pradesh, India. Patient had received the rst dose of COVISHIELD vaccine 30 days prior to beginning of symptoms. Patient was examined by the rst author. Patient was fully alert, oriented and able to make intelligent conversation but lay listlessly in bed unable to ambulate. Patient weighed 42 Kg and exhibited severe muscle wasting and marked kyphosis. Vital signs revealed temperature, 102 o Fahrenheit; heart rate, 100 per minute; blood pressure, 90/60 mm of Hg; and respiratory rate, 22 per minute. Mucosa were moist and mild pallor was present. There was no jugular venous distention or pedal edema. Chest examination revealed bilateral coarse rales especially prominent at both lung bases but no wheezes. Abdomen, cardiovascular and neurological examinations were unremarkable. Patient was not taking any medications.
Past medical history included hypertension for over 40 years; Hashimoto's thyroiditis with thyrotoxicosis for over 30 years treated with radioiodine therapy in 1999; severe osteoporosis with kyphosis; coronary artery disease leading to acute myocardial infarction and cardiac arrest in 2015 which required emergency coronary angioplasty and stent placement; chronic kidney disease with estimated glomerular ltration rate of about 20 mL/min. Nasopharyngeal and oropharyngeal swabs were positive for SARS-CoV-2 infection by RNA PCR with cycle threshold (Ct range < 20 cycles). Pulse oximetry revealed oxygen saturation of about 85-88%.
Patient was admitted on April 9, 2021 to Medanta Hospital, Indore, Madhya Pradesh, India. CT scan revealed moderate multifocal, patchy ground glass opacities, and consolidation. There was septal thickening in the central and peripheral subpleural aspect of both lung parenchyma. Serial laboratory examinations during the course of the illness are listed in Table 1.
During the hospital stay the patient was treated with intravenous remdesivir, antibiotics and methylprednisolone. In addition, prophylactic low-molecular weight heparin was administered subcutaneously in a dose of 40 mg twice a day. Patient required high-ow nasal oxygen during the hospital stay. Patient continued to have fever, cough, shortness of breath, diarrhea and profound weakness during the hospital stay. Oxygen saturation on room air ranged between 82-86%. After a hospital stay of 5 days the patient was discharged upon her request on April 14, 2021. Discharge medications included oral oseltamivir, doxycycline, vitamin C, aspirin 75 mg once a day, 5 mg prednisolone, vitamin D 3 , and nebulization with budesonide and salbutamol twice daily. Supportive management with betadine gargles, steam inhalation and breathing exercises was continued.
On April 15, the day after discharge from the hospital, the patient had fever with a temperature of 101 o Fahrenheit. Pulse oximetry revealed an oxygen saturation of 82-84%. Patient was profoundly weak and unable to get out of bed without assistance. At this time all drugs including low-dose aspirin were discontinued and the patient was started on ramatroban in a dose of 75 mg tablet, one-half tablet (37.5 mg) twice daily. Within 36 hours, after having received three doses of ramatroban, patient's oxygen saturation improved to 90% on room air and there was progressive improvement in her general condition.
The dose of ramatroban was increased to 37.5 mg in the morning and 75 mg at bedtime. Patient had complete resolution of cough and diarrhea over a period of next 3 days, and started ambulating independently without assistance. Patient had recovered almost completely by April 22, 2021.

Discussion
The rst case presented above is consistent with classical clinical manifestations of moderate to severe COVID-19 disease including hypoxia, lymphopenia, neutrophilia, and increased plasma levels of Creactive protein and D-dimers. This case represents the trilogy of maladaptive in ammatory response, immunosuppression and thromboin ammation in COVID-19, which is fueled by a proin ammatory lipid storm with massive generation of thromboxane A 2 and PGD 2 . 17 It has been proposed that early administration of well-tolerated antagonist of PGD 2 /DPr2 and thromboxane A 2 /TPr signaling may limit progression to severe COVID- 19. 21 Ramatroban is the only approved dual receptor antagonist of the DPr2 and TPr receptors. Ramatroban (Baynas®, Bayer Yakuhin Ltd., Japan) has been safely used for the treatment of allergic rhinitis in Japan since 2000. 23 The usual adult oral dose of 75 mg twice daily achieves an average plasma concentration of about 0.1 mg/L or 240 nM which is su cient to inhibit platelet activation since the IC 50 for human platelet aggregation is only about 30 nM. 23 Ramatroban exhibits surmountable binding to thromboxane A 2 receptors. 24 Ramatroban is 100 times more potent than aspirin in inhibiting platelet aggregation and P-selectin expression. 23,25 With a plasma half-life of about 2 hours the antiplatelet action of ramatroban is reversible. 23 This is of advantage in the event of bleeding complications which have been reported in > 5% of critically ill COVID-19 patients. 26 Ramatroban is primarily metabolized in the liver through the acyl glucuronic acid conjugate metabolic pathway, is excreted mainly in the bile; and therefore can be safely administered in the presence of acute kidney injury or chronic kidney disease. 27 The blood concentration of ramatroban in the elderly (≥ 65 years of age) is estimated to be higher than in the non-elderly population, and hence the starting dose in the elderly is 50 mg twice a day. 27 Ramatroban improves vascular responsiveness; while inhibiting endothelial surface expression of ICAM-1 and VCAM-1; inhibiting MCP-1 expression in response to TNF-α or platelet activating factor; and inhibiting macrophage in ltration. 23 In a rat model of endotoxic shock, ramatroban prevented hypotension, reduced plasma TNF levels by over 90%, and markedly reduced myeloperoxidase levels in lungs, ileum and heart, suggesting end-organ protection by mitigating thromboxane A 2 mediated platelet-polymorphonuclear leukocyte activation, and improved survival by 45%. 28 In rats with splanchnic artery ischemia-reperfusion injury, while plasma levels of thromboxane B 2 were increased about 7-fold, ramatroban prevented hypotension, improved survival, restored phagocytic function of peritoneal macrophages partially, inhibited plasma myocardial depressant factor activity about 50%, and inhibited tissue in ltration by neutrophils as measured by decline in ilium myeloperoxidase activity > 50% and lung myeloperoxidase activity > 80%. 29 Notably, myeloperoxidase is signi cantly increased in COVID-19, is abundant in NETs and regulates NET formation via synergy with neutrophil elastase. 30,31 Therefore, ramatroban is remarkably effective in both endotoxin and ischemia reperfusion injury induced shock states which share common pathogenetic mechanisms with severe COVID-19.
These are the rst reported cases of COVID-19 treated with ramatroban. The rapid recovery in both cases including the frail, 88 years old patient with chronic kidney disease (eGFR of about 20 mL/minute) and coronary artery disease, despite failure of conventional treatment regimen with remdesivir and steroids, appears to coincide with institution of ramatroban. Such a clinical response is consistent with the potent immunomodulatory and antithrombotic actions of ramatroban observed in preclinical studies. Therefore, in patients with COVID-19, ramatroban appears to have the potential to prevent microvascular thrombosis, 28,32 limit viral load by promoting interferon lambda (IFN-λ) response, 19 and reduce disease severity by mitigating the maladaptive shift from Th1 to Th2 immune responses. 11 The clinical response to ramatroban in the two cases reported, its established safety pro le and its oral bioavailability support further clinical trials of this drug as a potential treatment for COVID-19. Declarations Consent: The patient consented to participate and wishes to publish their clinical data.

Con ict of Interest:
There was no con ict of interest of the authors responsible for the management of the patient including the use of ramatroban.