Ramatroban as a Novel Immunotherapy for COVID-19

Ajay Gupta^1*, Kamyar Kalantar-Zadeh¹ and Srinivasa T. Reddy^2
*Correspondence: Dr. Ajay Gupta, Division of Nephrology, Hypertension and Kidney Transplantation, Department of Medicine, University of California Irvine (UCI) School of Medicine Orange, CA 92868, USA, Email:

Keywords

SARS-CoV-2 Virus • Ramatroban • COVID-19 • Immune Function

Abbreviations

PGD₂: Prostaglandin D₂ • DP₁: D-Prostanoid Receptor 1 • DP₂: D-Prostanoid Receptor 2 • TP Receptor: Thromboxane Receptor • IFN-λ: Interferon-λ [interleukin-28A/B (IL-28A/B)] • IP3: Inositol Trisphosphate ILC2, Group 2 Innate Lymphoid Cells • M-MDSC: Monocytic Myeloid-Derived Suppressor Cells •COX: Cyclo-Oxygenase • Phospholipase A₂: (PLA2) Group IID (PLA₂G2D) • rDC: Respiratory Dendritic Cell

Introduction

Novel coronavirus disease 2019, also known as COVID-19, is a highly infectious, rapidly spreading viral disease with very high morbidity and mortality [1]. The death rate with COVID-19 increases with advancing age, which could be due to several factors including progressive decline in immune function with aging [2]. Although SARS-CoV-2 and the 2003 SARS-CoV infections share a number of common clinical manifestations, SARS-CoV-2 virus appears to be highly efficient in person-to- person transmission and frequently cause asymptomatic infections, but the underlying mechanisms that confer the above characteristics of COVID-19 disease remain incompletely understood [3].

Literature Review

SARS-CoV-2 and SARS-CoV are similar in cell tropism, with both targeting types I and II pneumocytes, and alveolar macrophages. 3 In mouse models of viral respiratory infections, airway epithelial cells produce prostaglandin D₂ (PGD₂) in an age dependent manner, with higher levels in older mice [4]. There is increasing evidence that following respiratory virus infection, PGD₂ mediates airway inflammation while suppressing the host immune response to the virus [5]. We propose the hypotheses that higher basal production of PGD₂ in the airways of the elderly is causally linked to more severe disease with COVID-19; and that PGD₂ suppresses the innate and adaptive immune responses to SARS-CoV2 allowing robust unchecked viral replication and virulence. We present evidence that Ramatroban, a small molecule antagonist of PGD₂ and thromboxane A2, currently approved and used in Japan for the treatment of allergic rhinitis, merits immediate investigation as a potential therapeutic agent against SARS-CoV2.

Respiratory viruses’ upregulate PGD₂ production in lungs and airways, and PGD₂ regulates host immune responses to the virus

SARS-CoV-2 was found to be capable of infecting and replicating about 3 times more robustly than SARS-CoV in ex-vivo studies of human lung tissues. 3 The first line of defense against respiratory viruses, including influenza, rhino, respiratory syncytial, SARS-CoV and SARS-CoV-2 viruses are Type III interferons, including interferon-λ (IFN-λ) [interleukin-28A/B (IL-28A/B)] [6]. In the respiratory tract, IFN-λ expression is selective to mostly respiratory epithelial cells and dendritic cells. Both in vitro and in vivo studies have demonstrated that IFN-λ is as effective as Type I interferons in anti-viral activity [7]. The role of interferons in differential replication rates has been studied. The 2003 SARS-CoV infection resulted in significant upregulation of types I (IFNβ), II (IFNγ), and III (IFNλ1, IFNλ2, and IFNλ3) IFNs in human lung tissues, but SARS-CoV-2 infection did not significantly trigger the expression of any IFN [3,8]. Recent studies have demonstrated that interferon response to viral infections is regulated by local production of prostaglandins, especially prostaglandin D₂ [9].

Prostaglandins are known to play a key role in immune and inflammatory responses [10]. Respiratory viruses, including respiratory syncytial and SARS-CoV viruses, increase prostaglandin D₂ (PGD₂) in the airways in pathogen dependent manner [4,9]. Infection with respiratory syncytial virus increased PGD₂ release by cultured human primary airway epithelial cells [9]. Intranasal administration of SARS-CoV virus to mice resulted in marked increase in PGD₂ levels in the lungs [11]. Moreover, PGD₂ production was elevated in nasopharyngeal samples from young infants hospitalized with RSV bronchiolitis, compared to healthy controls [9]. To the best of our knowledge PGD₂ levels have not been examined in lung, airways or hematopoietic cells from patients with SARS-CoV or SARS-CoV-2 infections.

The increased generation of PGD₂ in response to viral infections is primarily mediated by upregulation of COX-2, phospholipase A2 and PGD₂ synthase as reviewed here. Transcriptional activation of COX-2 is mediated by multiple mechanisms. Intratracheal administration of a synthetic double stranded viral RNA (dsRNA) to mimic viral infection induced gene expression of cyclooxygenase-2 (COX-2) by direct binding to the COX-2 promoter [5]. Additionally, the nucleocapsid protein of SARS-CoV activates the expression of COX-2 by binding directly to the regulatory elements for nuclear factor-kappa B and CCAAT/enhancer binding protein [12]. HIV- 2 efficiently induces COX-2 transcription in human astrocytes through regulation of NF- κB p65/relA phosphorylation and transactivation [13]. COX-2 transcription is also induced directly or indirectly by inflammatory molecules released either as a result of viral infection per se or the host response to the virus [12]. Intranasal administration of SARS-CoV virus to mice resulted in marked increase in phospholipase A2 expression and PGD₂ levels in the lungs.11 Infection with respiratory syncytial virus upregulated hematopoietic prostaglandin D synthase expression in cultured human primary airway epithelial cells [9]. Therefore, as reviewed here, it has been conclusively demonstrated that respiratory viruses upregulate PGD₂ production in the airways and the lungs.

Studies over the past decade have investigated the role of PGD₂ in regulating the innate and adaptive immune responses to the SARS-CoV viruses. PGD₂ signals primarily through three G-protein coupled receptors, first the D-prostanoid receptor 1 (DP₁); second, prostanoid receptor 2 (DP₂) which was identified previously as the “chemoattractant receptorhomologous molecule expressed on Th2 cells” (CRTH); and third, the thromboxane receptor (TP) [14,15]. PGD₂ effects on cellular elements in the respiratory tree including pulmonary capillary endothelial cells, airway epithelial cells and cells of the innate and adaptive immune system are diverse and cell specific [4].

Werder RB, et al. have demonstrated in a neonatal mouse model of severe viral bronchiolitis that production of IFN-λ is dependent on PGD₂/DP₂ signaling; PGD₂/DP₂ antagonism decreases viral load, immunopathology, and morbidity [9]. The beneficial effects of DP₂ blockade were associated with increased IFN-λ (IL-28A/B) expression and were lost upon IFN-λ neutralization [9]. This suggests that PGD₂/ DP₂ antagonists may be useful antivirals for the treatment of respiratory infections including SARS-CoV2. T cells are necessary for viral clearance, and, the development of robust T cell responses in the lung requires well-functioning respiratory dendritic cells (rDC) to process and present antigens, migrate to draining lymph nodes and stimulate adaptive immunity against the virus, including cell and humoral mediated immune responses. Experimental data suggests that increase in PGD₂ expression in mouse lungs following viral infection leads to impairment in rDC migration to mediastinal lymph nodes [4]. The production of PGD₂ increases with aging, which results in diminished T cell responses and a more severe clinical disease in older mice infected with respiratory viruses [4]. Furthermore, PGD₂ drives ‘group 2 innate lymphoid cells’ (ILC2) to secrete interleukin-13 (IL-13), which activates “monocytic myeloid-derived suppressor cells (M-MDSCs)” to suppress downstream immune responses [16]. Blocking the PGD₂ pathway by a specific antagonist of the DP₂ receptor led to a decrease in ILC2 and M-MDSC cells, demonstrating that the ILC2/M-MDSC immunosuppressive axis is partly driven by high PGD₂ concentrations acting upon the DP₂ receptor on ILC2 cells (Figure 1).

It is important to note that production of PGD₂ by airway epithelial cells upon infection with respiratory viruses has salutary effects through the other PGD₂ receptor, D-prostanoid receptor (DP₁), in that PGD₂/DP₁ signaling upregulates interferon and accelerates viral clearance. There may be beneficial effects of PGD₂/DP₁ system in acute lung injury. PGD₂/ DP₁ signaling tightens endothelial barrier function in lipopolysaccharide (LPS) induced acute lung injury, while DP₂ antagonism was not harmful [17]. The biological effects of PGD₂/DP₁ axis in the respiratory system seem to be largely anti-inflammatory and opposed to the effects of PGD₂/ DP₂ signaling which has deleterious effects, as stated above [9,16]. Ramatroban is a selective blocker of DP₂ and TP receptors, and does not affect PGD₂ signaling through DP₁ receptor

PGD₂, a potential mediator of increased morbidity and mortality in COVID-19 disease

It is well known that during the aging process, immune functions decline, rendering the host more vulnerable to certain viruses. The morbidity and mortality from SARS-CoV-2 also increases with both aging and lymphopenia [18,19]. The mechanisms underlying this age-dependent susceptibility to viral infections are an active area of research. Stanley Perlman and colleagues have demonstrated that with aging, mice exhibit a higher basal PGD₂ levels in the airways and lungs, with 22-months old mice exhibiting PGD₂ levels that are 4-5 fold higher, compared to the 6-weeks old mice [4]. They further infected mice of various ages with SARS-CoV intranasally. While all 8 of the 22-month old mice died, all 14 of the 6-week old mice survived. Furthermore, increase in PGD₂ expression in mouse lungs upon aging correlated with a progressive impairment in respiratory dendritic cell (rDC) migration to mediastinal lymph nodes resulting in diminished T cell responses and more severe clinical disease in older mice [4]. Vijay et al. have subsequently demonstrated that secreted phospholipase A₂ (PLA₂) group IID (PLA₂G2D) is critical in determining the impact of age on host susceptibility to SARS-CoV [11].

Furthermore, PGD₂ via DP₂ signaling may have a pro-inflammatory effect by increasing the production of monocyte chemoattractant protein 1 } (MCP-1) and interleukin-6 (IL-6), while IL-6 may promote virus survival and/ or exacerbation of clinical disease [20,21].

Ramatroban, an antagonist of PGD₂/DP₂ and Thromboxane/TP axis, as a novel immunotherapeutic drug for COVID-19

Downregulation of the innate and adaptive immune responses to respiratory viruses is mediated by PGD₂/ DP₂ signaling, as discussed above. This suggests that a selective blockade of PGD₂/DP₂ signaling without blocking DP₁, if effectively operational, can favorably accelerate viral clearance and reduce immunopathology and morbidity. Ramatroban is a potent but reversible antagonist of PGD₂/ DP₂ receptors, while sparing the DP₁ receptors [14,22,23]. Ramatroban is also a potent antagonist of thromboxane receptors (TP), and inhibits tumor necrosis factor and platelet activating factor induced expression and production of monocyte chemoattractant protein-1 (MCP-1), and expression of adhesion molecules in human endothelial cells, while reducing inflammation [22]. Ramatroban enhances vascular response to acetylcholine, and has an inhibitory effect on vascular smooth muscle contraction and platelet aggregation [22]. Ramatroban reduces myocardial infarct size, and prevents neointimal formation after balloon arterial injury in hypercholesterolemic rabbits [22,24].

Romatroban exhibits a large safety factor. The usual dose of romatroban is 50 to 150 mg orally, twice a day. The intravenous LD₅₀ values in mice and rabbits were > 600 and > 100 mg/kg respectively, while no dogs died with an intravenous dose of 250 mg/kg [22]. In the 12-months toxicity study in dogs, no toxicologically important changes were observed in any dog given up to 30 mg/kg/day of ramatroban. In this study plasma concentration of ramatroban in animals at 2-hours after oral administration of 30 mg/kg of the drug was between 11.9 to 32.7 mg/mL, while C_max in healthy adult male volunteers given 75 mg of ramatroban twice daily (usual clinical dose) was about 0.4 mg/mL. Accordingly, the doses tested were judged to be sufficiently high to indicate clinical safety of ramatroban in humans. As a thromboxane A₂ antagonist, romatroban has been used for the treatment of allergic diseases. The clinical efficacy and safety of ramatroban have been demonstrated in clinical studies as treatment of allergic rhinitis, and for the past 2 decades ramatroban (Baynas®) has been marketed in Japan for this indication. COVID-19 is characterized by a prothrombotic state including disseminated intravascular coagulopathy (DIC), thrombosis and infarctions which are associated with poor outcomes and higher mortality [25,26]. The anti-platelet action of ramatroban, as a thromboxane A₂ antagonist, could potentially reduce thrombotic events in patients with COVID-19 disease. Therefore, ramatroban by its DP₂ antagonism can potentially help restore the IFN-λ, T and B cell responses that have been suppressed by the SARSCo- V2 virus in COVID-19 disease, especially in the elderly; and help control inflammation by inhibiting production of IL-6. Furthermore, ramatroban, as a potent thromboxane A₂ antagonist can potentially reduce the severity of coagulopathy, a sequela of severe COVID-19 disease.

Discussion and Conclusion

COVID-19 disease in more severe cases is characterized by immune suppression, inflammation and a prothrombotic state (Figure 1). Prostaglandin D₂ has been found to be a key mediator of immunosuppressive effects in animal models of SARS-CoV infection. Ramatroban selectively antagonizes the actions of thromboxane A₂ via the TP receptors and PGD₂ via the DP₂ and TP receptors while sparing PGD₂/DP₁ signaling (Table 1). Hence, Ramatroban, holds great potential for restoring or enhancing immune function in patients with COVID-19 disease, especially in the elderly patients. Ramatroban is expected to decrease inflammation, improve endothelial function, inhibit thrombosis and improve outcomes. Ramatroban has excellent safety profile. Therefore, Ramatroban is a highly promising therapy for patients with COVID-19 infection. Urgent and fast-track clinical trials are needed to investigate the efficacy and safety of Ramatroban across different levels of severity of COVID-19 disease. This requires cooperation between scientists, industry and governmental agencies globally.

Acknowledgement

The authors thank Ms. Kate Chander Chiang for her assistance in drafting the figure, and online submission of the manuscript.

Author Contributions

A. Gupta conceptualized, created the inventive concept and the framework for the manuscript; K. Kalanter-Zadeh and A. Gupta wrote the original draft; and all authors reviewed and edited.

Disclosure of Potential Sources of Conflict of Interest

The authors declare there is no conflict of interest.

References

1. Grasselli, Giacomo, Alberto Zangrillo, Alberto Zanella and Massimo Antonelli, et al. “Baseline Characteristics and Outcomes of 1591 Patients Infected with SARS-CoV-2 Admitted to ICUs of the Lombardy Region, Italy”. JAMA 323(2020): 1574-1581.
2. Montecino-Rodriguez, Encarnacion, Beata Berent-Maoz, and Kenneth Dorshkind. “Causes, Consequences, and Reversal of Immune System Aging”. J Clin Invest 123(2013): 958-965.
3. Chu, Hin, Jasper Fuk-Woo Chan, Yixin Wang and Terrence Tsz-Tai Yuen, et al. “Comparative Replication and Immune Activation Profiles of SARS-CoV-2 and SARS-CoV in Human Lungs: An ex vivo Study with Implications for the Pathogenesis of COVID-19”. Clin Infect Dis 2(2020): 2-4.
4. Zhao, Jincun, Jingxian Zhao, Kevin Legge and Stanley Perlman. “Age-Related Increases in PGD(2) Expression Impair Respiratory DC Migration, Resulting in Diminished T Cell Responses upon Respiratory Virus Infection in Mice”. J Clin Invest 121(2011): 4921-4930.
5. Shiraishi, Yoshiki, Koichiro Asano, Kyoko Niimi and Koichi Fukunaga, et al. “Cyclooxygenase-2/Prostaglandin D2/CRTH2 Pathway Mediates Double-Stranded RNA-Induced Enhancement of Allergic Airway Inflammation”. J Immunol 180(2008): 541-549.
6. Andreakos, Evangelos, Maria Salagianni, Ioanna E Galani and Ourania Koltsida. “Interferon-Lambdas: Front-Line Guardians of Immunity and Homeostasis in the Respiratory Tract”. Front Immunol 8(2017): 1232.
7. Lazear, Eric, Reza Ghasemi, Sarah M Hein and John Westwick, et al. “Targeting of IL-2 to Cytotoxic Lymphocytes as an Improved Method of Cytokine-Driven Immunotherapy”. Oncoimmunol 6(2017): e1265721.
8. O'Brien, TR, DL Thomas, SS Jackson and L Prokunina-Olsson, et al. “Weak Induction of Interferon Expression by SARS-CoV-2 Supports Clinical Trials of Interferon Lambda to Treat Early COVID-19”. Clin Infect Dis 2(2020): 2-4.
9. Werder, Rhiannon B, Jason P Lynch, Jennifer C Simpson and Vivian Zhang, et al. “PGD2/DP2 Receptor Activation Promotes Severe Viral Bronchiolitis by Suppressing IFN-Lambda Production”. Sci Transl Med 10(2018): 2-4.
10. Ricciotti, Emanuela and Garret A FitzGerald. “Prostaglandins and Inflammation”. Arterioscler Thromb Vasc Biol 31(2011): 986-1000.
11. Vijay, Rahul, Xiaoyang Hua, David K Meyerholz and Yoshimi Miki, et al. “Critical Role of Phospholipase A2 Group IID in Age-Related Susceptibility to Severe Acute Respiratory Syndrome-CoV Infection”. J Exp Med 212(2015): 1851-1868.
12. Yan, Xiaohong, Qian Hao, Yongxin Mu and Khalid Amine Timani, et al. “Nucleocapsid Protein of SARS-CoV Activates the Expression of Cyclooxygenase-2 by Binding Directly to Regulatory Elements for Nuclear Factor-Kappa B and CCAAT/Enhancer Binding Protein”. Int J Biochem Cell Biol 38(2006): 1417-1428.
13. Álvarez, Susana, Almudena Blanco, Florian Kern and Manuel Fresno, et al. “HIV-2 Induces NF-κB Activation and Cyclooxygenase-2 Expression In Human Astroglial Cells”. Virol 380(2008): 144-151.
14. Kupczyk, Maciej and Piotr Kuna. “Targeting the PGD2/CRTH2/DP1 Signaling Pathway in Asthma and Allergic Disease: Current Status and Future Perspectives”. Drugs 77(2017): 1281-1294.
15. Brightling, Christopher E, Guy Brusselle and Pablo Altman. “The impact of the Prostaglandin D 2 receptor 2 and its Downstream Effects on the Pathophysiology of Asthma”. Allergy 75(2020): 761-768.
16. Trabanelli, Sara, Mathieu F Chevalier, Amaia Martinez-Usatorre and Alejandra Gomez-Cadena, et al. “Tumour-Derived PGD2 and NKp30-B7H6 Engagement Drives an Immunosuppressive ILC2-MDSC Axis”. Nat Commun 8(2017): 593.
17. Murata, Takahisa, Kosuke Aritake, Yoshiki Tsubosaka and Toshihiko Maruyama, et al. “Anti-inflammatory Role of PGD2 in Acute Lung Inflammation and Therapeutic Application of its Signal Enhancement”. Proc Natl Acad Sci USA 110(2013): 5205-5210.
18. Mahase, Elisabeth. “COVID-19: Death Rate is 0.66% and Increases with Age, Study Estimates”. BMJ 2(2020): m1327.
19. Tan, Li, Qi Wang, Duanyang Zhang and Jinya Ding, et al. “Lymphopenia Predicts Disease Severity of COVID-19: A Descriptive and Predictive Study”. Sig Transd Targeted Ther 5(2020): 1-3.
20. Peeraully, Muhammad R, Henning Sievert, Mònica Bulló and Bohan Wang, et al. “Prostaglandin D2 and J2-series (PGJ2, Δ12-PGJ2) Prostaglandins Stimulate IL-6 and MCP-1, but Inhibit Leptin, Expression and Secretion by 3T3-L1 Adipocytes”. Pflügers Archiv (2006): 177-187.
21. Velazquez-Salinas, Lauro, Antonio Verdugo-Rodriguez, Luis L Rodriguez and Manuel V Borca. “The Role of Interleukin 6 during Viral Infections”. Front Microbiol 10(2019): 1057.
22. Ishizuka, Toshiaki, Takemi Matsui, Yasuhiro Okamoto and Atsuko Ohta, et al. “(BAY u 3405): A Novel Dual Antagonist of TXA2 Receptor and CRTh2, A Newly Identified Prostaglandin D2 Receptor”. Cardiovasc Drug Rev 22(2004): 71-90.
23. Ulven, Trond and Evi Kostenis. “Minor Structural Modifications Convert the Dual TP/CRTH2 Antagonist Ramatroban into a Highly Selective and Potent CRTH2 Antagonist”. J Med Chem 48(2005): 897-900.
24. Fiedler, Volker B, Elisabeth Perzborn and Friedel Seuter. “Protective Effect of a Novel Thromboxane Antagonist, BAY-U3405, on Canine Myocardial Damage after Coronary Artery Occlusion and Reperfusion”. Pharmacother 11(1991): 77-84.
25. Wang, Janice, Negin Hajizadeh, Ernest E Moore and Robert C McIntyre, et al. “Tissue Plasminogen Activator (tPA) Treatment for COVID-19 Associated Acute Respiratory Distress Syndrome (ARDS): A Case Series”. J Thromb Haemost 2(2020): 2-4.
26. Zhang, Yan, Meng Xiao, Shulan Zhang and Peng Xia, et al. “Coagulopathy and Antiphospholipid Antibodies in Patients with COVID-19”. N Engl J Med 382(2020): e38.