Pentoxifylline: An Immunomodulatory Drug for the Treatment of COVID-19

Rapidly spreading outbreak of the novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is causing serious health concerns worldwide. It started as an epidemic in Wuhan, Hubei province, central China, and has now become a pandemic, spreading over most of the continents of the planet. The major clinical symptoms of the infection are dry cough, fever, pneumonia, respiratory failure, hypoxia, and in certain cases, even death. Alveolar damage and respiratory system failure are observed in severe cases. Initial mild infection leads to activation of the immune system in the lungs and accumulation of various inflammatory cells and molecules. At a later phase during the infection, a “cytokine storm” causes an Acute Respiratory Distress Syndrome (ARDS), leading to an increase in the production of pro-inflammatory cytokines, migration of a large number of immune cells to the site of infection, and ultimately pulmonary damage. The rapid and uncontrolled outbreak requires putative therapeutic drugs for treatment of patients suffering from COVID-19. Amongst the currently used antiviral drugs, such as hydroxychloroquine, lopinavir, remdesivir etc. we would like to present an update on another effective drug, pentoxifylline. Pentoxifylline has anti-inflammatory, immunomodulatory, anti-viral, and bronchodilatory properties. Pentoxifylline is known to reduce cytokine production, immune cell migration, and suppress certain signal transduction pathways (e.g. NF-κβ and STAT3). Thus, it minimizes inflammatory damage in the lung tissues.


INTRODuCTION
The recent outbreak of the novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) as Corona Virus Disease-2019 (COVID- 19)  is causing serious health concerns worldwide.COVID-19, which started as an epidemic (the rapid spread of disease to a large number of people within a community, population, or region) has now been declared as a pandemic (an epidemic that spreads over multiple countries or continents) by the World Health Organization.Since 12 December 2019, 2,774,135 laboratory cases have been confirmed including 190,871 deaths worldwide as reported on 26 April 2020 1,2 .
S A R S -C o V -2 b e l o n g s t o t h e Betacoronavirus genera of the Coronaviridae family.SARS-CoV-2 is the third outbreak of the Betacoronavirus within the last two decades, including the SARS-CoV outbreak in 2003, and the MERS-CoV (Middle Eastern respiratory syndrome coronavirus) outbreak in 2012 3,4 .Coronaviruses are enveloped, non-fragmented, single-stranded, and contain positive-sense RNA as their genetic material 4 .This virus can be easily transmitted from one person to another through droplets generated by coughing or sneezing, through personal contact, or by touching contaminated surfaces or equipments.
Several anti-viral drugs are proposed and tested for the treatment of COVID-19.Lopinavir, along with a booster dose of ritonavir, is a drug widely used for the treatment of HIV.Despite the treatment being beyond standard care, no positive effect was observed when the COVID-19 patients were treated with lopinavir-ritonavir 5 .Another popular drug remdesivir, which is a broad-spectrum antiviral drug, is able to inhibit viral replication of multiple genetically unrelated RNA viruses 6 .Although treatment with remdesivir leads to a significant reduction of the viral titer in the lungs, it failed to improve the pulmonary damage that occurred in the later stages of MERS, SARS, and COVID-19 7 .Since SARS-CoV-2 infection is driven by both the virus and the host immune response, use of only an anti-viral drug, such as remdesivir may not be able to restore pulmonary homeostasis.Currently, hydroxycholoroquine (an anti-malarial drug) has gained much attention for the treatment of COVID-19 patients due to its anti-viral and immunomodulatory properties 8 .However, hydroxychloroquine is known for its relatively narrow therapeutic index, cardiac toxicity, prolonged QT (interval between ventricular depolarization and repolarization), and sodium channel inhibition, which results in ventricular arrhythmias, conduction blockade, and cardiovascular collapse 9 .Considering efficacies and limitations of the aforementioned drugs, some important features of pentoxifylline are highlighted in this review.

COVID-19 and its Immunopathology
COVID-19 with symptoms similar to pneumonia started in Wuhan, Hubei province, central China and has now spread to almost 213 countries 2 .Typical clinical features of the patients infected with this virus include fever, fatigue, dry cough, nausea, chest tightness, dyspnoea (breathing difficulties), headache, pneumonia, and sometimes death 2,10 .Alveolar damage and respiratory system failure are the most severe pathologies of this disease.Lung autopsies of the patients suffering from SARS showed flooding of the alveolar lumina mixed with inflammatory cells, increased alveolar macrophage population, and hemophagocytosis 11 .A close resemblance has been found between the pathogenicity and the pattern of inflammatory damage caused by SARS-CoV and SARS-CoV-2.
The human airway epithelium is an important physical barrier against external pathogens and allergens.It also regulates pulmonary inflammation.SARS-CoV-2 infection induces interferon-stimulated genes that inhibit viral replication.As a response to the anti-viral activity of the host, the viral system encodes various antagonist molecules to modulate the interferon activity 3 .Epithelial cells produce various cytokines and chemokines in response to pathogens and generate inflammatory reactions associated with a variety of lung diseases.The cytokine network includes alveolar macrophages, eosinophils, mast cells, and T lymphocytes 12 .Biological effects of cytokines are dependent on their concentration.At a lower concentration, many cytokines regulate normal physiological processes while at higher concentrations, they mediate local or systemic inflammatory responses.At the highest concentration, cytokines exert adverse effects to the host causing tissue damage 13 .Increased levels of proinflammatory cytokines (e.g.interferon gamma (IFNγ), interleukin (IL)-1β, IL-2, IL-6, IL-8, tumor necrosis factor alpha (TNFα), IFNγ induced protein-10 (IP10), macrophage inflammatory protein-1 (MIP1), granulocyte-colony stimulating factor (GCSF), and monocyte chemoattractant protein-1 (MCP1)) are responsible for pulmonary inflammation and extensive lung damage 14,15 .Expression of IL-8 and MCP-1 by the pulmonary epithelial cells facilitate migration of inflammatory and activated cells to the target tissue 16 .Depletion of CD4+ and CD8+ T lymphocytes in the patients infected with SARS is also associated with the adverse outcomes of the disease 15 .Thus, two main features of the immune dysregulations generated by SARS-CoV-2 are: (a) overproduction of proinflammatory cytokines by monocytes at the site of the infection, and (b) dysregulation of lymphocytes such as CD4+ T-cells and CD8+ T-cells, and (Natural Killer) NK cell count depletion .Chronic inflammation, which occurs at a later phase of the disease, is the main cause of Acute Respiratory Distress Syndrome (ARDS).ARDS is a secondary clinical manifestation of the COVID-19 infection, caused by pneumonia and sepsis due to "cytokine storm", in which the immune and non-immune cells associated with inflammation release huge amounts of pro-inflammatory cytokines that cause damage to the host 18 .
Nuclear factor kappa-light-chainenhancer of activated B cells (NF-κβ) is an important ubiquitous transcription factor and a pleotropic regulator of many genes involved in inflammatory response and immunoregulatory activities 19 .The major pathway that functions after the patient is infected with coronavirus is the activation of the NF-κβ pathway through myeloid differentiation factor 88 (MyD88) leading to the induction of a variety of pro-inflammatory cytokines including IL-6 and TNF-α 20 .IL-6 in turn activates signal transducer and activator of transcription 3 (STAT3), which is required for complete activation of the NF-κβ pathway.NF-κβ and STAT3 together activate the IL-6 amplifier leading to multiple inflammatory and autoimmune diseases 21 .IL-6 induces various pro-inflammatory cytokines and recruits various inflammatory cells to the target tissues.IL-6 is an important marker of cellular senescence.The amount of cellular IL-6 amplifier increases with age, and this might be one of the possible reasons for age dependent mortality of COVID-19 18 .
Cytokines up-regulate the expression of leukocyte adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) on the epithelial and endothelial cells of the lung airway 22 .ICAM-1 in turn mediates binding of activated T lymphocytes to the alveolar epithelial cells and retains the activated T lymphocytes 23 .T lymphocyte derived cytokine, IFN-β, is known for its cytotoxic effects and it participates in pulmonary inflammation by inducing the expression of ICAM-1 in the epithelial cells 12 .
P ro d u c t i o n a n d a c t i v i t y o f t h e inflammatory cytokines at the site of infection may help the host by promoting the accumulation of polymorphonuclear leukocytes (PMNs) and activating their anti-pathogenic functions.However, prolonged and excessive activation of PMNs can initiate microvascular injury, resulting in increased vasopermeability, hemorrhage, and thrombosis 13 .It altogether causes fever, dry cough, and pneumonia in the COVID-19 patients.

Pentoxifylline
Pentoxifylline (Oxpentifylline) (Fig. 1) is a methylxanthine derivative, also known as 1-(5-Oxohexyl)-3,7-dimethylxanthine, 1-(5-Oxohexyl)-theobromine.Its pharmacological properties are similar to other known xanthines such as theobromine, caffeine, and theophylline 24 .It has anti-viral, immunomodulatory, antiinflammatory, and bronchodilator effects [25][26][27][28] .As a bronchodilator it reduces the thickness of blood, increases the red blood cell flexibility so they can migrate through narrow capillaries more rapidly and as a result eases blood circulation 24 .Pentoxifylline has a broad spectrum of antiviral activity, as it is used against almost eight viruses namely herpes simplex virus, vaccinia virus, rotavirus, tick-borne encephalitis virus, HIV, hepatitis JA virus, vesicular stomatitis virus, and West Nile virus 26 .The anti-inflammatory activity of pentoxifylline is associated with its ability to inhibit the production and/or function of proinflammatory cytokines 28 .Pentoxifylline is comparatively inexpensive and has fewer side effects compared to the other anti-inflammatory and anti-viral drugs 29 .It can be administered through oral or intravenous routes, and can be metabolized by the red blood cells and the liver.It has an elimination half-life of 3.4 hours 24 .Moreover, research suggests that pentoxifylline can inhibit microvascular constriction, block red blood cell and platelet aggregation, decrease plasma fibrinogen levels, stimulate fibrinolysis, and suppress leukocyte deformability 30,31 .
Pentoxifylline is able to inhibit the actions of IL-1, TNF-α, and TNF-β, reduce ICAM-1 expression, and reduce IL-8 and MCP-1 production in vivo 12,29 .Dose-dependent pentoxifylline induced suppression of TNF-α has been observed in human trials and inhibition of spontaneous production of TNF-α was also demonstrated in early studies 29 .Blockage of adhesion molecules and chemotactic activities can reduce migration of inflammatory cells in the lung airway and present potent and possible targets for future anti-inflammatory therapies in airway diseases 12 .Pentoxifylline as a phosphodiesterase inhibitor raises cyclic AMP (cAMP) levels in PMNs, resulting in decreased PMN chemotaxis towards the site of inflammation and thus, reduced microvascular injury, vasopermeability, hemorrhage, and thrombosis 13,32,33 .
Pentoxifylline is non-specific inhibitor of phosphodiesterase (PDE) 19 .PDE is present in almost every pro-inflammatory and immune cell and it regulates the metabolism of cAMP 26 .However, a specific type of PDE isoenzyme regulates only a specific type of inflammatory cytokine biosynthesis 34 .Since lung tissues consist of a variety of PDE isoenzymes (I, II, III, V, and VII) and the isoenzymes that are activated by the coronavirus infection are unknown, the use of non-specific inhibitor, preferably pentoxifylline could have a broader range of inhibitory activity in SARS-CoV-2 19 .
Two different studies conducted on invitro cultured smooth muscle cells (SMC) have shown the inhibitory action of pentoxifylline on the activity of NF-κβ 35,36 .Inhibition of NF-κβ activity may down-regulate production and secretion of IL-6 and TNF-α.Experiments on A375 human melanoma cell-line and A549 lung cancer cellline have shown dose-dependent suppressed phosphorylation and DNA binding of STAT3, as well as limited secretion of IL-6 37,38 .-Inhibits phosphodiesterase and thus, decreases production immune and pro-inflammatory cells, leading of pro-inflammatory cytokines to production of cytokines Moreover, pentoxifylline has a very low toxicity level and has minimal side effects.However, its long-term use may cause dizziness, headache, nausea, or stomach discomfort in some patients 26 .It is highly advisable to take this drug only as prescribed by the physician.

DISCuSSION
COVID-19 is a life-threatening disease that has spread worldwide and infected millions of people within a short period of time.With an average reproduction number of 3.28 (R 0 , an indication of transmissibility of a virus, representing the average number of new infections generated by a single infectious person in a totally naive population), novel SARS-CoV-2 is rapidly spreading in all the countries of the world 39 .Almost all of the SARS-CoV-2 patients with pneumonia, who develop severe respiratory failure, are diagnosed with hyper-inflammatory response, increased pro-inflammatory cytokine production, and dysregulation of lymphocytes.Moreover, severe consequences of the disease are diffused alveolar damage, hyaline membrane formation, inflammation in the alveolar walls, desquamation of pneumocytes, and patients also develop secondary bacterial pneumonia in case of further complications 4 .Large amounts of proinflammatory cytokines released by the alveolar macrophages and other activated cells have a prominent role in the pathology of SARS 11 .The above mentioned clinical manifestations suggest the use of immunomodulatory drugs for treatment of patients suffering from COVID-19.
Anti-inflammatory, immunomodulatory, anti-viral, and bronchodilatory properties of pentoxifylline makes it the most suitable drug for the treatment of COVID-19.Pentoxifylline may have a possible anti-viral activity against SARS-CoV-2, along with a cytokine-modulation activity, but not as immunosuppressant as observed in the case of corticoids.It down-regulates only the cytokine activity and leaves the rest of immune response functional.It is an inexpensive drug, has a very low cytotoxicity, and has minimal side effects.Safe use of pentoxifylline has been shown in a number of clinical trials.Thus, all the characteristics of pentoxifylline make it a promising and efficient therapeutic drug to treat patients suffering from COVID-19.However, clinical trials and approvals are required before incorporating pentoxifylline as a routine treatment of COVID-19.

CONCluSION
N e i t h e r v a c c i n e s n o r p r e c i s e pharmaceutical medicines for treatment of COVID-19 are formulated till date.The recent strategy to be considered is to study the properties and effectiveness of available pharmacological medicines and to use one or a combination of two or more drugs to overcome the manifestations of the disease.Existing evidences strongly support an important role of abnormal immune response in the pathogenesis of SARS-CoV-2.Thus, immunomodulatory drugs could be a better alternative along with an antiviral therapy.The immunomodulatory drug pentoxifylline is inexpensive and has no defined adverse effects.Thus, it can be an effective therapeutic agent.However, clinical experiments are yet to be performed to study the effects of pentoxifylline on COVID-19 patients.

Table 1 .
Summary of the action of pentoxifylline against clinical manifestations of SARS-CoV-2 in COVID-19 patients Clinical manifestations of COVID-19 Effectiveness of pentoxifylline Viral infection and proliferation in lung -Demonstrates anti-viral activity against 8 deadly viruses tissues and may effectively inhibit SARS-CoV-2 replication Production of TNF-α and IL-1 by alveolar -Inhibits production of TNF-α, and IL-1 from macrophages macrophages and other cells Increased expression of surface ICAM-1 on -Reduces the expression of ICAM-1 and thus, decreases the lung epithelial and endothelial cells, binding of T-cells leading to binding of more T-cells Accumulation and prolonged activation of -Increases the cAMP levels in PMNs, thus decreasing PMNs leading to increased microvascular chemotaxis towards the inflammatory sites and results in injuries, hemorrhage, and thrombosis decreased microvascular injuries, hemorrhage, and thrombosis Expression of IL-8 and MCP-1; increased -Reduces production of IL-8 and MCP-1; decreased migration migration of other inflammatory cells to the of inflammatory cells to the target tissue target tissue Promotes signaling through STAT3 and NF--Suppresses signaling through STAT3 and NF-κβ leading to κβ for production of pro-inflammatory cytokines decreased production of IL-6, TNF-α, and other cytokines Activates phosphodiesterase in various

Fig. 2 .
Fig. 2. Coronavirus cycle and the hypothetical activity of pentoxifylline