key: cord-1053010-eym2rh8v
authors: El Tabaa, Manar Mohammed; El Tabaa, Maram Mohammed
title: New putative insights into neprilysin (NEP)-dependent pharmacotherapeutic role of roflumilast in treating COVID-19
date: 2020-10-01
journal: Eur J Pharmacol
DOI: 10.1016/j.ejphar.2020.173615
sha: 8f9ccdfbcdf5c73768c3b4b4b7662eba44a456c1
doc_id: 1053010
cord_uid: eym2rh8v

Nowadays, coronavirus disease 2019 (COVID-19) represents the most serious inflammatory respiratory disease worldwide. Despite many proposed therapies, no effective medication has yet been approved. Neutrophils appear to be the key mediator for COVID-19-associated inflammatory immunopathologic, thromboembolic and fibrotic complications. Thus, for any therapeutic agent to be effective, it should greatly block the neutrophilic component of COVID-19. One of the effective therapeutic approaches investigated to reduce neutrophil-associated inflammatory lung diseases with few adverse effects was roflumilast. Being a highly selective phosphodiesterase-4 inhibitors (PDE4i), roflumilast acts by enhancing the level of cyclic adenosine monophosphate (cAMP), that probably potentiates its anti-inflammatory action via increasing neprilysin (NEP) activity. Because activating NEP was previously reported to mitigate several airway inflammatory ailments; this review thoroughly discusses the proposed NEP-based therapeutic properties of roflumilast, which may be of great importance in curing COVID-19. However, further clinical studies are required to confirm this strategy and to evaluate its in vivo preventive and therapeutic efficacy against COVID-19.

Simultaneously, ACE-2 protein was also detected to be distributed in various 135 human organs other than lungs involving oral and nasal mucosa, gastrointestinal 136 tract (GIT), skin, heart, liver, kidney, and brain (Hamming et al., 2004) ; elucidating 137

the reason for developing other extra-pulmonary manifestations associated with 138

COVID-19 infection. 139

Binding of SARS-CoV-2 with ACE-2 may downregulate ACE-2 and subsequently, 140

inhibit the ACE-2-regulated generation of angiotensin (1-7) peptide which can, via 141

Mas receptor, perform several beneficial activities as vasodilator, anti-142

inflammatory, anti-hypertrophy, anti-proliferative, anti-fibrosis and antioxidant 143 (Kuba et al., 2005) . 144

Concerning the pulmonary RAS, cutting off the ACE-2 / angiotensin (1-7) / Mas 145 receptor axis will activate the vasopressor ACE / angiotensin (Ang) II / angiotensin 146 II type 1 receptor (AT1) axis on the other side. The axis which may drive the 147 airway inflammatory cascades, because of significant increase in Ang II level. Ang 148 II, through activating angiotensin II type 1 receptor, could promote the release of 149 multiple inflammatory cytokines especially TNF-α, IL-6, GM-CSF and MCP-1 150 (Sprague and Khalil, 2009) . 151

Cytokine storm is a fierce interplay of cytokines that can occur in numerous 153

infectious and non-infectious diseases (Teijaro, 2017) . It is considered as a 154

potentially fatal immune reaction that consists of a positive feedback loop between 155 cytokines and immune cells. When the immune system is fighting pathogens, 156 cytokines signal immune cells, such as T cells and macrophages can travel to the 157 site of infection, where they will be activated and stimulated to produce more 158

cytokines. This positive feedback loop reaction becomes uncontrolled and then, too 159 many immune cells are activated in a single place. Consequently, cytokine storm 160 J o u r n a l P r e -p r o o f will have the potential to significantly damage body tissues and organs (Tisoncik et 161 al., 2012) . 162

In the lungs, for example, increasing the release of cytokines such as interleukin- 6 163 (IL-6) will trigger the fluids and immune cells to be accumulated, eventually block 164 off the airways, and potentially lead to death (Rincon and Irvin, 2012) . This is 165 obviously detected in seriously ill COVID-19 patients who showed high levels of 166 IL-6 (Dal Moro and Livi, 2020) . 167

Because of the positive correlation between high IL-6 level and COVID-19 168 severity, IL-6 is specifically suggested to be the master marker used for monitoring 169 disease progression (T. . There is a growing evidence that IL-6 can 170 play a crucial part in the uncontrolled intestinal inflammatory process, proving its 171 role in the pathogenesis of COVID-19-asociated diarrhea. However, another 172

causing factor may be attributed to the direct viral invasion of gut epithelial cells 173

via ACE-2 (Mudter and Neurath, 2007) . 174

As previously reported, IL-6 could prohibit the olfactory signal pathway; proposing 175 that anosmia detected in COVID-19 patients may be due to IL-6-mediated 176

inflammation of the nasal mucosa (Henkin et al., 2013; Luërs et al., 2020) . Besides, 177

other additional elements supporting that SARS-CoV-2 may have a neuro-invasive 178

propensity to invade the central olfactory pathway causing olfactory dysfunction 179 (Marinosci et al., 2020) . Jointly, IL-6 was also found to be extremely involved 180

in promoting the ocular inflammation; matching with conjunctivitis that is recently 181

reported to be linked with COVID-19 infection (Ghasemi, 2018) . 182

In addition to the direct role of SARS-CoV-2/ACE-2 interaction in inducing the 184 endothelial dysfunction (Y. , IL-6 was also reported to interrupt 185 the normal function of endothelial cells (ECs) through inactivating the endothelial 186 nitric oxide synthase (eNOS) which in turn could decrease NO production with 187 J o u r n a l P r e -p r o o f subsequent induction of an oxidative stress state leading to impairment in 188 endothelial responses (Hung et al., 2010) . 189

As a consequence, disrupting the endothelial cell function either by SARS-CoV-2 190

itself or IL-6 could activate the platelets and stimulate their adhesion and 191 aggregation; resulting in a pulmonary specific vasculopathy termed pulmonary 192 intravascular coagulopathy (PIC) (Aird, 2003; Levi and van der Poll, 2017; 193 McGonagle et al., 2020) . 194

Most anatomical studies of COVID-19 victims demonstrate the formation of blood 195 thrombus (fibrin clot) in their pulmonary vessels, in addition to deep vein 196 thrombosis that increases the risk for developing pulmonary embolism (Cui et al., 197 2020; Klok et al., 2020) . These clots result in a compensatory increase of 198 plasminogen (fibrinolysin) but, with disease progression, it fails to break down 199 these fibrin deposits reflected in elevated D-dimer (DD) levels, which is reported to 200 be associated with the severity of COVID-19 infection and may be also correlated 201

with activation of the pro-inflammatory cytokine cascade (Belen-Apak and 202

Sarıalioğlu, Leonard-Lorant et al., 2020) . 203

Emerging data suggest that COVID-19-associated endothelial dysfunction could 204

induce several structural and functional changes resulting in leukocyte trafficking, 205 which in turn, may shift the vascular equilibrium towards triggering more 206

inflammation (Aird, 2003) . Although leukocyte trafficking was known to play an 207 essential part in the protective responses against any infection or injury, it may also 208 lead to extensive tissue damage as shown in numerous inflammatory disorders 209 (Chen et al., 2018) . One of the most abundant leukocytes being assured in 19 are neutrophils that represent the first line of defense in the innate immune 211 system. 212

With the continual reduction detected in lymphocytes count of COVID-19 patients, 214

they become more prone for secondary infections with the risk of high mortality 215

rate. This occurs due to loss of all lymphocyte effector cells that possess the 216 essential antiviral activity, including CD8+ or cytotoxic lymphocytes and natural 217 killer cells, as well as B cells, which able to form the specific antibodies targeted 218 for inactivating the virus (Dallan et al., 2020; Remy et al., 2020) . 219

Therefore, developing severe lymphopenia will effectively inhibit the stimulation 220 of adaptive cell-mediated immune response and consequently, facilitate the 221 inflammation-mediated neutrophil response which could be started with their 222 chemotaxis and recruitment, followed by degranulation (Didangelos, 2020; Hyun 223 and Hong, 2017) . Neutrophils possess an arsenal of proteases such as (elastase, 224

proteinase-3 and cathepsin G), inflammatory mediators such as (TNF-α and IL-6), 225

and toxic oxidants that do not kill phagocytosed pathogens only, but also can 226 damage the host tissue (Gernez et al., 2010) . 227

In response to high neutrophilia with progressive lymphopenia established in 229 COVID-19, viral sepsis may be promoted as a result of systemic uncontrolled 230 inflammation induced by neutrophils with further worsening of tissue injury (Hui 231 Li et al., 2020) , that is consistent with the final diagnosis emphasizing the existence 232 of a septic shock among COVID-19 patients with profound lymphopenia (Dallan et 233 al., 2020) . 234

Sepsis is a syndrome that has attracted the attention worldwide because of its high 235 mortality rate of about 50-80%. It is widely recognized as a systemic inflammatory 236 response syndrome, that had been defined as a complex disorder arising from the 237 dysregulation of an inflammatory response of the entire organism to an infection or 238

to circulating bacterial products, rather than infection (Bone et al., 1992) . However, 239 J o u r n a l P r e -p r o o f sepsis has been now redefined as a life-threatening organ dysfunction due to a 240 dysregulated response of the host to infection (Singer et al., 2016) . 241

Sepsis itself may share in the subsequent release of inflammatory factors (IL-6 and 242

TNF-α) that could eventually aggravate the existing inflammation (Molano Franco 243 et al., 2019) and thus, could lead to multiple organ dysfunction, shock, and even 244 death, which are not caused directly by the invading pathogens; but as a result of 245

inflammation (Crowther, 2001; Mantzarlis et al., 2017) . 246

During sepsis, there is an extensive crosslink between increased inflammation, 247 endothelial dysfunction and hyper-coagulopathy, in which the microvascular 248 dysfunction was documented to be one of important sepsis hallmarks (Schouten et 249 al., 2008) . 250

Given the reported evidence of induced endothelial dysfunction, pulmonary 252 fibrosis may be also prompted as a substantial problem during COVID-19 253 infection, to the extent that pulmonary post-mortem findings in fatal cases of 254 COVID-19 revealed the presence of extensive fibrotic features as myofibroblastic 255

proliferation or organizing pneumonia (George et al., 2020) . The vascular 256 endothelial dysfunction could stimulate the fibrotic consequences via secreting a 257 peptide, namely endothelin-1 (ET-1) (Elshazly et al., 2013) , which could induce 258 the release of transforming growth factor-β1 (TGF-β1), a fibrogenic cytokine 259 mainly implicated in driving the pulmonary fibrosis development (Wermuth et al., 260 2016) . 261

Surprisingly, ET-1 is also suggested to exaggerate the inflammation via inhibiting 263 adenylyl cyclase (AC) activity and thereby, cAMP accumulation (Insel et al., 264 2012) .Within the immune system, cAMP is synthesized from ATP by the action of 265 AC to regulate the anti-inflammatory effects (Gentile et al., 1988) . As reported, 266 J o u r n a l P r e -p r o o f cAMP could decrease the production of pro-inflammatory mediators as well as 267 enhance the production of anti-inflammatory factors in various immune cells 268 (Raker et al., 2016) . Meanwhile, cAMP was concluded to promote ATP production 269 that is described to potentially improve the efficiency of innate and adaptive 270

immune systems for fighting off COVID-19 (De Rasmo et al., 2016; Taghizadeh-271 Hesary and Akbari, 2020) . 272

Consistent with these findings, it was reported that COVID-19 may be more fatal 273

in the elderly-population than in children, as with increasing the age, there is a 274 gradual decline in the cellular ATP and subsequent ATP-induced cAMP 275

accumulation (Srivastava, 2017) . Furthermore, tobacco smokers, who suffer from a 276 decreased content of ATP in immune cells, are also found to be more susceptible 277

for COVID-19 infection (Malińska et al., 2019) . 278

Regardless of age, males are generally more prone to die by COVID-19 than 279

females . The finding which can be attributed to sex hormone 280 differences, since estrogen was recorded to potentially induce ATP production 281 during the inflammation than androgens (Kassi and Moutsatsou, 2010) . 282

Additionally, the same strategy could be particularly relevant for patients with 283 serious medical conditions, who showed an immune dysregulation as a result of 284 ATP-depletion (Zhou et al., 2020) . 285

With extremely rapid increase in the number of SARS-CoV-2-infected cases 287

globally, there is unfortunately sufficient time for discovering a newly therapeutic 288 agent. Taken together, directing most efforts towards vaccine production may be of 289 no avail at least nowadays, since millions of people everywhere have been already 290

infected with COVID-19, and they are in urgent need for rapid treatment in order to 291 prevent the disease progression. In addition, developing anti-viral drugs needs a 292 long way to go. Therefore, the best choice may be repurposing the currently 293 available drugs which may greatly save time and money as well as secure many 294 people from death. 295

World Health Organization (WHO) reported that COVID-19 now becomes much 296 more than a health crisis. Till present, curing COVID-19 remains elusive, in spite 297 of the great efforts directed by the researchers towards understanding and 298

identifying the disease mechanisms. There is no doubt that COVID-19 can trigger 299 airway inflammatory reactions, in which neutrophils play the major role in 300

increasing the severity by inducing COVID-19-associated coagulopathy (Zuo et al., 301 2020) . In that context, several therapeutic strategies have been proposed to control 302 -19 (Cascella et al., 2020) . 303

The most common one involves the use of hydroxychloroquine (HCQ) as the first-305 line therapy because of its anti-inflammatory and immunomodulatory effects (Hu 306 et al., 2017) . Based on the international guidelines, HCQ is reported to be utilized 307 either alone or in combination with other drugs including, systemic corticosteroids, 308 tocilizumab (TCZ), macrolide azithromycin, antiviral lopinavir/ritonavir and 309 anticoagulant enoxaparin (Mehra et al., 2020; Rosenberg et al., 2020) . However, 310 the use of HCQ is lately recorded to have many restrictions due to increased risk of 311 serious cardiac arrhythmias (Nguyen et al., 2020) . Additionally, both HCQ and 312 chloroquine (CQ) are no longer authorized by FDA to treat COVID-19 313 (FDA.,2020) . 314

Moreover, current COVID-19 treatment protocol also recommends the use of oral 315

anti-inflammatory steroids such as dexamethasone or inhaled corticosteroid such as 316

ciclesonide. Ciclesonide was reported to exhibit both antiviral and anti-317

inflammatory actions with less systemic immunosuppressive effects (Matsuyama et 318 al., 2020) . However, further studies are needed to confirm its potential effect 319

against COVID-19 (Iwabuchi et al., 2020) .

Controversially, using steroids may paradoxically exaggerate the COVID-19-321 associated neutrophilia (Fukakusa et al., 2005) . In addition, steroids should be 322 taken with caution in vulnerable patients with pre-existing hypertension, diabetes, 323 or cardiovascular diseases, which, at the same time, represent the highest risk 324

group of COVID-19 (Varga et al., 2020) . diseases (Rao et al., 2015) . Furthermore, anti-interleukin therapy is expected to 336

worsen the post-COVID-19 pulmonary fibrosis (George et al., Silva et al., 337 2020) . 338

As regards to azithromycin, pieces of clinical evidence revealed that it could exert 339 a great role against both SARS and Middle East Respiratory Syndrome (MERS), 340

that prompted scientists to strongly suggest it as a potential treatment for COVID-341

19. Azithromycin was detected to possess anti-inflammatory and 342

immunomodulating actions in addition to antiviral properties because of its ability 343

to minimize the production of pro-inflammatory cytokines particularly IL-6 and 344

TNF-α, noxious oxidative radicals as well as to improve T-helper cell functions. 345

However, the preliminary studies have demonstrated that using azithromycin 346

should be in caution due to its potential arrhythmogenic threat, especially in high 347 risk COVID-19 patients (Pani et al., 2020) . Ebola virus (EBOV), MERS-CoV, and SARS-CoV-1 . 363

A novel originally developed broad-spectrum antiviral drug, favipiravir, has been 364 also experimentally tested against COVID-19. Favipiravir is a pyrazine 365 carboxamide derivative that can selectively block influenza viral replication via 366

inhibiting the viral RNA-dependent RNA polymerase (Cai et al., 2020) . 367

Additionally, nafamostat, an oral serine protease inhibitor, was reported to 368 significantly inhibit SARS-CoV-2 infection in lung-derived human cell line Calu-3 369 (Hoffmann et al., 2020) . Regarding the efficacy and safety of nafamostat, a 370

prospective clinical trial (NCT04352400) is being conducted to evaluate its 371 possible role against COVID-19 (Azimi, 2020) . 372

Another repurposed drug suggested for treating COVID-19 because of its potential 373 antiviral activity was famotidine. Using famotidine, a histamine-2 (H2RA) receptor 374

antagonist among the hospitalized COVID-19 patients was documented to reduce 375 the mortality rate. Famotidine may interfere with SARS-CoV-2 maturation by inhibiting the activity of 3CLpro. However, its therapeutic role against COVID-19 377 is still at nascent stage and randomized controlled trials are urgently needed 378

(Aguila and Cua, 2020). 379

Considering ACE-2 to be the only viral receptors, a new study has proposed that 381 lactoferrin, an orally nutritional supplement, may be potentially useful against 382 COVID-19. In addition to its unique immunomodulatory and anti-inflammatory 383 effects, lactoferrin has been described to possibly occupy angiotensin-converting 384 enzyme ACE-2 receptors preventing SARS-CoV-2 from attaching to the host cells 385 (Kell et al., 2020) , however it is not proved till now. 386

Most of the repurposed drugs used for treating COVID-19 are directed mainly 387 towards blocking the induced cytokine storm, however this COVID-19-related 388 sepsis argues now for investigating a different therapeutic approach (Remy et al., 389 2020) . 390

Since the morbidity/mortality rate in septic patients was reported to be correlated 391

with the plasma level of ET-1, reducing its level may minimize all unwanted 392 reactions mediated by endothelin ET-1 receptors. The observation that may explain 393 why anti-inflammatory drugs like anti-TNF-α and IL-1-based therapies have failed 394

in treating sepsis, opposite to clinical trials that indicated the application of 395 endothelin ET-1 receptor blockers as an effective strategy (Kowalczyk et al., 396 2015) . In addition, decreasing ET-1 level may interrupt the fibrotic pathway 397 regulated by TGF-β1, thus inhibiting the induction of pulmonary fibrosis. 398

Because ET-1 was previously reported to be one of the substrates that could be 399 potentially degraded by endogenous NEP (neutral endopeptidase) (Abassi et al., 400 1992) , that pushed us to predict that enhancing NEP activity may become a 401

prerequisite to defeat COVID-19 ghost (El Tabaa and El Tabaa, 2020) . 402 endothelium (Li et al., 1995) as well as in many inflammatory cells including 405 neutrophils (Connelly et al., 1985) . In the airways, NEP has been found to be 406 expressed in the epithelium (Sont et al., 1997) , smooth muscle cells (Di Maria et 407 al., 1998) , and fibroblasts (Kletsas et al., 1998) . 408

NEP was also found to degrade the endogenous vasoactive peptides including atrial 409 natriuretic peptide (ANP). Thus, inhibiting NEP can prolong and potentiate their 410 natriuretic actions. That action pushed clinicians to use NEP inhibitors (e.g. 411

Sacubitril) in a combination with ACE inhibitors (e.g. valsartan) for lowering 412 blood pressure and treating heart failure (Bratsos, 2019). 413

Furthermore, a high cleaving affinity of NEP towards some potent inflammatory 414

such as bradykinins (BKs) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine 415

(fMLP) emphasized its potential role in alleviating the airway inflammatory 416

processes (Connelly et al., 1985; Shimamoto et al., 1994) . 417

Several studies ensured that destroying or down-regulating NEP may lead to 418 further pathophysiological changes. This involves an increase in vascular 419 permeability, recruitment, and activation of inflammatory cells, particularly 420 neutrophils. Neutrophil chemotaxis will lead to the release of neutrophil elastase 421 enzymes (e.g., cathepsin G), which may exert further destructive effects on airway 422

tissues, leading to worsening and progression of the disease (Borson, 1991) . 423

Therefore, reducing NEP activity either by cigarette smoking (Dusser et al., 1989) Along with this line, Rolipram, an investigative PDE4i, has also been examined, 440

since the increase in intracellular cAMP levels correlate directly with enhanced 441 NEP activity, which in turn may prolong and potentiate the cAMP-mediated short-442 term anti-inflammatory mechanism (Ayoub and Melzig, 2008; Graf et al., 1995) . 443

This outcome implies that another selective PDE4i, roflumilast, could exert 444 efficient anti-inflammatory effect via elevating cAMP level as well as NEP 445

activity. Accordingly, we predict that roflumilast may be one of the most useful 446 drugs that is expected to play a great role in treating COVID-19. However, until 447 this moment, no study has indicated the potential fundamental pathways 448

contributing to relying roflumilast on NEP activity. 449

Roflumilast is recorded to be a highly selective long-acting inhibitor of PDE4 451

isoenzyme, to which its use will be surely accompanied with an increase in the 452 level of intracellular cAMP (Rabe, 2011) . 453

Phosphodiesterase enzymes ( basophils (Halpin, 2008; van Schalkwyk et al., 2005) . 471

Notably, cAMP has a direct significant role in different inflammatory pathways via 472

inhibiting ROS generation and pro-inflammatory cytokine production, mainly 473

TNF-α and IL-6 (Isoni et al., 2009; Shames et al., 2001) . cAMP could also promote 474 the production of anti-inflammatory mediators such as IL-10 which was identified 475 as a "cytokine synthesis inhibitory factor", and acted as a principal regulator in the 476 JAK-STAT signaling pathway (Redford et al., 2011) . Therefore, elevating cAMP 477 level within the pulmonary tissue, vascular and inflammatory cells can provide an 478 efficient anti-inflammatory action (Li et al., 2018) . 479

On the other hand, it was found that the capacity of PDEs for cAMP hydrolysis is 480 greater than the maximum rate of its synthesis. Therefore, minute reduction in 481

PDEs activity can result in a high elevation in cAMP level with significant changes 482 in the activity of its dependent protein kinase (Halpin, 2008) . That notice pushed 483

scientists since 1970 to investigate the potential therapeutic importance of 484

inhibiting PDE4 activity (Weiss and Hait, 1977) .

Because of the involvement of cAMP signaling in the pathophysiology of many 487 inflammatory diseases, it has been proved that targeting PDE4 will resemble an 488 effective therapeutic strategy for different inflammatory conditions, such as chronic 489

obstructive pulmonary disease (COPD), asthma, atopic dermatitis (AD), 490

inflammatory bowel diseases (IBD), rheumatic arthritis (RA), lupus and 491

neuroinflammation (Li et al., 2018) . 492

Early, non-selective PDE inhibitors were discovered including theophylline and 493

doxofylline, but, because of their associated significant adverse effects, their use 494 Kumar et al., 2013) . 499

Since 2011, roflumilast has been approved by FDA as an anti-inflammatory drug 501 specifically designed for many respiratory disorders mainly COPD and asthma. By 502 time, roflumilast has been reported to exert different pharmacological activities, 503

Figure 2 and Table 1 (Li et al., 2018) . 504

Clinical trials have shown that that oral administration of roflumilast could 506 suppress airway inflammation and improve lung function of COPD patients. In 507 addition, it is documented to be effective in reducing the frequency of disease 508 exacerbations when given as add-on to inhaled therapy in patients with moderate or 509

severe COPD (Shen et al., 2018) . As regards asthmatic patients, roflumilast could 510 also significantly increase the Forced expiratory volume in 1 s (FEV 1 ) and 511

improved airway inflammation (Bateman et al., 2006) . inhibit neutrophil adhesion to endothelial cells (Sanz et al., 2007) . Additionally, 535

results from in vitro studies of human neutrophils showed that roflumilast could 536 prevent the release of neutrophil elastase, matrix metalloproteinase and 537 myeloperoxidase, inhibiting neutrophil function (Jones et al., 2005) A synergistic effect of roflumilast with other anti-inflammatory agents such as 539 corticosteroids or long-acting β2-agonists have been demonstrated 540 (Kawamatawong, 2017) . It was concluded that roflumilast-N-oxide (RNO), the 541 active metabolite of roflumilast, could enhance the anti-inflammatory effect of 542 dexamethasone in airway smooth muscle cells in vitro (Patel et al., 2017) . At the 543 same time, roflumilast was reported to reverse the corticosteroid-associated 544 insensitivity towards neutrophils in COPD patients (Milara et al., 2015b) . As well, 545

other study revealed the great value of roflumilast in restoring the glucocorticoid 546 sensitivity in glucocorticoid-resistant patients through blocking the downregulation 547 of glucocorticoid receptor (GRα) alpha, which was known to be responsible for 548 glucocorticoid resistance (Reddy et al., 2020) . 549

Neutrophils be the main pathological reaction of sepsis and the major cause for associated 576 multiple organ failure (Schouten et al., 2008) . Therefore, reducing inflammation 577 could be the key for treating sepsis. organ dysfunction through the above-referred anti-inflammatory and anti-583 thrombotic activities (Hattori et al., 2017) . 584

Because of the potential effect of anti-inflammatory treatment to mitigate airway 586 fibrotic remodeling, roflumilast might play anti-fibrotic role due to its well-known 587

anti-inflammatory action (Hatzelmann et al., 2010) . 588

Roflumilast was found to have the ability to prevent the progressive airway 589 fibrosis, as a result of antagonizing fibroblast activity, which could be mediated by 590 J o u r n a l P r e -p r o o f TGF-β1, an essential regulator of immune responses related to fibrosis (Togo et al., 591 2009 ). Anti-fibrotic profile of roflumilast could be also explained by its ability to 592 reduce the expression of upregulated NADPH oxidase 4 (NOX4) (Milara et al., 593 2015c), which was indicated to be critical for pulmonary fibrotic remodeling 594

( Amara et al., 2010) . 595

Within this regard, roflumilast could also normalize most of increased metabolic 596 changes like alterations in oxidative equilibrium, increased collagen, and protein 597 synthesis, resulting in decline in the fibrotic score. Simultaneously, reduced lung 598 tissue pH has been proposed as a risk factor for lung fibrosis development, which 599

was also reported to be corrected by roflumilast in bleomycin model of pulmonary 600

fibrosis (Milara et al., 2015a) . 601

Roflumilast can be safely administered as it is not associated with the parlous 603 induction of adverse effects involving seizures and cardiac arrhythmias; in 604 addition, its elimination is not significantly altered by several drug classes or even 605 by food and tobacco smoking (Gupta and O'Mahony, 2008) . 606

However, results from clinical trials demonstrated that the anti-inflammatory dose 607 of roflumilast in human was reported to be associated with a set of minor side 608 effects such as nausea, vomiting, diarrhea, weight loss and headache (Baye, 2012). 609

These effects appeared to be dose-dependent and transient, which in turn did not 610 need treatment discontinuation (van Schalkwyk et al., 2005) . As such, the newly 611 drug developing strategies are being directed to improve the therapeutic index of 612

Great efforts have been made to limit the gastrointestinal adverse reactions and to 614 provide a better benefit (Li et al., 2018) . Thus, for improving patient tolerability, a 615 study in the allergen-challenged Brown Norway rats, has been performed to evaluate the efficacy of inhaled roflumilast given either intratracheally or by nasal 617

inhalation. As concluded, the inhaled form showed a powerful effect on improving 618 the lung function (Chapman et al., 2007) , supporting the therapeutic importance of 619 using inhaled PDE4i against inflammatory lung diseases, which may be then more 620 efficacious with fewer adverse effects than its oral forms, however it is still under 621 clinical trial (Rhee and Kim, 2020) . For cardiovascular safety, roflumilast showed a lower rate of major adverse 645 cardiovascular events in treated COPD patients, supposing its potential 646 cardiovascular benefits (Rogliani et al., 2016; White et al., 2013) . 647

The rationale for selecting PDE4i for COVID-19 may be based on the previous 649 findings demonstrating that inhibiting the activity of PDE4 will suppress a myriad 650 of pro-inflammatory responses (Press and Banner, 2009) . Inhibiting PDE4 will 651 specifically prevent cAMP degradation, which in turn will decrease airway 652

inflammation via preventing the activation and recruitment of inflammatory cells, 653 specifically neutrophils as well as cytokines production (Barnette, 1999). That 654 observation drives scientists to attractively target PDE4 for treating COVID-19. 655

In addition to its anti-inflammatory, anti-coagulant and anti-diabetic roles, 656

roflumilast could be used safely in a combination with corticosteroids, 657 recommended to be used effectively against COVID-19 infection, by improving 658 their compromised anti-inflammatory properties and their resistance effect (Milara 659 et al., 2015b; Wang et al., 2016) . 660

At the same time, azithromycin, a macrolide antibiotic suggested for COVID-19 661 treatment, was documented to exhibit a lower affinity for cytochrome P-450A 662

(CYP) 3A4 CYP 3A4. Thus, azithromycin would poorly interact with roflumilast 663 because this cytochrome member resembles the main metabolic pathway for 664 roflumilast (Westphal, 2000) . 665

A little while ago, roflumilast was predicted to exert anti-viral effect similar to that 666 of lopinavir/ritonavir via binding very close to the middle pocket of SARS-CoV-2 667 3CLpro and thereby, interfering with its activity . Then, 668

roflumilast can deprive the virus from hydrolyzing the polyprotein into functional 669 J o u r n a l P r e -p r o o f proteins required for its replication, Figure 3 . However, the 670 preventive and therapeutic effectiveness of roflumilast against COVID-19 and its 671 pharmacological mechanisms have not been yet extensively studied. 672

One of the proposed NEP-dependent mechanisms for blocking the airway 674 inflammation is to cleave the neutrophil-released cathepsin G, that is documented 675 to convert both angiotensinogen and angiotensin I into angiotensin II, (Fig. 4 ) 676

( Meyer-Hoffert, 2009; Pham, 2006; Wintroub et al., 1984) . 677

In response to severe COVID-19 infection, ang II is reported to be continuously 678 generated to probably lead to the systemic cytokine storm (Xiong et al., 2020) . 679

Among the released cytokines, IL-6 will play a vital role in the progression of 680

numerous inflammatory reactions as well as endothelial dysfunction and platelet 681

activation (Funakoshi et al., 1999; Y. Liu et al., 2020) . Therefore, cleaving 682 cathepsin G by NEP with reducing associated Ang II formation may be a logical 683

commentary for the suppressed IL-6 expression detected following roflumilast 684 treatment (Feng et al., 2017) . 685

Postulating that IL-6 may be a key regulator of COVID-19 pathogenesis (T. Liu et 686 al., 2020) , decreasing its level by roflumilast will be of great importance. First, 687

roflumilast can stop IL-6-mediated intestinal, olfactory, and ocular inflammation 688 and consequently, inhibit the induction of anosmia, diarrhea, and conjunctivitis, 689

respectively. Second, roflumilast may suppress the endothelial activation and 690 inflammatory thrombocytosis prompted by IL-6 release. 691

As a result of the endothelial dysfunction, neutrophils trafficking has also been 692 implicated in the pathogenesis of COVID-19, since their activation and 693

accumulation are reported to be associated with tissue damage, exaggerated 694

inflammation and disordered tissue repair (Tay et al., 2020) . As such, NEP can 695 degrade the chemoattractant fMLP, which was known to be involved in neutrophil 696 J o u r n a l P r e -p r o o f chemotaxis. Hence, NEP may specifically prevent the recruitment of neutrophils 697 across the endothelial barrier from the blood circulation into the infected tissues 698 (Sato et al., 2013) . In particular, the potential role of roflumilast in inhibiting the 699 adhesion and transmigration of neutrophils and their subsequent inflammatory 700 sepsis may be attributable to increased NEP activity (Hui Li et al., 2020; Sanz et 701 al., 2007) . 702

Additionally, NEP was reported to effectively breakdown the endothelium-derived 703

ET-1; preventing the activation and aggregation of platelets as a result of 704

prohibiting the synthesis of PAF (Mustafa et al., 1995; Rao and White, 1982) , 705

which was previously demonstrated to be also suppressed by the action of PDE4i 706 (Tenor et al., 1996) . Accordingly, this observation may reflect the potential NEP-707 dependent anti-coagulant role of roflumilast against the thromboembolic events in 708 COVID-19; empowering it to restrain the development of PIC which is the initial 709 step for evolving stroke in COVID-19 patients (Avula et al., 2020) . 710

In line, it was also shown that COVID-19 patients may show pulmonary fibrosis, 711 from which NEP may protect lungs by stopping the ET-1-induced TGF-β1, 712

ensuring the concept that roflumilast may have the potential to attenuate the 713 fibroblast activities and thereby, the ability to function as anti-fibrotic agent via 714 blocking the fibrosis driven by TGF-β1 (Dunkern et al., 2007; Togo et al., 2009) . 715

Additionally, breaking ET-1 by NEP will prolong the anti-inflammatory effect of 716 roflumilast via maintaining the high cAMP level which is underscored to play an 717 important role in improving the immune system of highly risk COVID-19 groups 718 (Graf et al., 1995; Raker et al., 2016) . 719

Furthermore, enhancing NEP activity may explain the potential cardiovascular 720 benefits of roflumilast. During the airway inflammation, NEP itself may act 721

indirectly to decrease the blood pressure via degrading cathepsin G, that 722

consequently inhibits the formation of angiotensin II. Decreasing angiotensin II 723 J o u r n a l P r e -p r o o f level will direct the pulmonary renin angiotensinogen system (RAS) for generating 724 more angiotensin (1-7) which, via Mas receptor, can induce natriuresis/diuresis 725 (Shah et al., 2010) and trigger the endothelial nitric oxide synthase (eNOS) to 726 stimulate nitric oxide (NO) release, promoting blood vessel relaxation (Fraga-Silva 727 et al., 2008; Patel and Schultz, 2013) . 728

Accordingly, we recommend that future clinical efforts should be driven towards 729

ensuring the NEP-mediated pharmacotherapeutic mechanisms of roflumilast 730

proposed for counteracting COVID-19 infection. 731

Reducing the patient's risk of COVID-19 progression is assumed to be biologically 733 roflumilast has been reported to be the most selective and effective drug submitted 743

for treating many neutrophils-mediated airway inflammatory disorders. 744

Furthermore, roflumilast has been recently reported to behave as a potential 745

inhibitor of 3CLpro, which is a proteolytic enzyme required for viral replication 746

within the host cells. 747

Considering COVID-19 treatment, roflumilast may also have additive advantages 748

to the concurrent protocol, since it had been reported to be used safely in 749 combination with either corticosteroids, azithromycin and recommended vitamins 750 Binding of Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) with angiotensin converting enzyme-2 (ACE-2) may downregulate it; inhibiting the ACE-2 / angiotensin (1-7) / Mas receptor axis and subsequently, activating the ACE / angiotensin (Ang) II / angiotensin II type 1 (AT1) receptor axis on the other side, that may lead to an increase in the level of angiotensin II. Angiotensin II could promote the release of multiple inflammatory cytokines particularly, interleukin-6 (IL-6), which could play a crucial role in inducing intestinal, olfactory and ocular inflammation, in addition to disrupting the function of endothelial cells. SARS-CoV-2 itself can also induce endothelial dysfunction; resulting in platelet activation and aggregation. Moreover, endothelial dysfunction may trigger more inflammation through trafficking more neutrophils with subsequent inflammatory sepsis. Simultaneously, secreting endothelin-1 (ET-1) as a result of endothelial dysfunction could stimulate the fibrotic consequences via persuading the release of transforming growth factor-β1 (TGF-β1), developing pulmonary fibrosis. In addition, ET-1 could also exaggerate the inflammation via decreasing the level of cyclic adenosine monophosphate (cAMP).

J o u r n a l P r e -p r o o f For SARS-CoV-2 to be replicated inside the cytoplasmic membranes, its viral polyprotein chains should be firstly hydrolyzed into functional proteins either by papain like protease, 3C-like protease (3CLpro), RNA-dependent RNA polymerase (RdRp), helicase, or endoribonuclease. Roflumilast is predicted to specifically bind very close to the middle pocket of SARS-CoV-2 3CLprotease and thereby, may interfere with its proteolytic activity; preventing viral replication.

J o u r n a l P r e -p r o o f Being a highly selective phosphodiesterase-4 inhibitor (PDE4i), roflumilast acts by enhancing cyclic adenosine monophosphate (cAMP) level, which in turn will increase neprilysin (NEP) activity. Once NEP is activated, it can cleave the neutrophil-released cathepsin G and consequently, prevent angiotensin II formation. That will be accompanied by a decrease in the level of released interleukin-6 (IL-6) and its associated olfactory, intestinal and ocular inflammatory reactions as well as IL-6 -mediated endothelial dysfunction and platelet activation. Moreover, NEP can degrade the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), prohibiting neutrophil recruitment and chemotaxis and hence, their subsequent inflammatory sepsis. Therefore, NEP can participate in reducing the induction of endothelial dysfunction and platelet activation. Additionally, NEP can breakdown endothelin-1 (ET-1); preventing the synthesis of platelet activating factor (PAF) and accordingly, the activation and aggregation of platelets as well as pulmonary intravascular coagulopathy (PIC) development. Degrading ET-1 can also inhibit pulmonary fibrosis via blocking the ET-1-induced transforming growth factor-β1 (TGF-β1), and at the same time, maintain the high level of cAMP which may contribute for long-term anti-inflammatory effect of roflumilast.

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