Assessment the Activity Value of Isotretinoin (13- Cis-Retinoic Acid ) in the Treatment of COVID-19 ( Isotretinoin in Treatment of COVID-19) (Randomized)
Assessment the Activity Value of Isotretinoin (13- Cis-Retinoic Acid ) in the Treatment of COVID-19 ( Isotretinoin in Treatment of COVID-19) (Randomized)
Assessment the Activity Value of Isotretinoin (13- Cis-Retinoic Acid ) in the Treatment of
 COVID-19 ( Isotretinoin in Treatment of COVID-19)

 Mahmoud ELkazzaz(1),Tamer Haydara(2), Mohamed Abdelaal(3), Abedelaziz Elsayed(4) ,Yousry
 Abo-amer(5), Hesham Attia(6), Quan Liu(7)

 1. Department of chemistry and biochemistry, Faculty of Science, Damietta University,
 Egypt.

 2. Department of Internal Medicine, Faculty of Medicine, Kafrelsheikh University, Egypt

 3. Department of Cardiothoracic Surgery, Faculty of Medicine, Kafrelsheikh University,
 Egypt

 4. Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tanta University,
 Egypt.

 5. Hepatology,Gastroenterology and Infectious Diseases Department, Mahala Hepatology
 Teaching Hospital, Egypt

 6. Department of Immunology and Parasitology, Faculty of Science, Cairo University, Egypt.

 7. School of Life Sciences and Engineering, Foshan University, Foshan, Guangdong Province;
 Laboratory of Emerging Infectious Disease, Institute of Translational Medicine, The
 First Hospital of Jilin University, Changchun, China.

 -  Very Important Note

 -  This clinical study is the first clinical study in literature (submitted on 20
 April, 2020) which demonstrated that Isotretinoin will provide complete
 protection against covid-19

 Abstract

 The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
 has infected over 100 million people causing over 2.4 million deaths over the world, and it
 is still expanding. There is an urgent need for targeted and effective COVID-19 treatments
 which has put great pressure on researchers across the world for developing effective drugs.
 New drug discovery could require several years with no guarantee but repurposing established
 drugs may be useful to treat the infection. In this clinical study we attempt to demonstrate
 Isotretinoin could be an effective and promising treatment for SARS-CoV-2 based on the
 intracellular mechanism of SARS-CoV-2 transmission and consequences caused. Isotretinoin
 could strongly inhibit both inflammation and viral entry in severe acute respiratory syndrome
 coronavirus 2 infection via decreasing the overproduction of early response proinflammatory
 cytokines (interleukin-6 ) which are over expressed in COVID-19 and contributed to disease
 progression, poor outcomes, vascular hyper permeability and multiorgan failure in patients
 infected with COVID-19. It could also block the entry of COVID-19 by inhibiting androgenic
 factors that induce serine 2 transmembrane protease (TMPRSS2) expressions.. In addition to
 inhibiting of Angiotensin-converting enzyme-2 (ACE2), Angiotensin T1 protein and Angiotensin
 II-mediated intracellular calcium release pathway which is responsible for COVID-19 cell
 fusion and entry, ACE2-expressing cells are prone to SARS-CoV-2 infection as ACE2 receptor
 facilitates cellular viral entry and invasion. Moreover, isotretinoin is a potential
 repressor and inhibitor of papain-like protease (PLpro), which is a lethal protein expressed
 by COVID-19 genes and is an enzyme of dubiquitination which facilitates virus replication in
 patients with COVID-19.The genome of Middle East Respiratory Syndrome Coronavirus is
 recognized by melanoma differentiation-associated protein-5 (MDA5), retinoic acid inducible
 gene-1 (RIG-1) and endosomal toll-like receptor 3 (TLR3) as pathogen-associated molecular
 patterns. This recognition resulted in the formation of type-1 interferon (IFN1). As an
 evasion mechanism, virus synthesize proteins that hinder the production IFN1 in the pathway.
 13-cis retinoic acid induced significant upregulation of toll-like receptor 3 (TLR3),
 mitochondrial antiviral-signaling protein (MAVS) and (RIG-I) and IFN regulatory factor 1
 expression in a time-dependent. Furthermore, 13 cis Retinoic Acid (13 cis RA) could be an
 effective and promising treatment for SARS-CoV-2 owing to its ability to increase CD4 cells
 and induce mucosal IgA antibodies that are less prone to Antibody Dependent Enhancement
 process (ADE) and responsible for passive mucosal immunity in the respiratory tract. ADE is a
 phenomenon in which antiviral antibodies facilitate viral infection of target immune cells
 and, in some cases, make a second infection worse, such as dengue fever (dengue virus), By
 inducing IgA antibodies, 13 cis retinoic acid enhances mucosal immunity and is known to be a
 potent IgA isotype.13 Cis retinoic acid induced significant upregulation of toll-like
 receptor 3 an immune boosting action that may result in an immune response to dsRNA
 intermediate leading to the production of type I IFNs which is important to enhance the
 release of antiviral proteins for the protection of uninfected cells. Isotretinoin therapy
 has furthermore proven anti-platelet and fibrinolytic activities which may protect patients
 infected with covid-19 from widespread blood clots. From this point, we suggest that
 isotretinon will be the Immunity passport" in the context of COVID-19
The study is a randomized interventional comparative Phase III trial. 10000 adult male and
 female patients with positive COVID-19 diagnosis and fulfilling the below outlined inclusion
 criteria will be enrolled into the study.

 Isotretinoin(13cis RA) may be able to inhibit COVID 2019 entry via down regulation of ACE2 ,
 AT1 protein and Ang II-mediated intracellular calcium release rather than inhibition of
 interleukin-6 (IL-6) and this is discussed as follow :

 The COVID-19 pandemic caused by SARS-COV-2 has infected over 2,000,000 people causing over
 150,000 deaths. A key host cellular protein required for the virus entry is
 angiotensin-converting enzyme 2 (ACE2) whose expression has been demonstrated in many tissues
 including alveolar epithelial type II cells in lungs, oral mucosa ,intestine, heart, kidney,
 endothelium and skin. ACE2-expressing cells can act as home cells and are prone to SARS-CoV-2
 infection as ACE2 receptor facilitates cellular viral entry and replication. A study
 demonestrated that patients with hypertension and diabetes mellitus may be at higher risk of
 SARS-CoV-2 infection, as these patients are often treated with ACE inhibitors (ACEIs) or
 angiotensin II type-I receptor blockers (ARBs), which have been previously suggested to
 increase ACE2 expression. In another study by Sinha et al who analyzed a publicly available
 Connectivity Map (CMAP) dataset of pre/post transcriptomic profiles for drug treatment in
 cell lines for over 20,000 small molecules, isotretinoin was the strongest down-regulator of
 ACE 2 receptors. On the other hand, they found 6 drugs in CMAP that are currently being
 investigated in clinical trials for treating COVID-19 (chloroquine, thalidomide,
 methylprednisolone, losartan, lopinavir and ritonavir, from clinicaltrials.gov), none of
 which was found to significantly alter ACE2 expression (P>0.1) Moreover, another study
 demonstrated that isotretinoin is a potential papain like protease (PLpro) inhibitor which is
 a protein encoded by SARS-CoV-2 genes and considered one of the proteins that should be
 targeted in COVID-19 treatment by performing target-based virtual ligand screening.

 Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) and
 SARS-CoV FP FP have shown that calcium (Ca2+) plays an important role for fusogenic activity
 via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D)
 residuesdemonstrated that intracellular Ca2+ enhances MERS-CoV WT PPs infection by
 approximately two-fold and that E891 is a crucial residue for Ca2+interaction. Electron spin
 resonance revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP
 fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry titration showed that
 MERS-CoV FP binds one Ca2+, as opposed to SARS-CoV FP which binds to two Ca2+ ion.

 Angiotensin II increases the intracellular calcium activity in podocytes of the intact
 glomerulus. The L-type Ca2+ channel blocker nicardipine did not influence the Ang II-mediated
 [Ca2+] increase and it has been postulated that SARS-CoV-2 binding to ACE2 may attenuate
 residual ACE2 activity, skewing the ACE/ACE2 balance to a state of heightened angiotensin II
 activity leading to pulmonary vasoconstriction and inflammatory and oxidative organ damage,
 which increases the risk for acute lung injury (ALI). AngII via AT1 receptors upregulates
 many proinflammatory genes, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular
 adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6).30 but 13cis RA specifically
 down-regulated the AT1 protein in a dose- and time-dependent manner. Down-regulation of the
 AT1 expression leads to reduced AngII-mediated intracellular calcium release Similarly with
 receptor down-regulation, Treatment with 13cRA resulted in a significant reduction in AT1
 mRNA .13cRA has a glucose- and RAR/RXR independent mechanism for transcriptional inhibition
 of AT1,

 Isotretinoin(13cis RA) and ATRA may be able to inhibit COVID 2019 infection via inducing the
 antiviral immunity and this is discussed as follow :

 Since effective immune response against viral infections depends on the activation of
 cytotoxic T cells that can clear infection by killing virus-infected cells , boosting the
 numbers and function of T cells in COVID-19 patients is critical for successful recovery. A
 recent study reported that the 82.1% of COVID-19 cases displayed low circulating lymphocyte
 counts. A CoV infects macrophages, and then macrophages present CoV antigens to T cells. This
 process leads to T cell activation and differentiation, including the production of cytokines
 associated with the different T cell subsets (Th17), followed by a massive release of
 cytokines for immune response amplification. The continued production of these mediators due
 to viral persistence has a negative effect on NK, and CD8 T cell activation. However, CD8 T
 cells produce very effective mediators to clear CoV However, the factors which might cause
 the reduction in count, and the activation status of T cells in COVID-19 patients, remain
 uninvestigated.

 Recent study of 522 COVID patients and 40 healthy controls from two hospitals in Wuhan, China
 demonstrated that T cell numbers are negatively correlated to serum IL-6, IL-10 and TNF-α
 concentration, with patients in decline period showing reduced IL-6, IL-10 and TNF-α
 concentrations and restored T cell counts. T cells from COVID-19 patients have significantly
 higher levels of the exhausted marker programmed cell death protein(PD-1) as compared to
 health controls. Moreover, increasing PD-1 and Tim-3 expression on T cells could be seen as
 patients progressed from prodromal to overtly symptomatic stages, further indicative of T
 cell exhaustion. T cell exhaustion is a progressive loss of effector function due to
 prolonged antigen stimulation, characteristic of chronic infections. Dendritic cell
 inhibition is connected to exhaustion of CD8+ T cell polyfunctionality during chronic
 hepatitis C virus infection. CD8 T cells produce very effective mediators to clear nCoV2019.

 Dendritic cells (DCs) play a key role in innate immune and adaptive immune responses. As the
 strongest antigen presenting cells in the organism, they effectively stimulate the activation
 of T lymphocytes and B lymphocytes, thus combining innate and adaptive immunity. Immature DCs
 have strong migration ability, and mature DCs can effectively activate T cells in the central
 link of startup, regulation, and maintenance of immune responses. Thus, once the maturation
 process of DCs is blocked, it directly affects the initiation of subsequent adaptive immune
 response. MERS-CoV-2 is able to affect human dendritic cells and macrophages in-vitro
 .Productive replication of Middle East respiratory syndrome coronavirus in monocyte-derived
 dendritic cells modulates innate immune response.

 Another study proposed that C -C chemokine receptor type 4 (CCR4) contributes to T cell lung
 homing imprinting. It was found that lung DCs induce the expression of CCR4 on T cells. Lung
 DCs-activated T cells traffic more efficiently into the lung and protect against influenza
 more effectively compared with T cells activated by DCs from other tissues. Lim and
 colleagues suggested that CXCR4 plays a role in CD8+ T cell migration to airway tissues

 Dendritic cell inhibition is connected to exhaustion of CD8+ T cell polyfunctionality during
 chronic hepatitis C virus infection. CD8 T cells produce very effective mediators to clear
 nCoV2019

 Presence of RA in different tissues is very imprtant for immune induction and fighting viral
 infection, for example, RA is present at high concentrations in the small intestine due to
 metabolizing dietary vitamin A by gut epithelial cells. In this local environment, RA
 activates and primes dendritic cells (DCs) to become CD103+ DCs that produce RA.3, 4CD103+
 DCs are migratory cells that activate naive T cells in mesenteric lymph nodes to become
 effector T cells that contribute to both intestinal homeostasis and immunity.and also RA is
 an important signal that induces IgA-producing B cells. The gut homing T cells and B cells
 play essential roles in protecting the digestive tract from pathogens.

 Retinoic acid (atRA) can inhibit the spontaneous apoptosis of activated human T lymphocytes
 in vitro. 13-cis RA activates Th2 cytokine production Enhanced circulating dendritic cell
 numbers.

 So, according to this previous studies the principal investigator suggests that T cells
 lymphopenia and exhaustion may be resulted by Dendritic cells (DCs) infection and inhibition
 by MERS-CoV-2.

 Retinoic acid has profound effects on cellular proliferation and differentiation. Moreover,
 it has been reported that ATRA exhibits both anti-inflammatory and immunoregulatory effects.
 Recent studies have shown that FOXP3 expression and the immune function of Regulatory T cells
 (Tregs ) can be enhanced by ATRA in in both patients and mouse models. .13Retinoic acid (RA)
 is produced by a number of cell types, including macrophages and dendritic cells, which
 express retinal dehydrogenases that convert vitamin A to its main biologically active
 metabolite, all-trans RA. All-trans RA binds to its nuclear retinoic acid receptors that are
 expressed in lymphoid cells and act as transcription factors to regulate cell homing and
 differentiation. RA production by CD103+ dendritic cells and alveolar macrophages functions
 with TGF-b to promote conversion of naive T cells into Foxp3+ regulatory T cells and,
 Thereby, maintain mucosal tolerance

 So, principal investigator expects high inducing of Dendritic cells (DCs) by retinoic acid
 treatment which will lead to T cells activation and migration with less exhaustion
 phenomenon.

 According to this protocol treatment with retinoic acid will induce FOXP3 and
 CD8+,CD4+,CD25+,FOXP3+ Tregs which their absence during acute infection alters the ability of
 the host to limit tissue damage and inducesT cells which were dramatically reduced in
 COVID-19 patients to exert its antiviral and anti-inflammatory effect protecting lung cells
 and neural cells from the inflammatory and the destructive effect of IL-6, IL-1, and TNF-α
 which are induced and highly expressed in COVID 2019 patients.

 Researchers from Wenzhou, China looked at clinical laboratory features including lipid levels
 of patients with COVID 19. They found dramatic reductions in the cholesterol levels of
 patients infected with COVID 19, compared with healthy controls .The study provides data to
 suggest that cholesterol levels decline quite rapidly during the early stages of infection
 and increase as the patient starts to recover. Therefore, indicating that cholesterol may
 have an important role to play in defending the body against such infections According to our
 protocol depending on previous studies the principal investigator demonstrated that there is
 a strong relation between immune system and cholesterol levels .When the cholesterol level is
 low specifically in the case of viral infection like COVID 2019 infection the immune system
 is impaired and the antiviral immune cells will be declined and inhibited :-

 Cellular cholesterol is a component of the plasma membrane and is also essential in cell
 proliferation. Regulation of intracellular cholesterol levels has been proposed as a
 mechanism to regulate T cell and macrophages proliferation. Intracellular cholesterol level
 is regulated by two competing pathways, cholesterol uptake and efflux, and ABCA1 plays a
 major role in the cholesterol efflux pathway. The ATRA induces ABCA1 expression and
 ABCA1-dependent cholesterol efflux in activated primary human CD4+ T cells implying that RA
 could affect T cell functions by regulating the cellular cholesterol levels.

 ATRA upregulates ABCA1 expression only in activated CD4+ T cells, indicating that induction
 of ABCA1 by ATRA and 13 cis Retinoic Acid may play an important role in immune response.

 Retinoic acid and liver X receptor agonist synergistically inhibit HIV infection in CD4+ T
 cells by up-regulating ABCA1-mediated cholesterol efflux.

 So, the principal investigator expects that retinoic acid treatment will highly induce T
 cells and anti-inflammatory regulatory T cells, T helper via cholesterol efflux and inducing
 ATP-binding cassette transporter (ABCA1) and protect lung and neural cells and inhibit COVID
 2019 infection.

 The genome of Middle East Respiratory Syndrome Coronavirus is recognized by melanoma
 differentiation-associated protein-5 (MDA5), retinoic acid inducible gene-1 (RIG-1) and
 endosomal toll-like receptor 3 (TLR3) as pathogen-associated molecular patterns. This
 recognition resulted in the formation of type-1 interferon (IFN1). As an evasion mechanism,
 virus synthesize proteins that hinder the production IFN1 in the pathway.

 RA also acts directly on macrophages at both mucosal sites and other immunological sites.
 AtRA modulates peritoneal macrophage activation by endotoxin and IFN-γ by suppressing TNF
 production and nitric oxide (NO) synthesis .In addition, at RA inhibits the expression of
 PGE2 and COX-2 and the release of TNF, which are induced by bacterial lipopolysaccharide
 (LPS) in murine peritoneal macrophages .

 A study reported recently that substance (ATRA) have preventive effects on pulmonary fibrosis
 by inhibiting IL-6-dependent proliferation and TGF-β1-dependent trans differentiation of lung
 fibroblasts. Also, another studies demonstrated that 13-cis-retinoic acid and other retinoid
 analogs inhibit IL-1-induced IL-6 production and that this effect is analog-specific and, at
 least partially, transcriptionally mediated. This effect was dose-dependent with an IC50 of
 10(-7) M RA and significant inhibition being noted with doses of RA as low as 10(-8) M. IL-10
 production was inhibited by ATRA administration.

 A study demonstrated that TLR3(-/-), TLR4(-/-), and TRAM(-/-) mice are more susceptible to
 SARS-CoV than wild-type mice but experience only transient weight loss with no mortality in
 response to infection. In contrast, mice deficient in the TLR3/TLR4 adaptor TRIF are highly
 susceptible to SARS-CoV infection, showing increased weight loss, mortality, reduced lung
 function, increased lung pathology, and higher viral titers . New studies showed that the
 high level of IFN-α/β produced from the TLR3-IRF3/IRF7 pathway and IFN-β is the reason for
 inhibiting DENV replication. 13Cis retinoic Acid induced significant upregulation of
 toll-like receptor 3 (TLR3) resulting in an immune response to dsRNA intermediate which can
 be partially generated during CoV-2 replicationTLR3 sensitized by dsRNA and cascades of
 signaling pathways (IRFs and NFκB activation, respectively) are activated to produce type I
 IFNs. The production of type I IFNs is important to enhance the release of antiviral proteins
 for the protection of uninfected cells. Sometimes, accessory proteins of CoV can interfere
 with TLR3 signaling and bind the dsRNA of CoV during replication to prevent TLR3 activation
 and evade the immune response. 13Cis retinoic Acid induced significant upregulation of
 toll-like receptor 3 (TLR3) , mitochondrial antiviral-signaling protein (MAVS) and
 retinoid-induced gene I (RIG-I) and IFN regulatory factor 1 expression in a time-dependent.

 In further research, Tsai .and Chen showed the high level of IFN-α/β produced from the
 TLR3-IRF3/IRF7 pathway and IFN-β is the reason for inhibiting DENV replication. In HUH-7
 cells, huTLR3 can recognize DENV-1 and induce the expression of IFN-β, which can enhance the
 expression of huTLR3 on the contrary . TLR3 also induces type I IFN during WNV.

 Doctors treating the sickest Covid-19 patients have zeroed in on a new phenomenon: Some
 people have developed widespread blood clots, their lungs peppered with tiny blockages that
 prevent oxygen from pumping into the bloodstream and body.As with so much else about the
 Covid-19 response, health experts are learning about the symptom on the fly. Blood clots are
 common in patients who are immobilized, but they seem to be smaller and cause far more severe
 damage in some Covid-19 patients. Doctors have said they see patients with blood clots
 forming not only in their lungs, but also in blood vessels. Autopsies have also revealed
 blood clots in kidneys and other organs, which some experts say suggests an overwhelming
 immune system response to the virus that inflicts harm on the body

 Retinoic acid, is known to possess in vivo anti-inflammatory, anti-platelet and fibrinolytic
 activities. A study investigated the in vitro thrombin and platelet aggregation inhibitory
 activities of retinoic acid and retinaldehyde.Retinoic acid, retinaldehyde and retinol
 exhibited potent inhibition of thrombin, with IC50 values of 67μg/ml, 74μg/ml and 152μg/ml,
 respectively for the inhibition of thrombin (Sigma); and 49μg/ml, 74μg/ml and 178μg/ml,
 respectively for the inhibition of thrombin (plasma). Amongst vitamin A and its derivatives,
 retinoic acid showed the highest inhibition of both the forms of thrombin.

 Isotretinoin(13cis RA) may be able to inhibit COVID 2019 infection via reversIing the
 androgenic induction and activation effect of (DHT) on TMPRSS2 expression and helps to
 prevent cleaving and activating both the spike protein (S) of COVID 2019 and the viral
 receptor, and this is discussed as follow :

 TMPRSS2 is both the most frequently altered gene in primary prostate cancer and a critical
 factor enabling cellular infection by coronaviruses, including SARS-CoV-2. The modulation of
 its expression by steroids could contribute to the male predominance of severe infections and
 given that TMPRSS2 has no known indispensable functions, and inhibitors are available, it is
 an appealing target for prevention or treatment of respiratory viral infections

 TMPRSS2, a key regulator in prostate cancerTMPRSS2 was first identified in prostate cancer
 shortly after the gene had been originally cloned. Prostate cancer cell lines strongly
 upregulated TMPRSS2 expression in response to androgens . TMPRSS2 is expressed on the luminal
 side of the prostate epithelium, and its expression is increased in prostate cancer tissue
 compared to non-cancerous prostate tissue. Notably, the TMPRSS2 gene is a partner in one of
 the most common gene fusion eventsin solid tumors: somatic gene rearrangements involving
 TMPRSS2 witha member of the ETS family of oncogenic transcription factors, most commonly ERG.
 This fusion occurs in approximately 50% of primary prostate cancers among men of European
 ancestry.While ERG is not normally regulated by androgen, the gene fusion juxtaposes the
 androgen receptor regulatory elements of TMPRSS2 with the ERG gene. The ERG gene is
 consequently controlled by androgen receptor signaling and expressed highly in prostate
 cancers harboring the TMPRSS2: ERG fusion. Intriguingly, the prevalence of the TMPRSS2: ERG
 fusion is lower in prostate tumors of both black and Asian men. The relevance of this to the
 current COVID-19 pandemic is unclear.TMPRSS2: ERG fusion- cancers also have a distinct set of
 risk factors related to hormonal signaling. For example, men with higher genetically
 determined transcriptional activity of the androgen receptor have a higher risk of TMPRSS2:
 ERG fusion-positive prostate cancer but not of fusion-negative prostate cancer

 TMPRSS2 is an androgen receptor signaling target gene and an androgen-regulated cell-surface
 serine protease expressed predominantly in prostate and lung epithelial cell TMPRSS2 is
 normally expressed several fold higher in the prostate relative to any other human tissue,
 though the normal physiological function(s) remains unknown. Importantly, unlike other TTSPs,
 TMPRSS2 transcription is regulated by androgenic ligands and the androgen receptor (AR).
 There is a positive correlation between AR and TMPRSS2 in microdissected primary tumor
 epithelium (r2 = 0.39 ; p <0.001).

 Dihydrotestosterone (DHT) significantly and dramatically induced the expression of TMPRSS2
 protein with two molecular masses of 60 (full-length) and 38 kDa (N-terminus) in a dose
 responsive manner

 Data from Chinese outbreak show death rates for men almost 50 per cent higher than for women
 show that Early research from China suggests women and children are less likely to die than
 men if they catch the coronavirus. Death rates for Covid-19, the disease those infected with
 the coronavirus develop, are low for everyone: only 2.4 per cent of the 44,672 people in the
 Chinese study died. But although roughly even numbers of men and women catch the disease, men
 are more likely to develop such a serious case of Covid-19 they die.

 More than 70 percent of Italy's coronavirus deaths have been among men but scientists there
 admit they are mystified by the gender gap. At least 3,400 people in Italy have died of the
 devastating disease - it yesterday announced it had a higher death Toll than China - but less
 than 1,000 of them have been women. Men are also more likely to pick up the infection in the
 first place and account for 60 percent of confirmed cases, according to Italy's public health
 research agency. An earlier analysis found that 80 per cent of the deaths were in men and
 just 20 per cent were in women - but the gap has narrowed over time

 According to this data the principal investigator thinks that there is a strong relation
 between high mortality in males and androgenic effect specifically the effect of DHT on
 TMPRSS2 protein which is used by covid 2019 in cell invasion and entry and depending on this
 data related to six hormones specifically (DHT) , The investigator was able to discover whey
 women and children less likely to die from illness than men. So, the investigator divided
 infected patients according to their six hormone because TMPRSS2 is an androgen-regulated
 cell-surface serine protease expressed predominantly in prostate and lung epithelial cell
 TMPRSS2.

 Androgen(DHT) potential effect on TMPRSS2 expression in children is less than its effect in
 females and males followed by viral severity and vigrousity in men compared with children and
 women. .

 Androgen (DHT) potential effect on TMPRSS2 expression in females is less than in males
 followed by viral severity and vigrousity in men compared with females .

 So, the principal investigator thinks that when some researchers investigated the role of sex
 steroids in SARS-CoV pathogenesis by comparing gonadectomized and control counterparts after
 infection. Gonadectomy or treatment with flutamide, a non-steroidal anti-androgen did not
 affect morbidity and mortality in male mice following lethal MA15 infection, They may be were
 wrong in their conclusions in suggesting that androgens do not play a role in SARS-CoV
 pathogenesis because Gonadectomy or treatment with flutamide will not completely affect or
 inhibit DHT and its derivatives(5α-Androstan-3α,17β-Diol) concentration in tissues and blood
 because after inhibiting testosterone with flutamide . the pathway of DHT formation will be
 activated to compensate the inhibited testosterone levels so the TMPRSS2 expression will be
 significantly induced by DHT and the treated animals will not be affected in case of
 flutamide treatment but in case of Gonadectomy the expression of TMPRSS2 will be decreased by
 DHT inhibition only if along time has passed on Gonadectomy in order to make sure that DHT
 and its derivatives completely declined in levels that will not allow it to affect on
 expression of TMPRSS2 and in female mice after blocking estrogen receptors it died because
 increasing formation of androgenic hormones.

 A study demonstrated that 13- cis -Retinoic acid competitively and reversibly inhibits
 Dihydrotestosterone So, the principal investigator expects a significant modulation of
 TMPRSS2 expression after treating with 13- cis -Retinoic acid via temporary preventing the
 effect of dihydrotestosterone(DHT) on TMPRSS2 promoting and expression. And the type II
 transmembrane serine proteases TMPRSS2 which can cleave and activate the spike protein (S) of
 the severe acute respiratory syndrome coronavirus (SARS-CoV) for membrane fusion. In
 addition, these proteases cleave the viral receptor, the carboxypeptidase
 angiotensin-converting enzyme 2 (ACE2), and it was proposed that ACE2 cleavage augments viral
 infectivity.

 A study demonstrated that COVID-19 reduces testosterone levels in men by altering the
 functioning of the gonads. So could the increased severity of the disease in men be due to
 lowered testosterone. But according to the principal investigator explanation COVID-19
 reduces testosterone levels because there is a dramatic reductions in the cholesterol levels
 of patients infected with COVID 19, compared with healthy controls . Cholesterol levels
 decline quite rapidly during the early stages of infection and increase as the patient starts
 to recover.Therefore, indicating that cholesterol may have an important role to play in
 defending the body against such infections and depending on the principal investigator
 explanation, Testosterone is synthesized starting from cholesterol through a
 well-characterized steroid biosynthetic pathway involving the sequential action of multiple
 enzymes So, when cholesterol levels are decreased, this decrease will followed by decreasing
 in testosterone level and according to this explanation testosterone therapy in COVID 2019 is
 not recommended but temporary inhibitor of DHT is recommended such as Isotretinoin because
 this treatment by testosterone will inhibit cholesterol synthesis by feedback inhibition and
 decrease cholesterol uptake by Leydig cells in testis and this also will lead to over
 increase in DHT lvels and its derivatives in different tissues, which will induce TMPRSS2.
 because DHT is a potent activator of TMPRSS2 and this will be followed by processing and
 activation of COVID2019 spike protein to bined its ACE2 receptors in lung and kidney leading
 to their damage specifically in testis because it contains high levels of proteases and ACE2.
 Serine proteases are emerging as important contributors to the production, maturation, and
 functional competence of spermatozoa.

 Depending on this data and according to this protocol, The principal investigator expects and
 suggests that retinoic acid is specific modulator of androgens specifically DHT not
 testosterone and could reverse the androgenic induction and activation effect of (DHT) on
 TMPRSS2 expression and prevent cleaving and activating both the spike protein (S) of COVID
 2019 and the viral receptor, the carboxypeptidase angiotensin-converting enzyme 2 (ACE2).
 (All ideas and michanisms specifically role of Androgen in TMPRSS2 activation in COVID-19:
 Serendipity or opportunity for intervention and the inhibition of ACE2 ,AT1 protein and Ang
 II-mediated intracellular calcium release pathway which is responsible for SARS-CoV-2 cell
 fusion and entry included in the research protocol were submitted to Academy of scientific
 research and technology - Egypt- on 1 April , 2020. in Call no. 2/2019/ASRT- Ideation Fund)

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 ___________________________________________________________________________

 Promising features of COVID 2019 treatment according Principal Investigator Protocol:

 1. This medication have the feature of Aerosolized Drug Delivery to increase its efficacy
 beside Oral administration, Which makes it distinct from other medication in which
 should dose be only given orally. A study demonstrated that treating with 13 cis
 retinoic acid aerosolized via inhalation rout did not cause any damage in lung cells.
 Repeated high doses of 13 cis retinoic by inhalation resulted in moderate loss of body
 weight, but microscopic investigation of ten tissues including lung and oesophagus did
 not detect any significant aerosol-induced damage. The results suggest that
 administration of isotretinoin via powder aerosol inhalation is probably superior to its
 application via the oral route in terms of achieving efficacious drug concentrations in
 the lung.

 2. Inhaled isotretinoin might provide sufficient drug to the target cells for efficacy
 while avoiding systemic toxicity.

 3. A study demonstrated that 13 cis retinoic is used in treating Emphysema (emphysema is a
 lung condition that causes shortness of breath)

 4. ATRA has been reported to induce formation of new alveoli and returns elastic recoil in
 the lung to approximately normal values in animal models of emphysema.

 5. Strong expectation of complete COVID 2019 blockade from cell entry and infection
 depending on strong ethics, researches and references.

 6. Availability of our compounds.

 7. Ease of application.

 8. Expectation of COVID 2019 treating of by more than one distinct mechanism.

 9. Expectation of High induction of anti- inflammatory T cells and significant inhibition
 of IL-6 at low concentrations.

 10. Controlling Accompanying cytokine storm.

 11. No interactions with Egyptian protocol drugs were found.
Inclusion Criteria:

 Adult SARI patients with 2019-ncov infection confirmed by PCR; Absolute value of
 lymphocytes < 0. 6x 109/L; Severe respiratory failure within 48 hours and requires
 admission to ICU. (severe respiratory failure was defined as PaO2/FiO2 < 200 mmHg and was
 supported by positive pressure mechanical ventilation (including non-invasive and invasive
 mechanical ventilation, PEEP>=5cmH2O))

 Exclusion Criteria:

 Age < 18 Pregnant Allergic to experimental drugs and patients have the following
 conditions:

 1. Hypercholesterolemia

 2. Hypertriglyceridemia

 3. Liver disease

 4. Renal disease

 5. Sjögren syndrome

 6. Pregnancy

 7. Lactation

 8. Depressive disorder

 9. Body mass index less than 18 points or higher than 25 points

 10. Contraindications for hormonal contraception or intrauterine device.

 11. Autoimmune diseases A history of organ, bone marrow or hematopoietic stem cell
 transplantation

 12. Patients receiving anti-hcv treatment

 13. Permanent blindness in one eye

 14. History of iritis, endophthalmitis, scleral inflammation or retinitis 15-90 days of
 retinal detachment or eye surgery

 16-The competent physician considered it inappropriate to participate in the study

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 Safety and promising features of isotretinoin in tne era of COVID 2019 according Principal
 Investigator Protocol:

 1. This medication have the feature of Aerosolized Drug Delivery to increase its efficacy
 beside Oral administration, Which makes it distinct from other medication in which
 should dose be only given orally. A study demonstrated that treating with 13 cis
 retinoic acid aerosolized via inhalation rout did not cause any damage in lung cells.

 2. Repeated high doses of 13 cis retinoic by inhalation resulted in moderate loss of body
 weight, but microscopic investigation of ten tissues including lung and oesophagus did
 not detect any significant aerosol-induced damage. The results suggest that
 administration of isotretinoin via powder aerosol inhalation is probably superior to
 its application via the oral route in terms of achieving efficacious drug
 concentrations in the lung.

 3. Inhaled isotretinoin might provide sufficient drug to the target cells for efficacy
 while avoiding systemic toxicity.

 4. A study demonstrated that 13 cis retinoic is used in treating Emphysema (emphysema is
 a lung condition that causes shortness of breath)

 5. RA has been reported to induce formation of new alveoli and returns elastic recoil in
 the lung to approximately normal values in animal models of emphysema.

 6. Strong expectation of complete COVID -19 blockade from cell entry and infection
 depending on strong ethics, researches and references.

 7. Availability of our compounds.

 8. Ease of application.

 9. Expectation of COVID -19 treating by isotretinoin via more than one distinct
 mechanism.

 10. Expectation of High induction of anti- inflammatory T cells and significant inhibition
 of IL-6 at low concentrations of isotretinoin.

 11. Controlling Accompanying cytokine storm.

 12. No interactions with Egyptian protocol drugs were found.

 13. (13- cis -Retinoic acid ) can be given in the form of aerosol to avoid these systemic
 side effects. A clinical trial conducted on 148 subject from 5 university hospitals to
 evaluate the possibility of retinoids in the treatment of emphysema. The patients,
 were randomized to receive 13-cis retinoic acid (1 mg/kg/day, daily or ATRA at either
 low dose (1 mg/kg/day for 4 days/wk) or high dose (2 mg/kg/day for 4 days/wk), placebo
 for six months, followed by a three-month crossover phase. then, they were observed
 for an additional nine months before the final evaluation. In the trial, retinoids(13
 cis retinoic acid ) were proven to be safe as the drug-related AEs were generally
 mild[188]. A study reported that the application of aerosolized RA system led to a
 rise of RA levels in lung, but not plasma. or liver. In lung concentration and levels
 of retinol, retinyl palmitate and retinyl stearate also showed to be unchanged [189] A
 study on rabbits demonstrated that 13 cis retinoic acid can be given in the form of
 aerosol without serious side effects In this study repeated elevated doses of 13 cis
 retinoic acid by inhalation caused moderate loss of body weight, but microscopic
 examination of ten tissues including oesophagus and lung did not found any significant
 inhalation-induced injury or damage therefore aerosolized 13 cis retinoic acid might
 provide sufficient therapy to the target cells in lung for efficacy while avoiding
 systemic toxicity © 2000 Cancer Research Campaign[190]