Combination Therapy With Isotretinoin and Tamoxifen Expected to Provide Complete Protection Against Severe Acute Respiratory Syndrome Coronavirus

The study is a randomized interventional comparative Phase II trial. The duration of the
trial for each subject is expected to be 3 months.160 adult male and female patients with
positive COVID-19 diagnosis and fulfilling the below outlined inclusion criteria will be
enrolled into the study. Trial population will consist of both genders.

Study Type:Interventional [Change...]

Primary Purpose:Treatment

Study Phase:Phase 2 Interventional Study Model:Sequential Assignment

Number of Arms:3

Masking: None (Open Label)

Allocation:Randomized

Enrollment:160 [Anticipated]

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 and 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) inhibitors 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.As
Principal Investigator discussed before that (13cRA) is the strongest down-regulator of ACE2.
and the principal investigator expects that 13cRA can inhibit or dowenrgulat ACE2 by direc
interaction and binding with the transmembrane ACE2, Suggesting its therapeutic potential in
preventing the entry of COVID 2019 to the host cell.

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) 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.

Tamoxifen may be able to inhibit COVID 2019 by inhibition acidification of the endosomes and
lysosomes rather than inhibition of lysosomal enzymes.

A study demonstrated that tamoxifen causes redistribution of weak base chemotherapeutics from
acidic organelles to the nucleus in drug-resistant cells. Agents that disrupt organelle
acidification (e.g., monensin, bafilomycin A1) cause a similar redistribution. Measurement of
cellular pH in several cell lines reveals that tamoxifen inhibits acidification of endosomes
and lysosomes without affecting cytoplasmic pH. Similar to monensin, tamoxifen decreased the
rate of vesicular transport though the recycling and secretory pathways. Organellar
acidification is required for many cellular functions, and its disruption could account for
many of the side effects of tamoxifen. A sudy demonstrated that the phagocytosis is inhabited
by tamoxifen and chloroquine in retinal epithelial cells A study demonstrated that Tamoxifen
have weak base property and increase endolysosomal pH and alter endosomal dynamics.
Importantly, TAM treatment enhanced survival of mice injected with a lethal dose of STx1 or
STx2,The protective effect was independent of estrogen receptors but dependent on the weak
base property of TAM, which allowed TAM to increase endolysosomal pH and alter endosomal
dynamics. A study demonstrated that Tamoxifen have antimalarial effect via treating mice
infected with P. berghei, which show lower levels of parasitaemia and do not develop signs of
cerebral malaria, Tamoxifen is found to prevent lung fibrosis and reduce serum TGFβ-1 levels.
Astudy demonstrated that Tamoxifen have endosomal and lysosomal cysteine proteases inhibitory
effect better than chloroquine , Cathepsins are endosomal and lysosomal cysteine proteases
that play important roles in protein degradation in various cellular processes including both
the endocytic pathway and autophagy. The role of cathepsins in viral infection was first
identified by Huang et al and they found that one cysteine proteases inhibitor E64d and a
specific cathepsin L inhibitor Z-FY(t-Bu)-DMK are able to block the SARS-CoV infection.
Cathepsin D was more sensitive to tamoxifen than to chloroquine. Tamoxifen exposures
decreased the cathepsin D activity at less than 10 pM concentrations. The effect of
chloroquine started at 15 pM

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