Efficacy and Safety of Drug Combination Therapy of Isotretinoin and Some Antifungal Drugs as A Potential Aerosol Therapy for COVID-19 : An Innovative Therapeutic Approach COVID-19

This is a small pilot study investigating whether there is any efficacy signal of combination
therapy of Isotretinoin and some Anti fungal Drugs in COVID-19 treatment that warrants a
larger Phase III trial, or any harm that suggests that such a trial should not be done. It is
expected to produce statistically significant results in the major endpoints. The
investigator will examine all of the biologic, physiological, and clinical data to determine
whether a Phase III trial is warranted.

Primary efficacy analysis will be carried only on patients receiving at least 4 doses of
active combination drug. Safety analysis will be carried out on all patients receiving at
least one dose of active drug.

introduction

In Wuhan, Hubei Province, China, cases of acute respiratory infection were reported in
December 2019.1,2 The Chinese Center for Disease Control and Prevention (CDC), initially
reported that the cause of this disease is severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2), then it is founded to be a novel coronavirus.3Now, This disease, is designated
by the WHO as coronavirus disease 2019 (COVID-19), which rapidly spreads to other cities of
China, and has become a public health emergency of international concern (PHEIC) following
its global spread. The clinical manifestations of COVID-19 are fever, cough, muscle pain,
fatigue, diarrhea and pneumonia, and can cause death in severe cases.4China has reported
72436 cases of confirmed COVID-19 and 1868 fatalities through February 18, 2020.5 The innate
immune system represents the first line of defense and initiates counteractive responses that
protect the host from viral infection through evolutionarily conserved pattern recognition
receptors (PRR) 6. PRRs include the membrane-bound Toll-like receptors (TLRs) and cytosolic
sensors, such as retinoic acidinducible gene-I (RIG-I)-like receptors (RLRs), which sense RNA
viruses 6. RIG-I specifically detects the intracellular double-stranded viral RNA bearing 5'
triphosphate and panhandle structures to activate antiviral signaling7,8. Once a host is
invaded by a virus, PRRs transmit signals to the downstream kinases that activate
transcription factors, including IFN regulatory factor-3 (IRF3), nuclear factor κB (NF-κB),
and ATF-2/c-jun, with the help of different adaptor molecules (MAVS/IPS-1/VISA/Cardif for
RIG-I, TRIF for TLR3, and MyD88 for TLR7/8/9) to activate IFN production9,10,11.Viruses have
evolved elaborate mechanisms to evade or inactivate the innate immune signaling pathway for
their replication 12. Severe acute respiratory syndrome (SARS) is a highly contagious
respiratory disease that appeared first in China in 2002 and has infected more than 8000
people worldwide and killed about 800 of those infected. SARS coronavirus (SARS-CoV) has a
singlestranded, positive sense RNA genome of approximately 29.7 kb 13,14. Numerous studies
have revealed that SARS-CoV develops an antagonistic mechanism to evade the antiviral
activities of IFN 15 Infection with SARS-CoV-2 can lead to excessive production of
pro-inflammatory cytokines, but the production of type I interferons, which are key antiviral
mediators, is reportedly blunted; Previous studies suggested that SARS-CoV papain-like
protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a
robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the
IRF3 pathway is not fully known.16 As key components of the immediate antiviral response,
type I interferons are crucial for restricting viral replication and spread, through
autocrine and paracrine type I IFN receptor (IFNAR) signalling. Type 1 interferons have a
broad antiviral activity in vitro and are currently evaluated in a clinical trial to treat
MERS-CoV However, minimal amounts of IFNs have been detected in the peripheral blood or lungs
of patients with severe COVID-19 17,18 In a mouse model of SARS-CoV infection, local IFN
responses in the lungs were delayed relative to peak viral replication, which impeded virus
clearance and was associated with the development of CRS19 . SARS-CoV-2 ORF3b is a potent
interferon inhebitor and antagonist 20 The dysregulated IFN responses are indicative of the
effective immunomodulatory strategies used by betacoronaviruses. During the incubation phase,
SARS-CoV-2 replicates stealthily in host cells without detectably triggering IFNs, leading to
high viral loads1. Coronaviruses are known to induce the formation of membranous compartments
dedicated to viral RNA synthesis and thereby conceal viral pathogen-associated molecular
patterns (PAMPs; for example, viral RNAs) from detection by host pattern recognition
receptors (PRRs), such as RIG-I and MDA5. Furthermore, several conserved betacoronavirus
proteins, predominantly non-structural proteins (nsps), are known to exert direct
IFN-antagonistic activities. Some modify specific features of the viral RNA (by catalysing
guanosine-N7 and ribose-2'-O methylation) to avoid recognition by specific PRRs (for example,
nsp14 and nsp16), while others, such as nsp3 and nsp1, inhibit the signal transduction
mediated by PRRs and by IFNAR, respectively19

--Impact of TLR3 on type I IFNs

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that initiate
innate immune response to invading pathogens by sensing conserved molecular patterns for
early immune recognition of a pathogens like viruses, bacteria, and fungi to initiate innate
immune response to invading pathogens . In addition ,TLRs play an important role in tissues
repair and tissues injury- induced inflammation. The emergence of highly pathogenic severe
acute respiratory syndrome coronaviruses (MERSCoV) is a concern for global public health, as
there is a lack of efficacious vaccine platforms and antiviral therapeutic strategies21. A
study demonstrated that pre-stimulation of TLR3 polyinosini- polycytidylic acid (poly I:C)
hindered MHV infection through induction of INF- β in macrophages 22 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 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..21

--Impact of isotretinoin 13 cis retinoic acid on TLR3 and TLR-2

Many of the significantly up-regulated genes are known to be retinoid-responsive genes
including: retinoic acid responder 1 [tazarotene induced gene 1 (TIG1)}, cellular retinol
binding protein 1 and cellular retinoic acid binding protein 2. In addition, calcium-binding
proteins (i.e. S100 proteins), serine proteases, serine protease inhibitors (serpins),
lipocalin and solute carriers were significantly affected by 13-cis RA25 In addition, Gene
expression analysis in SEB-1 sebocytes and HaCaT keratinocytes with 72 hour 13-cis RA
treatment. Revealed that there were changes in several genes involved in apoptosis and innate
immunity such as TNFα-induced protein 2, TRAIL, interferon regulatory factor 1 (IRF1),
interferon-induced proteins, NFκB, the death receptor, Fas and TIG3 (a.k.a. retinoic acid
inducible gene 1 (RIG1)). TIG3 encodes an RNA helicase and represents a key intracellular
protein that like the TLR3, can recognize viral double stranded RNA (dsRNA). 26 isotretinoin
can inhebit cytokine storm thorough inhebtion of TLR-2 astudy demonstrated that treatment of
patients with isotretinoin significantly decreased monocyte TLR-2 expression and subsequent
inflammatory cytokine response to P. acnes after 1 week of therapy. This effect was sustained
6 months following cessation of therapy, indicating that TLR-2 modulation may be involved in
the durable therapeutic response to isotretinoin.22 IN contrary, 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. 23 this can resulted 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. Interferon regulatory factor 1 (IRF1) was identified as a
critical factor in mediating TRAIL induction by retinoic acid in NB4 APL leukemia cells and
SK-BR-3 breast cancer cells. Interestingly, 13-cis RA significantly up-regulates IRF1 gene
expression (2.42 fold increase) in SEB-1 sebocytes 27

Impact of antifungal drugs on inducing isotretinoin effect and concentration in targeted
tissues

Itraconazole and fluconazole are triazole azole ring antifungal drugs, which are multiringed
synthetic compounds containing three nitrogen atoms On the other hand, azole antifungal drugs
have been demonstrated to inhibit cytochrome P450 28Thecytochrome P450 family (CYP26) enzymes
are responsible for RA clearance, and are potential drug targets to increase concentrations
of retinoid into targeted cells31 CYP26 Inhibitors Increases Retinoid Signaling in Intimal
Smooth Muscle Cells32 Itraconazole has less inhibitive properties than the imidazoles in rat
liver microsomes 29. ICZ interferes with the ergosterol synthesis pathway of the host cell
and inhibits cytochrome P450 enzymes, particularly cyp 34 enzyme that metabolizes retinoic
acid in human cells which increase retinoic acid concentration in to targeted mammalian
cells40 leading to activation of TOLL-like receptors inducing its antiviral mechanism to
induce type I IFN expression levels toward the induction of a preactivated state, thereby
accelerating the virus-induced host cell response41 IFNs can serve as the first line of
immune defense against viral infections.41 IFNs are very powerful cytokines, which play a key
role in combatting pathogenic infections by controlling inflammation and immune response by
directly inducing antipathogen molecular countermeasures.42There are three classes of IFNs:
type I, type II, and type III.. Even though fluconazole has been shown to be a potent
cytochrome P450 inhibitor 30 Here,we suggest that because antifungal drug. Fluconazol or
itraconazol can inhibit cytochrome P450 enzymes, especially cype 26 which control retinoic
acid concentration into human cells antifungal can enhance both isotretinoin effect and
Concentrations in Target Tissues This in turn lead to hyper interferon induction and
synthesis in case of COVID-19 Astudy found that fluconazole has been shown to be a potent
cytochrome P450 inhibitor32 Safety of Aerosolized Isotretinoin Isotretinoin (Accutane, Roche
Laboratories Inc, Nutley, NJ) is an important drug, not only for the treatment of severe
acne, but also for other diagnoses and in chemoprevention settings. Because the use of
isotretinoin is increasing, it is important for physicians to be aware of the adverse events,
toxicities, and management issues related to its use. The most important issue is that of
congenital defects, which has resulted in new pregnancy prevention policies and programs
implemented by the manufacturer. A relatively new concern is that of depression associated
with isotretinoin use, also resulting in new policies placed by the manufacturer and the
FDA., But here, in our review we highly recommend that any researcher who wants to apply
isotritinoin clinically covid-19 his clinical study must be a small pilot study investigating
whether there is any efficacy signal that warrants a larger Phase 2B trial, or any harm that
suggests that such a trial should not be done. The investigator will examine all of the
biologic, physiological, and clinical data to determine whether a Phase 2B trial is
warranted. And he also must abide by these rules :Isotretinoin must be give in the form of
aerosol thorough respiratory route in order for it to has an effect that is focused only on
lung cells so as not to affect any other organs. Aerosol inhalation can deposit drug directly
on the population of cells caught up in the early phase of cancer, potentially achieving much
more efficiency compared with reliance on diffusion from the blood. Direct application of
aerosolized retinoic acid to the lung epithelium may avoid much of the protein binding, and
serious side effects on the other organs especially, congenital organs thus greatly
increasing potency at the target site.35 Because a study on rabbits demonstrated that
isotretinoin can be given in the form of aerosol without serious side effects . Repeated high
doses of 13 cis retinoic by inhalation resulted in moderate loss of body weight, but
microscopic investigation of ten tissues inhalation rout without any damage in lung including
lung and oesophagus did not detect any significant aerosol-induced damage therefore inhaled
isotretinoin might provide sufficient drug to the target cells in lung for efficacy while
avoiding systemic toxicity © 2000 Cancer Research Campaign33 In 2006, a clinical trial
conducted to assess the feasibility of retinoids for the treatment of emphysema, the FORTE
study, was published (NCT00000621)33. In that study, 148 patients from five university
hospitals were recruited and randomized to receive ATRA at either low dose (1 mg/kg/day for 4
days/wk) or high dose (2 mg/kg/day for 4 days/wk), 13-cis retinoic acid (1 mg/kg/day, daily),
or placebo for 6 months, followed by a 3-month crossover phase. Subsequently, they were
observed for an additional 9 months before the final assessment. In the trial, retinoids(13
cis retinoic acid ) were proven to be safe as the drug-related AEs were generally mild.37 In
addition, aerosolized isotretinoin must be given with low dose in accordance to patient
weight and also it must be given gradually concentration thorough a short period not
exceeding 14 days from the date of giving the first dose In contrast to the long course of
treatment, which is considered chronic treatment and Aerosolized 13 cis retinoic acid must be
be given gradually in one daily dose increases from 0.2 mg/kg/day to 2 mg/kg/day as inhaled
13 cis retinoic acid therapy for 14 days A study demonstrated that the application of
aerosolized retinoic acid system led to a rise of Retinoic Acid levels in lung, but not liver
or plasma. Cellular lung levels of retinol, retinyl palmitate, and retinyl stearate also
appeared to be unaffected (245.6 ± 10.7, 47.4 ± 3.4, and 132.8 ± 7.7 ng•g-1 wet weight,
respectively). And also demonstrated that the application of this aerosolized Retinoic Acid
also induced a dose-dependent protein expression of the cellular retinol-binding protein 1
(CRBP-1) in lung, without apparent harmful side effects.38 Although, more than one clinical
study reported safety and non serious side effects of systemic isotretinon but we highly
recommend that isotretinoin must be given in the form of aerosol with short period of
tratment and with low gradual dose to target lung cells only for avoiding any potential side
effects. A clinical study applied on 1166 patients thorough phase III trial (NCI #I91-0001)
at a National Cancer Institute (NCI) demonstrated that after the median follow-up of 3.5
years, there were no statistically significant differences between the placebo and
isotretinoin arms with respect to the time to SPTs, recurrences, or mortality. In this study
Patients were randomly assigned to receive a placebo or the retinoid isotretinoin (30 mg/day)
for 3 years in a double-blind fashion. Patients were stratified at randomization by tumor
stage, histology, and smoking status. The primary endpoint (time to SPT) and the secondary
endpoints (times to recurrence and death) were analyzed by log-rank test and the Cox
proportional hazards model. All statistical tests were two-sided.34 A study conducted on 720
patients who had received one or more courses of oral isotretinoin treatment, and had a mean
follow up period of 4.9 years (range 2/12 years). Most patients (442) had received a total
cumulative dose of 120/200 mg/kg body weight. One hundred and sixty two patients received a
cumulative dose of <120 mg/kg body weight, and 116 received a cumulative dose <200 mg/kg.
Finally this study proved that oral isotretinoin in the treatment of acne is a safe drug,
with no serious long term side effects.36

Safety of Aerosolized fluconazole

A study demonstrated that fluconazole can be given via route of inhalation. Respiratory
fungal diseases therapy is still facing challenges as a result of increasing autoimmune
disorders, cancers, and immunosuppressive medication usage. Fluconazole is a wide spectrum
antifungal agent and is still used successfully in the treatment of opportunistic infections
in combination with other antifungal agents. Since, the treatment of respiratory fungal
diseases requires prolonged hospitalization; it may increase the chances of other
opportunistic infections. Considering the reported drug resistance and adverse effects of
systemic administration, it appears that localized pulmonary antifungal therapy may be a
suitable alternative route. According to the reported suitable inhalation properties of spray
dried powders; spray drying technique was used to prepare fluconazole powders.39