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
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
Efficacy and safety of Drug combination therapy of Isotretinoin and some Anti fungal Drugs as
 A potential Aerosol therapy for COVID-19 : An innovative therapeutic approach

 The pandemic of COVID-19 which is caused by severe acute respiratory syndrome coronavirus 2
 (SARS-COV-2) has infected over 2,000,000 people causing over 150,000 deaths.It hasno
 currently approved treatments.. Airborne SARS-CoV-2 infections in humans initiate from the
 virus entering nasal and airway epithelial cells through binding to angiotensin-converting
 enzyme 2 (ACE2). TMPRSS2, a cellular protease that activates the SARS-CoV-2 spike protein,
 colocalizes with ACE2 and can prime SARS-CoV-2 fusion directly at the plasma membrane. In the
 lungs, SARS-CoV-2 infects type I and type II alveolar epithelial cells, as well as alveolar
 macrophages that are among the first producers of pro-inflammatory cytokines. As key
 components of the immediate antiviral response, type I interferons (here after referred to as
 IFNs) are crucial for restricting viral replication and spread, through autocrine and
 paracrine type I IFN receptor (IFNAR) signalling. However, minimal amounts of IFNs have been
 detected in the peripheral blood or lungs of patients with severe COVID-19 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 CRS .
 SARS-CoV-2 ORF3b is a potent interferon inhebitor and antagonist Here, we review the
 molecular mechanisms by which Retinoic acid (isotretinoin) and antifungal drugs can cooperate
 to induce interferon in covid-19 infected patients A study reported that 13 Cis retinoic acid
 induced significant upregulation of toll-like receptor 3 resulting in an immune response to
 dsRNA intermediate which can be partially generated during CoV-2 replication . TLR3
 sensitized by dsRNA and cascades of signaling pathways (Interferon-regulatory factor 1 (IRFs)
 and Nuclear factor-κB (NFκB) activation, respectively) are activated to produce type I
 interferons. The production of type I IFNs is important to enhance the release of antiviral
 proteins for the protection of uninfected cells. RA can be generated in multiple forms as
 all-trans, 9-cis,and 13-cis retinoic acid. A study reported that Retinoic acid induces
 directly the expression of two transcription factors, Stat1 and IRF-1 which play central
 roles in the IFN signal transduction. In addition, RA induces IFN-a synthesis, IFNs can serve
 as the first line of immune defense against viral infections. 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.
 There are three classes of IFNs: type I, type II, and type III. Antifungal drug. Fluconazol
 or itraconazol can inhibit cytochrome P450 enzymes, especially cype 26 which control retinoic
 acid concentration into human cells enhance both isotretinoin effect and Concentrations in
 Target Tissues This in turn lead to hyper interferon induction and synthesis in case of
 COVID-19. Also a study demonstrated that isotretinoin can be given as aerosolized via
 inhalation rout without 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
 therefore inhaled isotretinoin might provide sufficient drug to the target cells in lung for
 efficacy while avoiding systemic toxicity. In conclusion,isotretinoin therapy has furthermore
 a proven anti-inflammatory, anti-platelet and fibrinolytic activities which may protect
 patients infected with covid-19 from widespread blood clots. From this point, we suggest that
 isotretinoin will be the immunity passport" in the context of 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
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

 -  The underlying disease is very serious and the expected survival time is less than 6
 months (such as advanced malignant tumor);

 -  COPD or end-stage lung disease requires home oxygen therapy

 -  Expected survival time not exceeding 48 hours

 -  Participated in other clinical intervention trials within the last 3 months

 -  Autoimmune diseases

 -  A history of organ, bone marrow or hematopoietic stem cell transplantation

 -  Received radiotherapy and chemotherapy for malignant tumor within 6 months

 -  HIV infected patients or diagnosed with acquired immunodeficiency within the past year
 (CD4 T cells <=200/mm3)

 -  Patients receiving anti-hcv treatment

 -  90 days of retinal detachment or eye surgery

 -  Permanent blindness in one eye

 -  History of iritis, endophthalmitis, scleral inflammation or retinitis

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