Investigating the potential role of Aerosolized retinoic acid, a potent Vitamin Ametabolite for treating COVID-19 Anosmia and retinoic acid insufficiency .A novelapproach for regaining Sense of Smell.Mahmoud ELkazzaz(1),Tamer Haydara(2), Abedelaziz Elsayed(3) ,Yousry Abo-amer(4), HeshamAttia(5), Quan Liu(6) and Amr Ahmed(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 Pharmaceutical Biotechnology, Faculty of Pharmacy, Tanta University, Egypt. 4. Hepatology,Gastroenterology and Infectious Diseases Department, Mahala Hepatology Teaching Hospital, Egypt 5. Department of Immunology and Parasitology, Faculty of Science, Cairo University, Egypt. 6. School of Life Sciences and Engineering, Foshan University, Laboratory of Emerging Infectious Disease, Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, China. 7. Director of tuberculosis program Ghubera, public health department ,First health cluster ,Ministry of health ,Saudia Arabia. - Very important Note: This clinical study is the first clinical study in literature (First posted August 12, 2021) which demonstrated depending on molecular findings that Vitamin A /Retinoic Acid will treat smell loss resulted by COVID-19Recent rapidly accumulating evidences and reports indicate that partial loss of the senseof smell or even total anosmia are early markers of SARS-CoV-2 infection and frequentlyreported symptoms associated with the COVID-19 pandemic (Lechien J. R et al., 2020)However, the cellular mechanisms of this phenomenon are unknown. The rates of insomniaand depression were 26.45% and 9.92% in the COVID-19 patients after recovery. Therefore,finding an effective treatment for COVID-19 Anosmia is a critical point. Although, ACE2has been identified as the principal host cell receptor of 2019-nCoV, and it is thoughtto play a critical role in the virus's entrance into the cell and subsequent infection,many cells can be infected by COVID-19 while also expressing little or no ACE2. Eventhough the COVID-19 entry receptor, angiotensin-converting enzyme 2 (ACE2), is notexpressed in the receptor of olfactory neurons, or its synthesis is limited to to a minorfraction of these neurons.of these neurons, COVID-19 infection causes a loss of smell(anosmia) (Katarzyna Bilinska et al.,2021). Our recent findings showed that COVID-19binds directly to STRA6 receptors of retinol leading to retinol depletion and retinoicacid insufficiency (M Elkazzaz et al,. 2021) . Retinoic acid insufficiency in theolfactory epithelium, both in mouse and chick models, causes progenitor cell maintenancefailure and, consequently, olfactory neurons differentiation is not maintained . Anexplant system, showed that renewal of olfactory neurons is inhibited if retinoic acidsynthesis was failed in the olfactory epithelium (Paschaki M et al., 2013) . It's worthnoting that vitamin A shortage also causes olfactory and taste problems, In a study byGarrett-Laster et al., (1984), the patients had vitamin A deficiency because ofmalnutrition and alcoholic liver cirrhosis; they lost their sense of smell after thatdisorder. LaMantia and Rawson et al.,( 2007) reported that administration of retinoidacid after the damage of olfactory system motivates an immune response and produces amore quick recovery of olfactoryguided behavior. It was showed that Isotretinoin improvedthe significantly performance of patients in the olfactory test(Demet Kartal et al.,2017)Moreover, there is increasing evidence that retinoic acid (atRA) influences geneexpression of components of renin-angiotensin system (RAS), which plays a pivotal role inthe pathophysiology of essential hypertension. Retinoic acid induced ACE2 expression indifferent animal models. Moreover, a study suggests that topical retinoids may haveapplicability in promoting sinus regeneration and wound healing. In a study comparingtreated and untreated nasal mucosa ,untreated regenerated mucosa showed expected changesof submucosal gland loss, basal lamina and lamina propria fibrosis and loss of cilia.Reinoic acid treatment appeared to result in better mucosal regeneration marked by lesscellular atypia and fibrosis(Mendy S. Maccabee et al,. 2003).. Aerosolized retinoic acidwill have an effective role in treating post COVID-19 anosmia (loss of smell) viaupregulating ACE2, STRA 6 and regenerating of olfactory receptors and olfactory sensorycells and neurons.
The study is a randomized interventional comparative and multicenter Phase IIII trial.
10000 adult male and female patients with post COVID-19 anosmia (loss of smell) and
fulfilling the below outlined inclusion criteria will be enrolled into the study
Retinoic acids,STRA6, COVID-19, ocular and olfactory nervous system
Pinkeye (conjunctivitis), which is also associated with vitamin A deficiency, is
prevalent symptom in patients with severe COVID-19 infection. (68,69).According to our
findings, the binding of COVID-19 spike protein to STRA6, which is one of the main
receptors for retinol cell entrance and retinoic acid production in the retina, strongly
explains this symptoms .It was showed that genetic null mutation of STRA6 in mice model
results in high retinoid reduction in the neurosensory retina and retinal pigment
epithelium , diminished eye morphology and visual responses , despite the fact the
last-mentioned problem is not as serious as in patients with mutant STRA6
(11).STRA6-mediated transport is especially significant in the eye and in the existence
of vitamin A deficiency in the diet (Probable). Retinoic acid isn't transported. (70). A
study strongly suggested that STRA6 works as a retinol channel/transporter(70). Analysis
of function Loss- in embryos of zebrafishshowed that deficiency of Stra6 caused vitamin A
deprivation of the developing eyes(70). RA signaling pathway promotes normal development
of the optic nerve and ventral retina via its activities in the neural crest
cell-derivedperiocular mesenchyme (71)and its deficiency may leads to retinitis.
Although, several studies applied on COVID-19 patients have attempted to identify
parameters linked to the olfactory disorders and taste with Angiotensin-Converting Enzyme
2 (ACE2)receptors it is clear that it takes place via receptors of vitamin A . (72) It's
worth noting that vitamin A shortage also causes olfactory and taste problems, In a study
by Garrett-Laster et al.,(73)the patients had vitamin A deficiency because of
malnutrition and alcoholic liver cirrhosis; they lost their sense of smell after that
disorder. LaMantia and Rawson reported that administration of retinoid acid after the
damage of olfactory system motivates an immune response and produces a more quick
recovery of olfactoryguided behavior(74).
13 cis retinoic acid improved the sense of smell and the performance of the olfactory
test in acne patients (75) This also propose that insufficiency of vitamin A also rises
in COVID-19.
Therefore, we strongly suggest that loss of stra6 function thorough blocking it by
COVID-19 spike protein which binds to it with high affinity as a result it may hijack its
signaling pathway leading to retinoic acid synthesis disruption and vitamin A deficiency
. The symptoms and outcomes that arise in the eyes and nervous system of patients with
COVID-19 are with unknown etiology but the results of retinoic acid deficiency manifested
through vitamin A receptors. Ataxia , Headache , acute cerebrovascular disorder, impaired
and consciousness are observed in patients with COVID-19 as central nervous system
involvement and hyposmia, hypogeusia, neuralgia and hypopsia are seen as involvement of
the peripheral nervous system. Patients with muscle involvement were also
observed(76,77). COVID-19-related acute hemorrhagic necrotizing encephalopathy instances
have also been documented. The unenhanced cranial BT obtained in the patients revealed
hypodensity in both medial thalamuses (78). Similarly, this is a region with a lot of
Stra6 receptors of Vitamin A (58). Zhao et al (79) reported the first case of
Guillain-Barré syndrome linked to COVID-19.
Retinoic acids play a critical role in inducing neurogenesis and neuroplasticity. RA are
important for hypothalamus and the hippocampus that control alertness and mentality
.All-trans retinoic acid (atRA) can be formed from the vitamin A/ retinol in the brain.
This is important for long-term potentiation (LTP). Vitamin A insufficiency also leads to
circadian dysfunction. Cognitive dysfunction is also commonly showed(80,81).Pasutto et
al.(7) reported that STRA6 mutations associated with lung malformations and many heart ,
eye diaphragm as well as retardation in mentality as in syndrome of Matthew-Wood in
humans, validating its reported functions in vitamin A uptake by cells as vitamin
A/retinoic acid is very critical in the process of organogenesis.
For the adult brain, components of the retinoid metabolic pathways have been thoroughly
characterized(81). In some parts of the brain, all-trans-retinoic acid is synthesized..
Certain neuronal-specific genes contain recognition sequences for retinoid receptors and
can be arranged directly by retinoids. receptors of retinoid have a widespread
distribution in the human nervous system. This distribution differs significantly from
that seen during embryonic development, implying that retinoid signalling may have a
physiological role in the adult hypothalamus , cortex, striatum , amygdala, hippocampus,
and other brain regions (81,82).
Retinoid signal pathways disruption in models of rodent caused in disturbance in synaptic
plasticity, memory behaviors and learning . Signal pathways of retinoid also play a
critical role in the pathophysiology of schizophrenia , Alzheimer's disease, and
depression(81).
Retinoic acid regulates gonadotropin-releasing hormone (GnRH) and its receptor G-protein
coupled receptor (GnRH) an important action in smelling process .
The olfactory bulb (OB) is a conserved region found in brain that its main function is
receiving sensory neurons direct synaptic input in the nasal epithelium part and conveys
that instructions to the rest of the brain. (81). It gets instructions from the brain
regarding odours recognized by cells in the nasal cavity. Axons of the olfactory sensory
neurons extends to the region of the olfactory bulb, which is dedicated to process
odour-related instructions (82).The nervusterminalis, or zeroeth cranial nerve, contains
specific neurons that produce gonadotropin-releasing hormone (GnRH). All vertebrate
animals without sharks have a nervusterminalis, a chain of neurons implanted within
vomeronasal or olfactory nerves in the region of the nasal canal, where it is considered
a distinct nerve. The main role of the gonadotropin-releasing hormone ( GnRH) constituent
of the nervusterminalis is supposed to have neuromodulatory properties . (83). Numerous
studies suggested that the role of the intranasal gonadotropin-releasing hormone ( GnRH)
system is to adapt and modify olfactory information, maybe at opportune times for
reproduction(83). Gonadotropin-releasing hormone (GnRH), was showed to be expressed on 30
to 40 percent of neurons located in the region of the nervusterminalis and also, a small
dozen of these neurons may produce gonadotropin-releasing hormone (GnRH) directly into
blood veins underlying the olfactory epithelium (OE). (84). During prenatal GnRH neurons
emerge from the nasal placode until reach the brain(1). These neurons become critical
ingredients of the hypothalamic-pituitary-gonadal axis, which is required for activity of
reproduction, after they enter the brain. Hypogonadotropichypogonadism (HH) is caused
when this mechanism is disrupted (HH).
.The primary modulator of mammalian function of reproduction in both men and women is
gonadotropin-releasing hormone (GnRH). It acts via distinct receptors, G-protein coupled
receptor (GnRH) found in gonadotropes to induce production of the gonadotropin hormones,
follicle and luteinizing -stimulating hormones (FSH), (LH)(85). a study found that
congenital anosmia (loss of smell) is frequently linked with GnRH deficiency in human
patients, leading to the widely held belief that GnRH neurons rely on olfactory
structures to reach the brain, but this suggestion has yet to be proven(86).
Retinoic acid regulates both GnRH neurons and G-protein coupled receptor (GnRH-R).
Furthermore, retinoic acid plays critical physiological roles in synaptic plasticity,
learning and memory(27), hormone production(27,28) and adult neurogenesis(27) (15).They
also control a variety of processes in adults, including vision, cellular
differentiation, fertilization, and tissue homeostasis(87). Retinoids are therefore
essential for optimal physiology during both the early stages of development and
throughout maturity(87). The mammalian type I gonadotropin releasing hormone receptor
(GnRH-R) is a structurally unique G protein-coupled receptor (GPCR)(88). The majority of
hormones stimulates and mediates their signal transduction via G protein-coupled
receptors (GPCRs).(90) Retinoic acid induce expression of G protein-coupled receptor
called Retinoic Acid - Inducible G Protein-Coupled Receptors(89) . Studies show general
agreement that all-trans retinoic acid (atRA) has been linked to the regulation of G
protein-coupled receptor (GPCRs) signaling,(91,92,93). Retinoic acid induces expression
of G protein-coupled receptors that are used by Gonadotropin-releasing hormone (GnRH).
Retinoic acid (RA) appears to be a significant regulator of GnRH neurons in GT1-1 of rat
neuronal cells and hypothalamic fragments in vitro, according to a study. (93). In this
study during a short period (2hours), Retinoic acid raised gonadotropin-releasing hormone
(GnRH) production in a dose-dependent manner in addition, time-course tests revealed that
Retinoic acid speedily induced gonadotropin-releasing hormone (GnR0H release by thirty
min in both types of used cells. Furthermore, significant increase in mRNA levels of
gonadotropin-releasing hormone by Retinoic acid was observed within 12hours. (94). In
another study showed that that all-trans-RA controls gene expression and release of
gonadotropin-releasing hormone (GnRH) in neuronal cells and hypothalamic fragments of
rat. All-trans-RA increased GnRH transcription by activating functional retinoic acid
response elements (RARE) in the promoter's distal region of GnRH. (95).
.
Drug: Aerosolized 13 cis retinoic acid plus Vitamin D
Patients with Post COVID-19 Anosmia (Loss of Smell) will receive one dose daily of
Aerosolized 13 cis retinoic acid in gradual 2 divided doses increases from 0.2 mg/kg/day
to 4 mg/kg/day as inhaled retinoic acid therapy for 3 weeks. Furthermore, the patients
will receive Cholecalciferol(Vitamin D) Intramuscular injection of 600,000 units of
Cholecalciferol for 2 doses given at week 0 and week 4
Drug: Aerosolized All trans retinoic acid plus Vitamin D
Patients with Post COVID-19 Anosmia (Loss of Smell) will receive one dose daily of
Aerosolized All trans retinoic acid in gradual 2 divided doses increases from 0.2
mg/kg/day to 4 mg/kg/day as inhaled retinoic acid therapy for 3 weeks. Furthermore, the
patients will receive Cholecalciferol(Vitamin D) Intramuscular injection of 600,000 units
of Cholecalciferol for 2 doses given at week 0 and week 4
Other: Standard therapy
Standard therapy
Inclusion Criteria:
- adults 18 yrs or older patients
- confirmed case (+ve PCR),
- recovered/discharged (2 -ve PCR),
- suffered from sudden recent anosmia or hyposmia
Exclusion Criteria:
- Patients <18 years of age Patients who are unable to provide informed consent
- anosmia improved before COVID19 recovery,
- pregnancy
- patients who will not complete the follow up period.
- Patients without a positive COVID-19 PCR result obtained through nasopharyngeal
--swab - Patients with a COVID-19 diagnosis but without self-reported anosmia
--Patients with severe COVID-19 disease as defined by the Mouth Sinai Health System
--Treatment Guidelines for SARS-COV-2 (requiring high flow nasal cannula,
non-rebreather, CPAP/BIPAP, or mechanical ventilation OR patients requiring pressor
medication OR -----
- patients with evidence of end organ damage)
- Patients with pre-existing self-reported olfactory dysfunction
- Patients with a history of chronic nasal/sinus infections (rhinosinusitis) or
history of endoscopic sinus surgery
- Hypercholesterolemia
- Hypertriglyceridemia
- Patients using nasal steroid sprays or irrigations for any reason
- Patients who are prisoners of the state
- Patients who have psychiatric or developmental disorder conditions that may impair
ability to provide informed consent
- Permanent blindness in one eye
- History of iritis, endophthalmitis, scleral inflammation or retinitis 15-90 days of
retinal detachment or eye surgery
- The competent physician considered it inappropriate to participate in the study
Quan Liu
Foshan, Guangdong, China
Tamer Haydara
Kafr Ash Shaykh, Kafr Elshiekh, Egypt
Ministry of health.First health cluster ,Riaydh
Riyadh, Saudi Arabia
Mahmoud R Mahmoud, M.Sc of Biochemistry
+201090302015
mahmoudramadan2051@yahoo.com
Tamer Haydara, Ass/Prof of Internal medicine
00201142233340
tamerhaydara@yahoo.com