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Perspectives for the use of therapeutic Botulinum toxin as a multifaceted candidate drug to attenuate COVID-19

The recent outbreak of coronavirus disease (COVID-19) resulting from a distinctive severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to evolve in many countries and pose life-threatening clinical issues to global public health. While the lungs are the primary target for the SARS-CoV-2-mediated pathological consequence, the virus appears to invade the brain and cause unpredicted neurological deficits. In the later stage, COVID-19 can progress to pneumonia, acute respiratory failure, neurodegeneration and multi-organ dysfunctions leading to death. Though a significant portion of individuals with COVID-19 has been recovering from clinical symptoms, the pathological impact of the SARS-CoV-2 infection on the structural and functional properties of the lungs, heart, brain and other organs at the post-recovery state remains unknown. Presently, there is an urgent need for a remedial measure to combat this devastating COVID-19. Botulinum toxins (BoNTs) are potent neurotoxins that can induce paralysis of muscle and acute respiratory arrest in humans. However, a mild dose of the purified form of BoNT has been known to attenuate chronic cough, dyspnoea, pneumonia, acute respiratory failure, abnormal circulation, cardiac defects and various neurological deficits that have been recognised as the prominent clinical symptoms of COVID-19. Considering the fact, this review article provides 1) an overview of the SARS-CoV-2 mediated pathological impact on the lungs, heart and brain, 2) signifies the therapeutic uses of BoNTs against pulmonary failure, cardiac arrest and neurological deficits, and 3) emphasize the rationality for the possible use of BoNT to prevent SARS-CoV-2 infection and manage COVID-19.


Introduction
The ongoing outburst of coronavirus disease 2019  has rattled the entire human population as it potentiates the life-threatening acute medical complications and death worldwide. A distinctive coronavirus designated as severe acute respiratory syndrome (SARS)-CoV-2, has been determined to be accountable for the ongoing COVID-19 [1]. The SARS-CoV-2 responsible for the COVID-19 in human is proposed to be originated in bats and pangolins [2]. The first incidence of the transmission from animal to humans has been known to have occurred in Wuhan, Hubei Province, China in late 2019 [3][4][5]. Since then, COVID-19 continues to emerge all over the world through the person-to-person transmission of viral droplets. The victims encountered by the SARS-CoV-2 have been reported to develop a wide range of clinical symptoms that include dry cough, sore throat, fever, body pain, headache, abdominal pain, diarrhoea and fatigue [4,5]. In the severe stage, the SARS-CoV-2 infection is known to cause pneumonia, acute respiratory failure, encephalopathy and multi-organ dysfunctions, thereby leading to death [4][5][6]. In general, coronaviruses have been known to cross the blood-brain barrier (BBB) and affect the central nervous system (CNS) [7]. A growing J o u r n a l P r e -p r o o f 2. The pathological impact of COVID-19 on the lungs, heart and brain The SARS-CoV-2 belongs to a family of positive-strand RNA viruses of the genus Betacoronavirus and it shares genetic similarity with two viral strains that appear to have originated in bats [2,18]. The angiotensin-converting enzyme 2 (ACE2) has been recognized as a receptor for the SARS-CoV-2 [19]. Thus, cells that express ACE2 are vulnerable to the infection and are considered as the main target of the SARS-CoV-2 [20,21]. The ACE2 gene is highly expressed in the respiratory tract and thus the lungs are the major hotspot of SARS-CoV-2 infection and its pathogenic events [22]. However, SARS-CoV2 infection appears to takes place also in the gastrointestinal tract, cardiovascular system, brain and other organs that are known to express the ACE2 [19,20,23]. Individuals with diabetes, obesity, hypertension, and cardiovascular disorders have been recognised to be highly susceptible to SARS-CoV2 infection leading to the development of COVID-19 [1,6,24]. The abnormal expression and functions of the ACE2 have been linked to diabetes, obesity, hypertension, and cardiovascular disorders [4,20,25]. Moreover, recent findings suggest that the density of the ACE2 on the cells increases upon ageing and its expression is found to be higher in men than women since SARS-CoV-2 appears to affect men than women in many countries [26]. While SARS-CoV-2 infection develops various symptoms in the host, acute respiratory distress syndrome (ARDS) represents a pathological hallmark of COVID- 19 [27]. In many cases, pneumonia appears to be the major clinical consequence of SARS-CoV-2 infection due to prominent proinflammatory process in the alveoli of the lungs [2,26,27]. Many reports from in vivo and in vitro studies and histopathological findings on the post-mortem human J o u r n a l P r e -p r o o f lungs suggest that SARS-CoVs causes apoptosis in alveolar cells and degeneration of small blood vessels of the lungs [1,6,[26][27][28]. In the intensive conditions, respiratory arrest has been a major cause of COVID-19 associated death [2,5,26]. In addition, the available clinical data suggests that a significant portion of COVID-19 patients have hypertension and cardiovascular disease [6,21,24,29]. The medications that are used to manage the symptoms of hypertension and cardiovascular disorders in association with diabetes have been known to induce the expression of ACE-2 [20,22,23]. Thus, it has been correlated that individuals with hypertension, diabetes, cardiovascular disorders are at high risk of being infected with SARS-CoV-2 and developing COVID-19 [24,29].
Recent reports on the blood parameters indicated the elevated levels of creatine kinase and lactate dehydrogenase in a significant proportion of individuals with COVID-19 [30,31]. The elevated levels of creatine kinase and lactate dehydrogenase in the circulation have been known to be the biomarkers of the muscle degeneration and tissue damage in various organs including the heart and liver. Moreover, cardiac arrest has increasingly been noticed as one of the reasons for the mortality associated with COVID-J o u r n a l P r e -p r o o f that SARS-CoVs can invade the brain through the olfactory epithelium and can induce neuronal death in a transgenic mouse model that expresses the human ACE2 gene [38].
Though human subjects with autoimmune disorders like multiple sclerosis have been reported to be highly susceptible to get SARS-CoV2 infection, neurological abnormalities can increasingly be expected in the critical stage of any individuals with COVID-19 [39].
The viral infections mediated neuropathogenicity in the brain regions that regulate the respiratory, cardiac, gustatory and olfactory functions might be highly relevant for the COVID-19 cases. In addition, COVID-19 may exhibit abnormal regulations of HPA in association with panic, anxiety and depression-like disorders which may need an additional line of scientific focus. Considering the fact, the SARS-CoV-2 infection might impair the brain function at the level of impaired synaptic plasticity, neuroinflammation, neurodegeneration and abnormal neurogenesis, gliosis leading to cognitive impairment and dementia similar to that of other neuro-invasive viruses including herpes simplex virus, poliovirus and HIV [40]. Moreover, the ACE2 gene expression and functions have been linked to cerebral blood flow (CBF) in physiological state, while it can also be expected that SARS-CoV-2 infection can mediate abnormal regulation of the ACE2 gene [41]. A recent study revealed a significant portion of COVD-19 victims has been overlapped with signs of cerebrovascular disease [5,7,42]. Taken

Clinical uses of botulinum toxins
BoNTs are the most harmful biological substance currently known to human [12,13].
BoNTs are mainly produced by strains of anaerobic spore-forming bacterium, Clostridium botulinum, [11,43]. Seven major serotypes of BoNTs have been characterized and designated as type A to G [44]. Among them, BoNTs types A, B, E and F have directly been liked to induce lethal human diseases [11,12]. Structurally, BoNTs consist of a heavy and a light polypeptide chains of about 100 and 50 kDa in size respectively, coupled by a disulfide bridge [11,12]. The heavy chain of BoNTs binds to its receptors at the presynaptic terminals of the cholinergic neurons and enters into the cytoplasm of the neurons as receptor-mediated endocytosis, where the disulphide bond between two polypeptide chains of the BoNTs disassociates and gains their activated form [11,12].
The light chain binds to inactivate the proteins like synaptosomal-associated protein (SNAP) 25, vesicle-associated membrane protein (VAMP) and syntaxin that are responsible for the fusion of neurotransmitter containing vesicles with lipid bilayer in the synaptic cleft, thereby preventing the release of acetylcholine (ACh) in cholinergic, parasympathetic and postganglionic sympathetic nerve endings. BoNTs-mediated blockades of the ACh release and subsequent arrest in the cholinergic signalling at the synaptic junctions have been regarded to induce dreadful muscle paralysis and death [11,12]. BoNTs are known to induce symptoms like dryness of the mouth and eyes, urinary retention, fluctuations of the blood pressure, abnormal heart rate, fatigue and dyspnoea [13,14,45,12,11]. The lungs are the primary target organ when BoNTs are inhaled as it induces acute pneumonitis and paralysis of breathing muscle leading to respiratory arrest [11][12][13]45]. However, mild dose of the purified forms of BoNT has extensively been implemented to treat a wide range of clinical symptoms [11][12][13].
Notably, therapeutic BoNT has been implemented to reduce the severity of disease and J o u r n a l P r e -p r o o f provide a cure clinical conditions that include hyperhidrosis, chronic migraine, strabismus, hemifacial spasms, blepharospasm, abnormal heart rate, dysphagia and urinary bladder dysfunctions [11][12][13]17,45]. Moreover, mild therapeutic dose of BoNT has clearly been shown to provide beneficial effect against the conditions with obstruction of the lung functions like chronic cough, bronchitis, asthma and dyspnoea [11,12,14,16,17,46].
Though the causative factors and clinical symptoms of the aforementioned disorders are highly dissimilar, therapeutic BoNTs appear to yield beneficial effect regardless of aetiological differences. Taken together, insight into the potential molecular targets of therapeutic BoNTs would be of great help to extend its curative potential to combat the refractory ongoing diseases in human.

Proposed basis of the repurposing of botulinum toxin to combat COVID-19
Thus far, SARS-CoV-2 induced acute ARDS responsible for pneumonia and respiratory arrest has been implicated as a key determining factor for the extending casualties of the COVID-19 crisis worldwide [4-6,10,27,31]. Meanwhile, cardiac arrest resulting from the SARS-CoV-2 associated heart failure has also been linked to mortality in individuals with J o u r n a l P r e -p r o o f COVID- 19 [24]. Besides, SARS-CoV-2 appears to enter the brain and elicit the neuropathogenicity in various brain regions that controls body temperature, reparation and heart beat [10]. Studies on the neuropathological studies derived from both patients and experimental animals have strongly indicated that SARS-CoV-2 can also induce neuropathological outcomes as like other neuro-invasive viruses [8,10,34,42,48]. Owing to the aforementioned fact, Li YC and co-workers have recently reported that patients with COVID-19 exhibit many neurologic signs and further indicated that the brainstem is highly vulnerable in subjects with COVID19 [10]. Moreover, the CT and MRI based findings revealed that the thalamus, medial temporal lobes and subinsular region are affected and development of encephalopathy is apparently evident in the brain of a subject with COVID-19 [49]. Considering the fact, it can be expected that individuals who have recovered from COVID-19 are at high risk of the onset of neurological, motor, cognitive deficits and psychiatric symptoms. Therefore, in addition to aiming the druggable strategies at peripheral and systemic levels, the brain represents one of the major therapeutic targets to manage the symptoms and to evade COVID-19 related mortalities. Though inhibition of the expression and functions of the ACE2 gene using inhibitors may be of help in prevention, it would not yield an ultimate cure for positive cases with COVID-19 [50]. Apart from the vaccination based prevention of COVID-19 in the unaffected population, a number of medical measures may be considered in order to manage and provide effectual defence against COVID-19, as follows 1) reduction of the viral load by pharmacological, genetic interference-based interventions or therapies to boost immunity against SARS-CoV-2 in positive cases, followed by 2) assigning antiinflammatory regimes to reduce the SARS-CoV-2 mediated chronic proinflammatory responses, 3) supplementation of cytoprotective agents and 4) implementation of neurotrophic support to sustain the neuroplasticity. While many drugs can individually J o u r n a l P r e -p r o o f meet any one of the aforementioned strategies, identification of a single drug or establishment of a strategy with multipurpose therapeutic potentials might be highly appropriate for the moment.
The therapeutic forms of BoNT have been approved by the Food and Drug Administration (FDA) for the treatment of many clinical conditions including chronic headache and dystonia [11][12][13]. Besides, BoNTs have been licensed for its use as antiageing cosmetics [12,13,51]. In particular, mild therapeutic BoNT injection has been proven to be beneficial against headache, body pain, persistent fever, chronic cough, pneumonia and dyspnoea [11][12][13][14]16,17,46], while those are considered as the general clinical symptoms of COVID-19 [3][4][5][6]21,26,27,30,42,48,49]. Notably, a significant portion of patients with COVID-19 has been reported to exhibit lymphopenia, a condition of having a low level of lymphocytes in the blood [52]. Therapeutic BoNT injections appear to elevate immune cell counts including platelets counts in the blood which might help fight the SARS-CoV-2 as it can enhance antigen presentation and the macrophagesmediated phagocytosis to eliminate the virulent factors [53,54]. Moreover, therapeutic BoNT has been known to avert the irregular heart rhythm even after open heart surgery [55]. Therapeutic BoNT injection has been reported to improve blood circulation and oxygen supply thereby improving the survival rate of subjects with ischemic conditions [12,[56][57][58]. Besides, implementation of therapeutic BoNT is an eminent clinical practice to manage movement disorders in a wide range of neurological deficits [11][12][13].
Remarkably, therapeutic BoNT appear to provide neuroprotection against cerebral ischaemic insults [59]. Eventually, the existing list of diseases treated by therapeutic BoNT are associated with increased levels of ACE2 in the respective organs or in the circulation. For example, abnormal levels and functions of ACE2 have been recognized J o u r n a l P r e -p r o o f as a biomarker and therapeutic target for chronic headache, dyspnoea, bronchitis, cardiovascular disorders, hypertension, neuroinflammation and neurological disorders [60][61][62][63][64]. While the underlying mechanisms of therapeutic BoNT on the recovery of the aforementioned clinical complications remains unclear, therapeutic BoNT can be proposed to suppress the expression of the ACE2 [37,58]. Taken together, therapeutic BoNTs may also be used as a preventive measure against SARS-CoV-2 infection in population at risk.
Recently, rigorous implementation of many drugs and therapeutic strategies has been envisaged against COVID-19. While the prediction of the clinical outcome of the proposed drugs and therapies are the major focus, the underlying mode of action and possible adverse effects associated with such therapeutic approaches may not be excluded. For example, the hydroxychloroquine has been proposed as a treatment regime for COVID-19 along with azithromycin [65]. However, there exist some considerable remarks that the antiviral mechanism of action of these drugs against SARS-CoV-2 or their probable effects to reduce the pathological events of COVID-19 remains unknown. Besides, the use of both drugs has been reported to be associated with some fatal side effects [66,67]. While the transplantation of different types of stem cells has been proposed to improve the outcome of COVID-19 [68], the use of stem cells has been known to be associated with the risk of developing tumor growth and metastasis [69]. However, there has been some mounting optimism generated on the implementation of the aforementioned drugs and cell-based therapeutics against COVID-

J o u r n a l P r e -p r o o f
In 2008, the FDA indicated the potential dangers of the BoNT as a therapeutic agent and it further claimed that BoNT could spread beyond the injection site [70,71]. Notably, high doses of BoNT have been known to elicit adverse effects on the CNS [13]. Toxic effect, if any, found due to the treatment using BoNT could be neutralized using available antibodies against BoNT [72]. Besides, the implementation of antioxidants along with the BoNT treatment has also been reported to minimize its side effects [73]. Moreover, it has been clearly reported that the adverse effects of therapeutic BoNT are temporary and reversible [74]. The use of BoNT at a very low dose has been reported to be comparatively safe [12,13,47,53]. It has been repeatedly demonstrated that very mild doses of BoNT can attenuate many human diseases even at 10 to 20 units [12,13,45,75].
Eventually, a mild dose of BoNT injection has been an accepted practice for the aesthetic procedure even in healthy individuals despite some rare reports on its adverse effects depending upon some criteria [11,13,51,53]. A very recent retrospective study by Chiu SY in 2020 indicates that the FDA approved 400 units of BoNT as a maximum dose per treatment session to be implemented for the patients with neurological deficits with a 3month interval [76]. Even such a high-end dose of BoNT has been reported to improve the functional outcome in patients without any obvious adverse effects [76]. Therefore, a mild dose of BoNT can be translated for the benefit of individuals with COVID-19.

Conclusion
The present exponential incessant in COVID-19 requires ultimate eradication therapy.
While plans like nation-wide lockdown, social distancing and self-quarantine have been imposed by many countries to avoid the spread of SARS-CoV-2, the establishment of vaccination, identifying the pharmacological strategies and disease modifying therapies J o u r n a l P r e -p r o o f treatment like Ivermectin [78] have gained the front-line attention but their therapeutic efficacy needs to be tested against COVID-19. Adding to that, therapeutic BoNT, regarded as a magic bullet appears to have a proven curative effect against many explicated symptoms of COVID-19 including the respiratory failure, cardiac defects and neurological deficits. Importantly, therapeutic BoNT has the capacity to migrate from intramuscular injection site to the brain and other organs [71]. Therapeutic BoNT is relatively safe and the beneficial effect of a single therapeutic dose of BoNT appears to be long lasting [79]. The therapeutic forms of BoNT are available under the brand names like BOTOX® or OnabotulinumtoxinA, Dysport, Bocouture and Jeuveau in all countries.
Thus, this article strongly proposes the conception that therapeutic forms of BoNT can also be considered as a potent treatment against COVID-19, however with high precaution measures.

Declaration of Competing Interest
The author declares no competing interest.