Toremifene-Melatonin The Gold Standard for COVID-19 Treatment

By Doris Loh

There is currently no gold standard treatment for SARS-CoV-2 infections. Several vaccines have completed Phase III trials with impressive success rates. Whether these vaccines will be as effective in the face of new strains with new mutation sites is a question yet to be answered. Meanwhile, two major clinical trials for COVID-19 vaccines and a promising COVID-19 drug have been paused due to safety concerns [1-3]. The combination of ascorbic acid and melatonin could potentially be extremely effective in the treatment for COVID-19. However, most hospitals and medical professionals may not be able to approve their sole use, despite mounting evidence of their efficacy. 

For those infected by SARS-CoV-2 who do not wish to use pharmaceutical drugs, they are left with natural alternatives that are often as effective if not more. However, when a COVID-19 patient is hospitalized, or placed under a physician's care, what safe and effective choices are available to treat COVID-19 patients? 

The purpose of this article is to crucially examine a unique combination featuring a repurposed breast cancer drug called toremifene and the ancient molecule melatonin. This article will compare the potentials of the combined effects of toremifene and melatonin as well as the independent ability of melatonin against some known drugs that are being considered or used as treatment for SARS-CoV-2 infections.

In the selection of repurposed drugs for COVID-19, efficacy and safety are the first considerations. One of the more promising drugs with an established safety profile that can be used to treat COVID-19 is Ivermectin.

ivermectin

Ivermectin is a well-known anthelmintic agent from the late-1970s with relatively low toxicity, and is generally well tolerated. Ivermectin has been shown to be effective against flaviviruses that cause dengue fever, and certain types of encephalitis viruses [4]. To date, only one in vitro study examined the antiviral effects of ivermectin against SARS-CoV-2 infection [5].

In their experiment, Caly et al. infected true cells with the SARS-CoV-2 isolate Australia / VIC01 / 2020 [5]. This sample of the SARS-CoV2 virus was isolated in January 2020 from a man from Wuhan. This man was the first patient diagnosed with COVID-19 in Australia [6]. As we now know, the predominant global strain of SARS-CoV-2 is quite different from those from January as the result of selective mutations [7]. Thus the results from the Caly study require confirmation using the predominant strains in circulation today. 

Ivermectin Mechanism of Action in SARS-CoV-2

Ivermectin has been shown to disrupt SARS-CoV-2 replication and survival in vitro [5]. Ivermectin accomplishes this by inhibiting the host importin α / β transporter protein. The inhibition of this protein reduces the translocation of the SARS-CoV-2 structural protein called nucleocapsid (NCP) from the cytoplasm to the nucleus [8]. Viruses such as HIV-1 and dengue that rely on importin α / β nuclear import for replication have been successfully inhibited by ivermectin [9]. However, this feature is not unique to ivermectin.
Bharat et al. (2020) used in silico models to compare interactions between importin α3 (IMA3), Ivermectin and 20 plant molecules [10]. Bharat et al. found that all 20 plant molecules investigated showed better interactions with IMA3 than ivermectin, with one plant molecule ATR1001 showing exceptional superiority to Ivermectin in its ability to inhibit importian α3, effectively blocking nuclear import of SARS-CoV-2 proteins.

The plant molecule ATR1001 was the most potent inhibitor of IMA3 among all molecules tested, including ivermectin, because it exhibited the lowest binding energy of -7.290 Kcal / mol. The lower the binding energy, the more effective the inhibitor. Ivermectin also had a low binding energy, but was only detected at -4.946 Kcal / mol at its lowest energy conformation without any hydrogen bonds [10]. 

 In addition to spike protein (SP) and nucleocapsid (NCP), SARS-CoV-2 uses many other non-structural proteins including open reading frames (ORFs), for fusion, entry, and replication processes [11]. The inhibition of NCP by ivermectin may not be adequate to completely reverse all symptoms associated with COVID-19 infections, especially in light of the D614G mutation that may change the conformation of the spike protein, resulting in increased infectivity and alteration of antigenicity [12] . 

To date, there are no peer-reviewed studies that can definitively show the binding of ivermectin to SARS-CoV-2 proteins. A preprint uploaded in April explored the potential of ivermectin to bind two proteins of SARS-CoV-2 simultaneously via molecular docking modeling technique [13]. Another preprint from May 2020 examined the docking of ivermectin to spike protein, but the authors are still attempting to validate their results by computational models [14]. As of the publication of this article, plant molecules with higher binding efficiencies have not been tested against SARS-CoV-2 inhibition in vitro or in vivo either. 

The in vitro results obtained by Caly et al. has sparked an unprecedented level of interest in many nations including Bangladesh, Bolivia, Guatemala, Peru, India and even the United States to explore the drug as a preventative against SARS-CoV-2 infections [15]. The fact that ivermectin is an inexpensive, readily available drug that has been used for decades to treat livestock and humans as an antiparasitic makes it ever more attractive. 

It is noteworthy that the Indian Council of Medical Research (ICMR) has not recommended the use of ivermectin for the treatment of COVID-19, citing lack of evidence from RCT results [16], despite the encouraging results published in a recent pre-print (Nov, 2020) detailing a hospital-based matched case-control study involving 186 case-control paired healthcare workers from Bhubaneswar, India from September to October 2020. The authors of the pre-print found that a two-dose ivermectin prophylaxis at 300 μg / kg (72-hour gap between doses) was associated with a 73% reduction of COVID-19 infection among cohorts for one month following administration [17]. 

The reluctance of the ICMR to include ivermectin in their national COVID-19 guidelines may be due in part to concerns regarding the potential side effects of Ivermectin in humans. 

Biological Effects and Safety of Ivermectin

Traditionally, ivermectin (IVM) is viewed as an exceptionally safe drug for use in humans [18] even though some earlier in vitro studies have found IVM in concentrations of 1-280 μΜ to inhibit mammalian cell viability [19, 20]. 

Recently, Li et al. (2020) found IVM to inhibit cell proliferation and cell cycle progression by promoting apoptosis via the regulation of energy metabolism [21]. Another peer-reviewed paper published at Chemosphere by Zhang et al. (2020) was able to show for the first time that ivermectin can induce cytotoxic effects that may be potentially dangerous to human health [22]. 

Using a colony formation assay, Zhang et al. demonstrated that IVM treated cells exhibited decreased colony formation in a dose-dependent manner, showing IVM can inhibit proliferation of human cells [22]. IVM also was observed to induce DNA oxidative damage and DNA double-strand breaks [22]. The genotoxicity and cytotoxicity in cells treated with IVM exhibited increased expression of autophagic proteins, leading to mitochondrial damage, membrane permeability, and decreased ATP production that eventually resulted in cell death [22]. 

Ivermectin, Autophagy, and SARS-CoV-2 Replication

The fact that Zhang et al. demonstrated that IVM can induce increased autophagy in HeLa cells via the AMPK / mTOR pathway leading to apoptosis [22] now casts certain doubts on the exact role of IVM in SARS-CoV-2 replication.

The serine / threonine protein kinase ULK1 mediates the formation of autophagosomes, a key initial event in autophagy [23]. SARS-CoV-2 has been observed during in vitro cellular cleavage assay experiments to use ULK1 as a novel bona fide substrate for papain-like protease. ULK1 therefore, is able to enhance viral replication in early stages. However, whether ULK1 is pro-viral or antiviral during late stages of viral infection remains unclear since ULK1 has previously been reported to regulate antiviral innate immune signaling [24, 25]. 

Two well-known drugs currently explored as treatment for SARS-CoV-2 infections, chloroquine and hydroxychloroquine, are known autophagy inhibitors [26]. Yang et al. (2020) found that the autophagy process and endocytic pathway play key roles in facilitating viral entry for coronaviruses, including SARS-CoV-2 [27]. 

Since the role of autophagy in SARS-CoV-2 viral replication is not clearly understood, there is an urgent need to clarify the correct timing in the use of drugs like ivermectin and hydroxychloroquine with opposite effects on autophagy, in order to harness optimal inhibition of viral replication cycles. 

Hydroxychloroquine, Remdesivir, Dexamethasone & Methylprednisolone

Hydroxychloroquine: Is Autophagy Inhibition a Double-Edged Sword in COVID-19?

Hydroxychloroquine (HCQ) is a well-established antimalarial drug that is also frequently used to treat COVID-19. Like Ivermectin, HCQ has been shown to be an inhibitor of the dengue virus (flavivirus) [28], but no study has shown the exact mechanism (s) employed by HCQ against SARS-CoV-2. 

One study comparing the ability of hydroxychloroquine and chloroquine (CQ) to inhibit SARS-CoV-2 replication in Vero cells found HCQ to be less toxic than CQ [29]. The authors also confirmed that both HCQ and CQ could disrupt autophagic processes by elevating the pH of acidic intracellular organelles endosomes and lysosomes [29]. 

During autophagy, lysosomes are responsible for the clearance and recycling of abnormal proteins or organelles. Deregulated autophagy is often associated with many pathologies, including cancer. Hence the use of autophagy inhibitors like HCQ has emerged as viable cancer treatment alternatives [30]. 

The disruption of the endocytosis / lysosome pathways has been shown to be effective in deterring SARS-CoV-2 replication. SARS-CoV-2 typically enters host cells via endocytosis. Upon exit from endosomes, the virus replicates in the cytoplasm where it assembles and matures in trans-Golgi, and subsequently released via secretory vesicles. HCQ blocks viral entry and post-translational modifications by de-acidifying these organelles which are dependent upon an acidic environment for optimal functioning [31]. 

In fact, during autophagy, acidic pH is essential for the degradation of cytosolic proteins and damaged organelles via the formation of autophagosomes and the fusion with late endosomes / lysosomes. Chloroquine is able to de-acidify organelles involved in autophagy, additionally, it has been shown to inhibit autophagy directly by preventing autophagosome fusion with lysosomes [32]. Since autophagy is necessary to maintain cellular homeostasis [33], it is not surprising to find current research identifying the combination of autophagy inducers and autophagy inhibitors as the smarter strategy in the treatment of cancer [34].

Even though SARS-CoV-2 is dependent upon an acidic environment for host cell entry and replication, the use of HCQ and other autophagy inhibitors that can de-acidify endolysosomes and Golgi may underlie immunosuppressive effects that could lead to counterproductive effects. 

Some of the cited potential adverse effects of autophagy inhibition by HCQ and CQ include renal tubular dysfunction as direct result from the accumulation of damaged mitochondria due to lack of clearance through mitophagy and increased oxidative stress [35, 36]. In other preclinical studies, CQ inhibition of autophagy worsened the outcomes in ischemic cardiac injury and sepsis-induced liver or lung injury [37-39]. 

It is apparent from numerous cell biology studies that de-acidification can result in significant changes in fundamental cellular functions, including profound modifications in the structure, function, and cellular positioning of endolysosomes and Golgi, as well as the intracellular signaling between these organelles [31 ]. To what extent HCQ and CQ affect organelles and cell biology is an unanswered question that requires urgent clarification.

RdRp Inhibition

In silico molecular docking experiments show that HCQ can bind to the RNA dependent RNA polymerase (RdRp) active site of SARS-CoV-2 [40]. SARS-CoV-2 is dependent upon RdRp for the replication and transcription of its genome [41]. Studies using computational analysis demonstrated that inhibition of RdRp active sites are therapeutic options to suppress SARS-CoV-2 viral replication [42]. Cryo-electron microscopy studies revealed that the antiviral drug Remdesivir can potentially also target RdRp enzymes in SARS-CoV2 [43].

redevive 

Remdesivir was originally designed as an antiviral against the Ebola virus. A trial involving 1,062 COVID-19 patients showed shorter recovery time in patients who received remdesivir than controls [44], but the evidence on reduced mortality was lacking. Results from a large, randomized, mortality trial (SOLIDARITY trial) conducted in 405 hospitals from 30 countries involving 11,266 patients on repurposed antiviral drugs that included remdesivir and hydroxychloroquine found little or no effect on mortality, initiation of ventilation and duration of hospital stay in study participants treated with these drugs [45]. 

Dexamethasone and Methylprednisolone

Dexamethasone and methylprednisolone are synthetic corticosteroids with functions and features similar to natural glucocorticoids. Even though these two drugs are currently used to treat COVID-19 patients, they have not been shown to be able to inhibit SARS-CoV-2 viral replication. The association of dexamethasone with reduced mortality rates in COVID-19 patients is controversial in that the drug has been found in one study to lower mortality rates only in patients receiving invasive mechanical ventilation [46, 47]. In addition, both of these drugs when administered at high doses are known to cause liver damage and toxicity [48, 49]. Interestingly, one mortality study on survival rates in COVID-19 and non-COVID-19 patients receiving intubation or mechanical ventilation found the use of methylprednisolone to be significantly associated with negative outcomes [47].

Traditionally, dexamethasone is used for the attenuation of hemolysis and thrombocytopenia [50, 51]. Both of these conditions are extremely relevant in SARS-CoV-2 disease progression involving the complement system and vascular dysfunctions [52. 53]. Although dexamethasone may be able to attenuate SARS-CoV-2 symptoms, the use of this drug is known to induce bone loss by increasing the expression of 4-hydroxylase (CYP24A1).

Dexamethasone and Osteoporosis

Chronic use of glucocorticoids such as dexamethasone and methylprednisolone can cause rapid bone loss and clinical osteoporosis. Dexamethasone is a potent glucocorticoid that can significantly enhance renal expression of vitamin D-24-hydroxylase, which degrades vitamin D metabolites such as 25-hydroxyvitamin D3 and 1alpha, 25-dihydroxyvitamin D3 (1,25 [OH] 2D3), the active hormonal form of vitamin D3 [54]. Glucocorticoid-induced osteoporosis has been demonstrated to be caused by direct action of the steroids on bone [55]. Administration of glucocorticoids such as dexamethasone and methylprednisolone in older patients vulnerable to increased bone loss should warrant greater caution.  

It is unfortunate that most pharmaceutical drugs, however effective, usually have side effects. Sometimes these effects may even be cytotoxic and irreversible. The choice for an ideal treatment for SARS-CoV-2 would be a drug that can both inhibit replication of the virus and be enhanced by a natural, safe adjuvant that can ideally attenuate all the known symptoms caused by the virus, with the added ability to ameliorate and / or reverse potential side effects of the antiviral drug at the same time. 

Does such an ideal combination even exist? 

The Toremifene-Melatonin Clinical Trial 

In September, a good-sized clinical trial called "SENTINEL"(ClinicalTrials.gov Identifier: NCT04531748) involving 390 participants, sponsored by Reena Mehra, MD (the Cleveland Clinic) quietly began without much attention from the media [56]. What is unusual about this randomized, double-blind, controlled clinical trial is that the study seeks to evaluate the effects of the ancient molecule melatonin administered together with an FDA-approved anticancer drug called toremifene that has been repurposed for COVID-19. The trial will enlist adults displaying mild COVID-19 symptoms and treat them over a 14-day intervention period [56]. 

During the trial, the oral dosage for toremifene is 60 mg / day for 14 days. The oral dosage for melatonin is an eye-opening 100 mg for Days 1 and 2 (40mg in the morning and 60mg in the evening), and 60 mg on Days 3 to 14, (20mg in the morning and 40mg in the evening). The active comparator arm will receive melatonin in the exact same dosages, plus a placebo for toremifene. There will also be a comparator placebo arm as control. This is an extremely well-designed study that may reveal exactly how effective melatonin on its own, or in combination with toremifene may be against SARS-CoV-2 infections.

Almost everyone has heard of using melatonin for COVID-19 treatment. The use of melatonin as safe and effective treatment for COVID-19 infection has been extensively researched and documented. These peer-reviewed studies present detailed analysis of various mechanisms that render melatonin to be an ideal candidate that can address the comprehensive array of pathophysiologies from SARS-CoV-2 infections [57-67].

On the other hand, the anti-cancer drug torimefene is rarely known outside of oncology. Why would an anticancer drug be repurposed for COVID-19 and used in combination with melatonin? To me, combining toremifene with melatonin is truly a brilliant strategy that should be able to achieve high success rates. 

This article will explain why this combination can be a winning formula against SARS-CoV-2 infections and be established as the gold-standard treatment of choice for SARS-CoV-2 going forward.

What is Toremifene?

Toremifene, marketed under the name of Fareston, was first approved for use by the FDA in 1997 as a drug based on its in vitro activity against breast cancer. Toremifene is a nonsteroidal triphenylethylene derivative that is structurally similar to tamoxifen, a well-known breast cancer drug. Toremifene is a SERM (Selective Estrogen Receptor Modulator) as it is both an estrogen agonist and antagonist. However, toremifene has lesser uterotrophic effect than tamoxifen, and is therefore better tolerated than tamoxifen [68, 69]. 

Estrogen & Viral Inhibition: The Untold Stories

An overexpression of estrogen receptors plays a critical role in the inhibition of viral replication and infectious processes. In 2016, changes in estrogenic signaling after the use of selective estrogen receptor modulators (SERMs) were shown to successfully inhibit replication of influenza A virus in female subjects. SERMS bind to estrogen receptors to cause conformational changes that can both initiate induction or suppression of target gene transcription. As early as 2008, estrogen was already found to be able to regulate antiviral responses [70-72].  

Toremifene is a first-generation nonsteroidal selective estrogen-receptor modulator. This drug is FDA-approved for the treatment of advanced breast cancer in postmenopausal women. Toremifene is 99% bound to plasma protein with good bioavailability [73]. In addition to breast cancer, the drug has also been used to treat men with prostate cancer with some unexpected results.

Even though a large-scale 3-year phase III clinical trial using 20 mg. toremifene citrate showed no significant risk reduction in prostate cancer [74], another large-scale phase III study found 60 mg. toremifene to markedly increase bone mineral density of the hip and spine in men with prostate cancer [75]. Similarly, a study on postmenopausal women with breast cancer found that administration of toremifene (40 mg / day) prevented the age-associated reduction in bone mineral density [76].

So unlike dexamethasone that can induce bone loss, using toremifene could actually prevent bone loss in susceptible patients. But what is the rationale behind the use of toremifene to treat SARS-CoV-2 infections?

Toremifene Inhibits Ebola, MERS-CoV, SARS-CoV and Filoviruses

The classical estrogen receptor-related antiviral pathway regulates immune cells and signaling [77], whereas toremifene may actually be able to prevent viral fusion with host cells by interacting and destabilizing the virus membrane glycoprotein, effectively blocking viral replication. 

A study in 2016 demonstrated that toremifene could bind to the glycoproteins of the Ebola virus. The interactions between toremifene and attachment protein GP1 and fusion protein GP2 in Ebola virus decreased the stability of the viral glycoproteins and prevented viral fusion and replication [78]. Other studies showed toremifene to be extremely effective in the inhibition of MERS-CoV, SARS-CoV-1, as well as filoviruses [79-81].

Even though the effectiveness of toremifene against Ebola, MERS-CoV, SARS-CoV-1, as well as filoviruses have been extensively documented [79-81], is toremifene an effective antiviral for SARS-CoV-2? 

Toremifene Inhibits SARS-CoV-2 Viral Replication

The ground-breaking study by Martin and Cheng (September 2020) identified toremifene as a strong candidate for the potential treatment of SARS-CoV-2 [82]. Why?

The SARS-CoV-2 genome encodes 29 different proteins. 4 are structural proteins, 16 are non-structural proteins, and 9 are accessory proteins. The spike glycoprotein is an important structural protein involved in binding to host cell receptors, while the other three structural proteins (Envelope, Membrane and Nucleocapsid) are important for viral assembly and replication [83].

In their peer-reviewed study published in September 2020, Martin and Cheng used homology modeling, molecular docking, molecular dynamics simulation, and binding affinity calculations to show that toremifene has the potential to inhibit the important SARS-CoV-2 structural spike glycoprotein and prevent the fusion of viral membrane to host cells via a perturbation to the fusion core by changing configurations of key structural regions such as heptad repeat 1 (HR1) within the fusion core [82]. 

Toremifene Binds to Structural Spike Glycoprotein

[Source: Martin WR, Cheng F. Repurposing of FDA-Approved Toremifene to Treat COVID-19 by Blocking the Spike Glycoprotein and NSP14 of SARS-CoV-2. J Proteome Res. 2020; 19 (11): 4670-4677. doi: 10.1021 / acs.jproteome.0c00397]

 

Stick Representation of Final Pose for Toremifene with the Spike Glycoprotein at 500 nanoseconds of Simulation

[Source: Martin WR, Cheng F. Repurposing of FDA-Approved Toremifene to Treat COVID-19 by Blocking the Spike Glycoprotein and NSP14 of SARS-CoV-2. J Proteome Res. 2020; 19 (11): 4670-4677. doi: 10.1021 / acs.jproteome.0c00397]

In addition, Martin and Cheng found that toremifene exhibited an extremely strong interaction between the nonstructural methyltransferase protein NSP14 that may actually inhibit viral replication. NSP14 is an important nonstructural protein in SARS-CoV-2 responsible for viral replication and transcription. NSP14 is the exonuclease (ExoN) domain that proofreads nascent RNA strands and excises misincorporated nucleotides [84]. Inhibition of NSP14 by toremifene may prove to be an effective target that can potentially disrupt SARS-CoV-2 viral replication. 

Toremifene Binds to Nonstructural NSP14

[Source: Martin WR, Cheng F. Repurposing of FDA-Approved Toremifene to Treat COVID-19 by Blocking the Spike Glycoprotein and NSP14 of SARS-CoV-2. J Proteome Res. 2020; 19 (11): 4670-4677. doi: 10.1021 / acs.jproteome.0c00397]

 

Stick Representation of Final Pose for Toremifene with NSP14 at 500 Nanoseconds of Simulation

 

[Source: Martin WR, Cheng F. Repurposing of FDA-Approved Toremifene to Treat COVID-19 by Blocking the Spike Glycoprotein and NSP14 of SARS-CoV-2. J Proteome Res. 2020; 19 (11): 4670-4677. doi: 10.1021 / acs.jproteome.0c00397]

Now that we know some of the mechanisms used by toremifene to inhibit SARS-CoV-2 viral replication, the next question is, how safe is toremifene?  

Safety of Toremifene

Toremifene when administered at typical oral dosage of 60 mg daily is generally considered to be safe [73]. However, all pharmaceutical interventions almost always will have side effects, and toremifene is no exception.

This is where the combined use of toremifene and melatonin becomes intriguing because these two molecules are synergistic. In addition to toremifene, melatonin has been shown to reduce toxicity and increase the efficacy of a large number of drugs, including antiretroviral therapies for HIV patients [85]. Let us find out how melatonin can significantly reduce safety risks and enhance the antiviral effects of toremifene.

Toremifene and Melatonin are Synergistic

A network-based analysis (Cheng et al. 2020) on different approaches that combine anti-inflammatory and antiviral therapeutics identified a remarkable synergistic effect between toremifene and melatonin for patients infected with SARS-CoV-2. The authors of the study believe that the combined anti-inflammatory and antiviral effects of melatonin and toremifene may reduce viral infection and replication, stabilizing aberrant host inflammatory responses, and rescuing dangerous pulmonary and cardiovascular conditions often found in COVID-19 infections [86].

[Source: Cheng F, Rao S, Mehra R. COVID-19 treatment: Combining anti-inflammatory and antiviral therapeutics using a network-based approach. Cleve Clin J Med. 2020 Jun 30. doi: 10.3949 / ccjm.87a.ccc037]

Let us explore this concept in detail.

Combined Use of Melatonin and Toremifene May Reduce Side Effects

Potential Elevation of Liver Enzyme

The use of toremifene has been associated with mild-to-moderate elevation of OTHER or AST liver enzymes. The increased liver enzymes are usually transient and reversed upon cessation of treatment [87]. Long-term use (60 months) of toremifene has been shown to induce fatty liver disease together with elevated ALT, especially in Asian women. After discontinuation of treatment, elevated serum ALT normalized in over 92% of patients without any liver-related death or progression to liver cirrhosis [88]. 

Using melatonin in combination with toremifene addresses this issue, as melatonin has been demonstrated in a clinical trial to significantly decrease elevated serum ALT enzymes in NAFLD patients [89].

Potential Single Strand DNA Damage

Toremifene is extensively metabolized by CYP3A4, an enzyme belonging to the P450 superfamily of monooxygenase proteins that catalyze a variety of reactions involved in drug metabolism. One of the major metabolites of toremifene is 3,4-dihydroxytoremifene.

In a cytotoxicity study on toremifene and tamoxifen, the authors discovered that catechols of 3,4-dihydroxytoremifene could induce a higher level of single strand DNA breaks compared with the phenols in the different cell lines examined. Even though catechols represented a minor role in cytotoxic effects compared with their phenol analogues, the catechols induced more DNA damage at nontoxic doses in breast cancer cells. The authors of the study believed that the o-quinones formed from catechols may have contributed to genotoxicity in vivo [90]. 

Melatonin, DNA Strand Breaks & O-quinone Detoxification: The NQO2 Connection

DNA single ‐ strand breaks (SSBs) are the most common forms of DNA damage, and are usually repaired by the BER pathway [91] In animal models, melatonin administered at 1 mg / kg body weight blocked DNA single‐ and double ‐ strand breaks in brain cells induced by 60 ‐ Hz magnetic fields [92]. Melatonin has been extensively documented to protect against DNA damage caused by different mechanisms. Melatonin is recognized for its ability to protect against DNA damage not only through its antioxidant features, but its extremely versatile interactions with various components of the DNA damage response at different transduction, mediation, as well as functional levels [93].  

Melatonin may exert an additional layer of DNA damage protection during toremifene administration through its interactions with the NRH: quinone oxidoreductase 2 (NQO2), a class of two-electron quinone reductase that is responsible for the detoxification of catechol quinones that may be formed as metabolites of toremifene [94].

Even though NQO2 has been regarded by some as the third binding site for melatonin (MT3) [95], its affinity to the enzyme is much lower when compared to potent flavonoid inhibitors like quercetin, resveratrol, and pharmaceuticals such as chloroquine [96, 97 ]. Even though melatonin has been indicated to inhibit NQO2, it has been demonstrated that melatonin would only bind to the free oxidized (FAD) form of NQO2 [97], possibly in the capacity of a reducing agent (electron donor). Indeed, Tan et al. (2007) proposed that melatonin could be a naturally occurring co-substrate for NQO2 [98]. Additional research will greatly assist in the clarification of melatonin's role for the detoxification of catechol quinones from toremifene metabolism through NQO2.

Potential QT prolongation

QT extension is a measure of delayed ventricular repolarization, which means the heart muscle takes longer than normal to recharge between beats. It is an electrical disturbance which can be seen on an electrocardiogram (ECG). Excessive QT prolongation can trigger tachycardias such as torsades de pointes (TdP) [99].

Toremifene has been shown to prolong the QTc interval in a dose- and concentration-related manner [100]. Variations in the KCNQ1 and KCNH2 genes have been associated with long QT syndrome [101, 102]

In pinealectomized rats, administration of melatonin prevented QTc prolongation and decreased KCNH2 gene expression levels [103].

Potential Thromboembolic Events  

Long-term administration of toremifene at 40 mg to 60 mg over a 3 to 5 year period has been associated with adverse thromboembolic events such as deep vein thrombosis, cerebrovascular accident, and pulmonary embolism in breast cancer patients [104, 105]. Short-term administration of toremifene during viral infections may not have the same thromboembolic effects, especially when combined with high-dose melatonin.
Melatonin has been studied extensively for its cardioprotective and neuroprotective effects [106-108]. The importance of melatonin in stroke management and prevention of recurrent strokes has been exemplified in studies demonstrating the significant degree of influence over the development of cerebrovascular accidents in young adults [109].  

The study of melatonin as a therapeutic agent in the prevention of thromboembolic events can be traced as far back as 2011 when melatonin was shown in vitro to significantly stimulate the secretion of TFPI in vascular endothelial cells [110]. Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits early phases of the procoagulant response [111]. 

COVID-19 patients are often found to be challenged by higher disease severity and morbidity due to development of prothrombotic events that may be caused by dysregulation in coagulation pathways.

Platelets and the regulation of their activation play important roles in many thromboembolic events including deep vein thrombosis [112,113].

An important study (Manne et al. 2020) demonstrated altered platelet gene expression and functional responses in patients infected with SARS-CoV-2 via RNA sequencing techniques. Protein ubiquitination, antigen presentation, and mitochondrial dysfunction were among the major pathways observed to be affected by the altered gene expressions in platelets. In addition, the authors of the study detected the presence of SARS-CoV-2 N1 protein mRNA in platelets of some COVID-19 patients, indicating possible direct damage from viral fusion and replication [114].

Zhang et al. (2020) found increased mean platelet volume (MPV) and decreased overall platelet count to be correlated with platelet hyperactivity and detectable SARS-CoV-2 RNA in blood stream of critically ill patients [115]. The authors believed that the virus spike protein directly stimulated platelets to cause coagulation and platelet aggregation enhancing the severity of thrombosis in patients [115]. 

Melatonin is a unique pleiotropic molecule that is often found to assume contradictory roles. One one hand, melatonin has been shown to suppress platelet activation and hyperactivity. Yet melatonin is also able to protect platelets from thrombocytopenia (decreased platelets) [116-118]. 

It is therefore not surprising to find extensive discussions in peer-reviewed literature on the use of melatonin as an effective and safe therapeutic agent for the treatment of COVID-19 associated myocardial injury and thromboembolic events [59, 61, 119, 120].

The short-term 14 day administration of toremifene (60 mg / day) for COVID-19 treatment is unlikely to induce adverse thromboembolic events. However, the virus SARS-CoV-2 has been demonstrated to cause extensive endothelial damage, myocardial injury and thrombotic complications in severe COVID-19 patients [121-124]. The timely administration of melatonin may be an efficacious treatment alternative, especially when combined with toremifene.

In addition to the anti-inflammatory and immunomodulatory effects of melatonin, melatonin may actually be able to inhibit viral fusion and replication. In this respect, the results from the Toremifene-Melatonin SENTINEL trial will be extremely revealing. The SENTINEL trial, using high-dose melatonin at 100 mg for the first two days (40mg in the morning and 60mg in the evening), and 60 mg for the remaining 12 days (20mg in the morning and 40mg in the evening), may show once and for all whether melatonin on its own can be the 'silver bullet' against SARS-CoV-2 [125]. 

Melatonin - The 'Silver Bullet' for SARS-CoV-2

A team of scientists from the Cleveland Clinic published a peer-reviewed study at Plos Biology on November 6, 2020. This landmark study showed for the first time, the existence of a definitive correlation between the active use of melatonin and reduced positive laboratory test results for SARS-CoV-2 [126]. To identify specific drug-outcome relationships for COVID-19, the team led by Zhou et al. performed extensive analysis of COVID-19 registry data obtained from the Cleveland Clinic Health System in Ohio and Florida on 26,779 individuals tested for COVID-19 between March 8 to July 27, 2020. Patients who were actively taking melatonin at the time of testing were found to be associated with a 28% reduced likelihood of positive laboratory test result of SARS-CoV-2 confirmed by rtPCR assays. Importantly, there was a 52% reduction of positive laboratory test results associated with African Americans who were actively taking melatonin at time of testing [126].

The authors of the study concluded that melatonin had the potential to both PREVENT and TREAT SARS-CoV-2 infections [126].

The Cleveland Clinic study became the focus of major news media, as well as the target for critics who pointed out that correlation does not imply causation. I believe the critics are incorrect this time. Why

Melatonin May Inhibit SARS-CoV-2 to Prevent Disease Progression

A rather unexpected feature of melatonin that is rarely discussed, but has been observed and documented, is its ability to inhibit viral replication. The fact that the Cleveland Clinic study observed patients who were actively taking melatonin had reduced positive COVID-19 rtPCR results supports this theory. How does melatonin exert its antiviral effects?

MEL Inhibits Viral Replication via EGFR

Growth factor receptors (GFRs) are transmembrane proteins expressed in all eukaryotic cells. Their primary function is to bind to extracellular polypeptide growth factors and initiate a cascade of signaling events for the regulation of cell growth. 

What is less known about GFRs is that they are involved in the process of viral infections. Many viruses use GFRs for host cell membrane binding, internalization and replication. GFRs have been identified as necessary for the entry and replication of many viruses, including coronaviruses such as SARS-CoV-2 [127]. 

Recently, Klann et al. (2020) identified extensive changes in the phosphorylation of host and viral proteins after SARS-CoV-2 infection. By inhibiting growth factor receptor downstream signaling, the team was actually able to prevent SARS-CoV-2 replication [128]. 

Melatonin may actually be extremely effective in halting the viral infection progression because melatonin can regulate growth factor receptor signaling that is most critical during viral replication processes, including those of SARS-CoV-2 [129]. 

In 2017, scientists showed that hyperactive host responses to lung injury in SARS-CoV infections were mediated by epidermal growth factor receptor (EGFR) signaling, and that inhibiting EGFR signaling could effectively prevent an excessive fibrotic response to SARS-CoV and other respiratory viral infections [130].

An in vitro study on proliferative vitreoretinopathy (PVR) found melatonin to dose-dependently inhibit epidermal growth factor (EGF) -induced proliferation of human ARPE-19 cells. In addition, melatonin significantly reduced EGF-induced motility by suppressing cathepsin S (CTSS) expression in retinal pigment epithelial cells [131]. 

Melatonin Suppresses Cysteine ​​Proteases to Inhibit Viral Replication

SARS-CoV-2 uses two main routes of entry into host cells for replication. The structural spike protein can be activated at host cell membranes, resulting in fusion of the viral membrane with host cell plasma membrane [132]. An alternative route is through endocytosis where the virus enters host cells through the endosomal pathway facilitated by cysteine ​​proteases such as cathepsins L and cathepsin B. In vitro treatment with cathepsin L inhibitors has been shown to effectively decrease the entry of SARS-CoV-2 pseudovirion entry into cells by over 76% [133].

Cathepsins are Major Facilitators of Viral Replication in Endocytosis

Cathepsins such as cathepsin L (CatL) are endosomal cysteine ​​proteases that facilitate viral entry into human host cells by activating SARS-CoV-2 spike protein subunit 1 (S1) via cleavage. Upon successful cleavage of S1, the virus fuses with endosome membranes and viral RNA is subsequently released for replication. Hence the inhibition of cathepsins is now recognized as an effective target to block SARS-CoV-2 replication and infection [134,135].

 

Cathepsin L is a Major Protease that Cleaves SARS-CoV-2 Subunit 1 within intracellular Endosomes

[Source: Pišlar A, Mitrović A, Sabotič J, et al. The role of cysteine ​​peptidases in coronavirus cell entry and replication: The therapeutic potential of cathepsin inhibitors. PLoS Pathog. 2020; 16 (11): e1009013. Published 2020 Nov 2. doi: 10.1371 / journal.ppat.1009013]

 

Spike Protein Activation by Cathepsin L during Endocytosis

[Source: Pišlar A, Mitrović A, Sabotič J, et al. The role of cysteine ​​peptidases in coronavirus cell entry and replication: The therapeutic potential of cathepsin inhibitors. PLoS Pathog. 2020; 16 (11): e1009013. Published 2020 Nov 2. doi: 10.1371 / journal.ppat.1009013]

 

In addition to cathepsin L, cathepsin B has actually been demonstrated to be even more effective than cathepsin L in the cleavage of the S1 subunit in SARS-CoV-2 spike glycoproteins using proteolytic cleavage assays [136]

Melatonin is a known inhibitor of cysteine ​​proteases, including cathepsin L and cathepsin B [137, 138], therefore, it is entirely possible that melatonin may play an active role in the suppression of viral activation during SARS-CoV-2 infections. 

Melatonin Inhibits SARS-CoV-2 Main Protease (Mpro)

Coronaviruses, including SARS-CoV-2 contain a well-conserved main protease enzyme Mpro, which is vital during the replication life cycle of the virus. After viral RNA genomic particles (ppla and pplab from ORF1a and 1b) are released into the cytoplasm, they are cleaved by Mpro and PLpro. The successful cleavage of the RNA particles by Mpro and PLpro results in 15 new non-structural proteins (NSPs) that are integral to the replication-transcription complex. Without these new NSPs, the viral genome will not be able to replicate and generate individual sub-genomic mRNA templates required for the translation of the viral structural and accessory units [139]. 

Feitosa et al. (2020) identified 59 compounds that are able to bind and interact energetically with 74 Mpro-ligand complexes using molecular docking studies. Of these compounds, melatonin showed the greatest potential in its pharmacological properties against COVID-19. Melatonin was demonstrated in docking studies to reveal higher levels of energy interaction with Mpro compared to other candidates. The fact that melatonin can substantially reduce oxidative stress and inhibit cytokine storms as a result of its important immunomodulatory and anti-inflammatory features led the authors to reason that melatonin can be potentially effective in both early stage viral infection / replication inhibitor from Mpro inhibition, as well as later more severe stages of disease progression against hyper-inflammation [139]. 

Clinical trials using high-dose melatonin such as the toremifene-melatonin SENTINEL trial may be able to reveal exactly how melatonin can effectively inhibit viral replication on its own. 

Aside from Cleveland Clinic study by Zhou et al. in November 2020 [126] that showed a clear association of reduced positive laboratory results and melatonin intake, do we have other indications that melatonin is an effective viral replication inhibitor?

We do, and it is a most curious story about three children in Australia infected with SARS-CoV-2 from their parents.

The Curious Case Observations of Three Australian Children Infected by SARS-CoV-2

In March 2020, two parents attended a 3-hour wedding that was later identified to be responsible for a SARS-CoV-2 outbreak. After returning home 3 days later, the parents developed cough, congestion, fever, and headache. On day 7 after the parent's onset of symptoms, 2 of their 3 children aged 9 and 7 developed similar symptoms, while the 3rd child aged 5, was asymptomatic. The entire family was tested for SARS-CoV-2 when official notification of the wedding outbreak was released the day after the onset of symptoms in the 2 children (day 8). Both parents were SARS-CoV-2 PCR positive on nasopharyngeal (NP) swabs, while all 3 children were negative for SARS-CoV-2 on repeated NP swabs. 

The three children lived in close proximity to the parents. The asymptomatic 5 yr-old child even slept with the parents the entire time while the parents were sick. Researchers, intrigued by these children, analyzed blood samples from all family members and published their findings in a peer-reviewed study in Nature Communications [140]. 

The authors found that despite having no virological evidence of infection, all three children developed antibody and immune responses against SARS-CoV-2 specific epitopes. The 5 yr-old girl exhibited the strongest antibody / immune response despite being absolutely asymptomatic.

What is even more fascinating is that the active immune responses in the children were not accompanied by elevated plasma cytokine levels [140]. The authors of the study concluded that unique immune responses in children were responsible for preventing the establishment of SARS-CoV-2 infection. 

How could the 5 year old mount the strongest response yet remain completely asymptomatic? The girl lived in the same conditions and was exposed and infected just like her siblings di lei. 

The answer may lie in the chart below. 

In fact, children from ages 1-3 are often found with the highest levels of nocturnal serum melatonin in the ranges of 329.5 +/- 42.0 pg / mL. This is more than 10 times the amount found in individuals between the ages of 70-90 [141]. 

 

Melatonin is a known modulator of the immune system, exerting both pro as well as anti-inflammatory effects [142]. The high level of melatonin ensured a robust immune response in all three children without elevating pro-inflammatory cytokines that could initiate dangerous cytokine storms [140]. The potentially high melatonin in the youngest child may also explain the complete lack of symptoms di lei compared to her siblings di lei. It is possible that the youngest child had triple the level of melatonin. 

I believe that the high melatonin levels in all three children could have been responsible for the inhibition of viral replication because all three children showed repeated negative PCR results by nasopharyngeal (NP) swabs, even though they were positive for antibody and immune responses to the SARS -CoV-2 virus. Additionally, all children showed COVID-19-related symptoms except for the youngest child with the highest level of melatonin. The two parents, mother 38 years, and father 47 years, both tested positive for SARS-CoV-2 PCR by nasopharyngeal (NP) swabs [140]. It is entirely possible that inadequate melatonin levels was the reason for increased viral production. 

This curious incident of viral inhibition and immunity highlights a central issue in the prospect of using vaccines against the spread of SARS-CoV-2. 

Will Vaccines be Effective Against SARS-CoV-2?

Vaccines are designed to stimulate a person's immune system in response to a specific disease. An efficacious vaccine can theoretically protect the person from the disease because the person's immune system has been primed for the specific virus. It is believed that a primed infection will allow the adaptive immune system to develop appropriate immunity to viruses such as SARS-CoV-2. [143]. 

However, we currently lack thorough understanding regarding the different immune responses to SARS-CoV-2 infections, in that vaccine-induced protective immunity may actually differ from natural immunity due to immune-evasion strategies of the virus, as well as the extensive variations in immune responses between asymptomatic, mild, and severe cases during early and late stages of infections [144].

Human and viral proteins are homologous. That means the similarity in structure, relative position, and relationship can cause autoimmune issues. In fact, homology between human and viral proteins targeted by vaccines is a known factor in viral or vaccine-induced autoimmunity. The possibility of pathogenic priming from the use of vaccines leading to severe critical illness and mortality in COVID-19 as the result of autoimmune reactions has been raised, and warrants further attention [145]. 

An effective vaccine candidate against SARS-Co-2 could potentially act against infection, disease, or transmission. If a vaccine is capable of reducing any of these elements, it could theoretically contribute to the control of the spread of the disease. However, one of the most important efficacy endpoints, that of protection against severe disease and death is often difficult to assess in phase III clinical trials [146].

Another important consideration is how the mutation and phylogenetic diversification of the virus will affect efficacy of the candidate vaccine. Currently, experimental and in silico models suggest that potential vaccine candidates will likely be unaffected by the dominant global variant D614G mutation in SARS-CoV-2 spike protein [147]. The D614G variation took place in the important spike protein where the key amino acid changed from aspartic acid (D) to glycine (G) at position 614 [148]. The “G clade” mutation was rare in January, but gained global prominence beginning in Europe around late February, early March [149].

In theory, an efficacious vaccine and / or successful host adaptive immune responses could exert selective pressure for viruses to mutate, or what is commonly known as immune escape. Viral immune escape is most effective at intermediate values ​​of immune strength [150]. A new variant of SARS-CoV-2 known as 20A.EU1 emerged from Spain late June and subsequently spread to multiple European countries. The frequency of this variant has been detected to have increased from very low values ​​prior to 15th July to 40-70% in Switzerland, Ireland, and the United Kingdom in September. It is also found to be prevalent in Norway, Latvia, the Netherlands, and France [151].

Very little is known about this new variant, except that this cluster differs from ancestral sequences at 6 or more positions, including the mutation A222V in the spike protein and A220V in the nucleoprotein [151].

If the mutation is the result of selective pressure against successful adaptive immunity, then the daily infection chart for Sweden is confirmation. If Sweden has achieved herd immunity against the original virus and subsequent D614G mutations, the recent spike in infection rates may indicate a new variant with different infectivity has taken hold.

The pattern of a sudden explosion in both infectivity and death rates for SARS-CoV-2 globally does support the hypothesis that a new variant is challenging existing established adaptive immunity. 

Worldwide Daily New Cases and Daily Deaths as of December 2, 2020

Whether current vaccine candidates will be effective against new mutations that can cause increased infectivity and deaths is unknown. What we do know is vaccines themselves do not target and contain virus replication. Pharmacological interventions such as toremifene or natural molecules like melatonin may be just as effective as vaccines due to their ability to inhibit and / or disrupt viral fusion and replication. Melatonin may be a potent antiviral that may be extremely useful during early stages of viral infections, while its ability to control hyper-inflammation with its immunomodulatory and anti-inflammatory effects renders it indispensable during later stage disease progression that are more severe. 

Without additional clarity on the efficacies and potential negative effects of vaccines, the use of relatively safe and non-invasive therapeutic treatments such as toremifene and / or melatonin becomes an increasingly attractive prospect that must be duly considered. 

I await the results of the SENTINEL trial with much anticipation. In the meantime, have you had your AA and MEL today?

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