Could dantrolene be explored as a repurposed drug to treat COVID-19 patients by restoring intracellular calcium homeostasis?

Abstract

Dantrolene, an FDA approved drug to treat malignant hyperthermia and muscle spasm, has been demonstrated to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediated toxicity of host cells. Ryanodine receptor overactivation and associated disruption of intracellular Ca²⁺ homeostasis play important roles in SARS-CoV-2 infection and replication of host cells. Dantrolene, as an inhibitor of RyRs, is expected to ameliorate these detrimental effects of SARS-CoV-2 in host cells. Additionally, dantrolene has also been shown to inhibit multiple cell or organ damage induced by hypoxia/ischemia, mitochondria damage, oxidative stresses, inflammation, impairment of autophagy and apoptosis, etc., which are often the causes of severity and mortality of COVID-19 patients. We have repurposed that dantrolene has a high potential at treating COVID-19 patients and reducing its morbidity and mortality.

Introduction

The epidemic of coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has lasted more than half a year with varied population-level case fatality ratio ranged between 2–8%¹, though much lower after adjusting for demography and under-ascertainment, which is still higher in aged groups (≥60 years: 6·4%, ≥80 years: 13·4%)¹. Nevertheless, there is still a lack of powerful drugs to treat COVID-19 patients, even with the promising drug Remdesivir, a nucleotide analog with broad-spectrum antiviral activity². Furthermore, randomized clinical trials have also shown disappointing findings of other drugs, including hydroxychloroquine³ and lopinavir-ritonavir⁴. In the setting of the absence of robust drug and vaccine, it may be beneficial to develop drugs that can reduce the infection and replication of SARS-CoV-2 and severity of the symptoms⁵, protect the organs, ameliorate the deterioration⁶ and reduce mortality in the critically ill COVID-19 patients⁷. Considering its plausible ability to inhibit SARS-CoV-2 virus cytotoxicity of host cells⁸, cytoprotection⁹, and organ protection¹⁰ in a wide variety of models of stress and disease, we propose that dantrolene, an FDA approved drug to treat malignant hyperthermia and muscle spasm, could be repurposed as a potential adjuvant drug for the treatment of COVID-19 patients.

1. Potential and Proposed Mechanisms of dantrolene to inhibit SARS-CoV-2 Infection and/or Replication in the Host Cells

Infection and replication of SARS-CoV-2 (Figure 1) in the host cells initially require binding of the S1 domain of the virus spike protein (S protein) to angiotensin-converting enzyme 2 (ACE2) on the plasma membrane, followed by fusion with the plasma membrane mediated by S2 domain of S protein to make its entry^11,12.

Figure 1.

Dantrolene might inhibit infection and replication of SARS-CoV-2 and associated pathology. Cathepsin L, a protease on the plasma membrane of host cells, increases Ca²⁺ release from the endoplasmic reticulum (ER) via the ryanodine receptors (RyRs). The associated elevation of cytosolic Ca²⁺ concentration, in turn, increases cathepsin L activity. Cathepsin L promotes virus fusion with host cells by cleaving and activating the spike (S) protein. High levels of extracellular and cytosolic Ca²⁺ concentrations are also necessary for virus fusion and endocytosis. Cathepsin L in the endosome, under the condition of a high level of Ca²⁺ concentration, promotes virus RNA release into the cytosol. On the other hand, the increased cytosolic Ca²⁺ concentration due to the overactivation of RyRs activates calcineurin, which dephosphorylates NF-AT and translocates into the nucleus for promoting transcription and virus replication. Excess Ca²⁺ release from ER via overactivation of RyRs in AD cells results in depletion of ER Ca²⁺ and associated ER stress, as well as the overloading of mitochondria with Ca²⁺ and associated mitochondria damage. All of the above pathologies eventually result in impaired ATP production, oxidative stress damage, apoptosis, and cytokine storm, leading to final host cell damage. Dantrolene inhibits the infection and replication of the SARS-CoV-2 virus and host cell damage by inhibiting abnormal and excessive activation of RyRs and restoring the intracellular Ca²⁺ homeostasis.

The cleavage and activation of S protein by protease, especially cathepsin L¹³, provides a preliminary priming step of these enveloped viruses¹⁴. Meanwhile, cathepsin L promotes activation of the ryanodine receptors (RyRs)¹⁵, which results in an abnormal increase in cytosolic Calcium ions (Ca²⁺) concentration heightening the activity of Ca²⁺-dependent cathepsin L¹⁶. Further, the endosomes containing the virus enter cytosol via endocytosis¹², and the high Ca²⁺ concentration in the mature endosome activates cathepsin L^14,17. These processes finally release the virus RNA into the cytosol. Dantrolene inhibits the abnormal and excessive activation of RyRs and restores the intracellular Ca²⁺ homeostasis⁹, which breaks the pathological feedback between the cathepsin L and the Ca²⁺ and prevents the entry of the virus.

It was suggested that S-mediated membrane fusion was Ca²⁺-dependent (Figure 1)¹⁸. The Ca²⁺ binding to fusion peptides via conserved negatively charged residues are required to trigger the fusion process¹⁸. To promote fusion, the virus needs additional Ca²⁺ which is imported from the ER via RyRs (Figure 1) to the endosomes. So it is intriguing to note that amiodarone, a drug that blocks endosomal/lysosomal Ca²⁺ channels, inhibits SARS-CoV entry after endosomal uptake¹⁹. Commonly used Ca²⁺ channel blockers showed therapeutic effects in COVID-19 patients²⁰. Dantrolene may inhibit calcium influx from extracellular space and elevation of cytosolic Ca²⁺ primarily by reducing the capacitive calcium entry (CCE). The ability of dantrolene to inhibit L-type Ca²⁺ channel or NMDA glutamate receptor is not fully clear. Therefore, it is not surprising to demonstrate that SARS-CoV entry was inhibited by Ca²⁺ chelators such as BAPTA-AM at the cytosol and endosomes¹⁸. The critical initiation of infection and subsequent virus replication depends on the presence of Ca²⁺, especially the intracellular Ca²⁺ concentration^14,18. Dantrolene is expected to ameliorate SARS-CoV-2 mediated over activation of RyRs and associated disruption of intracellular Ca²⁺ homeostasis (Figure 1)⁹. These effects, in turn, are expected to inhibit the SARS-CoV-2 virus infection of the host cells. Likewise, inspiring news demonstrated the in vitro antiviral cytotoxicity activity of dantrolene against SARS-CoV-2, in clinically relevant concentrations and duration, with minimal cytotoxicity of dantrolene itself⁸. Furthermore, the abnormal increase in cytosolic Ca²⁺ concentration via over activation of RyRs on the ER membrane enhances the calcineurin’s activity, which promotes NF-AT nucleus translocation and transcription, leading to the subsequent promotion of virus replication in the cytosol (Figure 1)¹⁶. So, as an antagonist of RyRs, dantrolene is theoretically expected to inhibit the replication of SARS-CoV-2, although it needs to be investigated in future studies.

2. Proposed Mechanisms of Dantrolene to Reduce Cell Stress and Damage

1). Dantrolene Reduces Pathological Inflammation

Both SARS-CoV-2 and SARS-CoV are characterized by a pathological inflammatory response. The host inflammatory response is a major cause of tissue damage and subsequent mortality. Increased inflammatory response and elevated levels of cytokines (IL-1β, IL-6, IL-8, MCP-1, IP-10, TNFα, IFNγ, et al) have been observed in patients with COVID-19, which implied potential of a cytokine storm^21–24. In an animal study, the cytokine and IFNγ were also detected in the lungs of the SARS-CoV-2-infected animals, which suggested that SARS-CoV-2 triggered the innate immune response and the activation of inflammation²⁵. Furthermore, the SARS-CoV E protein forms a Ca²⁺ permeable channel in ERGIC/Golgi membranes. The channel activity alters Ca²⁺ homeostasis within cells and boosts the activation of the NLRP3 inflammasome, which leads to the overproduction of IL-1β²⁶. The development of an uncontrolled inflammatory response can thus lead to detrimental outcomes such as diffused alveolar damage and fibrosis, progressive respiratory failure, and multiple organ damage and dysfunction. Additionally, inflammation and SARS-CoV proteins cause ER stress, which consequently leads to dysregulation of Ca²⁺ homeostasis^27,28.

Intracellular Ca²⁺ signalling is essential in the release of pro-inflammatory cytokines and the elevation of the intracellular Ca²⁺ has been suggested to be a critical event in sepsis²⁹. Calcium influx may play a partial role in promoting the plasma levels of cytokines, because the calcium channel blockers have been demonstrated to ameliorate excessive inflammation³⁰. Subsequently, calcium channel blockers have been proposed to treat COVID-19 patients³¹. With its ability to ameliorate Ca²⁺ dysregulation by inhibiting over activation of RyRs (Figure 1), dantrolene has been demonstrated to suppress plasma and tissue concentration of IL-6³², IL-8³³, IL-1β, TNF-α^34,35, and IFN-γ³⁶ in vivo and in vitro. Consequently, dantrolene inhibited ER-mediated Ca²⁺ release and ameliorated ER stress³⁷.

2). Dantrolene Reduces Pathological Oxidative Stress

Oxidative stress generated from SARS-CoV-2, might further exacerbate the pro-inflammatory epigenetic changes and result in a vicious circle of cytokine response. At the same time, response to SARS-CoV-2 infection, DNA methylation defect exacerbated by oxidative stress will further enhance viral entry through epigenetic de-repression of ACE2 and increased ACE2 expression³⁸. As for SARS-CoV, oxidative stress-sensitive genes were upregulated in peripheral blood mononuclear cells of patients³⁹. Alterations of reactive oxygen species (ROS) production that are caused by respiratory viral infections are implicated in inflammation, lung epithelial disruption, tissue damage, and even pulmonary fibrosis⁴⁰.

Given SARS-CoV induced oxidative stress cell damage, anti-oxidative treatment may play a role in the SARS-CoV treatment. Dantrolene was reported to protect cells against oxidative stress by elevating the levels of GSH and GSH/GSSG^41,42. Calcium release from the ER was associated with the generation of ROS⁴³, which was inhibited by dantrolene via lowering mitochondrial superoxide, ROS⁴⁴.

3). Dantrolene Inhibits Cell Death By Apoptosis

Apoptosis is induced as one of the host antiviral responses to limit virus replication and production during viral infections. Lymphopenia was common in SARS-CoV-2 infected patients, probably due to lymphocyte apoptosis^21,24,45. Also, laboratory research in peripheral blood mononuclear cells demonstrated that TP53, an important gene in the process of apoptosis, showed an increasing trend in patients infected with SARS-CoV-2²⁴. In SARS-CoV-2 infected animals, apoptosis has been found in the respiratory tract and TUNEL staining showed the diffused signals in the lungs, bronchiolar lumen cell debris, and collapsed alveolar walls²⁵. The release of Ca²⁺ from ER has been proposed to be involved in the induction of apoptosis by oxidative stress, which is also a pathological process induced by SARS-CoV-2⁴³.

Apoptosis contributes to SARS-CoV-2 virus pathogenesis, and inhibition of apoptosis may protect host cells against damage. Abnormal Ca²⁺ release from the ER and consequent increase in cytosolic and mitochondria Ca²⁺ levels play pivotal roles in inducing cell apoptosis in a variety of cell types⁴⁶. Thus, dantrolene can suppress apoptosis through inhibiting RyR-mediated abnormal and excessive Ca²⁺ release^47,48. Moreover, dantrolene can ameliorate apoptosis by directly inhibiting nuclear condensation and fragmentation^49,50.

4). Dantrolene Ameliorates Impairment of Autophagy

SARS-CoV has the potential to inhibit the autophagy process. An analysis of a relatively wide database of SARS-CoV-2 genomes of worldwide isolates representative of COVID-19 has revealed two synonymous mutations, of which one is non-structural viral proteins 6 (NSP6)⁵¹. NSP6 is a common component of both α and β-coronaviruses, which locates to the ER and generates autophagosomes⁵². It has been shown that NSP6 and ER binding may favor coronavirus infection by compromising the ability of autophagosomes to deliver viral components to lysosomes for degradation^53,54. Thus, this would limit autophagosome expansion and activity⁵⁵. Moreover, overexpression of membrane-associated papain-like protease PLP2 of SARS-CoV and MERS-CoV led to blockage of autophagosomes-lysosomes fusion and suppression of the autophagic flux⁵⁶. It has been shown that high cytosolic Ca²⁺ concentration suppressed vesicle fusion, and calcium channel blockers can promote autophagosome-lysosome fusion⁵⁷. Dantrolene, as a calcium channel blocker, through inhibition of the RyRs in ER, has been reported to promote autophagy activity by inducing autophagy induction^58,59 and, therefore, potentially ameliorating the impaired autophagy function mediated by SARS-CoV-2 viruses.

3. Dantrolene Potentially Ameliorates the Multiple Organ Damages in COVID-19 Patients

COVID-19 typically demonstrates severe progressive lung injury, multi-organ failure, and death^3,60,61. Although SARS-CoV-2 initially infects the lungs and causes lung damage, the virus eventually reaches many organs, resulting in multiple organ damage⁶². Critically ill patients are typically found to have systemic multiple-organ damage and dysfunction^63,64.

1). Lung

Acute respiratory distress syndrome (ARDS) is often seen in critically ill COVID-19 patients, which is usually life-threatening because it is associated with progressive hypoxia and associated multiple organ damage^3,60,65. Pulmonary hypertension (PH) is a recognized consequence of AR-DS and a severe condition with a very poor survival rate^66,67, which was presented in COVID-19 patients⁶⁸. Pulmonary vasoconstriction due to hypoxia and inflammation constitutes the majority of the underlying mechanisms of PH⁶⁹. It has been proposed that the correction of abdominal pH by reducing hypoxic pulmonary vasoconstriction could benefit COVID-19 patients.

RyRs play an important role in hypoxia-induced Ca²⁺ release and contraction⁷⁰, which contributes significantly to the development of pulmonary hypertension⁷¹. Chronic hypoxia increases RyR2 expression and further induces pulmonary hypertension⁷². Dantrolene can inhibit hypoxia-induced Ca²⁺ release in the pulmonary arterial smooth muscle cell and vasoconstriction of the pulmonary artery^70,73,74, which reverses the hypoxic vasoconstriction⁷⁵. In light of this beneficial effect, dantrolene may be a potential adjunctive countermeasure.

Moreover, in the airway smooth muscle, RyRs also mediate the Ca²⁺ response and thus bronchoconstriction, which can be attenuated by dantrolene⁷⁶. This potentially mitigates the high airway pressure, which might result in the pneumothorax of COVID-19 patients⁷⁷.

2). Cardiovascular System

Cardiac injury in COVID-19 patients was more likely related to multiple stress factors rather than direct damage by the virus⁶⁸. Therefore, the goal is to minimize the myocardial ischemia and ischemia-reperfusion injury (IRI) in these patients.

Cytosolic Ca²⁺ overload plays a major role in the development of irreversible injury during myocardial ischemia, while the abnormal Ca²⁺ release from the sarcoplasmic reticulum contributes to this damage significantly⁷⁸. Dantrolene reduced ischemic injury even at concentrations that did not affect contractile performance in the heart⁷⁹. In vitro studies showed that dantrolene attenuated the lethal cellular injury⁸⁰, reduced infarct damage^79–81, protected cardiac function^79,82,83, and was even antiarrhythmic⁸³ under IRI.

Cardiac arrhythmia and associated cardiac arrest are often seen in COVID-19 patients³. In heart failure, arrhythmogenic Ca²⁺ release and chronic Ca²⁺ depletion arise due to the altered function of the RyR Ca²⁺ release channel⁸⁴. Dantrolene has been demonstrated to have antiarrhythmic effects against Ca²⁺ overload mediated arrhythmias^85,86, while at the same time preserving inotropy⁸⁴. Dantrolene can also improve survival after ventricular fibrillation by mitigating impaired Ca²⁺ handling in animal models⁸⁷, and prevent catecholaminergic polymorphic ventricular tachycardia⁸⁸.

3). Brain

The expression and distribution of ACE2 in the brain⁸⁹ suggest that the SARS-CoV-2 may cause some neurologic manifestations through direct⁹⁰ or indirect mechanisms⁹¹. The infection itself has also been described as a risk factor for stroke⁹². The ischemia of the brain seems to be a severe threat to COVID-19 patients.

One approach to protect the brain against ischemia is to reduce the tissue’s functional activity to preserve energy for the metabolic processes that are essential to viability⁹³. The neuroprotective effect of dantrolene, which inhibits abnormal Ca²⁺ release from ER, and then contributes to the large reversible reductions in O₂ consumption, glycolysis, and electrophysiological function⁹³, appears rather consistent across multiple cells and animal models of neurological injury that include excitotoxicity^94–98, oxygen-glucose deprivation (OGD), forebrain ischemia^104–107, focal ischemia¹⁰⁸, global ischemia^109,100, and traumatic injury¹¹¹. In humans, dantrolene is capable of attenuating cerebral vasospasm¹¹² and providing neuroprotection¹¹³.

4). Liver

Many patients with COVID-19 range from differing degrees of liver damage and function abnormality⁶¹. Pneumonia-associated hypoxia and immune-mediated inflammation, such as cytokine storm, might contribute to liver injury or even develop into liver failure in patients who are critically ill¹¹⁴.

It was reported that dantrolene offered significant functional and structural protection of the ischemic liver, by decreasing TNF-α but increasing IL-10 and was also associated with better liver function tests and less necrosis during ischemia in rat livers¹¹⁵.

5). Kidney

Kidney failure may be part of whole-body events in COVID-19 patients⁶². Renal ischemia/reperfusion injury is a common cause of acute renal failure¹¹⁶ and induces renal tubule apoptosis, which is associated with the elevation of the cytosolic calcium concentration¹¹⁷. The renal tubular cell injury can be attenuated by dantrolene¹¹⁸.

6). Pathological Inflammation and Cytokine Storm

In COVID-19, higher plasma levels of cytokines including IL-6, IL-2, IL-7, IL-10, granulocyte-colony stimulating factor, interferon-γ–inducible protein, monocyte chemoattractant protein, macrophage inflammatory protein 1α, and TNF-α were found in ICU patients, which implied that a cytokine storm occurred^21,61. For COVID-19 patients, cytokine storms are a major reason that some require intensive care and ventilation. Dantrolene has been shown to inhibit various cytokine release and inflammation in various animal models^34–36. It was reported that dantrolene decreased TNF-α in the lung (26.1%), liver (29.4%), and spleen (35.4%) and IL-1α in the lung (30.0%) and liver (25.4%)³⁴. These beneficial effects of dantrolene make it potentially effective at ameliorating cytokine-mediated pathological inflammatory reaction and associated cytokine storm in COVID-19 patients.

Conclusions

In such a global pandemic, little is known for certain. Besides direct antivirus treatment, attention should also be paid to reducing the severity of the symptoms, protecting the organs, and ameliorating the deterioration. Based on previous studies illustrating the dantrolene protective effects against SARS-CoV-2 virus cytotoxicity in host cells, cell or organ damage induced by hypoxia/ischemia, mitochondrial damage, oxidative stresses, inflammation, impaired autophagy function, etc., we propose that dantrolene might be a potential repurposed drug for the treatment of COVID-19 patients (Figure 2), with an expectation to assist in reducing mortality. Further studies at the varied molecular, cellular, animal, and patient levels are important and recommended.

Figure 2.

Dantrolene is expected to protect cell and organ damage induced by multiple pathological stresses in COVID-19 patients.