We are being confronted with the most consequential pandemic since the Spanish flu of 1918‑1920 to the extent that never before have 4 billion people quarantined simultaneously; to address this global challenge we bring to the forefront the options for medical treatment and summarize SARS‑CoV2 structure and functions, immune responses and known treatments. Based on literature and our own experience we propose new interventions, including the use of amiodarone, simvastatin, pioglitazone and curcumin. In mild infections (sore throat, cough) we advocate prompt local treatment for the naso‑pharynx (inhalations; aerosols; nebulizers); for moderate to severe infections we propose a tried‑and‑true treatment: the combination of arginine and ascorbate, administered orally or intravenously. The material is organized in three sections: i) Clinical aspects of COVID‑19; acute respiratory distress syndrome (ARDS); known treatments; ii) Structure and functions of SARS‑CoV2 and proposed antiviral drugs; iii) The combination of arginine‑ascorbate.
The combination of arginine-ascorbate
There is ample data suggesting that the severity of the COVID-19 is less related to the viral replication itself than to the host responses to the infection: delayed IFN production, increased neutrophils and cytokines in the lung creating a pro-inflammatory, pro-apoptotic milieu, combined with lymphopenia, acidosis, coagulation and vascular endothelium modifications and defective tissue repair with fibrosis in the lungs. Most treatments are focused on blocking viral replication, however, in fatal cases irreversible changes and deterioration occur even though viral replication is essentially blocked. Therapeutic interventions which combine agents for pleiotropic actions rather than single agents acting on well-defined pathways are more likely to improve patient outcomes.
Analyzing genes with modified expression in ARDS evidenced 201 genes, predominantly from pathways modulating inflammation, innate immunity, reactive oxygen species, and endothelial vascular signaling; all these pathways are modulated by NO generated from arginine, the only physiological substrate for NO.
Arginine-ascorbate has several important benefits in CoV infection: firstly, direct antiviral actions, secondly, improvement of leukocyte function and number, especially important in patients with neutrophilia and lymphopenia and finally it provides essential components and stimulates the mechanisms of tissue repair.
a) Antiviral actions. During the 2003 SARS epidemic medicinal NO gas, a mixture of 0.8% NO and 99.2% N2 was administered by Keyaerts et al (123) and followed by prompt improvement in oxygenation and sustained patient benefit; following-up on this observation it was shown that NO donor compounds inhibited SARS-CoV replication in vitro, a finding replicated by another team who also observed that besides S-nitroso-N-acetylpenicillamine (SNAP), IFN also inhibited SARS-CoV replication in vitro (124), confirming results obtained by another team who showed that IFN stimulates iNOS and NO production for prompt antiviral action.
Interestingly, IFN-γ stimulates NO production, and inhibition of NOS in mice resulted in conversion of a resolving infection by the ectromelia virus into fulminant mousepox.
NO production from arginine by the inducible NO synthase (iNOS) was shown to be essential for native immune responses in many infections and arginine availability is a critical factor for host resistance to infection. NO can pass through membranes, unlike complement and antibody, and is especially useful in syncytia, it can act on multiple targets increasing efficacy and preventing developing of resistance, and do not require recognition of infected cell by the immune system, which can be limited by pathogens or tissue-specific differences.
Reducing arginine availability either by nutrient deprivation or by specific pathogens significantly blunts the immune response, the former via impaired TLR4/MAPK pathway signalling, the latter by inducing arginase 1 via other TLRs in macrophages in an autocrine-paracrine manner involving cytokines, an immune evasion mechanism shown with mycobacteria and probably also in SARS-CoV2 infection.
Arginine also directly stimulates transcription/translation of the iNOS gene leading to de novo NOS protein synthesis, and this sheds light on the 'arginine paradox' where intracellular NO production is directly related to the extracellular arginine concentration even though endogenous synthesis of arginine provides approximately half of the arginine needed by cells. To ensure the increased need for intracellular arginine, the cationic amino acid transporter CAT2B is induced by IFN-γ, which is stimulated by ascorbate.
Reciprocally, the pH buffering action of arginine is increasing the cellular uptake of ascorbate by improving the activity of the cellular transporter of ascorbate, SVCT2, which has a pH optimum of 7.5 and is reduced to ~50% when pH is 5.5.
Ascorbate is actively accumulated in phagocytic cells where its concentration is 70-100 times higher than in plasma; it enhances chemotaxis, ROS generation, phagocytosis and viral clearance, important for minimising necrosis and tissue damage.
Ascorbate stimulates the synthesis and actions of antiviral NO indirectly in multiple ways, one via IFN-γ stimulation, another via inhibition of HIF-1α and correction of hypoxia, which significantly inhibits the availability of intracellular arginine and NO production; and also by synergizing with the apoptotic effects of NO in virus-infected cells, and thus inhibiting viral replication. It is also important to note that all three of NOS as well as IDO are heme-containing proteins which require ascorbate or another intracellular reducing agent for their activation. In this context, the observation that three accessory proteins of SARS-CoV2 - orf1ab, ORF3a, ORF10 - bind to heme followed by Fe inactivation and sequestration deserve to be further investigated for specific anti-NOS and other IDO activities.
In the case of SARS-CoV2 infection which directly antagonizes NO production, arginine provides both the substrate for iNOS and activates its synthesis; together with ascorbate, which is necessary for iNOS activation, it prepares a new functional enzyme necessary for NO production.
b) Improving lymphocyte function and number. Lymphopenia is present in a significant number of severe COVID-19 patients and its causes are strongly linked to arginine and ascorbate deficiencies.
Arginine is known to improve lymphocyte-based immunity. Arginine depletion due to upregulation of Arg1 in myeloid-derived suppressor cells induces T-cell anergy, decreased proliferation of T cells, low expression of CD3 ζ-chain T-cell receptor, and impairments of production of T cell cytokines and of upregulation of cyclin D3 and cyclin-dependent kinase 4 (cdk4). L-arginine is essential for maturation of CD3+ and the proliferation of CD8+ T lymphocytes. Arginine is also critical for B-lymphocyte differentiation; in transgenic mice over-expressing arginase in intestinal cells there was an impaired transition of pro- to pre-B cells in the bone marrow with marked decrease in B cell cellularity, serum IgM levels and number and size of Peyer's patches; this was reversed by arginine supplementation.
Noting the essential role of arginine for immunity, an arginine deficiency syndrome (ADS) was proposed, defined by pathological increase in arginase, decreased NO production, decreased arginine availability, abnormal T cell function including loss of ζ-chain, and presence of one or more of: trauma, cancer, chronic infection, liver necrosis, pulmonary hypertension; treatment for ADS include L-arginine and the arginase inhibitors N-hydroxy-arginine (NOHA) and COX-2 inhibitors.
Ascorbate is also important for adaptive immune responses, starting with the observations that reducing environments make immune responses more efficient; it increases the levels of IFN produced by activated fibroblasts; improves neutrophil function by preserving/restoring function of their myeloperoxidase; is essential for lymphocyte development, stimulating the proliferation of NK cells; and has ample epigenetic effects through dioxygenases TET (ten-eleven translocases) and Jumonji C (JmjC)-domain-containing histone demethylases.
A comprehensive review of the actions of ascorbate including those on the immune system concludes that enhancement of B- and T-cell differentiation and proliferation is likely due to its effects on gene regulation; an epigenetic effect (DNA demethylation) of ascorbate on the CD8 receptor gene was also inferred in a series of lab experiments which concluded that ascorbate is necessary and sufficient for the CD8a gene transcriptional activation. A graphic representation of these actions are given.
Multiple authors noted that administration of ascorbate in chronic granulomatous disease led to consistent improvements; more recent research showed that in human granulomas depletion of arginine by arginases from macrophages was associated with T-cell suppression without affecting their viability, via downregulation of T-cells receptors.
Both arginine and ascorbate stimulate production of NO, which inhibits IDO, and its suppressive effects on lymphocyte proliferation; the simultaneous action of arginine (NO donor) and ascorbate (catalase inhibitor) is synergical for leukocytes function and especially apoptosis, yet another example of the arginine-ascorbate antimicrobial synergy.
Synergistic action of arginine-ascorbate for inhibiting viral replication and achieving viral clearance is shown by the combined effect of IFN-α (induced by NO) and IFN-γ (induced by ascorbate) in inducing the antiviral response via ISGs, which is much enhanced when compared to either one alone. This was also seen with IFN-β1 and IFN-γ in improved apoptosis, RNA degradation and inflammatory response; and also with TNF-α and IFN-β which together induced a novel synergistic antiviral state, highly distinct from that induced by either cytokine alone and involved >850 novel host cell genes; this was necessary and sufficient to completely block the replication and spread of myxoma virus in human fibroblasts and to block the spread of vaccinia virus and tanapox virus to neighboring cells.
Nitric oxide from arginine directly enhances the stimulation of ascorbic acid on T cell proliferation, and this interaction is especially important in T cell recovery after stem cell transplantation, where generation and proliferation of T cells is greatly improved by ascorbate and blocking of NOS diminished this effect.
To simplify the arginine-ascorbate actions in the immune response by the way of dichotomy, arginine is an essential factor for the native immune response via NO production and activation of IFN-α/β (type I) while ascorbate is critical for the efficacy of the adaptive immunity via T cell proliferation and function via IFN-γ (type II); their synergy ensures both decreased viral replication and clearance.
c) Stimulation of tissue repair. It is known for decades that L-arginine supplementation (3-10 g/day) speeds wound healing and recovery after surgery; we also know that ascorbate is an essential cofactor for collagen synthesis, meaning that both arginine-ascorbate are required for tissue repair; additional molecules and pathways are involved in the immune response in viral infection and individual variability can also have an important role.
In a first step for tissue repair arginine is metabolized to ornithine by arginases (Arg1/2), resulting in both proline (collagen synthesis) and biologically active amines (putresceine, spermidine, spermine) essential for cell repair and proliferation; the balance iNOS/Arg1 determines the pro- or anti-inflammatory environment and intra-cellular use of arginine. Depending on this, macrophages are polarised in M1 (proinflamamtory, cytotoxic, NO) and M2 (anti-inflammatory, proliferative, Arg1), and excessive activity of either can create pathologies; surprisingly the suppression of M2 activity in mice resulted in reduced lung fibrotic lesions in SARS-CoV infection, suggesting the virus produces a shift to M2 activities, despite the high levels of inflammatory cytokines.
Arginine corrects the pH and its increased extracellular concentration helps cause a shift towards M2, and is the substrate for arginase, which is stimulated by uric acid. Arginases are activated by Mn2+ in a pH-dependent fashion, Arg1/2 have an alkaline optimum activity of 9.0-9.5. NOHA, the arginase inhibitor, can also be oxidized to NO by many heme proteins, including hemoglobin, peroxidase, cytochromes P-450, and catalase, which is inhibited by ascorbate, which is another mechanism by which ascorbate modulates redox and metabolic reactions and NO production.
While NO donors (SNAP, molsidomine) or iNOS gene transfer enhance wound collagen synthesis in a dose-dependent manner, high levels of NO strongly impair collagen synthesis probably due to inhibition of arginase.
Ascorbate helps reduce inflammation by limiting expression of pro-inflammatory cytokines, reducing CRP in individuals with CRP >1.0 mg/l; it scavenges ROS and reduces the oxidative stress on mitochondria, improves intracellular pH via decreasing lactate, decreases cell apoptosis and tissue necrosis also by inhibiting HIF-1α, which is stimulated by IFN-I; hypoxia by inhibiting arginine intracellular synthesis is a deleterious factor for tissue regeneration.
To get a good perspective on the dose needed of arginine-ascorbate, we note that the daily plasma flux of arginine is about 22-25 g with 5-6 g from diet, the majority of arginine is synthesized in the liver and kidney (15-20 g daily) from citrulline in the urea cycle, ~10% is used for creatine synthesis and 1% for NO generation. Pharmacological doses of up to 30 g/day for 2 weeks administered either intravenously or enteral in multiple clinical studies appear to be well tolerated by healthy individuals and cancer patients without major side effects; significant elevation in IGF1 was the only modified biomarker.
Ascorbate is sufficient in normal situations in doses of 50-250 mg daily for maintaining plasma levels >30 µg/ml, however, during stress and infections the need for ascorbate intake exceeds 1,000 mg/day and its intestinal absorbtion is optimal when administration is fractionated at ~250-350 mg per dose or in extended release forms (calcium ascorbate and slow-release micronised preparations).
In our experience an effective antiviral regimen is the combination arginine+extended release vitamin C, in doses of 1,000 mg L-arginine and 250-500 mg calcium/sodium ascorbate (Ester-C) or Cetebe (GSK) or other modified release vitamin C, at least 3 times a day each.
We have found this treatment effective in many types of viral infections, including common cold and flu (many of which are caused by coronaviruses), herpes labialis, zona zoster, and hepatitis C, including a chronic hepatitis C patient who had significant viral loads after 2 treatments with ribavirin+interferon, and undetectable at 5 years after intravenous arginine-ascorbate.
Arginine-ascorbate in severely ill patients. Metabolic acidosis is associated with hyper-inflammation in ARDS patients, and administration of sodium bicarbonate for balancing pH can be limited by electrolyte imbalance and/or cardiovascular pathology. Arginine administration can improve pH via the buffering action of NH+ groups and additionally can provide an immunity boost to septic patients, however, the formation of NO (nitric oxide), which may induce relaxation of the vascular smooth muscle has raised concerns.
A very thorough review of arginine use in critical care has analyzed both the mechanistic concern of possible NO-induced vasodilatation, alongside other actions that arginine has in the critically ill patient. This study has importantly discussed the multiple clinical trials and reports of arginine use in septic patients. During critical illness there is a severe arginine deficit following a 2/3 reduction in arginine synthesis and increased degradation due to a 4-fold increase of arginase activity, so that arginine becomes an essential need for supplementation. Decreased arginine is especially deleterious for the immune system, which produce excessive myeloid suppressor cells which induce a pro-inflammatory state and suppress T lymphocytes, with reduced number of CD4+ cells which are also functionally deficient, as they lack the ζ-chain of its receptor.
Clinical trials found that arginine shortened hospitalizations and decreased infections, and in severe sepsis improved outcomes and decreased mortality; it also reversed septic shock without hemodynamic instability.
A better assessment of septic shock patients can be done by using the ratio of arginine and its metabolite asymmetric dimethyl arginine (ADMA), which can block NO production and act as vasoconstrictor; a declining arginine/ADMA ratio was associated with increased mortality; while an increasing arginine/ADMA ratio in septic patients improved mortality. Also important is a report showing that intravenous administration in critically ill patients of supra-physiologic doses of arginine did not produce hemodynamic instability; this may be due to a preferential uptake in septic patients of arginine by leukocytes compared to endothelial cells.
The review concludes that arginine is safe to administer and beneficial in critically ill patients, contributing to resolution of infection and improving morbidity and mortality.
Adding ascorbate makes administration of arginine even safer; in septic mice ascorbate administration prevented the impaired vasoconstriction and angiotensin actions and excessive vasodilation as much as the iNOS gene knockout, and greatly increased survival, seemingly by inhibiting excessive NO production.
An important fact is that ascorbate, which is known to be decreased in septic patients, is a cofactor required for adrenal synthesis of hormones and neurotransmitters including cathecholamines (norepinephrine) and vasopressin, physiological vasopressors which otherwise need to be administered.
In septic patients ascorbate was shown to decrease mortality especially in the hyperinflammatory subphenotype characterized by high leukocyte count (>15,000/ml) and fever (>37.5°C).A meta-analysis including 1,210 patients with sepsis showed that different daily doses of intravenous ascorbate were associated with different results, and only a dose between 3-10 g/day resulted in decreased mortality, decreased need for vasopressor and mechanical ventilation, but not lower or higher doses.
In a prospective randomised placebo-controlled double blind trial - CITRIS-ALI - ascorbate 50 mg/kg or placebo was given q 6 h intravenously for 96 h to 167 patients with sepsis and ARDS; no significant difference was seen at 96 h in organ dysfunction scores (SOFA), inflammation marker CRP or thrombomodulin (for vascular integrity). However, patients treated with vitamin C had a significant benefit in 28-day mortality, transfer from ICU by hour 168, number of ICU-free days by day 28, and number of hospital-free days.
Hence in ascorbate deficiency, it is likely that when administered intravenously to hypotensive patients, ascorbate should be administered first, followed by a lower dose of arginine (ex 1500 mg ascorbate in 100-200 ml normal saline followed by 1000 mg L-arginine in 100 ml saline administered at 1 ml/min).
Other medication should be added depending on the clinical and laboratory signs: for the ′ground glass′ image on imagistics, which signals presence of intra-alveolar- and intravascular emboli, especially when D-dimer is elevated: administration of anticoagulant, heparin or fractionated heparin; for signs of porphyria (spontaneous ecchymosis which do not change color) - chloroquine, amodiaquine, curcumin; for tachycardic, tachyarrhythmic patients amiodarone 400 mg qd for a maximum 7 days; for all patients and especially with high cholesterol, simvastatin for asthma patients, Montelukast and/or fexofenadine or other lipophilic antihistaminic and anti- inflammatory, preferably curcumin; for anti-inflammatory, analgesic actions - PPAR-α/γ agonists fenofibrate/pioglitasone or ABDH6 inhibitors KT185 and KT203 (which decrease macrophage activation and pain-inflicting CCXl-2 with allodynia); rabeprazole for preventing gastritis and for possible antiviral effects; methylprednisolone was shown to be of benefit in COVID-19, and even with associated immune depression favoring viral replication, it can be considered for short-term in severely ill.
Practical measures - prevention and early interventions. A majority of SARS-CoV2 patients (85-90%) have mild to moderate forms of disease, however 10-15% develop rapidly deteriorating forms with approximately half of those dying, and it is difficult to predict accurately the individual risk for developing severe disease besides advanced age and existing co-morbid conditions.
Unopposed or excessive viral replication is an important pathogenic mechanism through which the virus overwhelms the natural defenses of the body; besides a weakened immune response associated with chronic pathologies (cardiovascular, renal, hepatic, autoimmune and neoplasic), the virus has an advantage in tired or stressed individuals (with high cortisol and lactate/LDH levels) and by repeated exposure which increases the dose of the virus (doctors, nurses, medical personnel). Protective measures are very important for limiting exposure to the pathogen in vulnerable populations and also testing and treating early, as soon as possible after respiratory symptoms.
After infection and respiratory symptoms, prompt local treatment is very important: aerosol inhalation, nasal drops, throat sprays are very useful treatments. Water vapors hydrate the airway mucosa, increase the volume of ASL and help the natural clearance of the virus via mucus shedding. Adding to the inhaled vapors natural antimicrobials such as rosemary, basil, tea tree, mint, eucalyptus, thyme, onion, in fresh or dry form; throat or nasal sprays with propolis, argentum, basil, thyme, cumin will also help viral inactivation and decrease inflammation.
We propose arginine-ascorbate as the mainstay of antiviral treatment for SARS-CoV2 and other coronaviruses, administered from the initial signs of infection until its resolution.
As much as NSAIDs (non-steroidal antiinflammatory drugs) are not recommended (aspirin, ibuprofen, naproxene and indomethacin), possibly because of concomitant inhibition of PGD2, known to be elevated in elderly patients, and further elevated by SARS-CoV infection and in more severe forms of COVID-19; however PGD2 has protective, anti-inflammatory actions, and blocking cyclooxygenase (COX) also inhibits PGD2, which further imbalances an already malfunctioning immune response.
Interestingly, NSAID-mediated curcumin (also a main protease inhibitor) is useful for alleviating inflammation and pain, and if rhinitis and conjunctive inflammation are present, an antihistaminic can be of help (fexofenadine also inhibits the main COVID-19 protease). We have successfully used the curcumin/antihistaminic combination for pain of various types from sore throat to allodynia, in patients with allergies, increased PGD2, rheumatoid arthritis, and also for porphyria. Patients who cannot tolerate curcumin may use instead extracts of Boswellia serrata (400 mg tid) as anti-inflammatory and analgesic; artemisinin can be a good substitute for chloroquine or amiodarone in patients with prolonged QT interval, COPD or lung fibrosis.
Risk evaluation of CoV infected patients. Assessing the risk for progression to severe forms early on during CoV infection is important for prompt individual treatment and medical resource management, and also to avoid imposing restriction on the whole population, knowing that 90% of infections are asymptomatic or can be treated outside hospital; this may allow shifting isolation measures from whole populations to truly at-risk populations.
A two-pronged strategy of risk evaluation can be employed based on age and exposure risk: in younger individuals (≤55 years old) a specific panel for genetic risk factors which predispose to fatal CoV infections (rs78142040, rs9605146, rs3848719; NAMPT - 1001G allele, POPDC3, PDE4B, ABCC1, TNFRS11 and DD genotype of ACE) may be more useful, especially in individuals with potential increased exposure to the virus (eg., healthcare professionals). However, in patients most at-risk - elderly and with chronic co-morbidities, evaluation with different testing panels can reasonably assess their risk for severe COVID19, starting with the evaluation of oxidative/peroxisomal status (catalase, malondialdehyde), inflammation - CRP, fibrinogen - and metabolism - bicarbonate, HgbA1c, lipid profile, and immune balance - PGD2 and serum protein electrophoresis - for asymptomatic patients, and adding for symptomatic patients the CD3+, CD4+, CD8+ and NK counts (predictors of need for intensive care), LD1 isoensyme, which were good predictors for intensive care during the SARS epidemic; for hyper-inflammatory status also useful are markers of neutrophil activation - MMP-9, LTB4, ferritin, CXCl-2.
Another approach to risk evaluation of severe COV infection is measuring the high-sensitivity cardiac troponin (hs-cTnT), shown with data from the ARIC study to be a marker for end-organ damage in patients with chronic pathologies; elevated troponin levels were also seen in some COVID-19 patients.
Our efforts as front-line defenders against this invisible foe, SARS-CoV, and possible others to come, are essential not only from a medical perspective but also towards fully regaining our individual and societal freedoms; through risk evaluation and prompt, effective treatments the burden of disease can be greatly diminished for both individuals and whole populations.
Reference & source information: https://www.spandidos-publications.com/
Read More on: