PMC:7408073 / 95877-112683 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7408073","sourcedb":"PMC","sourceid":"7408073","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7408073","text":"8. Hypothesizing Pharmacological Treatments for COVID-19\nBased on the above described hypothesis, inhibition of ACE2 pathway might be beneficial for COVID-19 patients. Indeed, the clinical picture as a whole is consistent with an ACE2 gain of function (possibly due to both circulating active forms of S1-sACE2 complexes and local forms of SARS-CoV-2-sACE2 complexes) rather than an ACE2 loss of function, as initially supposed. Therefore, inhibition of ACE2/Ang (1–7)/MasR axis or other ACE2 pathways to restore ACE/ACE2 balance might be needed, at least in the first phases of the disease when hypoxia is not yet induced. Different strategies could be pursued through ACE2 pathway inhibitors and/or MasR antagonists and/or renin inhibitors and/or metal chelators.\nSeveral different molecules have been designed to specifically inhibit human ACE2 enzyme (both membrane bound and soluble forms) or human MasR signal transduction, but only a few have been studied in vivo. Some ACE2 pathway inhibitors have been widely used in mouse/rat models, as control of human and mouse ACE2 activity or in mouse models of colitis. Thanks to mouse/rat in vivo experiments and clinical trial results in human participants it has been possible to infer toxicity/efficacy for some human ACE2 inhibitory molecules (MLN-4760/C16/GL1001/ORE1001, Dx600 and A779) that may be exploited to face this exceptionally dramatic situation. Unfortunately, to my knowledge, only one (MLN-4760/C16/GL1001/ORE1001) of the ACE2-specific inhibitors has been tested in vivo in humans in a Phase I clinical trial long time ago (http://oreholdings.com/wp-content/uploads/2013/06/09.02.09-S-4-A.pdf).\nHere, I have focused my attention on inhibitors of human ACE2 pathway that were consistently administered in vivo. Making use of published reports in which human/rodent ACE2 pathway inhibitors were administered in vivo, I have hypothesized a possible therapeutic pharmacological intervention through an inhibition strategy of the RAS pathways for COVID-19 in patients experiencing both mild and critical, advanced and untreatable stages of the disease (the most problematic cases to manage).\n\n8.1. Inhibition of ACE2: MLN-4760\nBriefly, one of the best candidates to treat COVID-19 patients, but not to prevent viral entry, is the small synthetic molecule MLN-4760 (specific ACE2 inhibitor, also known as C16, GL1001 or ORE1001, [113]) for the following reasons:(1) It has been shown to bind/inhibit ACE2 enzymatic activity even at low/acidic pH (pH 6.5, [115]) typical of hypercapnia (as it might occur in lungs of COVID-19 patients) when human ACE2 activity is maximal [79]; nevertheless, it retains its inhibitory effects on soluble ACE2 bound to spike proteins [24], indicating that it is able to bind and inhibit ACE2 activity regardless ACE2 binding to SARS-CoV-2 particles or to S1 fragments.\n(2) No significant adverse effects were described upon its chronic administration neither alone nor in combination with ACE2 activators (while inhibiting their activating effects) nor after inducing functional impairment of ACE2 activity in rodent experiments in vivo [52,117,118,122,183,184] nor in a clinical Phase I trial in humans (http://oreholdings.com/wp-content/uploads/2013/06/09.10.09-425.pdf);\n(3) Its administration by different route is well described in rodents and humans. In particular:(a) Chronic administration (about 4 weeks) of C-16/DLM-4760 in combination with ACE2 activating treatments was performed by daily intraperitoneal injection at a dose of 25mg/kg in distilled water (as a solution of 42 mg/mL) or 0.9% sterile saline (as a solution of 84 mg/mL using a 0.5-mL insulin syringe) freshly prepared [52,183,184].\n(b) Alternatively, chronic administration (about 8 days) of GL1001/DLM-4760 disodium salt in combination with an ACE2 activating treatment was performed by subcutaneous injection (5mL/kg) containing up to a dose of 300 mg/kg, twice a day, formulated in a vehicle solution [15% 2-hydroxypropyl-β-cyclodextrin (HPBDC)/85% H2O] [122]. Subchronic doses of GL1001 indicate no adverse effects up to 1,000 mg/kg (see [122]).\n(c) In humans ORE1001/GL1001/MLN-4760 was already proposed and tested in clinical trials. Its pharmaceutical indication was for digestive tract inflammations (Inflammatory bowel disease, gastritis and colitis) that are correlated with overexpression of ACE2. In a Phase I clinical testing up 14 days dosing, ORE1001 was well tolerated. Subjects received drug (dosing up to 2100 mg) with no side adverse effects reported. In particular, 47 subjects received single-dose from 2.1 to 2100 mg and 24 subjects received 14 day multiple doses from 50 mg to 1800 mg. All doses were well tolerated, with no significant side effects including blood pressure. Pharmacokinetics of orally administered capsules was consistent with once-daily dosing. (http://oreholdings.com/wp-content/uploads/2013/06/09.10.09-425.pdf). 300 mg (active drug) oral capsules were used in a Phase Ib/IIa clinical trial that was, however, abandoned. (https://clinicaltrials.gov/ct2/show/NCT01039597).\n(d) Finally, MLN-4760 was also administered (2.5 mg/kg per day) by nasal inhalation for 2–3 days in lung-infected mice by Pseudomonas bacteria [185]. Interestingly, the report underscores the role played by local concentration of molecules (ACE2) in modulating lung inflammation and disease. For these reasons, in diseases involving respiratory tract, like SARS, inhalation treatment is preferable, even for the lower concentration (and hopefully lower toxicity) of MLN-4760 needed for this route of treatment administration.\nDifferent routes of MLN-4760 treatment administration can be pursued depending on the hospital condition/expertise. In this exceptionally critical situation, it could be delivered to critical untreatable patients as a controlled “compassionate use”, in particular by inhalation. However, when, under hypoxic conditions, both arms of the RAS are upregulated, it might have a limited action and, by shifting the balance of ACE/ACE2 ratio in favour of ACE, might be even dangerous. Instead, specific inhibition of ACE2 enzymatic activity might effectively work in preventing the establishment of positive feedback loops in COVID-19 patients who are suffering from mild symptoms of the disease. MLN-4760 is sold by different companies, that, in case it works, could be encouraged to manufacture the molecule, actively contributing to face this global threat. On the other hand, the drug could be also synthesized in University chemistry labs because, to my knowledge, is no longer under patent restriction. MLN-4760, whose clinical development was abandoned after phase I trials (clinical name, ORE1001), is actually an interesting compound that could be useful for all patients in which a specific ACE2 upregulation is proven and associated to different (heart/lung/liver/intestine/kidney/immune system/blood/coagulation, etc.) pathological conditions related to ACE2 pathway downstream events, as it seems to occur in COVID-19 patients. Although in blood of normal subjects sACE2 activity is undetectable because its catalytic activity in human plasma is masked by an endogenous inhibitor [116] and chronic ACE2 activation may be deleterious, some patients might need of sACE2 activity to better face an acute pathological condition, therefore a careful monitoring of clinical parameters should be performed during patient treatment.\n\n8.2. Inhibition of Renin Activity: Aliskiren\nAn alternative approach is to inhibit the RAS pathway at its origin by inhibiting renin enzymatic activity. Regarding renin inhibitors, aliskiren is the sole compound in this class of drugs that was approved by the US Food and Drug Administration in March 2007 and commercialized for the management of hypertension [186]. Aliskiren may be used alone or in combination with other drugs to face COVID-19, in particular the severe forms of SARS-CoV-2 infection. Indeed, in patients with severe symptoms of COVID-19 hypoxia might have upregulated both arms of the RAS. Therefore, aliskiren might be a useful “tool” to reduce production of Ang I, the necessary fuel for both ACE and ACE2 hyperactivity and their detrimental effects. Aliskiren treatment in rats has been shown to upregulate both AT1R and MasR expression probably as consequence of a compensation mechanism when both Ang II and Ang (1–7) ligands are reduced by renin inhibition [187]. Of particular interest in the context of SARS-CoV-2 infection, the treatment has been shown to reduce expression of both AT2R and ACE2, thus possibly performing a multiple action in inhibiting both arms of the RAS [187]. Similarly, cation chelating agents such as CaNa2EDTA or nicotianamine might also work, although at different levels.\n\n8.3. Chelating Agents: CaNa2EDTA\nAs already mentioned, chelating agents, by limiting zinc cellular availability, might influence hyperactivity, synthesis and conformation of both ACE2 and ACE, which may impair not only ACE2 (and ACE) enzymatic activity but also its availability on cell surface for viral entry. For these reasons, chelating agents might be effective to counter SARS-CoV-2 infection, and in particular when (e.g., induced by hypoxia) both arms of the RAS are likely upregulated. Differently from renin inhibitors, more chelating agents are commercially available and both classes of drugs might be administered, alone or in combination with other therapies for COVID-19. Of note, these classes of drugs might be beneficial not only for SARS-CoV-2 infection but also for other disease in which both arms of the RAS (and of the inflammatory system) are upregulated, as for example ARDS, PAH and other pathologies associated to chronic hypoxia and cardiac failure. Indeed, both Ang-II and Ang-(1–7) upregulation and a cytokine storm are common aspects in patients not only with ARDS and PAH but also with critical coronary artery disease [182], suggesting an upregulation of both arms of the RAS and of the inflammatory system in several different dysfunctions. Notably, a number of clinical trials have indicated the utility of CaNa2EDTA in coronary heart disease. It was believed that chelation therapy might alter plaque morphology and volume, or improve endothelial function [123]. However, the evidence was not convincing [123]. Future clinical trials on selected patients with both ACE and ACE2 upregulation might be more convincing. It is clear that similar pathways of activation can lead to different outcomes depending on individual predisposition/background. Therefore, future efforts to understand the genetic, morpho-physiological, environmental and lifestyle factors that individually affect the predisposition to a specific disease with a precision medicine must be pursed.\nTo avoid disrupting extracellular calcium levels, CaNa2EDTA formulation is typically employed as an antidote for lead poisoning or as an extracellular zinc chelator [123]. CaNa2EDTA is poorly absorbed in the gastrointestinal tract therefore can be preferentially administered by parenteral route or by inhalation. Nevertheless, oral tablets (e.g., 250–500 mg/tablet) or suppositories of CaNa2EDTA might also work in reducing plasma zinc levels by inhibiting intestinal zinc absorption (the only physiological way of zinc uptake by the organism) [188,189]. In this regard, eight men showing elevated lead concentrations were given a seven-day course of CaNa2EDTA, 4 g/day in divided doses. Oral CaNa2EDTA caused a rise in lead excretion within few hours, suggesting that orally administered drug was partially absorbed [188]. Importantly, the trial in the eight lead workers demonstrated that oral administration of CaNa2EDTA was safe and produced no side-effects [188]. However, the FDA-approved method for delivery of CaNa2EDTA in treatment of lead poisoning is intravenously or intramuscularly and the recommended dose for asymptomatic adults and pediatric patients with elevated blood lead levels is 1000 g/m2/day (~0,05 g/kg/day) [190]. CaNa2EDTA was usually administered for 5–10 days followed by an interval of at least 5–10 days before a second administration. Unfortunately, intravenous administration is very painful. Administration of CaNa2EDTA via nebulizer has also been pursued for inhalation therapy in pulmonary metal poisonings to protect lung tissue and reduce its systemic uptaken [123]; moreover, nebulized solutions containing EDTA have been proven safe, not inducing adverse effects [191]. For the sake of completeness, the unconventional methods for administering chelating agents such as rectal or inhalatory administration are not FDA approved methods for delivery and they have not been studied in detail [192]. CaNa2EDTA diffuses mainly in the extracellular fluids, where it is not significantly metabolized, and it is excreted rapidly by glomerular filtration. The chelator has a half-life of 1.4 to 3 h in adults and is completely excreted within 24 h [123]. For all of the above reasons, CaNa2EDTA might be employed in therapy of COVID-19. However, before beginning any therapy with medication, particularly in the case of new diseases (such as COVID-19) or new treatments, a careful patient selection for these new treatments is essential to prevent unnecessary toxicity (“first do not harm”).\n\n8.4. Safety and Efficacy Concerns\nUnfortunately, diseases of different aetiologies may present similar final dysfunction/alterations (e.g., cardiac dysfunction/lung alterations/thrombosis are induced by excessive activation of either AT1R or MasR/AT2R pathways, see Figure 1), while diseases with same aetiologies may present different final dysfunction/alterations depending on patients (COVID-19 is a clear example). This confounding aspect of diseases can lead to erroneous interpretations, and only defining the correct and early molecular origin of diseases is possible to reach a successful outcome if a therapy is available.\nIn order to decide who to treat, and when to start treatment, it is important to define the entity of dysfunctions, probability of treatment success and probability to develop adverse effects. To promptly intervene in order to prevent severe forms of COVID-19, we need early markers of disease progression. To this regard, if plasma (free) Zn2+ (or total zinc/albumin molar ratio), albumin, IL-10, Ang (1–9), Ang (1–7), Ang (1–5) and/or bradykinin (1–7) will be proven to be reliable surrogate markers for ACE and/or ACE2 enzymatic activity in vivo, they could be exploited, together with circulating ACE and ACE2 activity evaluation, in order to decide who and when to start treating COVID-19 patients. Alternatively, eosinopaenia and hypotension may be also exploited as signs of disease progression. In any case, since the conditions of some patients can be critical, it is highly recommended to administer the minimal effective dose in order to reduce possible side effects, starting with low and increasing doses, while at the same time monitoring patient status and early signs of disease progression (e.g., blood pressure, eosinophil counts, molecular concentrations of the RAS peptides and other markers of inflammation). The aim of the project was not necessarily to stop viral entry (the epithelial cells take already care of it shedding ACE2) but to block positive feedback loops that follow the cellular response to SARS-CoV-2 infection, which are likely the main cause of the severe symptoms associated with COVID-19, doing so, the disease might hopefully become like a simple flu, a spontaneously eradicable disease.\nAt the moment, I am alone in proposing these new and non-conventional therapeutic approaches that may or may not work on COVID-19. In doing so, I have not only described a rationale/strategy to counter COVID-19, but also provided practical information to optimize the RAS inhibitors based on known experimental data. Before any in vivo drug administration (e.g., ACE2 inhibitor), it has to be checked the biological activity (e.g., ACE2) that is affected by the drug, considering the possible side effects. However, this is not always the case and, in some cases, patients are treated without a careful evaluation of the biological parameter that is modulated by the administered drug. In this regard, I have tried to address all aspects of the RAS inhibition in detail because my main concern is that the use of these approaches in a wrong way may lead to a failure or even to deleterious effects. That is why I have meticulously described all the steps before possible administration and provided the guideline suggestions and specific recommendations in order to increase the probability of success and to minimize the deleterious effects. The readers are, however, expected and encouraged to debate and make their own optimized 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