FIELD OF THE INVENTION

[001] The present invention relates to a novel combination therapy and to the use of this combination therapy for the prevention and/or treatment of Acute Respiratory Distress Syndrome (ARDS), a common cause of death in COVID-19 patients. The combination therapy of the present invention is also useful for the prevention of thrombotic complications observed in COVID-19 patients.

BACKGROUND OF THE INVENTION

[002] Acute respiratory distress syndrome (ARDS) is a severe lung condition it occurs when fluid accumulates in alveoli of the lungs. The accumulated fluid can lower the amount of oxygen entering the bloodstream and/or increase the amount of carbon dioxide in the bloodstream. This can ultimately lead to organ failure and significant morbidity and mortality.

[003] It is well documented that the COVID-19 (SARS-CoV-2) pandemic is causing significant morbidity and mortaility across the world, putting healthcare systems, and intensive care units in particular, under considerable strain. Vaccines provide promise for an end to the pandemic. However, it is becoming increasingly clear that a combination of new virus variants, rare but concerning vaccine complications, vaccine phobia and global production and logistical problems mean that complementary approaches to treating patients with the virus are going to be needed.

[004] ARDS is a common cause of death in COVID-19 patients. Certain sub-groups of patients infected with COVID-19 are at much higher risk of getting ARDS than others, and there is an urgent need to identify new and effective therapies for treating and/or preventing ARDS in patients infected with COVID-19. In addition, there is a need for new therapies for reducing thrombotic complications observed in COVID-19 patients, as well as alleviating pneumonia and symptoms such as anosmia, loss of taste and persistent cough.

[005] The present invention was devised with the foregoing in mind.

SUMMARY OF THE INVENTION

[006] The present invention resides in the recognition that a combination therapy involving the administration of dexamethasone and a mineralocorticoid receptor antagonist can provide an effective prevention and/or treatment for ARDS, particularly in patients with a coronavirus infection, such as COVID-19. The combination therapy of the present invention is also anticipated to be useful for the treatment and/or prevention of the thrombotic complications observed in patients suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection). The combination therapy of the present invention is also useful for the treatment and prevention of anosmia (loss of smell), loss of taste and/or cough (including persistent cough) observed in patients suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection).

Acute respiratory distress syndrome (ARDS)

[007] Thus, the present invention relates to dexamethasone, or a pharmaceutically acceptable salt thereof, for use in the treatment of acute respiratory distress syndrome (ARDS), wherein the dexamethasone, or a pharmaceutically acceptable salt thereof, is administered in combination with a mineralocorticoid receptor antagonist.

[008] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, for use in the treatment of acute respiratory distress syndrome (ARDS), wherein the mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[009] The present invention also relates to the use of dexamethasone, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of acute respiratory distress syndrome (ARDS), wherein the medicament is administered in combination with a mineralocorticoid receptor antagonist.

[0010] In another aspect, the present invention also relates to the use of a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of acute respiratory distress syndrome (ARDS), wherein the medicament is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[0011] The present invention also relates to a method of preventing and/or treating acute respiratory distress syndrome (ARDS), the method comprising administering a therapeutically effective amount of dexamethasone, or a pharmaceutically acceptable salt thereof, to a patient in combination with a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

Blood Clotting and Thrombotic Risk

[0012] The present invention also relates to dexamethasone, or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of thrombosis (blood clotting), or to reduce the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection), wherein the dexamethasone, ora pharmaceutically acceptable salt thereof, is administered in combination with a mineralocorticoid receptor antagonist.

[0013] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of thrombosis (blood clotting), or to reduce the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection), wherein the mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[0014] The present invention also relates to the use of dexamethasone, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention and/or treatment of thrombosis (blood clotting), or to reduce the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection), wherein the medicament is administered in combination with a mineralocorticoid receptor antagonist.

[0015] In another aspect, the present invention also relates to the use of a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention and/or treatment of thrombosis (blood clotting), or to reduce the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection), or a pharmaceutically acceptable salt thereof.

[0016] The present invention also relates to a method of preventing and/or treating thrombosis (blood clotting), or reducing the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection), the method comprising administering a therapeutically effective amount of dexamethasone, or a pharmaceutically acceptable salt thereof, to a patient in combination with a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

Anosmia (Loss of Smell), Loss of Taste and Cough (Including Persistent Cough)

[0017] The present invention also relates to dexamethasone, or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection), wherein the dexamethasone, or a pharmaceutically acceptable salt thereof, is administered in combination with a mineralocorticoid receptor antagonist.

[0018] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection), wherein the mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[0019] The present invention also relates to the use of dexamethasone, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection), wherein the medicament is administered in combination with a mineralocorticoid receptor antagonist.

[0020] In another aspect, the present invention also relates to the use of a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection), wherein the medicament is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[0021] The present invention also relates to a method of preventing and/or treating anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection) the method comprising administering a therapeutically effective amount of dexamethasone, or a pharmaceutically acceptable salt thereof, to a patient in combination with a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

Pneumonia

[0022] The present invention also relates to dexamethasone, or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of pneumonia in a patient suffering with a virus (e.g. a coronavirus/COVID-19 infection), wherein the dexamethasone, or a pharmaceutically acceptable salt thereof, is administered in combination with a mineralocorticoid receptor antagonist. [0023] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of pneumonia in a patient suffering with a virus (e.g. a coronavirus/COVID-19 infection), wherein the mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[0024] The present invention also relates to the use of dexamethasone, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention and/or treatment of pneumonia in a patient suffering with a virus (e.g. a coronavirus/COVID-19 infection), wherein the medicament is administered in combination with a mineralocorticoid receptor antagonist.

[0025] In another aspect, the present invention also relates to the use of a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention and/or treatment of pneumonia in a patient suffering with a virus (e.g. a coronavirus/COVID-19 infection), wherein the medicament is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[0026] The present invention also relates to a method of preventing and/or treating anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection) the method comprising administering a therapeutically effective amount of dexamethasone, or a pharmaceutically acceptable salt thereof, to a patient in combination with a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

Combination Products

[0027] In another aspect, the present invention relates to a combination comprising dexamethasone, or a pharmaceutically acceptable salt thereof, and a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

[0028] In another aspect the present invention relates to a pharmaceutical product comprising a combination as defined herein.

[0029] In another aspect, the present invention relates to a pharmaceutical composition comprising a combination as defined herein, and one or more pharmaceutically acceptable excipients. [0030] In another aspect, the present invention relates to a combination as defined herein, or a pharmaceutical product as defined herein, or a pharmaceutical composition as defined herein for use in therapy.

[0031] In another aspect, the present invention relates to a combination as defined herein, or a pharmaceutical product as defined herein, or a pharmaceutical composition as defined herein for use in the prevention and/or treatment of acute respiratory distress syndrome (ARDS).

[0032] In another aspect, the present invention relates to a use of a combination as defined herein in the manufacture of a medicament for preventing and/or treating of acute respiratory distress syndrome (ARDS).

[0033] In another aspect, the present invention relates to a method of preventing and/or treating acute respiratory distress syndrome (ARDS) in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a combination as defined herein.

[0034] In another aspect, the present invention relates to a combination as defined herein, or a pharmaceutical product as defined herein, or a pharmaceutical composition as defined herein for use in the prevention and/or treatment of thrombosis (blood clotting), or to reduce the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection).

[0035] In another aspect, the present invention relates to a use of a combination as defined herein in the manufacture of a medicament for use in the prevention and/or treatment of thrombosis (blood clotting), or to reduce the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection).

[0036] In another aspect, the present invention relates to a method of preventing and/or treating thrombosis (blood clotting), or reducing the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection), the method comprising administering to said subject a therapeutically effective amount of a combination as defined herein.

[0037] In another aspect, the present invention relates to a combination as defined herein, or a pharmaceutical product as defined herein, or a pharmaceutical composition as defined herein for use in the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection). [0038] In another aspect, the present invention relates to a use of a combination as defined herein in the manufacture of a medicament for use in the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection).

[0039] In another aspect, the present invention relates to a method of preventing and/or treating anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection), comprising administering to said subject a therapeutically effective amount of a combination as defined herein.

[0040] Suitably, the dexamethasone, or a pharmaceutically acceptable salt thereof, is administered simultaneously, sequentially or separately with the mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

[0041] Preferred, suitable, and optional features of any one particular aspect of the present invention described herein are also preferred, suitable, and optional features of any other aspect.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0042] Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

[0043] It is to be appreciated that references to “treating” or “treatment” include prophylaxis as well as the alleviation of established symptoms of a condition. “Treating” or “treatment” of a state, disorder or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.

[0044] A “therapeutically effective amount” means the amount of a compound that, when administered to a mammal for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated. [0045] References to “a pharmaceutically acceptable salt” of an inhibitor defined herein is refers to any salt form suitable for pharmaceutical use. Examples of pharmaceutically acceptable salts include an acid-addition salt of an inhibitor of the invention which is sufficiently basic, for example, an acid-addition salt with, for example, an inorganic or organic acid, for example hydrochloric, hydrobromic, sulfuric, phosphoric, trifluoracetic, formic, citric methane sulfonate or maleic acid. In addition, a suitable pharmaceutically acceptable salt of an inhibitor of the invention which is sufficiently acidic is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a pharmaceutically acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.

[0046] References herein to dexamethasone, or a pharmaceutically acceptable salt thereof, and a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, include, where appropriate, any isomeric, tautomeric, polymorphic, amorphous and solvate (e.g. hydrate) forms of the inhibitors. An inhibitor may also be administered in the form of a prodrug which is broken down in the human or animal body to release the active inhibitor. Examples of pro-drugs include in vivo cleavable ester derivatives of the inhibitors that may be formed at a carboxy group or a hydroxy group in an inhibitor compound and in-vivo cleavable amide derivatives that may be formed at a carboxy group or an amino group in an inhibitor compound. Various forms of pro-drug have been described, for example in the following documents :- a) Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985); b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985); c) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and

H. Bundgaard, Chapter 5 “Design and Application of Pro-drugs”, by H. Bundgaard p. US- 191 (1991); d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); e) H. Bundgaard, et ai, Journal of Pharmaceutical Sciences, 77, 285 (1988); f) N. Kakeya, et al., Chem. Pharm. Bull., 32, 692 (1984); g) T. Higuchi and V. Stella, “Pro-Drugs as Novel Delivery Systems”, A.C.S. Symposium

Series, Volume 14; and h) E. Roche (editor), “Bioreversible Carriers in Drug Design”, Pergamon Press, 1987. [0047] References herein to the dexamethasone, or a pharmaceutically acceptable salt thereof, being administered “in combination with” a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, or vice versa, unless otherwise stated otherwise, include the inhibitors being administered sequentially, separately or simultaneously with one another.

[0048] As used herein “simultaneous administration” refers to therapy in which the both agents (e.g. dexamethasone, or a pharmaceutically acceptable salt thereof, and a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof) are administered at the same time.

[0049] As used herein “sequential administration” means that one agent is administered after the other, however, the time period between the administration of each agent is such that both agents are capable of acting therapeutically concurrently. Thus, administration "sequentially" may permit one agent to be administered within seconds, minutes, or a matter of hours after the other provided the circulatory half-life of the first administered agent is such that they are both concurrently present in therapeutically effective amounts. The time delay between the administration of the agents may vary depending on the exact nature of the agents, the interaction there between, and their respective half-lives.

[0050] As used herein, "separate administration" means that one agent is administered after the other, however, the time period between administration is such that the first administered agent is no longer present a therapeutically effective amount when the second agent is administered. Accordingly, the two agents exert their therapeutic effects separately. Nevertheless, the overall therapeutic effect observed when the two agents separately act therapeutically may be greater than either agent used alone.

[0051] As used herein the, “subject(s)” and/or “patient(s)”, suitably refer to human(s).

[0052] As used herein, a “pharmaceutical product” refers to a product comprising a pharmaceutical. For instance, examples of a pharmaceutical product include a medical device, a pharmaceutical composition and a kit of parts suitably comprising one or more devices, containers and/or pharmaceuticals.

Combination Therapy

[0053] The present invention resides in the recognition that the administration of a combination therapy comprising dexamethasone and a mineralocorticoid receptor antagonist is a potentially useful therapy for the prevention/treatment of ARDS, particularly in patients suffering with COVID-19. [0054] The majority of patients infected with the COVID-19 virus are either asymptomatic or have a short-lasting clinical problem. However, a small number, and especially those with hypertension, cardiovascular disease, diabetes and hypercholesterolaemia are at much higher risk of progressing to the Acute Respiratory Distress Syndrome. These require hospitalisation, intensive care, oxygen therapy and often ventilation. It is the development of ARDS which is usually the major factor leading to death.

[0055] Patients infected with COVID-19 can also be susceptible to an increased thrombotic risk. The present invention also resides in the recognition that the administration of a combination therapy comprising dexamethasone and a mineralocorticoid receptor antagonist is a useful therapy for the prevention and/or treatment of thrombosis, or reducing the risk of thrombosis, particularly in patients suffering with COVID-19.

[0056] Common symptoms associated with COVID-19 infections include anosmia, loss of taste, and/or persistent cough. The present invention also resides in the recognition that the administration of a combination therapy comprising dexamethasone and a mineralocorticoid receptor antagonist is a useful therapy for the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough, particularly in patients suffering with COVID-19.

Rationale for the combination therapy of the present invention

[0057] The rationale behind the combination therapy of the present invention and the basis for the original hypothesis behind this combination therapy is set out below:

Description of Figures

Figure 1 shows signalling pathways associated with Angiotensin II and Angiotensin-(1-7).

Figure 2 shows the effect of aldosterone on ATP cellular release with consequential activation of purinergic receptors, entry of calcium into the cell, contraction and sodium channel opening

16

Figure 3A shows Endothelial cell Weibel-Palade bodies.

Figure 3B shows von Willebrand factor immunofluorescent staining of human umbilical vein endothelial cells showing the typical Weibel-Palade body distribution (courtesy of Dr Tom McKinnon and Prof. Anna Randi, Imperial College, London). Figure 4 shows data to indicate that Aldosterone stimulates the release of von Willebrand factor from endothelial cells. This can be blocked by MR antagonist spironolactone (Jeong et al 2009)

Figure 5 shows the results of Ukraine study 1. Clinical findings (%) in Group I given high dose dexamethasone (HIDEX) compared to low dose dexamethasone and spironolactone (SPIDEX) at baseline (lower bars) and after 5 days (upper bars).

Summary

[0058] The present inventor understands that patients with severe COVID-19 infection have covert Mineralocorticoid Receptor (MR) activation. Under normal circumstances the salt- retaining hormone aldosterone binds to MR and results in the opening of the sodium channel in key cells such as those in the kidney.

[0059] Rather surprisingly, the MR is non-selective and can bind both cortisol and aldosterone. Given that the levels of cortisol are much higher than those of aldosterone one would expect that the MR would always be occupied by cortisol. The fact that it is not is mainly due to a key protective enzyme in the cell which converts cortisol into inactive cortisone. This was described in a young man with very severe hypertension who lacked the ability to convert cortisol to cortisone. Cortisol bound to the MR and produced massive salt retention and potassium loss. Treatment with dexamethasone was dramatic. It binds preferentially to the Glucocorticoid Receptor (GR) rather than the MR. This suppresses the secretion of ACTH by the pituitary and hence cortisol by the adrenal.

[0060] The present inventors have found that levels of aldosterone were undetectable in two thirds of COVID-19 patients. As these patients are not salt depleted this suggests that cortisol is binding to the MR to produce sodium retention. This makes the suppression of cortisol a key objective in treatment.

[0061] It is therefore understood that in patients with severe COVID-19 infection there is covert activation of the Mineralocorticoid Receptor by cortisol and not aldosterone in both epithelial and endothelial cells. In the latter it is proposed this plays a key role in the exocytosis of Weibel-Palade Bodies (WP). These bodies contain angiopoietin-2 and von Willebrand Factor (VWF).

[0062] Angiopoietin-2 increases capillary permeability and is very high in patients with Acute Respiratory Distress Syndrome (ARDS), a common cause of death in COVID-19 patients.

[0063] VWF unfurls and produces large strings that bind platelets and are thus pro-thrombotic. [0064] Mineralocorticoid Receptor activation leads to ATP release from the cell which then acts back on purinergic receptors increasing intra-cellular calcium which stimulates exocytosis of WP bodies; this can be inhibited by spironolactone.

[0065] The present invention relates to the administration of a mineralocorticoid receptor antagonist (e.g. spironolactone) to block the Mineralocorticoid Receptor and dexamethasone to suppress cortisol secretion, to help to prevent the development of ARDS and thrombotic complications in COVID-19 infected patients.

COVID-19 and ARDS

[0066] Certain subgroups of patients infected with COVID-19 are at much higher risk of getting ARDS than others. It is understood that the major lung damage produced by the COVID-19 virus is due to a combination of its local pulmonary inflammatory effect and its inhibition of the function of the ACE-2 (Angiotensin Converting Enzyme-2) enzyme. The virus uses the ectodomain of the enzyme to enter the type 2 alveolar cell. It is not surprising, given the importance role of these cells in lung surfactant production and in alveolar fluid removal, that major emphasis of research has been on this. Animal models of ARDS, however, suggest that this may not be the main reason why some patients with COVID-19 develop ARDS.

[0067] Two mouse models of ARDS have involved either acid aspiration or sepsis-induction. Both produce acute lung injury with typical ARDS. Studies published in 2005 ¹ showed that ACE-2 played a major role in protecting the animals from acute lung failure. In animals where ACE-2 was knocked out and acid/sepsis given, there was progressive hypoxia associated with massive lung oedema and inflammatory cell infiltration. All wild type animals survived, but only two out of 10 ACE- knockout. These studies suggest that regardless of the cause of lung injury (e.g. virus, acid or sepsis) it is the loss of the ACE-2 enzyme that determines the severity of the outcome.

Role of ACE-2 enzyme :

[0068] ACE-2 converts Angiotensin II to Angiotensin (1-7) ((A) (1-7)) (Fig.1). The role of Angiotensin (1-7) has been well reviewed ² . A (1-7) binds to a G-protein coupled receptor Mas and is a key counter-regulatory pathway within the renin-angiotensin system. It is a vasodilator and anti-proliferative. Deletion of the Mas receptor produces hypertension, endothelial dysfunction and thrombogenesis. Xu et al studied endothelial function ³ , using TBARS levels as an indicator of oxidative stress. They found these were significantly elevated in KO mice. Nitric Oxide (NO) acts as an endogenous anti-oxidant by quenching O and protects against ROS. They found decreased NO. In addition, a subunit of NADPH oxidase, a key source of ROS was significantly higher in the KO animals and two important antioxidant enzymes (Superoxide Dismutase and catalase) were reduced. [0069] If the Angiotensin (1-7) KO results are similar to COVID-19 ACE-2 enzyme damage, this may explain why COVID infections produce major local tissue oxidative stress with a marked increase in Reactive Oxygen Species (ROS).

[0070] Studies have shown that Angiotensin (1-7) can attenuate Angiotensin II induced ROS generation in endothelial cells. In the absence of ACE-2 this would mean that there are higher local levels of Angiotensin II and lower levels of Angiotensin (1-7). This has profound effects on the renin-angiotensin axis. In the acid aspiration ARDS model mice have markedly increased angiotensin II levels in both lung and plasma ¹ . However, these levels are significantly higher in the lungs and plasma of acid-treated ACE-2 knockout mice. In mice recombinant ACE-2 can protect from severe acute lung injury.

Measurement of plasma aldosterone in COVID-19 patients:

[0071] Given the high levels of Angiotensin II in both the ACE-2 KO mice and in COVID-19 patients ⁴ it might be expected that aldosterone levels would be elevated. Plasma aldosterone was measured in anonymised samples from 20 unselected patients with COVID-19 infection admitted to hospital. In 15 patients the levels were undetectable and in the other 5 low normal (104-128 pmol/l).

[0072] This dissociation of the renin-angiotensin system from aldosterone secretion has been described by Findling et al ⁵ . They studied patients admitted to the Respiratory Intensive Care Unit. One group (Group I) had inappropriately low levels of aldosterone for their elevated Plasma Renin Activity. Group II had significantly higher aldosterone levels despite lower PRA. None of the patients had evidence of mineralocorticoid deficiency. Mortality was significantly higher in Group I (75%) than Group II (46%, p<0.001).

[0073] A possible explanation for this paradox between high levels of PRA/AII and aldosterone is that high levels of All stimulate Endogenous Ouabain (EO) release from the adrenal cortex ⁶ . This acts via the low affinity AT2R and inhibits the Na ⁺ /K ⁺ pump ot2 subunit. Ouabain is a potent inhibitor of aldosterone secretion in the human adrenal ⁷ . The relative or absolute deficiency of aldosterone secretion suggested by the results in COVID patients underlines the importance of an alternative agonist for the Mineralocorticoid Receptor (MR) which is most likely to be cortisol.

Control of local glucocorticoid levels:

[0074] The present inventors have demonstrated that in aldosterone-specific tissues such as the renal distal tubules the MR was protected from cortisol by an enzyme 11b-hydroxysteroid dehydrogenase type 2 (11b-H802) that converted cortisol to inactive cortisone ⁸ . This study reported a 21 year old man with very severe hypertension and hypokalaemia who was unable to convert cortisol to cortisone because of a genetic defect ⁹ . Treatment with dexamethasone lowered his blood pressure to normal and restored normal plasma potassium. The reason for this effect is that dexamethasone, unlike cortisol, binds more selectively to the Glucocorticoid Receptor (GR). It also suppresses the secretion of ACTH and hence cortisol.

[0075] John Funder published a review of “Aldosterone, mineralocorticoid receptors and vascular inflammation” ¹⁰ . This suggests that it is not only 11b-H8ϋ2 that protects the MR from cortisol but also the cofactor NADH. 11 b -HSD2 requires NAD to convert cortisol to cortisone and in the process produces NADH. NADH then binds to a C-terminal binding protein (CtBP) that then acts as a co-repressor of DNA transcription ¹¹ . Thus, it is suggested that the majority of cortisol is inactivated by conversion to cortisone; the residue binds the MR but is inactivated by NADH-CtBP. Further work has shown that normal intracellular glucocorticoid levels can activate the MR when aldosterone levels are low and 11b-H802 blocked (and thus low NADH).

[0076] Endothelial cells have Mineralocorticoid Receptors but very low or absent expression of 11 b-H8ϋ2 ¹² . Because of the high affinity of cortisol for the MR it is not surprising that these cells contain cortisol-MR complexes. Under normal circumstances these are not activated probably because of binding of NADH-CtBP to the complex. However, they can be activated by Reactive Oxygen Species (ROS) ^{13 10 14} . As discussed above ROS levels are very high in COVID-19 infected cells with ACE-2 depletion.

[0077] Abais and colleagues ¹⁵ have defined ‘kindling’ and ‘bonfire’ ROS. Intracellular and extracellular ROS are normally very tightly regulated (levels kept at less than 1% of produced ROS). These can activate the formation of the NLRP3 inflammasome. They showed that ROS derived from NADPH oxidase may serve as ‘kindling’ signalling molecules. However, when the NLRP3 inflammasome is activated by other stimuli such as DAMPS (Damage Associated Molecular Patterns) such as ATP, a local inflammatory response occurs. This then recruits macrophages and T cells which lead to a ‘bonfire’ production of ROS with damage to DNA, proteins and lipids.

Mechanism of action of aldosterone:

[0078] In 2005 it was found that aldosterone opened sodium channels by a novel mechanism ¹⁶ . After binding to the Mineralocorticoid Receptor (MR) ATP is released from the cell. The time course for this suggests that it is non-genomic. The released ATP acts back on the cell and adjacent cells to stimulate purinergic receptors (P2X4) that then increase calcium entry. This results in cell contraction with opening of the sodium channel (Fig.2).

[0079] In 2009 Jeong et al ¹⁷ showed that this mechanism was responsible for exocytosis of Weibel-Palade bodies from endothelial cells (Fig. 3, 4). These are critically important bodies which contain key factors involved in local thrombosis (von Willebrand factor), cell adhesion (P-selectin) and vascular permeability (Angiopoietin-2). The Weibel-Palade release could be inhibited by MR antagonist spironolactone (Fig. 4) .

[0080] The mechanism controlling W-P bodies release has been well reviewed by Schillemans et al ¹⁸ .

[0081] Kumpers and Lukasz reviewed the potential importance of this in their paper The curse of angiopoietin-2 in ARDS ¹⁹ . Angiopoietin-1 is an anti-permeability factor that protects the vasculature from plasma leakage. One source is alveolar type II cells that are damaged by COVID-19 using their ACE- receptors for cell entry. Angiopoietin-2 release from Weibel- Palade bodies in endothelial cells prevents Angiopoietin-1 from binding to its receptor thus promoting inflammation and vascular permeability. The plasma levels of Angiopoietin-2 are proportional to ARDS severity and rising levels predict a poor ARDS outcome.

[0082] The inventors have deduced that the administration of spironolactone or similar MR antagonists such as eplerenone could possibly prevent WP bodies being released from infected endothelial cells and hence reduce the increased capillary permeability and pro- thrombotic state of COVID-19 patients.

Learning from other models of Acute Respiratory Distress Syndrome:

[0083] In addition to animal models such as ACE-2 receptor KO ¹ it is interesting to look at conditions such as High Altitude Pulmonary Edema (HAPE). Solaimanzadeh has compared the clinical and radiological findings in HAPE and COVID-19 which appear to be very similar ²⁰ . He suggests that drugs used for HAPE such as amlodipine or nifedipine (Calcium Channel Blockers, CCBs) might be of value in COVID-19 ARDS. A retrospective review of COVID-19 patients over 65 admitted to hospital in New York examined the patient outcomes in those admitted on treatment with either nifedipine or amlodipine. Of the 24 CCB treated patients 12 (50%) survived. Of 41 not on CCBs only 6 (15%) survived. Only 1 patient on CCBs required ventilation as compared to 39% not on CCBs.

[0084] The comparison between HAPE and COVID-19 has been questioned by Luks et al ²¹ . Despite this warning it is worth asking if HAPE can inform us about COVID-19 ARDS. In addition to the clinical presentation with cough, fever and ground glass appearance on chest X-ray HAPE has remarkable similarities to COVI D when it comes to coagulation and fibrinolytic abnormalities ²² . D-Dimer levels in patients with HAPE were 3 times higher than unacclimatised controls: 97% of patients admitted to hospital with COVID have elevated D- Dimer. [0085] A key response to hypoxia is increased expression of lactate dehydrogenase ²³ which rapidly uses NADH to provide NAD for glycolysis and ATP generation. As discussed above NADH binds to C-terminal Binding Protein (CtBP) and produces a conformational change that promotes CtBP dimerization that is essential for co-repressor activity ^{11 24} . This then inhibits MR mediated gene expression. Lack of NADH will thus allow cortisol to activate the MR. This would be in keeping with studies showing that hypoxia stimulates the release of ATP from human endothelial cells ²⁵ . Hypoxia also releases Weibel-Palade bodies ²⁶ . Hypoxia has also been associated with a dissociation of renin and aldosterone secretion.

[0086] If HAPE and COVID-19 ARDS share a common mechanism resulting in purinergic activation and release of Weibel-Palade bodies then studies designed to prevent HAPE are of interest. Maggiorini et al ²⁷ studied mountaineers with previous HAPE. They were randomly allocated to dexamethasone (4mg bd) or placebo started 24 hours before the ascent to 4559m. Of nine mountaineers on placebo seven developed HAPE (clinically and radiologically). None of ten mountaineers on dexamethasone developed HAPE (p<0.001). The mechanism is unclear but could relate to cortisol suppression by dexamethasone which binds preferentially to the Glucocorticoid Receptor.

Angiotensin II and vascular damage:

[0087] Extensive data shows that elevated angiotensin II in animals on a high salt intake produces vascular injury (fibrinoid necrosis, perivascular inflammation, focal infarctions) ²⁸ which can be prevented by Mineralocorticoid Receptor blockade. This damage was found in all animals post-adrenalectomy given A II and aldosterone but only in 2 /10 in those post adrenalectomy on dexamethasone and given A II. This could be the result of having no circulating corticosterone to bind to the MR and thus unable to be activated by ROS as part of the inflammatory response.

Risk factors for the development of ARDS in COVID-19 infection:

[0088] Increasing age, male gender, certain coexisting conditions (hypertension, cardiovascular disease, diabetes, hypercholesterolaemia, obesity) and racial background (BAME) are thought to be important risk factors for COVID-19 associated ARDS.

[0089] If covert activation of the Mineralocorticoid Receptor-cortisol complex by ROS is playing a major role it is worth looking at age and disease associated changes in oxidative stress. Massudi et al detailed changes in age-related NAD ⁺ metabolism in human tissue ²⁹ . Oxidative DNA damage is a major factor associated with age-related diseases. This activates a DNA repair mechanism poly(ADP-ribose) polymerase (PARP). Essential to this enzyme is NAD ⁺ . Overactivity of this system can lead to severe NAD ⁺ depletion. [0090] The authors found a striking fall in tissue NAD ⁺ levels with increasing age (0-1 years 8.54 ng NAD ⁺ per mg protein, 30-50 years 2.74, 51-70 1.08, >71 1.06). This was inversely correlated with the progressive increase in PARP activity. There was an interesting gender- related difference in NAD ⁺ levels with aging. These were negatively correlated with age for males aged 0-77 years (p<0.0007). The age-related decline in females was less obvious but in those aged 37-76 was significant. The authors make the point that females may have a greater capacity to recycle NAD ⁺ from the PARP metabolite nicotinamide.

[0091] Cells infected with COVID-19 will have a very high level of ROS with likely increased DNA damage and hence PARP activity. In these cells the NAD level may be inadequate for the normal protection of the MR by 11b-H802 in the type II alveolar cells and also in the renal tubules.

[0092] NADPH oxidase is an important source of ROS. Not only is this activated by angiotensin II in COVID-19 infected cells but also by insulin and Advanced Glycosylation End products (AGEs) ³⁰ . A very similar process is closely linked to the development of atherosclerosis. This mechanism could underlie the increased risk of diabetic patients getting COVID-19 complications. In addition, patients with cardiovascular disease and those with diabetes have a change in their vascular basement membrane with collagen being replaced by fibronectin ³¹ . Shear stress in these vessels produces marked activation of the NFKB pathway. All forms of vascular remodelling are associated with increased oxidative stress.

[0093] Glutathione is the main defence mechanism against ROS. Normally Glucose-e- phosphate dehydrogenase (G6PD) in the pentose phosphate pathway converts glucose-e- phosphate to 6-phosphogluconolactone and NADP to NADPH. NADPH then maintains the reduced glutathione to mop up free radicals. G6PD deficiency is an X-linked disorder that affects about 400 million people globally and is especially common in the Black African Minority Ethnic (BAME) population. Studies have shown that G6PD deficiency activates endothelial cells by increasing levels of ROS, NOX-4, ICAM-1 whilst reducing Nitric Oxide. Thus the loss of the ACE-2 receptor in G6PD deficient endothelial cells would likely have a markedly adverse effect on endothelial cell function with activation of cortisol-Mineralocorticoid Receptor complexes ¹⁰ .

[0094] This could be a possible factor in why some COVID-19 infected BAME patients have a higher mortality. Public Health England reported that age-standardised death rates in England in confirmed cases of COVID-19 per 100,000 population were black ethnic groups 257 men, 119 women, Asian 163 men 78 women, white 70 males, 36 females. There has been no testing as yet for G6PD deficiency which could well be a factor, especially given it is X-linked. Female carriers of the mutation can be affected because of failure of X-chromosome Lyonisation.

The purinergic basis of COVID-19 complications:

[0095] As shown in epithelial cells ¹⁶ and by Jeong ¹⁷ in endothelial cells, activation of the Mineralocorticoid Receptor leads to the release of ATP from the cells which then acts back on local purinergic receptors. With COVID-19 infection this could play a major role in the clinical presentation. Failure to activate the MR could explain why many people, especially the young have asymptomatic infection. Thus, in alveolar cells infected with the virus but with high levels of NAD the MR could be protected from cortisol. In contrast with much lower levels of intracellular NAD this protection may be lost with MR activation and ATP release.

[0096] One of the most common early features of COVID-19 infection is a non-productive cough. Recent studies in patients with chronic cough have shown that this can be a consequence of ATP released from epithelial cells acting via purinergic receptors (P2X2/3) on lung vagal nerve fibres ³² . This has led to the successful introduction of a purinergic blocker Gabapentin for chronic refractory cough. This raises the interesting question as to whether the cough with COVID-19 can be reduced by MR receptor blockade via reduced local ATP release.

[0097] After the infection of the type II alveolar cells by COVID-19, the virus spreads to infect multiple cells with ACE-2 receptors and in particular endothelial cells. As discussed above, activation of the MR in these cells leads to the release of ATP and the exocytosis of the Weibel- Palade bodies.

Potential prophylactic treatment:

[0098] If the purinergic mechanism discussed could be blocked by inhibition of the Mineralocorticoid Receptor in endothelial cells this could have major benefit. It would seem likely that the most appropriate approach to COVID-19 would be to use a combination of a specific MR antagonist such as spironolactone with dexamethasone to inhibit ACTH and hence cortisol secretion.

[0099] It is of interest that dexamethasone, unlike methylprednisolone, has been shown to significantly reduce the mortality of patients with moderate to severe ARDS (26% in dexamethasone group, 36% in control group) ³³ . The rationale for dexamethasone was its anti inflammatory potency. The high dose used (20mg daily for 5 days and then 10mg daily) was associated with a high incidence of hyperglycaemia (76%).

[00100] Dexamethasone has also being tested as an anti-inflammatory drug in the Oxford PRINCIPLE trial in COVID-19 patients. They have used dexamethasone 6mg intravenously once per day. Their aim was to use this as an anti-inflammatory drug. This study found a major decrease in mortality of 35% in the low dose dexamethasone versus the control group. If the mechanism proposed plays a key role in pathogenesis then much lower doses of dexamethasone might be possible with measurement of plasma cortisol to show that it is suppressed.

[00101] It is important to consider the optimum time for introducing any treatment. In the Villar multi-centre study of the effect of dexamethasone the patients had established moderate to severe ARDS. In the High Altitude Pulmonary Edema (HAPE) study when dexamethasone was given to susceptible mountaineers prior to ascent it was strikingly successful (100% prevention) in comparison to lack of dexamethasone benefit in established Acute Mountain Sickness. It could well be argued that if persistent cough indicates purinergic activation then giving low dose dexamethasone at this stage might be most sensible. This could be particularly important for patients in the high-risk groups.

[00102] The discovery that plasma aldosterone levels are often undetectable in hospitalised COVID-19 patients suggests that measurement of plasma aldosterone may be a useful marker of covert mineralocorticoid excess and may thus suggest that dexamethasone and MR receptor blockade might be considered.

[00103] MR receptor antagonism has been very successful in preventing vascular damage in rats infused with Angiotensin II and salt-loaded ¹⁴ . It could be argued that this is the situation in COVID-19 patients with high levels of Angiotensin II and covert mineralocorticoid excess. Spironolactone has also been used successfully to treat acid- aspiration induced ARDS in rats. Again, this treatment was given at an early stage.

[00104] Against this background a proposal for a clinical trial of spironolactone and dexamethasone will be conducted. The study aims to recruit patients at high risk of developing ARDS who have cough, fever and/or shortness of breath. The patients would be treated with a loading dose of 100mg spironolactone and then a maintenance dose of 50mg twice daily plus dexamethasone at a dose of 2mg given at 09:00h and at 21 :00h. The aim is to prevent progression to critical care/ventilatory support. References:

1 Imai Y, Kuba K, Rao S, et al. Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature 2005; 436: 112-6.

2 Santos RAS, Ferreira AJ, Verano-Braga T, Bader M. Angiotensin-converting enzyme 2, angiotensin-(1-7) and Mas: New players of the renin-angiotensin system. J. Endocrinol. 2013; 216. DOI:10.1530/JOE-12-0341.

3 Xu P, Costa-Goncalves AC, Todiras M, et al. Endothelial dysfunction and elevated blood pressure in Mas gene-deleted mice. Hypertension. 2008; 51 : 574-80.

4 Xu XW, Wu XX, Jiang XG, et al. Clinical findings in a group of patients infected with the 2019 novel coronavirus (SARS-Cov-2) outside of Wuhan, China: Retrospective case series. BMJ 2020; 368: 1-7.

5 FINDLING JW, WATERS VO, RAFF H. The Dissociation of Renin and Aldosterone during Critical Illness*. J Clin Endocrinol Metab 1987; 64: 592-5.

6 Funder JW. Aldosterone and mineralocorticoid receptors — physiology and pathophysiology. Int J Mol Sci 2017; 18: 1-9.

7 Antonipillai I, Schick K, Horton R. Ouabain is a potent inhibitor of aldosterone secretion and angiotensin action in the human adrenal. J Clin Endocrinol Metab 1996. DOI:10.1210/jc.81.6.2335.

8 Edwards CR, Stewart PM, Burt D, et al. Localisation of 11 beta-hydroxysteroid dehydrogenase-tissue specific protector of the mineralocorticoid receptor. Lancet 1988; 2: 986-9.

9 Stewart PM, Corrie JE, Shackleton CH, Edwards CR. Syndrome of apparent mineralocorticoid excess. A defect in the cortisol-cortisone shuttle. J Clin Invest 1988; 82: 340-9.

10 Funder JW. Aldosterone, mineralocorticoid receptors and vascular inflammation. Mol Cell Endocrinol 2004; 217: 263-9.

11 Zhang Q, Piston DW, Goodman RH. Regulation of corepressor function by nuclear NADH. Science (80- ). 2002; 295: 1895-7.

12 Gong R, Morris DJ, Brem AS. Variable expression of 11 b Hydroxysteroid dehydrogenase (I ΐb-HSD) isoforms in vascular endothelial cells. Steroids 2008; 73: 1187— 96.

13 Ward MR, Kanellakis P, Ramsey D, Funder J, Bobik A. Eplerenone suppresses constrictive remodeling and collagen accumulation after angioplasty in porcine coronary arteries. Circulation 2001; 104: 467-72.

14 Wang H, Shimosawa T, Matsui H, et al. Paradoxical mineralocorticoid receptor activation and left ventricular diastolic dysfunction under high oxidative stress conditions. J Hypertens 2008. DOI : 10.1097/HJ H.0b013e328300a232.

15 Abais JM, Xia M, Zhang Y, Boini KM, Li PL. Redox Regulation of NLRP3 Inflammasomes: ROS as Trigger or Effector? Antioxidants Redox Signal. 2015; 22: 1111-29.

16 Gorelik J, Zhang Y, Sanchez D, etai. Aldosterone acts via an ATP autocrine/paracrine system: the Edelman ATP hypothesis revisited. Proc Natl Acad Sci U S A 2005; 102: 15000- 5.

17 Jeong Y, Chaupin DF, Matsushita K, et al. Aldosterone activates endothelial exocytosis. 2009; 106: 3782-7.

18 Schillemans M, Karampini E, Kat M, Bierings R. Exocytosis of Weibel-Palade bodies: how to unpack a vascular emergency kit. J. Thromb. Haemost. 2019; 17: 6-18.

19 Kumpers P, Lukasz A. The curse of angiopoietin-2 in ARDS: On stranger TI(E)des. Crit Care 2018; 22: 4-7.

20 Solaimanzadeh I. Acetazolamide, Nifedipine and Phosphodiesterase Inhibitors: Rationale for Their Utilization as Adjunctive Countermeasures in the T reatment of Coronavirus Disease 2019 (COVID-19). Cureus 2020. DOI:10.7759/cureus.7343.

21 Luks AM, Freer L, Grissom CK, et al. COVID-19 Lung Injury is Not High Altitude Pulmonary Edema. High Alt Med Biol 2020; on line Ap: 1-2.

22 Ren Y, Cui F, Lei Y, Fu Z, Wu Z, Cui B. High-altitude pulmonary edema is associated with coagulation and fibrinolytic abnormalities. Am J Med Sci 2012; 344: 186-9. 23 Firth JD, Ebert BL, Ratcliffe PJ. Hypoxic Regulation of Lactate Dehydrogenase A. J Biol Chem 1995; 270: 21021-7.

24 Nardini M, Spano S, Cericola C, etal. CtBP/BARS: A dual-function protein involved in transcription co-repression and Golgi membrane fission. EMBO J 2003; 22: 3122-30.

25 Lim To WK, Kumar P, Marshall JM. Hypoxia is an effective stimulus for vesicular release of ATP from human umbilical vein endothelial cells. Placenta 2015; 36: 759-66.

26 Pinsky DJ, NakaY, Liao H , etal. Hypoxia-induced exocytosis of endothelial cell weibel- palade bodies: A mechanism for rapid neutrophil recruitment after cardiac preservation. J Clin Invest 1996; 97: 493-500.

27 Maggiorini M, Brunner-La Rocca HP, Peth S, etal. Both tadalafil and dexamethasone may reduce the incidence of high-altitude pulmonary edema: A randomized trial. Ann. Intern. Med. 2006; 145: 497-506.

28 Rocha R, Martin-Berger CL, Yang P, Schemer R, Delyani J, McMahon E. Selective aldosterone blockade prevents angiotensin ll/salt-induced vascular inflammation in the rat heart. Endocrinology 2002; 143: 4828-36.

29 Massudi H, Grant R, Braidy N, Guest J, Farnsworth B, Guillemin GJ. Age-associated changes in oxidative stress and NAD+ metabolism in human tissue. PLoS One 2012; 7: 1-9.

30 Kaneto H, Katakami N, Matsuhisa M, Matsuoka TA. Role of reactive oxygen species in the progression of type 2 diabetes and atherosclerosis. Mediators Inflamm 2010; 2010. DOI:10.1155/2010/453892.

31 Schwartz MA, Vestweber D, Simons M. A unifying concept in vascular health and disease. Science (80- ). 2018; 360: 270-1.

32 Keller JA, Mcgovern AE, Mazzone SB. Translating Cough Mechanisms Into Better Cough Suppressants. Chest 2017; 152: 833-41.

33 Villar J, Fernando C, Martinez D, et al. Dexamethasone treatment for the acute respiratory distress syndrome: a multicentre, randomised controlled trial. Lancet Respir Med 2020; 8: 267-76.

Dexamethasone

[00105] Dexamethasone is classified as a glucocorticosteroid and is widely used clinically for its anti-inflammatory and anti-cancer properties.

[00106] Recent data arising from the Recovery Trial conducted in the UK also showed that the administration of 6 mg of dexamethasone intravenously daily significantly reduced mortality in COVID-19 patients receiving oxygen therapy or on ventilation.

[00107] Suitable dosage ranges and schedules for the administration of dexamethasone are known in the art.

[00108] For example, dexamethasone may be administered at a dosage within the range of 0.25 mg - 20 mg per day.

[00109] In the combination therapy of the present invention, the dose of dexamethasone is suitably within the range of 0.5 mg to 10 mg per day. More suitably, the dosage of dexamethasone is within the range of 0.5 mg to 6 mg per day.

[00110] In an embodiment of the invention, the dosage of dexamethasone is within the range of 1 mg to 10 mg per day. [00111] In an embodiment of the invention, the dosage of dexamethasone is within the range of 1 mg to 8 mg per day.

[00112] In an embodiment of the invention, the dosage of dexamethasone is within the range of 1 mg to 6 mg per day.

[00113] In an embodiment of the invention, the dosage of dexamethasone is within the range of 1 mg to 4 mg per day.

[00114] In an embodiment of the invention, the dosage of dexamethasone is 4 mg per day.

[00115] In another embodiment of the invention, the dosage of dexamethasone is 1.5 mg per day.

[00116] The daily dose of dexamethasone may be administered as a single dose or as divided doses.

[00117] In an embodiment of the invention, the dexamethasone is administered as a single daily dose.

[00118] More suitably, dexamethasone is administered in divided dosages, e.g.: a dosage of 0.25 mg to 6 mg given twice daily; a dosage of 0.25 mg to 5 mg given twice daily; a dosage of 0.25 mg to 4 mg given twice daily; a dosage of 0.25 mg to 3 mg given twice daily; a dosage of 0.5 mg to 2 mg given twice daily; a dosage of 1 mg to 3 mg given AM (in the morning) and 1 mg to 3 mg given PM (e.g. in the evening/at bedtime); a dosage of 2 mg given AM (in the morning) and 2 mg given PM (e.g. in the evening/at bedtime); a dosage of 0.5 mg to 1.5 mg given AM (in the morning) and 0.25 mg to 1 mg given PM (e.g. in the evening/at bedtime); or a dosage of 1 mg given AM (in the morning) and 0.5 mg given PM (e.g. in the evening/at bedtime).

[00119] In an embodiment of the invention, the dexamethasone is administered at a dosage of a dosage of 2 mg in the morning and 2 mg in the evening, optionally reduced to 1 mg in the morning and 0.5 mg in the evening depending on the severity of the symptoms. Mineralocorticoid receptor antagonists

[00120] Mineralocorticoid receptor antagonists (also known as aldosterone antagonists) of the present invention are well known in the art.

[00121] Suitably, the mineralocorticoid receptor antagonist is selected from spironolactone, eplerenone or canrenone, or pharmaceutically accpetable salts of these agents.

[00122] In a particular embodiment of the combination therapy of the present invention, the mineralocoricoid receptor antagonist is spironolactone, or a pharmaceutically acceptable salt thereof.

[00123] In another particular embodiment of the combination therapy of the present invention, the mineralocorticoid receptor antagonist is eplerenone, or a pharmaceutically acceptable salt thereof.

[00124] In another particular embodiment of the combination therapy of the present invention, the mineralocorticoid receptor antagonist is canrenone, or a pharmaceutically acceptable salt thereof.

Spironolactone

[00125] Spironolactone is a well known non-selective mineralocorticoid receptor antagonist, which has been used for many years for the treatment of conditions such as oedema, ascites, nephoritic syndrome, heart failure and resistant hypertension.

[00126] Suitable dosage ranges and schedules for the administration of spironolactone are known in the art.

[00127] For example, spironolactone may be administered at a dosage within the range of 25 mg - 500 mg per day.

[00128] In the combination therapy of the present invention, the dose of spironolactone is suitably within the range of 25 mg to 400 mg per day. More suitably, the dosage of spironolactone is within the range of 25 mg to 200 mg per day.

[00129] In an embodiment of the invention, the dosage of spironolactone is within the range of 25 mg to 400 mg per day.

[00130] In an embodiment of the invention, the dosage of spironolactone is within the range of 50 mg to 400 mg per day.

[00131] In an embodiment of the invention, the dosage of spironolactone is within the range of 25 mg to 200 mg per day. [00132] In an embodiment of the invention, the dosage of spironolactone is within the range of 50 mg to 200 mg per day.

[00133] In an embodiment of the invention, the dosage of spironolactone is 50 to 150 mg per day.

[00134] In an embodiment of the invention, the dosage of spironolactone is 100 to 150 mg per day.

[00135] In an embodiment of the invention, the dosage of spironolactone is 100mg per day.

[00136] The daily dose of spironolactone may be administered as a single dose or as divided doses.

[00137] In an embodiment of the invention, the spironolactone is administered as a single daily dose.

[00138] Alternatively, spironolactone is administered in divided dosages, e.g.:

25 mg to 400 mg given twice daily;

50 mg to 400 mg given twice daily;

25 mg to 200 mg given twice daily;

50 mg to 200 mg given twice daily;

50 mg to 150 mg given twice daily;

100 mg to 200 mg given twice daily; or 100 mg to 150 mg given twice daily.

[00139] In an embodiment of the invention, spironolactone is administered at an initial loading dosage of 50 mg to 400 mg followed by a maintenance dosage of 25 mg to 200 mg twice daily.

[00140] In another embodiment of the invention, spironolactone is administered at an initial loading dosage of 50 mg to 200 mg followed by a maintenance dosage of 25 mg to 100 mg twice daily.

[00141] In another embodiment of the invention, spironolactone is administered at an initial loading dosage of 50 mg to 200 mg followed by a maintenance dosage of 25 mg to 100 mg twice daily. [00142] In another embodiment of the invention, spironolactone is administered at an initial loading dosage of 50 mg to 150 mg followed by a maintenance dosage of 25 mg to 75 mg twice daily.

[00143] In another embodiment of the invention, spironolactone is administered at an initial loading dosage of 100 mg followed by a maintenance dosage of 50 mg twice daily.

[00144] Spironolactone is given orally.

[00145] Treatment will be typically continued for between 1 and 6 weeks depending on the patient’s symptoms. More typically, treatment will be continued for 1 to 3 weeks or 1 to 2 weeks.

Eplerenone

[00146] Eplerenone is a selective mineralocorticoid receptor antagonist, which is indicated for use in the treatment of hypertension and heart failure.

[00147] Suitable dosage ranges and schedules for the administration of eplerenone are known in the art.

[00148] For example, eplerenone may be administered at a dosage within the range of 10 mg - 100 mg per day.

[00149] In the combination therapy of the present invention, the dose of eplerenone is suitably within the range of 25 mg to 100 mg per day. More suitably, the dosage of eplerenone will be within the range of 25 mg to 50 mg per day.

[00150] In an embodiment of the invention, the dosage of eplerenone will be within the range of 25 mg to 100 mg per day.

[00151] In an embodiment of the invention, the dosage of eplerenone will be within the range of 25 mg to 50 mg per day.

[00152] In an embodiment of the invention, the dosage of eplerenone will be 25 mg per day.

[00153] In an embodiment of the invention, the dosage of eplerenone will be 50 mg per day.

[00154] In an embodiment of the invention, the dosage of eplerenone will be 100 mg per day.

[00155] The daily dose of eplerenone may be administered as a single dose or as divided doses. [00156] In an embodiment of the invention, the eplerenone is administered as a single daily dose, e.g. a single daily dose of 25 mg or 50 mg per day.

[00157] In another embodiment of the invention, the eplerenone is administered in divided doses, e.g. a dose of 25 mg or 50 mg given twice daily.

[00158] In an embodiment of the invention, eplerenone is administered at an initial dosage of 25 mg or 50 mg once a day and this titrated to a maintenance dosage of 25 mg once or twice daily.

[00159] In another embodiment of the invention, eplerenone is administered at an initial dosage of 50 mg once a day and this titrated to a maintenance dosage of 25 mg once daily.

[00160] In another embodiment of the invention, eplerenone is administered at an initial dosage of 25 mg once a day and this titrated to a maintenance dosage of 50 mg once daily.

[00161] Suitably, the initial dosage is administered for 3 to 10 days, most suitably for a week.

[00162] Eplerenone is given orally.

[00163] Treatment will be typically continued for between 1 and 6 weeks depending on the patient’s symptoms. More typically, treatment will be continued for 1 to 3 weeks or 1 to 2 weeks.

Canrenone

[00164] Canrenone is a metabolite of spironolactone which is used as a duiretic in a limited number of countries.

[00165] Suitable dosage ranges and schedules for the administration of canrenone are known in the art.

[00166] Canrenone can be given parenterally (e.g. intravenously) or orally.

[00167] Treatment will be typically continued for between 1 and 6 weeks depending on the patient’s symptoms. More typically, treatment will be continued for 1 to 3 weeks or 1 to 2 weeks.

Therapeutic Uses

[00168] The combination therapy of the present invention therefore provides a significant clinical benefit in:

(i) the treatmeant and/or prevention of ARDS in COVID-19 patients;

(ii) reducing the risk of thrombosis in COVID-19 patients; (iii) the prevention and/or treatment of anosmia, loss of taste, and/or persistent coughy in COVID-19 patients.

Acute respiratory distress syndrome ARDS

[00169] The present invention provides dexamethasone, or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of acute respiratory distress syndrome (ARDS), wherein the dexamethasone, or a pharmaceutically acceptable salt thereof, is administered in combination with a mineralocorticoid receptor antagonist.

[00170] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of acute respiratory distress syndrome (ARDS), wherein the mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[00171] The present invention also relates to the use of dexamethasone, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention and/or treatment of acute respiratory distress syndrome (ARDS), wherein the medicament is administered in combination with a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

[00172] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention and/or treatment of acute respiratory distress syndrome (ARDS), wherein the medicament is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[00173] The present invention also relates to a method of preventing and/or treating acute respiratory distress syndrome (ARDS), the method comprising administering a therapeutically effective amount of dexamethasone, or a pharmaceutically acceptable salt thereof, to a patient in combination with a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

[00174] Suitably, the therapy is intended for the prevention/treatment of ARDS in patients diagnosed with COVID-19, but it may also be used for the prevention/treatment of ARDS in patient suffering from any infection (e.g. a coronavirus infection) that causes ARDS. Thus, particular uses and methods of the invention, the prevention and/or treatment of ARDS is in patients suffering with an infection associated with an increased risk or incidence of ARDS (e.g. a coronavirus infection such as COVID-19). Suitably, the prevention or treatment of ARDS is in patients suffering with COVID-19.

[00175] Thus, in a particular aspect, the present invention relates to dexamethasone, or a pharmaceutically acceptable salt thereof, for use in the prevention/treatment of acute respiratory distress syndrome (ARDS) in patients suffering with a respiratory tract infection (e.g. COVID-19), wherein the dexamethasone, or a pharmaceutically acceptable salt thereof, is administered in combination with a mineralocorticoid receptor antagonist.

[00176] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, for use in the prevention/treatment of acute respiratory distress syndrome (ARDS) in patients suffering with a respiratory tract infection (e.g. COVID-19), wherein the mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[00177] The present invention also relates to the use of dexamethasone, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention/treatment of acute respiratory distress syndrome (ARDS) in patients suffering with a respiratory tract infection (e.g. COVID-19), wherein the medicament is administered in combination with a mineralocorticoid receptor antagonist.

[00178] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention/treatment of acute respiratory distress syndrome (ARDS) in patients suffering with a respiratory tract infection (e.g. COVID-19), wherein the medicament is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[00179] The present invention also relates to a method of preventing/treating acute respiratory distress syndrome (ARDS) in patients suffering with a respiratory tract infection (e.g. COVID-19), the method comprising administering a therapeutically effective amount of dexamethasone, or a pharmaceutically acceptable salt thereof, to a patient in combination with a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

Thrombotic Complications

[00180] The present invention also relates to dexamethasone, or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of thrombosis (blood clotting), or to reduce the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection), wherein the dexamethasone, ora pharmaceutically acceptable salt thereof, is administered in combination with a mineralocorticoid receptor antagonist.

[00181] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of thrombosis (blood clotting), or to reduce the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection), wherein the mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[00182] The present invention also relates to the use of dexamethasone, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention and/or treatment of thrombosis (blood clotting), or to reduce the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection), wherein the medicament is administered in combination with a mineralocorticoid receptor antagonist.

[00183] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention and/or treatment of thrombosis (blood clotting), or to reduce the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection), or a pharmaceutically acceptable salt thereof.

[00184] The present invention also relates to a method of preventing and/or treating thrombosis (blood clotting), or reducing the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection), the method comprising administering a therapeutically effective amount of dexamethasone, or a pharmaceutically acceptable salt thereof, to a patient in combination with a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

[00185] In particular uses and methods of the invention, the prevention and/or treatment of thrombosis (blood clotting), or reduction in the risk of thrombosis, is in patients suffering with an infection associated with an increased risk of thrombosis (e.g. a coronavirus infection such as COVID-19). Suitably, the prevention and/or treatment of thrombosis (blood clotting), or reduction in the risk of thrombosis, is in patients suffering with COVID-19.

[00186] The present invention also relates to dexamethasone, or a pharmaceutically acceptable salt thereof, for use in the prevention/treatment of the thrombotic complications associated with COVID-19 infection, wherein the dexamethasone, or a pharmaceutically acceptable salt thereof, is administered in combination with a mineralocorticoid receptor antagonist.

[00187] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, for use in the prevention/treatment of the thrombotic complications associated with COVID-19 infection, wherein the mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[00188] The present invention also relates to the use of dexamethasone, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention/treatment of the thrombotic complications associated with COVID-19 infection, wherein the medicament is administered in combination with a mineralocorticoid receptor antagonist.

[00189] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention/treatment of the thrombotic complications associated with COVID-19 infection, wherein the medicament is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[00190] The present invention also relates to a method of preventing/treating the thrombotic complications associated with COVID-19 infection, the method comprising administering a therapeutically effective amount of dexamethasone, or a pharmaceutically acceptable salt thereof, to a patient in combination with a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

[00191] Suitably, the therapy is intended for the prevention/treatment of the thrombotic complications associated with COVID-19 infection, but it may also be used for the prevention/treatment of thrombotic complications in patients suffering from any infection (e.g. another coronavirus infection) that causes thrombotic complications.

Loss of Smell, Loss of Taste and Cough

[00192] The present invention also relates to dexamethasone, or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection), wherein the dexamethasone, or a pharmaceutically acceptable salt thereof, is administered in combination with a mineralocorticoid receptor antagonist.

[00193] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, for use in the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection), wherein the mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[00194] The present invention also relates to the use of dexamethasone, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection), wherein the medicament is administered in combination with a mineralocorticoid receptor antagonist.

[00195] In another aspect, the present invention also relates to a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection), wherein the medicament is administered in combination with dexamethasone, or a pharmaceutically acceptable salt thereof.

[00196] The present invention also relates to a method of preventing and/or treating anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection) the method comprising administering a therapeutically effective amount of dexamethasone, or a pharmaceutically acceptable salt thereof, to a patient in combination with a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

[00197] In particular uses and methods of the invention, the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, is in a patient suffering with anosmia, loss of taste, and/or persistent cough as a consequence of a coronavirus/COVID-19 infection. Suitably, the prevention and/or treatment of t anosmia, loss of taste, and/or persistent cough, is in a patient suffering with COVID-19. [00198] In another aspect, the present invention relates to a combination comprising dexamethasone, or a pharmaceutically acceptable salt thereof, and a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

[00199] In another aspect the present invention relates to a pharmaceutical product comprising a combination as defined herein.

[00200] In another aspect, the present invention relates to a pharmaceutical composition comprising a combination as defined herein, and one or more pharmaceutically acceptable excipients.

[00201] In another aspect, the present invention relates to a combination as defined herein, or a pharmaceutical product as defined herein, or a pharmaceutical composition as defined herein for use in therapy.

[00202] In another aspect, the present invention relates to a combination as defined herein, or a pharmaceutical product as defined herein, or a pharmaceutical composition as defined herein for use in the prevention and/or treatment of acute respiratory distress syndrome (ARDS).

[00203] In another aspect, the present invention relates to a use of a combination as defined herein in the manufacture of a medicament for preventing and/or treating of acute respiratory distress syndrome (ARDS).

[00204] In another aspect, the present invention relates to a method of preventing and/or treating acute respiratory distress syndrome (ARDS) in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a combination as defined herein.

[00205] In another aspect, the present invention relates to a combination as defined herein, or a pharmaceutical product as defined herein, or a pharmaceutical composition as defined herein for use in the prevention and/or treatment of thrombosis (blood clotting), or to reduce the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection).

[00206] In another aspect, the present invention relates to a use of a combination as defined herein in the manufacture of a medicament for use in the prevention and/or treatment of thrombosis (blood clotting), or to reduce the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection).

[00207] In another aspect, the present invention relates to a method of preventing and/or treating thrombosis (blood clotting), or reducing the risk of thrombosis, in a patient suffering with an infection associated with an increased thrombotic risk (e.g. a coronavirus/COVID-19 infection), the method comprising administering to said subject a therapeutically effective amount of a combination as defined herein.

[00208] In another aspect, the present invention relates to a combination as defined herein, or a pharmaceutical product as defined herein, or a pharmaceutical composition as defined herein for use in the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection).

[00209] In another aspect, the present invention relates to a use of a combination as defined herein in the manufacture of a medicament for use in the prevention and/or treatment of anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection).

[00210] In another aspect, the present invention relates to a method of preventing and/or treating anosmia, loss of taste, and/or persistent cough, in a patient suffering with anosmia, loss of taste, and/or persistent cough (e.g. as a consequence of a coronavirus/COVID-19 infection), comprising administering to said subject a therapeutically effective amount of a combination as defined herein.

[00211] In one embodiment the separate formulations of dexamethasone, or a pharmaceutically acceptable salt thereof, and mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, are administered simultaneously (optionally repeatedly).

[00212] In one embodiment the separate formulations of dexamethasone, or a pharmaceutically acceptable salt thereof, and mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, are administered sequentially (optionally repeatedly).

[00213] In one embodiment the separate formulations of dexamethasone, or a pharmaceutically acceptable salt thereof, and mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, are administered separately (optionally repeatedly).

[00214] The skilled person will understand that where the separate formulations of dexamethasone, ora pharmaceutically acceptable salt thereof, and mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, are administered sequentially or serially that this could be administration of dexamethasone, or a pharmaceutically acceptable salt thereof, followed by a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, or vice versa. [00215] In one embodiment, separate formulations of dexamethasone, or a pharmaceutically acceptable salt thereof, and mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, may be administered in alternative dosing patterns. Where the administration of the separate formulations of dexamethasone, or a pharmaceutically acceptable salt thereof, and mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, is sequential or separate, the delay in administering the second formulation should not be such as to lose the beneficial effect of the combination therapy.

[00216] Suitably, the mineralocorticoid receptor antagonist is administered orally and the dexamethasone is administered orally or parenterally, preferably orally.

[00217] In one embodiment, the pharmaceutical product may comprise a kit of parts comprising separate formulations of dexamethasone, or a pharmaceutically acceptable salt thereof, and a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof. The separate formulations may be administered sequentially, separately and/or simultaneously.

[00218] In another embodiment the pharmaceutical product is a kit of parts which comprises: a first container comprising dexamethasone, or a pharmaceutically acceptable salt thereof; and a second container comprising a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof; and a container means for containing said first and second containers.

[00219] In one embodiment, the pharmaceutical product may comprise a one or more unit dosage forms (e.g. vials, tablets or capsules in a blister pack). In one embodiment, each unit dose comprises only one agent selected from the dexamethasone and the mineralocorticoid receptor antagonist. In another embodiment, the unit dosage form comprises both the dexamethasone and the a mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof.

[00220] In one embodiment the pharmaceutical product or kit of parts further comprises means for facilitating compliance with a dosage regimen, for instance instructions detailing how to administer the combination.

[00221] In one embodiment, the pharmaceutical product or kit of parts further comprises instructions indicating that the combination, as defined herein, can be used in the treatment of acute respiratory distress syndrome (ARDS). [00222] In one embodiment, the pharmaceutical product is a pharmaceutical composition.

Pharmaceutical Compositions

[00223] In one aspect the present invention relates to a pharmaceutical composition comprising a combination of dexamethasone, or a pharmaceutically acceptable salt thereof, and mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[00224] The pharmaceutical compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs).

[00225] The pharmaceutical compositions may be obtained by conventional procedures using conventional pharmaceutical excipients well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.

[00226] An effective amount of a combination of dexamethasone, or a pharmaceutically acceptable salt thereof, and mineralocorticoid receptor antagonist, or a pharmaceutically acceptable salt thereof, for use in the combination therapy of the invention is an amount sufficient to treat or prevent ARDS; treat or prevent blood clotting or reduce the thrombotic risk; or treat or prevent anosmia (loss of smell), loss of taste or cough (including persistent cough).

[00227] The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the individual treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.25 mg to 0.25 g of active agent (more suitably from 0.5 to 100 mg, for example from 1 to 30 mg) compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition.

[00228] The size of the dose for therapeutic or prophylactic purposes of a combination of the invention will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well-known principles of medicine.

[00229] In using a combination of the invention for therapeutic or prophylactic purposes it will generally be administered with a therapeutically effective dose of the dexamethasone and the mineralocorticoid receptor antagonist selected. These dosages are known in the art and will vary from one inhibitor to another. The dosage may, for example, be in the ranges indicated above, or within a general range of 0.1 mg/kg to 30 mg/kg body weight. The dosing schedule will also vary from mineraolcorticoid receptor antagonist to another. Suitable doing schedules are known in the art.

Combinations with additional therapeutic agents

[00230] The combination treatment defined herein may be applied as a sole therapy for the treatment of the specified condition or it may involve, in addition to the combination therapy of the present invention, one or more additional therapies (including treatment with another therapeutic agent, surgery or other therapeutic interventions).

[00231] Typically, the other therapeutic agent used in combination with the combination therapy of the present invention will be one or more therapeutic agents used as the standard of care for the treatment of the disease or condition concerned or various symptoms thereof. The other therapeutic agent may include, for example, another drug used for the treatment of the condition concerned, or an agent that modulates the biological response to the combination therapy of the invention, such as, for example, an immunomodulatory agent.

[00232] Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the compounds of this invention within the dosage range described hereinbefore and the other pharmaceutically-active agent within its approved dosage range.

EXAMPLES

Example 1 - Case report of a patient suffering COVID-19 symptoms and showing early signs of acute respiratory distress syndrome

[00233] A 50 year-old lady with classic COVID-19 symptoms of cough, fever and myalgia for 1 week then developed progressive shortness of breath. This was sufficiently severe for hospital admission to be arranged.

[00234] After discussion with her doctor it was agreed to start her on a combination of dexamethasone and spironolactone.

[00235] The basic rationale for this was that by blocking the mineralocorticoid receptor it might be possible to prevent the release of the Weibel-Palade bodies in endothelial cells that had been damaged by the virus. These bodies contain angiopoietin-2, a key molecule in producing increased capillary permeability and the von Willebrand factor which is pro- thrombotic.

[00236] The increased capillary permeability is a major part of the development of the Acute Respiratory Distress Syndrome which is responsible for about 50% mortality of COVID- 19 patients in Intensive care Units. The progressive rise of circulating level of angiopoietin-2 is predictive of death in these patients. The von Willebrand factor could be important in the thrombotic complications of COVID-19.

[00237] Within a few hours of starting the treatment the lady started to feel better and was able to avoid going into hospital. She then progressively improved over the next 12 days. She then ran out of spironolactone tablets and was off them for 24 hours. Her condition then markedly deteriorated. She restarted spironolactone and was better by the next day. Over the next month she returned to normal, able to stop spironolactone and dexamethasone after a further 2 weeks and is now back to her normal outdoor running routine

Example 2 - Clinical Study to investigate the effect of spironolactone and dexamethasone in preventing the acute respiratory distress syndrome (ARDS) in patients suffering with COVID-19

Aims

[00238] The aims of this study is to:

1. To recruit patients who have gone through the first 5-7 days of COVID-19 infection who then continue with fever, cough and progressive shortness of breath.

2. To determine if it is possible prevent such patients progressing to ARDS.

Background:

[00239] The majority of patients infected with the COVID-19 virus are either asymptomatic or have a short-lasting clinical problem. However, a small number, and especially those with hypertension, cardiovascular disease, diabetes and hypercholesterolaemia are at much higher risk of progressing to the Acute Respiratory Distress Syndrome. These require hospitalisation, intensive care, oxygen therapy and often ventilation. It is the development of ARDS which is usually the major factor leading to death.

[00240] Animal studies have used sepsis, acid aspiration and COVID-like viruses to produce ARDS. These models have shown that, regardless of the cause of the ARDS, that knocking out the ACE-2 receptor produces a dramatically worse outcome. This is the receptor used by the virus to get into the cell. The loss of this enzyme results in a major pro- inflammatory cascade. The enzyme normally converts angiotensin II into angiotensin (1-7) (A(1-7)). This is a major counter-regulatory hormone and loss of this produces high circulating levels of angiotensin II and aldosterone. It also decreases NO synthesis in platelets and the endothelium resulting in a pro-thrombotic tendency commonly seen in COVID patients.

[00241] A key molecule involved in the pathogenesis of ARDS is Angiopoietin-2. This is stored in Weibel-Palade bodies in vascular endothelial cells. Its release into the circulation produces a major increase in vascular permeability. Studies have shown that aldosterone stimulates release of Weibel-Palade bodies and this can be blocked by spironolactone. The mechanism involved is that aldosterone produces a rapid release of ATP from the cells. The ATP then acts on cell surface purinergic receptors and promotes calcium entry. It is the rise in intracellular calcium that stimulates exocytosis of the WP bodies.

[00242] A similar mechanism to this has been shown for the release of the NLRP3 inflammasome from type 1 pro-inflammatory macrophages.

Study design:

[00243] This study involves the use of two commonly prescribed drugs - spironolactone and dexamethasone. They act together to block the Mineralocorticoid Receptor (MR) and activate the Glucocorticoid Receptor (GR). Unlike in the kidney, the MR in the endothelial cells and in the macrophage is not protected and hence will bind both aldosterone and cortisol.

[00244] Using this combination of drugs, this study will determine if it is possible to block the release of Weibel-Palade bodies from endothelial cells and thus lower Angiopoietin-2 levels. In addition, the combination will have an anti-inflammatory effect and decrease the release/activation of inflammasomes from the macrophage.

[00245] If a major part of the damage produced by the virus is the loss of the ACE-2 enzyme then spironolactone therapy may have further benefit. In studies in heart failure patients low dose spironolactone for 1 month produced a 300% increase in ACE-2 expression in macrophages and 640% increase in ACE-2 mRNA.

Drug doses:

Spironolactone: 50 mg on waking, 50 mg on going to bed

Dexamethasone: 1 mg on going to bed, 2 mg on waking and 2 mg at 12 Midday.

Entry into study:

The proposal is to enter patients who are deteriorating after the end of the first phase of COVID-19 and have persistent cough, fever and increasing shortness of breath. Basic screening:

Blood sample screening:

Full Blood Count

Electrolytes

Creatinine

- LDH

C-Reactive Protein

- Aldosterone (store sample for subsequent assay) Patient measurements:

Body temperature Oximetry

Major end points:

Hospital admission Intensive Care

Mortality

Example 3 - Clinical Studies into Spironolactone/Dexamethasone Treatment of COVID- 19

Methods:

[00246] This study report presents the results of a proof of concept study in which a combination of spironolactone and dexamethasone has been used to block the mineralocorticoid receptor which has been activated by the virus. The study is in two parts. The first relates to 40 patients with COVID-19 admitted to hospital with pneumonia treated with conventional high dose dexamethasone (HIDEX) and antibiotic and compares this with 40 similar patients treated with spironolactone and low dose dexamethasone (SPIDEX) and antibiotic. In a second study SPIDEX was given to 20 COVI D-positive outpatients without pneumonia within 2-3 days of first symptoms of disease.

Findings:

[00247] On all parameters in study 1 (clinical, biochemical and radiological) SPIDEX was superior to HIDEX. In study 2 90% patients had resolution of symptoms and signs within 10 days of starting treatment. Only 2 required short hospital admission. Hyperkalaemia which has been suggested as a possible problem with this approach was not found.

Interpretation:

[00248] These results suggest that it may be possible to safely treat COVID-19 patients with two cheap, readily available drugs with potential major health and economic benefit. This was an open study. Randomised trials of this treatment are underway.

Introduction to Clinical Study

[00249] This study describes an approach which is based on increased understanding of the metabolic consequences of the SARS-CoV-2 viral infection.

[00250] A large number of studies have shown that the virus enters cells using the ACE2 receptor which is present on key epithelial cells such the nose, tongue, salivary glands and lungs. After the primary infection, patients with specific co-morbidities such as hypertension, cardiovascular disease and diabetes, are at risk of developing an endothelitis which plays a vital role in the development of the major complications of Acute Respiratory Distress Syndrome (ARDS) and microthrombi. Young people and those with normal microcirculation have very low expression of ACE2 receptors on their endothelial cells. In contrast those with vascular disease have high levels of expression induced by changes in blood flow, shear stress and change in basement membrane.

[00251] The loss of the ACE2 receptor results in a failure of the conversion of angiotensin II to angiotensin (1-7) (Fig.1).

Methods

Patient group description, treatments and biochemical testing

STUDY 1

[00252] This was a retrospective study of 80 patients with PCR-confirmed infection with SARS-CoV-2 admitted to Vinnytsia State Hospital, Vinnytsia, Ukraine with a radiologically confirmed diagnosis of bilateral pneumonia. 40 patients (Group 1 - HIDEX) were then given standard treatment with high dose dexamethasone (16mg/day), a mucolytic ambroxol 90mg/day and antibiotic ceftriaxone 2g/day. A further 40 patients (Group II - SPIDEX) were given low dose dexamethasone 4mg/day, spironolactone 100mg/day, ambroxol 90mg/day, ceftriaxone 2g/day. Both groups received treatment for 10 days. The treatment was not based on a randomised design and both groups were composed a posteriori.

[00253] Before treatment and during their stay in hospital all patients had routine clinical assessment: body temperature, blood pressure, heart rate, respiratory rate, blood oxygen saturation: routine biochemistry, C-reactive protein (CRP), D-dimer, fasting blood glucose:radiology, baseline and day 5 chest X-rays.

[00254] The age range of patients treated was from 28-84 years.

[00255] The patient characteristics, co-morbidities, and presenting symptoms are listed in Table 1.

Table 1. Study 1. Patients’ general characteristics, co-morbidities and clinical parameters at baseline

Study 2.

[00256] This was a study of the effectiveness and safety of treating 20 outpatients with PCR-confirmed COVID-19 patients. Treatment was started within 2-3 days of first symptoms. No patient had pneumonia. Dexamethasone was given in a dose of 4 mg/day, spironolactone 100mg/day: both drugs were given for 10 days.

[00257] The age range of patients treated was 21-58 years.

[00258] The patient characteristics, co-morbidities, and presenting symptoms are listed in Table 2. Table 2. Study 2. Patients’ general characteristics, co-morbidities and clinical parameters at baseline

Patient and other consents

[00259] The studies received approval from the Vinnytsia Hospital Ethics Committee and were carried out with informed consent from participants.

Statistical method

[00260] For normally distributed clinical characteristics and demographics data were expressed as the mean and standard deviation.

Findings

[00261] Study 1. The two groups did not significantly differ as judged by their age, sex, and clinical characteristics including their co-morbidities. Only 1 patient died: they were in group I given high dose dexamethasone (HIDEX). No patients required ventilation. Patients with oxygen saturation less than 92% were given oxygen by mask. The results of the clinical findings in the HIDEX and SPIDEX groups after 5 days of treatment are shown in Fig.7. [00262] Both treatment regimens produced clinical improvement but, as judged by every symptom and sign assessed at 5 days, this was greater on the SPIDEX than the HIDEX regimen. A strict comparison of the 2 groups at 10 days was not possible as more patients had been discharged before 10 days in the SPIDEX group.

[00263] Cardiovascular, respiratory and laboratory markers are included in Table 3. Table 3. Cardiovascular, respiratory and laboratory markers in Study 1 at baseline and after 5 days of treatment

Figures expressed as Mean and Standard Deviation

# - p<0,05 in comparison with baseline; * - p<0,05 in comparison with Group I Table 4: Dynamics of radiological indicators on the background of treatment

[00264] Systolic blood pressure was significantly higher in the HIDEX group compared to baseline in comparison with the SPIDEX group where it was significantly lower. The respiratory rate was significantly reduced by both treatments. Blood oxygen saturation rose in both groups. C-reactive protein was significantly lowered by both treatment regimens but was significantly lower at 5 days in the SPIDEX than the HIDEX group. The same pattern was found for D-Dimer levels. The fasting blood glucose was significantly elevated in the HIDEX group but not in the SPIDEX group. The SPIDEX fasting blood glucose was significantly lower than that of the HIDEX.

Radiological findings

[00265] All 80 patients had radiological evidence of bilateral pneumonia. At 5 days there was radiological evidence of resorption of the pneumonia in 24 of the 40 patients in the SPIDEX group and 15 of the 40 in the HIDEX group. In 5 patients in the HIDEX group there was deterioration of the Chest X-ray on day ⁵ .

STUDY 2

[00266] In this study of patients with a diagnosis of COVID-19 who were treated within 2-3 days of the onset of symptoms with low dose dexamethasone (4mg/day) and spironolactone (100mg/day) for 10 days (SPIDEX) only two patients required hospitalisation. The majority of patients showed major improvement after 5 days and over 90% were asymptomatic at 10 days. No patient became hyperkalaemic.

Table 5. Study 2. Clinical characteristics and plasma potassium levels before and after 5 and 10 days on low dose dexamethasone and spironolactone (SPIDEX) in 20 patients with positive COVID-19 PCR test where treatment was started within 2-3 days of symptom onset. Interpretation

[00267] This work was designed to test the hypothesis that corona virus-induced exocytosis of ATP from epithelial cells and Weibel-Palade bodies from endothelial cells is important in the pathogenesis of SARS-CoV-2 infection. This process results from oxidative stress activation of the mineralocorticoid receptor. If, as suggested, exocytosis of ATP plays a key role in the genesis of loss of smell/taste, cough and the inflammatory response it might be predicted that this could be inhibited by mineralocorticoid receptor blockade. This is supported by the results showing that SPIDEX (spironolactone and 4mg/day dexamethasone) produced major benefit with regard to recovery of taste/smell, cough and fever in comparison to HIDEX (dexamethasone 16mg/day). This suggests that the major effect of dexamethasone may well be via suppression of cortisol secretion and hence its activation of the MR rather than acting as an anti-inflammatory agent via the glucocorticoid receptor.

[00268] Endothellitis plays a major role in the pathogenesis of the key complications of COVID-19, the Acute Respiratory Distress Syndrome and the genesis of microthrombi. The latter are 9 times more common in the lungs of patients dying from SARS-CoV-2 infection than with influenza related pneumonia. A key indicator of thrombosis is D-dimer. Early studies from Wuhan showed that D-dimer levels were the strongest independent predictor of mortality in COVID-19 patients. In Study 1 , patients in both the HIDEX and the SPIDEX groups had a significant reduction in D-dimer levels. However, the levels in the SPIDEX treated patients were significantly lower than those on HIDEX. It is suggested that this is related to blockade of the exocytosis of Weibel-Palade bodies containing Von Willebrand Factor.

[00269] The high levels of co-morbidities in study 1 are typical of patients with severe COVID-19. These conditions all predispose to vascular disease which is key to the expression of the ACE2 receptors on vascular endothelium ² .

[00270] The more rapid resolution of pneumonia that was observed in study 1 SPIDEX patients might be expected if inhibition of endothelial exocytosis blocked the release of the capillary permeability factor angiopoietin ² . This appears to play an important role in the genesis of the Acute Respiratory Distress Syndrome (Kumpers, P. & Lukasz, A. The curse of angiopoietin-2 in ARDS: On stranger TI(E)des. Crit. Care 22, 4-7 (2018). Rising levels of angiopoietin are predictive of a poor outcome.

[00271] Study 2 was aimed attesting the effectiveness and safety of giving SPIRODEX to 20 outpatients within 2-3 days of the onset of symptoms. Only 2 of these patients required hospital admission within 10 days and that was brief. 30% had co-morbidities that might increase their potential for severe disease. There was marked improvement after 5 days and this was virtually complete after 10 days. At this time only 1 patient had loss of taste and smell compared to 18 pre-treatment. Two patients still had a dry cough and 2 were breathless. Of particular importance no patient had significant hyperkalaemia. Example section references:

1. Edwards, C. New Horizons: Does Mineralocorticoid Receptor Activation by Cortisol Cause ATP Release and COVID-19 Complications? J. Clin. Endocrinol. Metab. XX, 1-14 (2020).

2. Kaneko, N. et al. Flow-Mediated Susceptibility and Molecular Response of Cerebral Endothelia to SARS-CoV-2 Infection. Stroke (2021). doi: 10.1161 /STROKEAHA.120.032764

3. Funder, J. W. Mineralocorticoid receptor activation and oxidative stress. Hypertension 50, 840-841 (2007).

4. Gorelik, J. et al. Aldosterone acts via an ATP autocrine/paracrine system: the Edelman ATP hypothesis revisited. Proc. Natl. Acad. Sci. U. S. A. 102, 15000-5 (2005).

5. Jeong, Y. et al. Aldosterone activates endothelial exocytosis. Proc. Natl. Acad. Sci. U. S. A. 106, 3782-3787 (2009).

6. Li, Z. et al. Lysosome exocytosis is involved in astrocyte ATP release after oxidative stress induced by H202. Neuroscience Letters 705, 251-258 (2019).

7. Hegg, C. C., Greenwood, D., Hua, W., Han, P. & Lucero, M. T. Activation of purinergic receptor subtypes modulates odor sensitivity. J. Neurosci. 23, 8291-8301 (2003).

[00272] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).

[00273] All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

[00274] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise paragraphed. No language in the specification should be construed as indicating any non-paragraphed element as essential to the practice of the invention.

[00275] The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

[00276] This invention includes all modifications and equivalents of the subject matter recited in the paragraphs appended hereto as permitted by applicable law.