IE20140275A1 - Use of antagonist of the mineralocorticoid/aldosterone receptor as agent to prevent haemorrhaging and macrophage inflammatory effect if filovirus infection - Google Patents

Abstract

Use of antagonist of the mineralocorticoid/aldosterone receptor as agent to prevent haemorrhaging and macrophage inflammatory effect of filovirus infection The present invention is directed to therapeutic use of antagonists of the mineralocorticoid/aldosterone receptor for their previously unappreciated, prophylactic and therapeutic properties in preventing damage to the endothelial barrier that causes loss of vascular integrity resultant from filoviral infection of a patient and the effect of the Ebola viral coat glycoproteins GP1/2 attaching to the aldosterone receptor and causing the loss of endothelial cell attachment leading to characteristic haemorrhaging which results in hypovolemic shock to the patient. The invention also relates to compositions that comprise antagonist of the mineralocorticoid/aldosterone receptor and an excipient.

Description

Use of Antagonist of the Mineralocorticoid I aldosterone receptor as agent to prevent haemorrhaging and macrophage inflammatory effect of filovirus infection Description The present invention is directed to therapeutic use of antagonists of the mineralocorticoid I aldosterone receptor for their previously unappreciated, prophylactic and therapeutic properties in preventing damage to the endothelial barrier that causes loss of vascular integrity resultant from filoviral infection of a patient and the effect of the Ebola viral coat glycoproteins GP1 I 2 attaching to the aldosterone receptor and causing the loss of endothelial cell attachment leading to characteristic haemorrhaging which results in hypovolemic shock to the patient.

The invention also relates to compositions that comprise antagonist of the mineralocorticoid / aldosterone receptor and an excipient.

Accordingly it is an object of the present invention to provide a method and composition for preventing vascular instability and hypovolemic shock in Filoviral infected patients.

Aldosterone is a mineralocorticoid, a hormone that is produced by the adrenal glands. Aldosterone acts by stimulating the Mineralocorticoid Steroid Receptor which is present on cells of the immune system and endothelial cells.

It is believed that aldosterone increases the reabsorption of sodium (and the excretion of potassium) by the distal tubules of the kidney. The reabsorption of sodium results in an increased reabsorption of water that can result in hypertension. Low blood aldosterone levels are seen in Addison's disease and toxaemia of pregnancy. Higher levels can be seen in Cushing's syndrome, primary hyperaldosteronism, malignant hypertension, severe swelling in congestive heart failure, and nephrotic syndrome.

We have demonstrated that certain sequences present on the filoviral coat protein GP1 I GP2 have Aldosterone mimic characteristics which can modulate endothelial permeability, endothelial nitric oxide and reactive oxygen species and the actin -2cytoskeleton. By stimulating the mineralocorticoid receptor as a mimic Aldosterone the filovirus coat glycoproteins GP1 / GP2 can generate a situation similar to excessive Aldosterone exposure. This endothelial cell over exposure to aldosterone causes the production of inflammatory cytokines and nitrites and ROS to be released by both macrophages and endothelial cells of the Ebola patient. These inflammatory cytokines and chemical entities causes a disruption in cell adhesion that results in the loss of cell-cell contacts,as well as a loss of attachment and also integration of the organ effected. This loss of endothelial cell attachment is key to the generation of the hemorrhagic effects seen in Ebola infected patients and which can lead to hypovolemic shock.

By mimicking Aldosterone the Ebola virus can also cause apoptosis of macrophages and dendritic cells and cause the massive loss of immune cells early in infection which has been documented in patients suffering from Ebola infection. Many patients die from the infection without their immune system ever making an antibody to the Ebola virus and those that survive usually only present with high antibody titres to Ebola after recovery.

By administering compounds and antibodies which block the aldosterone attachment site on the mineralocorticoid receptor this effect of the Ebola virus can be neutralised and endothelial cell attachment will remain intact and macrophages and other immune cells will not be stimulated to apoptosis and as a result a proper immune response can be generated and viral clearance achieved in association with therapy with antiviral compounds and antibodies which can circulate normally within the body to assist the patient in normal recovery.

A number of drugs have been identified which can antagonise the mineralocorticoid / aldosterone receptor and inhibit the activity of aldosterone in the body by blocking Mineralocorticoid Steroid Receptor, these including spirolactones.

The term spirolactone indicates that a lactone ring (i.e., a cyclic ester) is attached to another ring structure in a spiro configuration (i.e., the lactone ring shares a single carbon atom with the other ring). Spirolactones, which are coupled to steroids, are the most important class of spirolactones from a pharmaceutical perspective, so they are widely referred to in the pharmaceutical art simply as spirolactones. As used -3herein, spirolactone refers to a molecule comprising a lactone structure coupled via a spiro configuration to a steroid structure or steroid derivative.

One particular spirolactone is called spironolactone, a synthetic steroid with an aldosterone-like structure, is classically described as an anti-mineralocorticoid compound or antagonist, acting functionally as a competitive inhibitor of the Mineralocorticoid Steroid Receptor which certain viral peptide can activate. We have identified other viral coat peptides which also utilise this strategy to both cause immuno-suppression and destruction of key immune cells needed initially to mount a successful attack on the infecting virus.(eg HIV).

Spironolactone belongs to a class of blood pressure lowering agents called potassium-sparing diuretics. It is used to treat essential hypertension, low potassium blood levels, and fluid retention and swelling associated with congestive heart failure, cirrhosis and other conditions in which a diuretic is needed without the associated decrease in potassium levels. It works by removing excess fluid from the body. Another use is in the diagnosis and treatment of primary hyperaldosteronism.

Spironolactone is marketed as an anti-hypertensive and diuretic drug by G. D. Searle (Skokie, HI.) under the trademarks Aldosteronectone and Aldactazide. Spironolactone is the name commonly used by chemists, the full chemical name is IT-hydroxy-T-alpha-mercapto-S-oxo-IT-alpha- pregn-4-ene-21 -carboxylic acid gamma - lactone acetate. This compound, its activities, and modes of synthesis and purification are described in a number of U.S. patents, U.S. Pat. No. 4,529,811 (Hill and Erickson 1985). Another spironolactone effective as an antagonist of the mineralocorticoid I aldosterone receptor is epoxymexrenone.

Epoxymexrenone possesses high mineralocorticoid receptor affinity (comparable to spironolactone) but with reduced binding affinity for androgen and progesterone receptors. Initial studies of this compound have demonstrated a Na * /K effect equipotent with spironolactone at a 50 mg dose. Antagonist of the mineralocorticoid / aldosterone receptor include analogous compounds of spironolactone are exemplified potassium canrenoate [Clin. Pharm. Ther. 21, 602 (1977)], potassium canrenoate [J. Pharmacol. Exp. Ther. 209, 144 (1979)], potassium prorenoate [Clin.

Pharm. Ther. 18, 391 (1975)], spirorenone [Japanese Unexamined Patent -4Publication No. 55- 162799], dihydrospirorenone, eplerenone [U.S. Pat No. 5981744 and WO00033847] and the like.

The present invention also provides novel spirolactones of FORMULA 1 described in Patent No 4,129,564 (1978) to prevent vascular instability and hypovolemic shock in Ebola viral infections.

FORMULA 1 R is a lower alkyl of up to 5 carbon atoms, and ire useful diuretics.

Lower alkyl residues include branched and un-branched groups, preferably methyl, ethyl and n-propyl.

Spironolactone is extensively metabolised {J. Clin. Pharmacol. 29, 342 (1989)]. Canrenone is thought to be the primary metabolite, but other metabolites include: 1alpha-thiospironolactone, 7-alpha-thiomethylspironolactone, 6-beta-hydroxy-7-alphathiospironolactone, and 6-beta-hydroxy-7-alpha-thiomethylspironolactone.

The present invention is directed to a method of treating an individual expected to be exposed to or infected with a lipid envelope filovirus comprising administering to that individual a therapeutically effective amount of one or more compounds of the present invention, to prevent vascular instability and hypovolemic shock by -5interactively blocking the Ebola virus surface glycoproteins binding to the mineralocorticoid receptor within the cell nucleus and activating cytotoxic and inflammatory signals.

The present invention relates to the use of antagonists of the mineralocorticoid / aldosterone receptor that inhibit filoviral effects of causing vascular instability and hypovolemic shock and the inflammatory cytokines cascade that destroys immune cells. It has been surprisingly found that Antagonists of the mineralocorticoid / aldosterone receptor have potent activity in preventing endothelial permeability collapse from filovirus infection. Furthermore, the present invention relates to the metabolic derivatives (or metabolites) of these antagonists of mineralocorticoid / aldosterone receptor that also prevent vascular instability and other inflammatory effects upon filovirus infection.

This inventions objectives include the provision of pharmaceutical formulations of antagonists of the mineralocorticoid / aldosterone receptor which are suitable for effective treatment of patients infected with or at risk of infection from a filovirus. Other objects are to provide methods to make and use the formulations.

In accordance with the present invention, a method is provided to treat or prevent hypovolemic shock resulting from a filoviral infection, comprising administering to a subject an effective amount of one or more compounds of the present invention.

The present invention also provides the use of one or more of the compounds of the present invention, for the manufacture of a medicament to protect from or prevent endothelial cell collapse and hypovolemic shock resulting from a filoviral infection.

The present invention also provides compounds of the present invention for use in a method of treatment to protect from or prevent endothelial collapse and hypovolemic shock and inflammatory cytokines cascade resulting from a filovirus infection, said method comprising administering one or more antagonists of the mineralocorticoid I aldosterone receptor to a subject.

In accordance with the objects of the present invention, is provided an improved pharmaceutical formulation for use (or method) in the prophylaxis and therapy of filoviral infection or a complication or consequence thereof. In particular the invention relates to the use of antagonist of the mineralocorticoid / aldosterone receptor and -6their metabolic derivatives in the prophylaxis and therapy of filoviral induced hypovolemic shock and cytokine cascade.

Other embodiments provide a pharmaceutical formulation or method to treat a filoviral infection to prevent vascular instability due to loss of or to ameliorate one or more symptoms associated with hypovolemic shock administering to an infected patient an effective amount of a antimineralocorticoid molecule or compound or a formulation as disclosed herein.

It is an object of the present invention to provide a composition, which includes at least one Antimineralocorticoid compound as treatment, therapeutic or prophylaxis, against a filoviral infection. The composition containing at least one Antimineralocorticoid compound is designed so that upon administration, it has maximum bioavailability.

The present invention is further directed to a pharmaceutical formulation or method in the prophylaxis and therapy against vascular endothelial instability and related syndromes of an infection with Filovirus.

Accordingly, the Antimineralocorticoid molecules and compounds disclosed herein provide a pharmaceutical formulation or a method for treating a filoviral infection, comprising administering to a patient in need thereof a prophylactically or therapeutically effective amount of a composition comprising at least one Antimineralocorticoid compound.

The advantage of this is that an effective prevention of filoviral infection leading to hypovolemic shock is provided that has minimal risk of generating toxixity to the infected patient and provides a competent circulatory blood system to allow anti-virai therapy to be administered.

In another embodiment the Antimineralocorticoid compound is, selected from the group consisting of spironolactone, spirorenone, 1 ,2-dihydro-spirorenone, 1,2amethylene-spirorenone, 7a-Acetylthio-3-oxo-4,15-androstadiene-[17{JS-1 ')-spiro-5'jperhydrofuran-2'- one 3-Oxo-7a-propionylthi0-4, 15-androstadiene-[17(/7-1')- spiro- 5']perhydrofuran-2'-one -76/?,7/?-Methylene-3-oxo-4', 15-androstadiene-[17(β-1 ')- spira-5']perhydrofuran-2'one 15a, 16a-Methylene-3-oxo-7a-propionylthio-4-androstene-[17 (/M')- spiro5']perhydrofuran-2'-one 63,7p,15al16a-Dimethylene-3-oxo-4-androstene-[17(P-T)spiro-5']perhydrofuran- 2'-one 7a-Acetyithio-15a, 16a-methylene-3-oxo-4-androstene[17(3-1')-spiro 5']perhydrofuran-2'-one 7a-Acetylthio-15β, 16β-methylene-3-oxo-4androstene-[17^-1 ')- spiro- 5']perhydrofuran-2'-one 15β,16β-ΜβίΗ^θηθ-3-οκο-7β~ propionylthio-4-androstene -[17(β-1')- spiro- 5'Jperhydrofuran-2'-one 6β,7β,15β,16βDinnethylene-3-oxo-4-anclrostene-[17(fi-T)- spiro-5']perhydrofuran~ 2'-one eplerenone, potassium canrenoate, canrenoate, canrenone and pharmaceutically acceptable salts thereof or their metabolites.

In another embodiment the Antimineralocorticoid compound is, selected from the group of progestogens with antimineralocorticoid activity consisting of progesterone, gestodene, drospirenone, dimethisterone, ethinyloestradiol, ethisterone, 11βhydroxyprogesterone, 17a-hydroxyprogesterone, 16a-methyl progesterone, hydroxyprogesterone caproate, medroxyprogesterone acetate, proligestone and pharmaceutically acceptable salts thereof or their metabolites, analogues and mimic molecules.

In another embodiment the antagonist of the mineralocorticoid / aldosterone receptor can be synthesized and administered as a prodrug.

In another embodiment, the antagonist of the mineralocorticoid / aldosterone receptor. Is 7 a acetylthio-4 pregnene3,20-dione represented by formula B CH2R In which Ri is hydrogen, hydroxy, hydroxy), a mineral acid ester such as sulfate, phosphate or nitrate group, or acyloxy-OR2, the acyl group R2 being derived from a carboxylic acid of the formula R4OOH which may have up to 12 carbon atoms, and in which R4 may be substituted or unsubstituted, saturated or unsaturated, straight chain or branched, alicyclic, aryl, heterocyclic or mixed and R3 is methyl. -8In one embodiment, Ri is hydroxyl or OR2 where R2 is derived from a carboxylic acid of the above type, but having one or more from 3 to 12 carbon atoms.

In another embodiment Ri is hydroxy, monocarboxylic, straight or branched-chain alkanooyloxy group having up to 12 carbon atoms.

In another embodiment Ri is hydrogen, hydroxy, acetoxy, propionyloxy, nbutyryloxy, trimethylacetoxy, n-valeroyloxy or n-heptanoyloxy. in another embodiment the Antimineralocorticoid compound is selected from the group comprising: 7a-acetylthio~4-pregnene-3,20-dione; 7a-acetylthia-21-hydroxy- 4pregnene-3,20-dione; 7a-acetylthio-21-acetoxy-4-pregnene~3,20-dione; 7aacetylthio-21-propJonyloxy-4-pregnene-3,20-dione; 7a-acetyfthio-21-n-butyryloxy-4pregnene-3,20-dione; 7a-acetylthio-21 -trimethylacetoxy-4-pregnene-3,20-dione; 7αacetylthio-21 -n-valeroyloxy-4-pregnene-3,20-dione; and 7a-acetyithio-21 heptanoyloxy-4-pregnene-3,20-dione.

In another embodiment the antagonist of the mineralocorticoid I aldosterone receptor is a 9,11-epoxy steroid compound, especially those of the 20-spiroxane series and their analogs.

In another embodiment, the antagonist of the mineralocorticoid / aldosterone receptor is halogenated in any position. The halogen can be selected from the group consisting of chlorine, bromine, fluorine and iodine.

In another embodiment the composition further includes a pharmaceutically acceptable carrier, which in one embodiment is cyclodextrin, preferably hydroxypropyl beta cyclodextrin.

The advantage of administering a pharmaceutical formulation containing at least one antagonist of the mineralocorticoid / aldosterone receptor with a suitable carrier is two fold: 1. The antagonist of the mineralocorticoid / aldosterone receptor eliminates the filovirus's ability to cause vascular endothelial instability within the patient and the resultant hypovolemic shock. 2. The antagonist of the mineralocorticoid / aldosterone receptor has immuno upregulatory properties, for example in macrophages and/or dendritic cells inflammatory cytokines Nitrites and ROS are inhibited and the immune cells do not undergo apoptosis from exposure to filovirus.

The compounds of the invention can be formulated and administered as free bases or in the form of their pharmaceutically acceptable salts for purposes of stability, convenience of crystallization, increased solubility, and the like.

One useful property of the Antimineralocorticoid compound and their metabolites is their ability to stabilize vascular endothelial cells, which is manifested, pursuant to one aspect of the present invention, in a broad-spectrum inhibition of hemorrhagic symptoms. Accordingly, the present invention contemplates administering daily to a subject an amount of at least one Antimineralocorticoid compound that is clinically effective in treating or preventing vascular endothelial instability, which the subject suffers or is at risk of due to Ebola viral infection.

Pursuant to a preferred embodiment of the invention, an effective amount of Antimineralocorticoid compounds thus administered is such as to produce a circulating concentration of the Antimineralocorticoid compounds and their metabolites sufficient to reduce viral hemorrhagic effect on the vascular endothelial system and immune effector cells such as macrophages / dendritic cells.

Treatment according to the present invention can be effected when the subject is a neonate. Administration is carried out prior to delivery of the neonate and/or during delivery of the neonate.

The Antimineralocorticoid compounds according to the present invention can be administered to a patient in any of a wide range of routes. Thus, with regard to the types of formulations in which the active compounds according to the present invention can be administered, as well as any additives can be included with the active compounds in the formulations, and the possible routes of administration, it is well known to those of skill in the art that such formulations can be provided in a wide variety of types, and it is within the skill of the ordinary artisans to select a specific formulation and route of administration and then test suitability for use. By way of example but not limitation, suitable routes include enteric, intravenous, parenteral, -10topical, oral, rectal, nasal or vaginal routes. Parenteral routes include subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal and sublingual administration. Also, compositions may be implanted into a patient or injected using a drug delivery system.

The pharmaceutical formulation according to the present invention may be administered locally or systemically. By systemic administration means any mode or route of administration that result in effective amounts of active ingredient appearing in the blood or at a site remote from the route of administration of the active 10 ingredient.

In the preferred embodiment of the invention, the Antimineralocorticoid compound is micronized. In accordance with the present invention, the expression micronized means that the compound has been micronized in accordance with any process for 15 micronizing, a number of which are known in the art. The micronized particles preferably include a percentage of particles, which are of a diameter, which is about 10 microns, or less, preferably 5 microns or less. For example, in this preferred aspect of the invention, at least 80% of the particles in a formulation of micronized particles have a diameter of less than 5 microns.

For oral administration, the Antimineralocorticoid compounds of this Patent can be formulated into solid or liquid preparations. Suitable formulations for oral administration include hard or soft gelatin capsules, dragees, pills, tablets, including soft-coated tablets, troches, lozenges, melts, powders, micronized particles, non25 micronized particles, solutions, emulsions, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.

One of the formulations of the present invention is that the compound is enterically coated and is administered orally. In another embodiment, the compound is 30 administered sub-lingually.

In one embodiment of the invention the enteric coating is made of a polymer, preferably selected from the group consisting of poly(lacttc-glyco!ic acid) polyester, cellulose acetate phthalate, hydroxypropyl-methyl cellulose phthalate poly(butyl 35 methacrylate), (2-dimethyl aminoethyl) methacrylate, and methyl methacrylate. - 11 Solid dosage forms in addition to those formulated for oral administration include rectal suppositories. According to a further aspect of the invention the compound is formulated in a liposome.

In another embodiment of the invention, a liposome's or cyclodextrim molecular cage are provided carrying the Antimineralocorticoid compounds targeted to Ebola infected cells by putting antibodies to the Ebola coat glycoproteins GP1 or GP2 on its surface. The advantage of this is that the liposome can selectively target Ebola infected cells. Suitable injectable solutions include intravenous, subcutaneous and intramuscular injectable solutions. Examples of injectable forms include solutions, suspensions and emulsions. Typically the compound(s) is injected in association with a pharmaceutical carrier such as normal saline, Ringers solution, dextrose solution and other aqueous carriers known in the art. Appropriate non-aqueous carriers may also be used and examples include cyclodextrin, preferably hydroxypropyl beta cyclodextrin, mixed oils (vitamin E oil), polyethylene glycol and ethyl oleate. A preferred carrier is cyclodextrin in water. Frequently, it is desirable to include additives in the carrier such as buffers and preservatives or other substances to enhance isotonicity and chemical stability. The composition may also be administered in the form of an implant. in another embodiment it may also be preferable to co-administer with at least one other anti-viral agent Examples of anti-virals include are inhibitors of Sadenosylhomocysteine hydrolase (SAH), but are not limited to: • Carbocyclic 3-deazaadenosine (C-c3Ado), • The R- and S-isomers of 6'-C-neplanocin A analogues • Various carbocyclic analogues of adenosine, • aristeromycin (carbocyclic adenosine) • carbocyclic 3-deazaadenosine • neplanocin A (NepA) • 3-deazaneplanocin A • 5'-nor derivatives of aristeromycin • carbocylic 3-deazaadenosine, • 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A • 9-(hydroxyalkenyl)purines (adenines and 3-deazaadenines), which are analogues of neplanocin A, • 3-deazaneplanocin A, • the 5'-nor derivatives of carbocyclic adenosine (C-Ado, aristeromycin), and the 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A. • 6'-C-methylneplanocin A (isomers I and II) • 5'-noraristeromycin • (S)-9-(2,3-dihydroxypropyl)adenine • 5'-nor derivatives of carbocyclic adenosine (C-Ado, aristeromycin), • 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A.

» A series of 9-(hydroxyalkyl)-3-deazaadenines, which are analogues of the carbocyclic derivative of 3-deazaadenosine (3-deaza-C-Ado) • (RS)-3-adenine-9-yl-2-hydroxypropanoic acid [(RS)-AHPA] isobutyl ester • 3-deaza-C-Ado • 4-Amino-1-(2,3-dihydroxy-1-propyl)imidazo[4,5-c]pyridine • Γ-, 2'-, and 3'-carbons of 3-deaza-C-Ado • 4-Amino-1-(4-hydroxy-1-butyl)imidazo[4,5-c]pyridine • 5~-deoxy-S'-S-isobutyladenosin~ (SI BA) • (S)-9-(2,3-dihydroxypropyl)adenine • ribavirin • vidarabine • pyrazofurin « tubercidin e carbodine * (S)-9-(2,3-dihydroxypropyl)adenine [(S) DHPA] * 3-deaza-adenosine (DZA) * 3-deaza-(+/-)aristeromycin (DZAri) • 2',3'-dideoxy-adenosine (ddAdo) • 2',3'-dideoxy-3-deaza-adenosine (ddDZA), • 2',3'-dideoxy-3-deaza-(+/-)aristeromycin (ddDZAri) • 3-deaza-5'-(+/-)noraristeromycin (DZNAri) • 3-deaza-neplanocin A (DZNep) • Homodimer enzyme inhibitory antibodies to SAH inhibitors The components of any of the pharmaceutical formulations disclosed herein can be administered simultaneously (in a combination formulation), essentially -13simultaneously (e.g., administration of each compound a few minutes or a few hours apart), or can be administered sequentially, e.g., several days apart, or more than a week apart. For example, a compound of the present invention, at least one Antimineralocorticoid compound can be administered together, or essentially simultaneously, e g., administration of each compound a few minutes or a few hours apart, or can be administered sequentially, e g., several days apart, or more than a week apart. All such variations in administration of the combination therapy are encompassed within the scope of the invention.

In another embodiment, the composition is incorporated in a pharmaceutically acceptable carrier, diluents, vehicles and the like for systemic administration by feeding. An example of such a carrier is cyclodextrin.

With regard to dosage and duration of treatment according to any aspect of the present invention, it is recognized that the ability of an artisan skilled in pharmaceutical administration of drugs to determine suitable dosages depending on many inter-related factors is well known, and skilled artisans are readily able to monitor patients to determine whether treatment should be started, continued, discontinued or resumed at any given time. For example, dosages of the compounds are suitably determined depending on the individual cases taking symptoms, age, weight and sex of the subject and the like into consideration. The amount of the compound to be incorporated into the pharmaceutical composition of the invention varies with dosage route, solubility of the compound, administration route, administration scheme and the like. An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient and the method, route and dose of administration. The clinician using parameters known in the art makes determination of the appropriate dose. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved. Suitable dosages can be determined by further taking into account relevant disclosure in the known art.

In general, the amount of compound delivered to the patient is sufficient to achieve a plasma concentration of from about 3 to 10 pg/ml to about 5000 pg/ml of plasma, typically about 3 to about 50 pg/ml or about 5 to about 25 pg/ml. However, when liposomes targeted to Ebola viral infected cells are used to administer the - 14Antimineralocorticoid compounds, high doses of 25 mg/ml are used. The effective amount is optionally administered in a dosage ranging between 10 yg/kg and about 20,000 yg/kg of body weight of the patient. Unit dosages for any of the conditions described in the disclosure will typically comprise about 1-1000 mg/day of an Antimineralocorticoid compound often about 5 to 500 mg/day, ideally 400 mg/day with an optimal blood plasma concentration of 60 yM in the blood all day. Preferable pediatric doses range from 0.001 to 100 mg/kg/day, with optimal doses in the range of 3 mg/kg/day.

Administration of the Antimineralocorticoid compounds has a direct effect, as reported herein to maintain vascular endothelial stability and prevent hypovolemic shock and immune cell viability in a patient infected by filovirus.

The Mineralocorticoid Steroid Receptor like most steroid hormone receptors, mediates its biological response by aldosterone crossing the plasma membranes of cells and interacting with Aldosterone Receptor proteins in the cytosol or nucleus, to form complexes. Mineralocorticoid Steroid Receptor has been shown to be a heterooligomeric complex that includes a 90 kDa heat shock protein (HSP90).

The aldosterone/ Mineralocorticoid Steroid Receptor complexes are then chaperoned by heat shock proteins to the nucleus where they then accumulate in the nucleus of cells where they bind to specific regulatory DNA sequences.

In publications Mineralocorticoid Steroid Receptor have been shown to be functionally present on lymphocytes (Leukemia (2000) 14, 1097-1104, Demonstration of the mineralocorticoid receptor hormone and action in human leukemic cell lines N Mirshahi, S Mirshahi, N Golestaneh, Z Mishal, C Nicolas, C Hecquet and MK Agrwal).

The soluable G glycoproteins Ebola GP1 & GP2 in this Patent have been demonstrated to act to mimic the aldosterone molecule, (Please see Figure 1 and Figure 2).

The Antimineralocorticoid compounds (aldosterone antagonist and/or molecules which mimic aldosterone structure) in particular compete with the Ebola viral glycoprotein GP1 / GP2 coat proteins involved in Mineralocorticoid Steroid Receptor -15attachment and reduce viral cell entry, vascular endothelial instability and apoptosis of macrophages and dendritic cells.

In another embodiment the anti-sense mineralocorticoid Steroid ReceptorNA and the 5 DNA are delivered to viral infected cells by incorporating the anti-sense mineralocorticoid Steroid ReceptorNA and the DNA into liposomes or carbohydrate vehicles targeted to infected viral cells by placing Ebola directed antibodies on the surface of these liposomes or vesicles.

In another embodiment of the invention, liposomes are provided carrying the the antisense mineralocorticoid Steroid ReceptorNA and the DNA targeted to Ebola infected cells by putting antibodies to the Ebola coat protein GP1 or GP2 on its surface. The advantage of this is that the liposome can selectively target Ebola infected cells.

The outer coat of a lipid envelope virus is constructed of several types of polypeptide chains often arranged in several layers. In many viruses, moreover, the protein capsid is further enclosed by a lipid bilayer membrane that contains proteins. Many of these enveloped proteins acquire this envelope in the process of budding from the plasma membrane. This budding process allows the virus particles to leave the cell without disrupting the plasma membrane and therefore not killing the cell. However, the coat protein of the lipid envelope virus is significantly different in phospholipid profile than that of the plasma membranes of the host cell. It is thought that this is due to selective sequestration of lipids occurring through the budding process, in which the Ebola viral proteins select specific domains within the host cell membrane) )5 through which to emerge during maturation. Aldosterone, is one hormone that is selected and sequestered during the budding process of the Ebola virus. It would seem that a high cholesterol/phospholipid ratio within viral envelopes is required for infectivity of filovirus.

Furthermore, the invention provides a method of rendering an Ebola virus or Filivirus not capable of generating hemorrhagic effects, but viable for immune clearance and immune memory cell development comprising deleting from its genome the code directed to the Hydrophobic amino acid sequences that bind the Ebola virus to the aldosterone binding site on the Mineralocorticoid Steroid Receptor. -16The term idiotypic antibody is an antibody raised against the antigen binding site of another antibody. The antibody is produced using the anti-idiotypic method. For example the monoclonal idiotypic antibody is anti-aldosterone antibody was raised against the aldosterone binding site of the anti-aldosterone antibody.

In general the term steroid is considered as a group name for lipids that contain a hydrogenated cyclopentanoperhydrophenanthrene ring system. The substances of particular interest to the present invention are the adrenocortical hormones, particularly aldosterone.

The term steroid hormone receptors are defined herein as nuclear receptors that are phosphoproteins that include receptors for mineralocorticoids, capable of binding to its DNA responsive element.

The terms comprise, comprised and comprising and the terms include, included and including are used interchangeably in this specification and are to be afforded the widest interpretation.

The invention is not limited to the embodiments described above, but may be varied 20 in both construction and detail within the scope of the claims.

Example 1.

The effects of aldosterone, Ebola Glycoprotein GP1 arid the combination of aldosterone and eplerenone, and F.bola Glycoprotein GP1 and eplerenone on NO 25 (Nitrouse Oxide) production in human macrophages (Figure 1) The effects of aldosterone, Ebola Glycoprotein GP1 and the combination of aldosterone and eplerenone, and Ebola Glycoprotein GP1 and eplerenone on ROS (Reactive Oxygen Species) production in human macrophages (Figure 2) Materials and Methods: Twelve healthy volunteers (6 men and 6 women) aged 18-30 years who were taking no drugs participated in the study. Peripheral blood mononuclear cells were 35 separated from blood samples by histopaque density gradient centrifugation. - 17Monocytes were isolated from the peripheral blood mononuclear cells by negative immunomagnetic separation using Pan-T and Pan-B Dynabeads (Dynal, Oslo, Norway). The isolated cells were labelled with monoclonal antibody (Dako, Glostrup, Denmark) against the monocyte-specific positive antigen CD14. The procedure yielded 92% CD14-positive cells in the isolated fraction. The viability of immunomagnetically isolated monocytes was > 98%, as assessed by the 0.1% trypan blue exclusion test to measure cell membrane integrity. Monocytes were suspended in RPMI 1640 medium supplement with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, 100pg/ml streptomycin and 10 pg/ml fungizone (Gibco, Grand Island, NY). The cells were counted and the number of monocytes was adjusted to 1 x 106/ml. Equal numbers of cells (1 x 10B monocytes per well) were placed in each well of a plastic 24-well plate (Becton-Dickenson, NJ, USA) and left intact for 2 h to allow them to adhere to the bottom. Then the medium was changed, and the cultures were incubated for 24 h. Incubation was performed in triplicate at 37 °C in a humidified atmosphere containing 5% CO2 in air. After a 24 h incubation, the supernatant was carefully removed and replaced with medium supplemented with aldosterone (10 7 M), eplerenone (10 5 M) (Sigma-Aldrich USA) and Ebola Glycoprotein GP1 (Sino Biologicals Inc China) for another 24 h. In wells containing both eplerenone and Ebola GP1 cells were pre-incubated eplerenone for 1 h before the Ebola Glycoprotein GP1 was added. Eplerenone was dissolved in DMSO (Sigma-Aldrich, USA) at an initial concentration of 10 μΜ. Further dilutions were performed using the appropriate medium. A corresponding amount of DMSO was added to negative controls. The final concentration in the medium did not exceed 0.05% and, and did not have any effect on the monocyte cultures. The cells were incubated in the described conditions for an additional 24 h. At the end of the incubation, the supernatant was collected and stored until used for nitrite measurements using colorimetric assay kits according to the manufacturer’s recommendations (Cayman Chemicals, Ann Arbor, USA). MTT conversion, ROS measurement were conducted in the cultures adhered to the bottom of the 24-well plates. Cell viability was determined using trypan and MTT (Sigma-Aldrich, USA).

MTT conversion The viability of monocytes treated with the studied compound and Ebola GP1 was evaluated using the MTT conversion method. MTT (2.5 pg/ml) was added to the medium 3 h before the scheduled end of the experiment, and then the cultures were - 18incubated at 37 °C in 5% 002/95% air. Then, the monocytes were lysed in lOOpLDMSO, which enabled the release of the blue reaction product, formazan (RT, 10 min in the dark).

Absorbance at the wavelength of 570nm was read using a microplate reader (Dynex Technologies, Chantilly, VA, USA), in three measurements per well.

Monocytes incubated in 24-well tissue culture plates (1x106 cells/well) were treated with the studied compound and Ebola GP1 (37 °C in 5% 002/95% air). After 24 h, the cells were resuspended in DMSO containing NBT (1 mg/ml), (Sigma10 Aldrich). Next, the monocytes were lysed using sonication, and NBT reduction was measured by absorbance at 550 nm in triplicate using a microplate reader.

Results: The effects of aldosterone, Ebola GP1, eplerenone, and the combination of 15 aldosterone with eplerenone plus Ebola GP1 with eplerenone on cell viability The concentration of mineralocorticoid modulators were selected based on a study conducted in human endothelial vein cells and the concentration of Ebola GP1 was based on a study performed on human monocytes earlier. Prior to the evaluation of 20 the MR blockade by the Ebola GP1 we determined whether the compounds used in this study affected the viability of the cells in our experimental settings. Non of the drugs utilized nor their combinations affected the cell viability, as determined by MTT and trypan blue exclusion assays.

The effects of aldosterone, Ebola GP1 and the combination of aldosterone and eplerenone, and Ebola GP1 and eplerenone on NO production in human macrophages (Figure 1) Treatment with 10'7 M aldosterone or the 20 micro grams of Ebola GP1 significantly 30 increased the production of NO.

In contrast, treatment with 107 M Aldosterone in combination with 10'5 M eplerenone, and the treatment of Ebola GP1 in combination with 10 5 M eplerenone, had a significant effect in reducing the NO production by the Aldosterone and most importantly demonstrates the ability of eplerenone to inhibit the aldosterone mimic 35 activity of Ebola GP1. - 19The effects of aldosterone, Ebola GP1 and the combination of aldosterone and eplerenone, and Ebola GP1 and eplerenone on ROS production in human macrophages (Figure 2) Treatment with 10’7 M aldosterone or the 20 micro grams of Ebola GP1 significantly increased ROS production.

In contrast, treatment with 10 z M Aldosterone in combination with 10'5 M eplerenone, had a significant effect on reducing ROS production. Treatment of the 20 micro grams of Ebola GP1 in combination with 10’5 M eplerenone, this combination had a significant effect on reducing ROS production.and most importantly again demonstrates the ability of eplerenone to inhibit the aldosterone mimic activity of Ebola GP1.

Claims (51)

Claims

1. A pharmaceutical formulation for treating or preventing vascular endothelial cell instability resultant from the actions of filoviral coat glycoproteins GP1 f 2 in causing the loss of endothelial cell attachment leading to characteristic haemorrhaging which results in hypovolemic shock, comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising at least one antagonist of the mineralocorticoid I aldosterone receptor.

2. A pharmaceutical formulation for treating or preventing apoptosis of immune cells particularly macrophage and dendritic cells resultant from filoviral infection causing the loss of initial immune response in an infected patient,comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising at least one antagonist of the mineralocorticoid I aldosterone receptor.

3. The pharmaceutical formulation as claimed in claim 1, wherein the antagonist of the mineralocorticoid receptor compound is selected from the group comprising spironolactone, spirorenone, 1 ,2-dihydro-spirorenone, 1 ,2a-methylenespirorenone, eplerenone, Drospirenone, potassium canrenoate, canrenoate, canrenone and pharmaceutically acceptable salts thereof or their metabolites.

4. The pharmaceutical formulation as claimed in claim 3, wherein the Antagonist of the mineralocorticoid / aldosterone receptor is spironolactone.

5. The pharmaceutical formulation as claimed in any preceding claim wherein the antagonist to the mineralocorticoid receptor is an anti-idiotypic humanized monoclonal to the aldosterone binding site.

6. A pharmaceutical formulation as claimed in any preceding claim wherein, the antagonist of the mineralocorticoid / aldosterone receptor is a 7 o-acetylthio-4pregnene-3,20-dione represented by formula B

7. A pharmaceutical formulation as claimed in claim 5 wherein, Ri is hydroxyl or OR 2 where R 2 is derived from a carboxylic acid of the formula R 4 OOH which may have up to 12 carbon atoms, and in which R 4 may be substituted or unsubstituted, saturated or unsaturated, straight chain or branched, alicyclic, aryl, heterocyclic or mixed, but having one or more from 3 to 12 carbon atoms.

8. A pharmaceutical formulation as claimed in claim 5 or 6 wherein Ri is hydroxy, monocarboxylic, straight or branched-chain alkanooyloxy group having up to 12 carbon atoms. y.

9. A pharmaceutical formulation as claimed in claim 6 or 7 wherein Ri is hydrogen, hydroxy, acetoxy, propionyloxy, n-butyryloxy, trimethylacetoxy, n- valeroyloxy or n-heptanoyloxy. 10. 5']perhydrofuran-2'-one, 6β,7β,15β,16β-Dimethyiene-3-oxo-4-androstene- [17(β1')- spiro-5']perhydrofuran-2'-one.

10. A pharmaceutical formulation as claimed in claim 6 or 7 or 8 wherein the antagonist of the mineralocorticoid / aldosterone receptor is selected from the group comprising: 7α- acetylthio-4-pregnene-3,20-dione; 7a-acetylthio-21 hydroxy-4-pregnene-3,2O- dione; 7a-acetylthio-21-acetoxy-4-pregnene-3,20dione; 7a-acetylthio-21- propionyloxy-4-pregnene-3,20-dione; 7a-acetylthio-21-nbutyryloxy-4-pregnene- 3,20-dione; 7a-acetylthio-21-trimethylacetoxy-4pregnene-3,20-dione; 7a-acetylthio-21 -n-valeroyloxy-4-pregnene-3,20-dione; 7αacetylthio-21- heptanoyloxy-4-pregnene-3,20-dione; 7a-Acetylthio-3-oxo-4, 15 -22androstadiene- [17(3-T)-spiro-5’]perhydrofuran-2'-one, 3-Oxo-7a-propionylthio-4, 15- androstadiene-[17(P-1')- spiro-5']perhydrofuran-2'-one, 6p,7p-Methylene-3oxo- 4,15-androstadiene-[17(P-1')- spiro-5']perhydrofuran“2'-one, 15a, 16aMethylene-3-oxo-7a-propionylthio-4-androstene-[17(β-1')- spiro5 5']perhydrofuran-2’-one, 6β,7β, 15a, 16a-Dimethylene-3-oxo-4-androstene-[17(βΓ)- spiro-5']perhydrofuran-2'-one, 7a-Acetylthio-15a, 16a-methylene-3- oxo-4androstene-[17^-1')- spiro-5']perhydrofuran-2'-one, 7a-Acetylthio- 15β,16βmethylene-3-oxo-4-androstene-[17(3-T)- spiro-5']perhydrofuran-2'- one, 15β,16βMethylene-3-oxo-73- propionylthio-4- androstene-[17(3-T)- spiro

11. A pharmaceutical formulation as claimed in any preceding claim wherein the antagonist of the mineralocorticoid / aldosterone receptor is a 9, 11-epoxy steroid

12. The pharmaceutical formulation as claimed in any proceeding claim wherein the composition further includes at least one antiviral agent known to inhibit filovirus replication.

13. The pharmaceutical formulation as claimed in claim 11 wherein the anti-viral agents are selected from the group comprising: Carbocyclic 3deazaadenosine (C-c 3 Ado), the R· and S-isomers of 6'-C-neplanocin A analogues, various carbocyclic analogues of adenosine, aristeromycin 25 (carbocyclic adenosine), carbocyclic 3-deazaadenosine, neplanocin A (NepA), 3 deazaneplanocin A, 5'-nor derivatives of aristeromycin, carbocylic 3 deazaadenosine, 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A, 9-(hydroxyalkenyl)purines (adenines and 3deazaadenines), which are analogues of neplanocin A, 3-deazaneplanocin A, the 30 5'-nor derivatives of carbocyclic adenosine (C-Ado, aristeromycin), and the 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A, 6'-Cmethylneplanocin A (isomers I and II), 5'-noraristeromycin, (S)-9-(2,3dihydroxypropyl)adenine, 5'-nor derivatives of carbocyclic adenosine (C-Ado, aristeromycin), 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives 35 of neplanocin A, A series of 9-(hydroxyalkyl)-3-deazaadenines, which are analogues of the carbocyclic derivative of 3-deazaadenosine (3-deaza-C-Ado),

14. The pharmaceutical formulation as claimed in any preceding claim, wherein the composition is administered Intra-venous, enterally, parenterally, topically, orally, rectally, nasally or vaginally.

15. The pharmaceutical formulation as claimed in any preceding claim, wherein the compositions are formulated into liposomes or carbohydrate or cyclodextrin vehicles. 15 compound, especially those of the 20-spiroxane series and their analogs.

16. The pharmaceutical formulation as claimed in claim 14, wherein the liposomes or carbohydrate vehicles are targeted to Ebola infected cells by putting antibodies directed to filovirus coat glycoproteins GP1 and/ or GP2 on its surface.

17. The pharmaceutical formulations as claimed in any preceding claim, wherein the composition is administered prophylactically to prevent loss of endothelial cell attachment leading to characteristic haemorrhaging which results in hypovolemic shock.

18. The pharmaceutical formulation as claimed in any preceding claim, wherein the formulation is a unit dose that comprises 5-500 mg of the antagonist of the mineralocorticoid I aldosterone receptor compound.

19. A pharmaceutical formulation as claimed in any preceding claim, wherein said patient is a neonate and said administering is effected prior to delivery of said neonate and/or during delivery of said neonate.

20. A pharmaceutical formulation as claimed in any preceding claim, wherein said antagonist of the mineralocorticoid I aldosterone receptor compound is manufactured as a Pro-Drug entity.

21. A pharmaceutical formulation for use in treating or preventing a future infection and/or minimizing the effects of a future infection by a Filoviridae virus comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising at least one antagonist of the mineralocorticoid / aldosterone receptor to prevent loss of endothelial cell attachment leading to characteristic haemorrhaging which results in hypovolemic shock. -21 CH 2 R! c=o in which R, is hydrogen, hydroxy, hydroxyl, a mineral acid ester such as sulfate, phosphate or nitrate group, or acyloxy-OR 2 , the acyl group R 2 being derived from a carboxylic acid of the formula R 4 OOH which may have up to 12 carbon atoms, and in which R 4 may be substituted or unsubstituted, saturated or unsaturated, straight chain or branched, alicyclic, aryl, heterocyclic or mixed and R s is methyl.

22. The pharmaceutical formulation as claimed in claim 20, wherein the antagonist of the mineralocorticoid / aldosterone receptor is selected from the group comprising spironolactone, spirorenone, 1,2-dihydro-spirorenone, 1 ,2αmethylene- spirorenone, eplerenone, Drospirenone, potassium canrenoate, canrenoate, canrenone and pharmaceutically acceptable salts thereof or their metabolites.

23. The pharmaceutical formulation as claimed in claim 21, wherein the antagonist of the mineralocorticoid / aldosterone receptor is spironolactone. -23(RS)-3-adenine-9-yl-2-hydroxypropanoic acid [(RS)-AHPA] isobutyl ester, 3deaza-C-Ado, 4-Amino-1-(2,3-dihydroxy-1-propyl)imidazo[4,5-c]pyridine, T-, 2'-, and 3’-carbons of 3-deaza-C-Ado, 4-Amino-1-(4-hydroxy-1-butyl)imidazo[4,5cjpyridine, 5~-deoxy-S’-S-isobutyladenosin~ (SIBA), (S)-9-(2,3dihydroxypropyl)adenine, ribavirin, vidarabine, pyrazofurin, tubercidin, carbodine, (S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA], 3-deaza-adenosine (DZA), 3deaza-(+/-)aristeromycin (DZAri), 2',3'-dideoxy-adenosine (ddAdo), 2',3'-dideoxy3-deaza-adenosine (ddDZA), 2',3'-dideoxy-3-deaza-(+/-)aristeromycin (ddDZAri), 3-deaza-5'-(+/-)noraristeromycin (DZNAri), 3-deaza-neplanocin A (DZNep), Homodimer enzyme inhibitory antibodies to SAH inhibitors.

24. The pharmaceutical formulation as claimed in claim 20, 21 or 22, wherein the antagonist of the mineralocorticoid / aldosterone receptor is Drospirenone.

25. The pharmaceutical formulation as claimed in any of claims 17 to 23, wherein the antagonist of the mineralocorticoid / aldosterone receptor is, selected from the group of prostogens with antimineralocorticoid activity consisting of progesterone, gestodene, dimethisterone, ethinyloestradiol, ethisterone, 11 βhydroxyprogesterone, 17α- hydroxyprogesterone, 16a-methyl progesterone, hydroxyprogesterone caproate, medroxyprogesterone acetate, proligestone and pharmaceutically acceptable salts thereof or their metabolites, analogues and mimic molecules.

26. A pharmaceutical formulation as claimed in any of claims 17 to 24, wherein the antagonist of the mineralocorticoid / aldosterone receptor is a 7 a-acetylthio-4pregnene-3,20-dione represented by formula B as defined in claim 5 in which Ri -25is hydrogen, hydroxy, hydroxyl, a mineral acid ester such as sulfate, phosphate or nitrate group, or acyloxy-OR 2 , the acyl group R 2 being derived from a carboxylic acid of the formula R,OOH which may have up to 12 carbon atoms, and in which R4 may be substituted or unsubstituted, saturated or unsaturated, straight chain or branched, alicyclic, aryl, heterocyclic or mixed and R 3 is methyl.

27. A pharmaceutical formulation as claimed in claim 25 wherein, Ri is hydroxyl or OR 2 where R 2 is derived from a carboxylic acid of the above type, but having one or more from 3 to 12 carbon atoms.

28. A pharmaceutical formulation as claimed in claim 25 or 26 wherein Ri is hydroxy, monocarboxylic, straight or branched-chain alkanooyloxy group having up to 12 carbon atoms.

29. A pharmaceutical formulation as claimed in claim 25 or 26, wherein R, is hydrogen, hydroxy, acetoxy, propionyloxy, n-butyryloxy, trimethylacetoxy, nvaleroyloxy or n-heptanoyloxy.

30. A pharmaceutical formulation as claimed in claim 25 or 26, wherein the antagonist of the mineralocorticoid / aldosterone receptor for prevention of vascular instability leading to hypovolemic shock from a filovirus infection is selected from the group comprising: 7a-acetylthio-4-pregnene-3,20-dione; 7αacetylthio-21-hydroxy-4-pregnene-· 3,20-dione; 7a-acetylthio-21 -acetoxy-4pregnene-3,20-dione; 7a-acetylthio-21 - propionyloxy-4-pregnene-3,20-dione: 7α acetylthio-21-n-butyryloxy-4-pregnene- 3,20-dione; 7a-acetylthio-21 trimethyiacetoxy-4-preg nene-3,20-dione; 7α- acetylthio-21 -n-valeroyloxy-4pregnene-3,20-dione; 7a-acetylthio-21 heptanoy!oxy-4-pregnene-3,20-dione; 7a-Acetylthio-3-oxo-4, 15- androstadiene-[17(p-1 ')-spiro-5']perhydrofuran-2'-one, 3-Oxo-7a- propionylthio-4, 15-androstadiene-[17(P-i spiro-5']perhydrofuran-2’one, 6p,7p-Methylene-3-oxo-4,15-androstadiene-[17(β-1')- spiro5']perhydrofuran-2'-one, 15a,16a-Methylene-3-oxo-7a-propionylthio-4- androstene-[17(β-1')- spiro-5']perhydrofuran-2'-one, 6p,7p,15a,16a- Dimethylene3-oxo-4-andΓostene-[17(β-Γ)- spiro-5']perhydrofuran-2'- one, 7a-Acetylthio-15a, 16a-methylene-3-oxo-4-androstene-[17(β-1')- spiro-5'Jperhydrofuran-2'-one, 7aAcetylthio-ΐδβ, 163-methylene-3-oxo-4- androstene-|17(6-1> spiro5']perhydrofuran-2'-one, 15β, Ιδβ-Methylene- 3-οχο-7β- propionylthio-4 -26androstene-[17(3-T)- spiro-5']perhydrofuran- 2 J -one, 6β,7β, 15β, 16βDimethylene-3-oxo-4-androstene-[17(β-1')- spiro- 5']perhydrofuran-2'-one.

31. A pharmaceutical formulation as claimed in any of claims 20 to 29, wherein the antagonist of the mineralocorticoid / aldosterone receptor is a 9,11-epoxy steroid compounds, especially those of the 20-spiroxane series and their analogs.

32. A pharmaceutical formulation as claimed in any of claims 20 to 30, wherein the antagonist of the mineralocorticoid / aldosterone receptor are halogenated in the 9-alpha position.

33. A pharmaceutical formulation as claimed in claim 31, wherein the halogen is selected from the group consisting of chlorine, bromine, fluorine and iodine.

34. The pharmaceutical formulation as claimed in any of claims 20 to 32, wherein the composition includes at least one anti-viral agent.

35. The pharmaceutical formulation as claimed in claim 33, wherein the coadministered anti-viral agents are selected from the group comprising: Carbocyclic 3-deazaadenosine (C-c 3 Ado), the R- and S-isomers of 6'-Cneplanocin A analogues, various carbocyclic analogues of adenosine, aristeromycin (carbocyclic adenosine), carbocyclic 3-deazaadenosine, neplanocin A (NepA), 3-deazaneplanocin A, 5'-nor derivatives of aristeromycin, carbocylic 3-deazaadenosine, 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-Rmethyl) derivatives of neplanocin A, 9-(hydroxyalkenyl)purines (adenines and 3deazaadenines), which are analogues of neplanocin A, 3-deazaneplanocin A, the 5'-nor derivatives of carbocyclic adenosine (C-Ado, aristeromycin), and the 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A, 6'-Cmethylneplanocin A (isomers I and II), 5'-noraristeromycin, (S)-9-(2,3dihydroxypropyl)adenine, 5’-nor derivatives of carbocyclic adenosine (C-Ado, aristeromycin), 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A, A series of 9-(hydroxyalkyl)-3-deazaadenines, which are analogues of the carbocyclic derivative of 3-deazaadenosine (3-deaza-C-Ado), (RS)-3-adenine-9-yl-2-hydroxypropanoic acid [(RS)-AHPA] isobutyl ester, 3deaza-C-Ado, 4-Amino-1-(2,3-dihydroxy-1-propyl)imidazo[4,5-c]pyridine, Γ-, 2'-, and 3-carbons of 3-deaza-C-Ado, 4-Amino-1-(4-hydroxy-1-butyl)imidazo[4,5- -27c]pyridine, 5~-deoxy-S’-S-isobutyladenosin~ (SIBA), (S)-9-(2,3dihydroxypropyl)adenine, ribavirin, vidarabine, pyrazofurin, tubercidin, carbodine, (S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPAJ, 3-deaza-adenosine (DZA), 3deaza-(+/-)aristeromycin (DZAri), 2',3'-dideoxy-adenosine (ddAdo), 2',3'-dideoxy3-deaza-adenosine (ddDZA), 2’,3 , -dideoxy-3-deaza-(+/-)aristeromycin (ddDZAri), 3-deaza-5'-(+/-)noraristeromycin (DZNAri), 3-deaza-neplanocin A (DZNep), Homodimer enzyme inhibitory antibodies to SAH inhibitors.

36. The pharmaceutical formulation as claimed in claim 33 or 34 wherein the antiviral agents are selected from the group comprising Abacavir; Acemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine; Alvircept Sudotox; Amantadine Hydrochloride; Aranotin; Arildone; Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline; Coviracil; Cytarabine Hydrochloride;. Delavirdine Mesylate; Desciclovir; Didanosine; Disoxaril; Edoxudine; Emivirine; Emtricitabine; Enviradene; Enviroxime; Epivir; Famciclovir; Famotine Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscarnet Sodium; Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium; Idoxuridine; Indinavir; Kethoxal; Lamivudine; Lobucavir; Lodenosine; Lopinavir, Memotine Hydrochloride; Methisazone; Nelfinavir; Nevirapine; Penciclovir; Pirodavir; Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate; Ritonavir; Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine; Tenofovir; Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride; Vidarabine; Vidarabine Phosphate; Vidarabine Sodium Phosphate; Tipranavir, Viroxime; Zalcitabine; Zidovudine and Zinviroxime.

37. The pharmaceutical formulations as claimed in any of claims 20 to 35, wherein the composition is administered enterally, parenterally, intravenous,topically, orally, rectally, nasally or vaginally.

38. The pharmaceutical formulations as claimed in any of claims 20 to 36, wherein the compositions are formulated into liposomes or carbohydrate or cyclodextrin vehicles.

39. The pharmceutical formulation as claimed in claim 37, wherein the liposomes or carbohydrate vehicles are targeted to Ebola infected cells by putting viral directed antibodies on its surface.

40. The pharmaceutical formulation as claimed in claim 38, wherein the viral antibodies are directed to the Ebola viral glycoproteins GP1 and/or GP2.

41. The pharmaceutical formulations as claimed in any of claims 20 to 39, wherein the composition is administered intermittently.

42. The pharmaceutical formulations as claimed in any of claims 20 to 40, wherein the formulation is a unit dose that comprises 5-500 mgs of antagonist of the mineralocorticoid / aldosterone receptor.

43. An pharmaceutical formulation as claimed in any of claims 20 to 41, wherein said patient is a neonate and said administering is effected prior to delivery of said neonate and/or during delivery of said neonate.

44. An pharmaceutical formulation as claimed in any of claims 20 to 42, wherein said antagonist of the mineralocorticoid / aldosterone receptor is synthesized as a Pro-Drug.

45. A method of rendering a Filoviridae virus not capable of endothelial cell damage causing haemorrhaging but viable for immune clearance and immune memory cell development by vaccination comprising deleting from its genome the code directed to the hydrophobic amino acid sequences listed that bind the virus to the aldosterone binding site on the Mineralocorticoid Steroid Receptor. Hydrophobic Amino Acid Sequences • DWYEDIIQAYREY • WIDSPFIWDNVMF • LTHWYAVDFNMWT . PWWYWEMRAFDAE • DWNIWDGWYREIY • RTHFEREFDDWFL

46. A method for the treatment and/or prophylaxis to prevent filoviral endothelial cell damage resulting in hypovolemic shock, the method comprising the steps of: • providing a pharmaceutical composition according to any one of claims 1 to 50, and • administering a therapeutically useful amount of said composition to a subject in need of treatment.

47. A method as claimed in claim 45, wherein the filoviridae virus is selected from the 5 group comprising: Ebola and Marburg.

48. Use of a pharmaceutical composition as claimed in any one of claims 1 to 47 in the preparation of a medicament for the treatment of or preventing vascular instability resultant from the actions of filoviral coat glycoproteins GP1 12 binding 10 to the aldosterone attachment site on the mineralocorticoid receptor and causing the loss of endothelial cell attachment leading to characteristic haemorrhaging which results in hypovolemic shock

49. Use of an antagonist of the mineralocorticoid / aldosterone receptor in the 15 preparation of a medicament for the treatment of vascular endothelial cell instability resulting from a filoviridae infection.

50. Use as claimed in claim 48 wherein the antagonist of the mineralocorticoid I aldosterone receptor is spironolactone or a Pro-Drug, derivative or analogue 20 thereof.

51. The preferred embodiment is where the antagonist of the mineralocorticoid I aldosterone receptor is micronized and delivered to the patient by IV administration either for prophylactic or therapeutic protection from vascular 25 instability resultant from the actions of filoviral coat glycoproteins GP1 I. 2 binding to the aldosterone binding site on the mineralocorticoid receptor and causing the loss of endothelial cell attachment leading to characteristic haemorrhaging which results in hypovolemic shock.