(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2016/063269 Al 28 April 2016 (28.04.2016) P O P C T

(51) International Patent Classification: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, A61K 31/4422 (2006.01) A61P 31/12 (2006.01) KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, A61K 31/56 (2006.01) MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (21) International Application Number: SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, PCT/IE20 15/0000 16 TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (22) International Filing Date: (84) Designated States (unless otherwise indicated, for every 20 October 2015 (20.10.201 5) kind of regional protection available): ARIPO (BW, GH, (25) Filing Language: English GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, (26) Publication Language: English TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (30) Priority Data: DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, 2014/0275 20 October 20 14 (20. 10.20 14) IE LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, 2014/03 10 11 December 2014 ( 11. 12.2014) IE SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, 2014/0324 23 December 2014 (23. 12.2014) IE GW, KM, ML, MR, NE, SN, TD, TG). 2015/0024 29 January 2015 (29.01.2015) IE Declarations under Rule 4.17 : 2015/0140 6 May 2015 (06.05.2015) IE — as to the identity of the inventor (Rule 4.1 7(Ϊ)) (72) Inventor; and — of inventorship (Rule 4.17(iv)) (71) Applicant : PRENDERGAST, Patrick T. [IE/AU]; Kenilworth Station, Byrock, New South Wales (AU). Published: (81) Designated States (unless otherwise indicated, for every — with international search report (Art. 21(3)) kind of national protection available): AE, AG, AL, AM, — before the expiration of the time limit for amending the AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, claims and to be republished in the event of receipt of BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, amendments (Rule 48.2(h)) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,

(54) Title: USE OF ANTAGONISTS TO THE NUCLEAR STEROID RECEPTOR ALONE OR IN COMBINATION A S DIRECT ANTIVIRAL AGENTS TO INHIBIT ALPHAVIRUS, TOGAVIRIDAE, ARENAVIRIDAE, FILOVIRIDAE, BUNYAVIRIDAE, FLAVIVIRIDAE AND RHAB- © DOVIRIDAE 2 7) Abstract: 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 GPl / 2 at - taching to the aldosterone receptor and causing the loss of endothelial cell attachment leading to characteristic haemorrhaging which results in hypovolemic shock to the patient. T e invention also relates to compositions that comprise antagonist of the mineralocor- ticoid / aldosterone receptor and an excipient. Title

Use of Antagonists to the Nuclear Steroid Receptor alone or in combination as direct antiviral agents to inhibit Alphavirus, Togaviridae, Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae and Rhabdoviridae.

Description

The nuclear steroid hormone receptor super family of ligand activated transcription factors are present in various tissues, and responsible for a broad range of effects in these tissues.

The nuclear steroid receptor (NR) super family presently comprises approximately 48 different proteins, most of which are understood to function as ligand activated transcription factors, exerting widely different biological responses by regulating gene expression. Members of this family include receptors for endogenous small, lipophilic molecules, such as steroid hormones, retinoids, vitamin D, calcium channel blockers, and thyroid hormone.

The nuclear steroid receptor (NR) super family includes the steroid nuclear receptor subfamily, such as the mineralocorticoid receptor (MR) (or aldosterone receptor), the estrogen receptors (ER), ER alpha and ER beta, the androgen receptor (AR), the progesterone receptors (PR), glucocorticoid receptors (GR) and others. Also closely related in structure are the estrogen related receptors (ERRs) ERR-alpha, ERR-beta and ERR-gamma. The steroid nuclear receptors perform many important functions in the body some of which are related to the transcriptional homeostasis of electrolyte and water balance, growth, development and wound healing, fertility, stress responses, immunological function, and cognitive functioning. The effects may be mediated by cytosolic or nuclear events. Accordingly, compounds that modulate (i.e.

Antagonist , agonise, partially antagonise, partially agonize) the activity of steroid nuclear receptors are important pharmaceutical agents that have specific utility in this incidence as antiviral agents against filoviruses and Alphavirus and other haemorrhagic viral families as will be outlined below in the attached antiviral screening results presented.

l Members of the steroid nuclear receptor sub-family exhibit significant homology to each other and possess closely related DNA and ligand binding domains hence the need in the formulation of the antiviral combination created for filovirus and Alphavirus infections to utilize both alone and in combination the specific ligands which antagonise at least one two or three of the receptor sub-families as outlined in this patent.

Given the very close similarity in ligand binding domains of the steroid nuclear receptors, it is not surprising that many naturally occurring and synthetic molecules possess the ability to modulate the activity of more than one steroid nuclear receptor.

The present invention is directed to providing therapeutic use of named nuclear steroid receptor antagonists alone and/ in combination of antagonists to the (1) mineralocorticoid / aldosterone receptor and the (2)oestrogen receptor and the (3) progesterone receptor and (4) glucocorticoid receptor and (5) integrin receptor and (6) androgen receptor (7) calcium channel blockers for their previously unappreciated, prophylactic and synergistic therapeutic properties as direct antivirals to filivirus, flavivirus and Alphavirus and other haemorrhagic viruses and also preventing damage to the endothelial barrier that causes loss of vascular integrity resultant from filoviral and or Alphavirus infection of a patient and the effect of the Ebola viral coat glycoproteins GP1 / 2 attaching to sections of the Nuclear receptor and causing the loss of endothelial cell attachment leading to characteristic haemorrhaging which results in hypovolemic shock to the filovirus infected patient. These prescription drugs with nuclear steroid receptor antagonists activity also in this patent have been demonstrated to inhibit the replication of Chikungunya virus of the Alphavirus family.( Togaviridae ) .

The invention also relates to compositions that comprise antagonist of the mineralocorticoid / aldosterone receptor and/ or the progesterone receptor and/ or the glucocorticoid receptor and the integrin receptor all of which may be combined in a therapeutic regime or formulation to inhibit the filivirus, flavivirus and Alphavirus replication direct and cause elimination of the viral load within the infected patient to enable recovery from either viral infection

Accordingly it is an object of the present invention to provide a method and composition principally to inhibit viral replication and also to prevent vascular instability and hypovolemic shock in Filoviral and other haemorrhagic virally infected patients and also to prevent and treat Chikungunya virus infection from the Alphavirus family.

Antagonist to the mineralocorticoid receptor within the Nuclear receptor family: 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) and enhances renal calcium re-absorption by the distal tubules of the kidney. The reabsorption of sodium and calcium 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, flavivirus and Alphavirus coat proteins have Aldosterone mimic characteristics or interfere in one of the lipophilic regions associated with the Nuclear receptor system which can modulate endothelial permeability, endothelial nitric oxide and reactive oxygen species and the actin cytoskeleton. By stimulating the mineralocorticoid receptor as a mimic Aldosterone molecule the filovirus and to a lesser extent with Alphavirus 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 and the long term muscle pain and rheumatic symptoms seen for prolonged periods by Chikungunya viral infected patients.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 integrality 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 other haemorrhagic viral infections which can lead to hypovolemic shock.

By mimicking Aldosterone as one ligand for the Nuclear receptor 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 ligand compounds and or antibodies which block the lipophilic attachment sites on the Nuclear receptor this effect of the Ebola virus and other haemorrhagic viruses can be neutralised and filivirus replication inhibited together with 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 these nuclear receptor antagonists of the nuclear receptor outlined are utilised as direct antiviral compounds and antibodies of this patent which can circulate normally within the body to assist the patient in recovery from filovirus and other haemorrhagic viral infection E.g. Dengue Hemorrhagic Fever (DHF) and also to Chikungunya virus infection.

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 herein, "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 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 Ebola ) 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 was 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. 4,529,81 (Hill and Erickson 1985). Another spironolactone effective as an antagonist of the mineralocorticoid / aldosterone receptor iseplerenone

Eplerenone 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 * IK 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 Publication 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 inhibit haemorrhagic viruses especially filoviral family and prevent vascular instability and hypovolemic shock in Ebola viral infections.

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: 7- alpha-thiospironolactone, 7-alpha-thiomethylspironolactone, 6-beta-hydroxy-7-alpha- thiospironolactone, and 6-beta-hydroxy-7-alpha-thiomethylspironolactone. A number of drugs have been identified which can antagonise the other domains of the nuclear receptor and inhibit filivirus and other haemorrhagic viral replication in the body by blocking Other ligand sites of the Nuclear Steroid Receptor, these including (2) estrogen receptor antagonists (3) progesterone receptor antagonists and (4) glucocorticoid receptor antagonists.(5) integrin receptor antagonists.(6) androgen receptor antagonists or modulators.

We also want to include in its entirety patent PCT/US201 2/0371 77 Integrin receptor antagonists and their methods of use by Allegro Pharmaceuticals Inc. Priority date May 9th 2011.

We also want to include in its entirety patent 8,475,804 Compositions and methods for treatment of filovirus-mediated diseases by U.S. Army Medical Research and Material Command. Filed February 22nd 2010.

The present invention is directed to a method of treating an individual expected to be exposed to or actually infected with a lipid envelope filovirus such as Ebola viral infection or Bunyaviridae viral infection such as Crimean-Congo hemorrhagic fever (CCHF) and or infection with Chikungunya virus of the Alphavirus family as example by administering to that individual a therapeutically effective amount of one or more compounds of the present invention, to inhibit replication of the virus and prevent vascular instability and hypovolemic shock by interactively blocking the Ebola virus and CCHF and other hemorrhagic viruses and inhibiting the replication of Alphavirus, flavivirus and preventing the muscle ache and rheumatic symptoms that occur with and long after Chikungunya viral infection binding to the mineralocorticoid receptor or other receptors within the cell nucleus and activating cytotoxic and inflammatory signals.

The present invention relates to the use of antagonists of the nuclear receptor family that inhibit filoviral and other hemorrhagic viruses such as Bunyaviridae (CCHF) and flavivirus replication causing vascular instability and hypovolemic shock and inhibit Chikungunya virus and the inflammatory cytokines cascade that destroys immune cells that creates the pain and muscular damage associated with these viral infections. It has been surprisingly found that Antagonists of the nuclear steroid receptor have potent activity as direct antiviral agents to prevent replication of filoviridae and Bunyaviridae (CCHF) and Alphavirus and in preventing endothelial permeability collapse from filovirus and other hemorrhagic viral infections and the muscle ache and rheumatic pain associated with Chikungunya virus infection. Also the present invention relates to the metabolic derivatives (or metabolites) of these antagonists of the nuclear steroid receptor family that inhibit filovirus and other hemorrhagic virus replication and also prevent vascular instability and other inflammatory effects upon filovirus, flavivirus and Alphavirus infection.

This inventions objectives include the provision of pharmaceutical formulations of antagonists of the nuclear steroid receptor family and specified calcium channel blockers which are suitable for effective treatment of patients infected with or at risk of infection from a filovirus or Bunyaviridae or Alphavirus a and other hemorrhagic viral family. 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 replication of all hemorrhagic viral entities eg Filovirus,Bunyaviridae and Alphavirus ( Chikungunya ) to prevent hypovolemic shock and other cytokine related symptoms resulting from these viral infections comprising administering to a subject an effective amount of one or more compounds outlined in 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 inhibit hemorrhagic viral Replication eg Ebola ,CCHF. replication and Alphavirus ( Chikungunya virus) to prevent endothelial cell collapse and hypovolemic shock and inflamitory cytokines resulting from a these viral infections

The present invention also provides compounds identified in the present invention for use in a method of treatment to inhibit hemorrhagic viral family replication and protect from or prevent endothelial collapse and hypovolemic shock and inflammatory cytokines cascade resulting from a filovirus or Bunyaviridae or Alphavirus or other hemorrhagic viral family infection, said method comprising administering one or more antagonists of the nuclear steroid receptor family to a subject. In accordance with the objects of the present invention, is provided an improved pharmaceutical formulation for use (or method) in the inhibition, prophylaxis and therapy of filoviral,Bunyaviridae and Alphavirus ( Chikungunya virus)and other hemorrhagic viral family infection or a complication or consequence thereof. In particular the invention relates to the use of antagonist of the nuclear steroid receptor family and their metabolic derivatives in the prophylaxis and therapy of filoviral,Bunyaviridae and Alphavirus or other hemorrhagic viral family induced hypovolemic shock and inflamitory cytokine cascade.

Other embodiments provide a pharmaceutical formulation or method to inhibit the replication of filoviral .Bunyaviridae and other hemorrhagic viral families and Chikungunya virus to prevent vascular instability due to loss of or to ameliorate one or more symptoms associated with hypovolemic shock resultant or rheumatic pains from these viral infections by administering to an infected patient an effective amount of a antagonist or modulator of the nuclear steroid receptor family all molecules or compounds or formulations as disclosed herein.

It is an object of the present invention to provide a composition, which includes at least one modulator or antagonist of the nuclear steroid receptor family and the use of these compounds outlined in the antiviral results included as direct antiviral treatment, therapeutic or prophylaxis, against a filoviral .Bunyaviridae and Alphavirus ( Chikungunya virus) or other hemorrhagic viral family infection. The composition containing at least one antagonist of the nuclear steroid receptor family and it's delivery 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, Bunyaviridae or other hemorrhagic viral family. This patent is also presenting a pharmaceutical formulation or method in the prophylaxis and therapy against muscle pain and rheumatic symptoms created by the Chikungunya virus infection.

Accordingly, the nuclear steroid receptor family antagonists molecules and and selected calcium channel blocker compounds disclosed herein provide a pharmaceutical formulation or a method for treating a filoviral .Bunyaviridae and Alphavirus or other hemorrhagic viral infection; comprising administering to a patient in need thereof a prophylactically or therapeutically effective amount of a composition comprising at least one Antagonist of the nuclear steroid receptor family of compounds disclosed herein.

The advantage of this is that a n effective antiviral treatment and / or prevention of filoviral , Bunyaviridae and Alphavirus or other hemorrhagic viral family infection is achieved preventing a situation leading to hypovolemic shock is provided that has minimal risk of generating toxicity to the infected patient and provides a competent circulatory blood system to allow these anti-viral therapy molecules of this patent to circulate around the infected patients body.

In another embodiment the antagonists of the nuclear steroid receptor family are selected from the group consisting of spironolactone, spirorenone, 1 ,2-dihydro- spirorenone, 1,2a- methylene-spirorenone,

7a-Acetylthio-3-oxo-4, 15-androstadiene-[17{JS-1 ')-spiro-5']perhydrofuran-2'- one

3-Oxo-7a-propionylthi0-4, 15-androstadiene-[17(/?-1 ')- spiro- 5']perhydrofuran-2'-one

6/?,7/?-Methylene-3-oxo-4\ 15-androstadiene-[1 7(β- 1 ')- spiro-5']perhydrofuran-2'- one 5a, 16a-Methylene-3-oxo-7a-propionylthio-4-androstene-[1 7(/M')- spiro-

5']perhydrofuran-2'-one63,7 , 5a, 16a-Dimethylene-3-oxo-4-androstene-[1 7 (β- 1')- spiro-5']perhydrofuran- 2'-one 7a-Acetyithio-15a, 16a-methylene-3-oxo-4-androstene-

[17 (3-1')-spiro 5']perhydrofuran-2'-one 7a-Acetylthio-15P, 16P-methylene-3-oxo-4- androstene-[17(p-1 ')- spiro- 5']perhydrofuran-2'-one 153,16p-Methylene-3-0K0-7p~ propionylthio-4-androstene -[17( β-1')- spiro- 5'Jperhydrofuran-2'-one 6 β,7β,15 β,16 β-

Dinnethylene-3-oxo-4-anclrostene-[17^-1')- 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, 1 β- hydroxy progesterone, 17a-hydroxyprogesterone, 16a-methyl progesterone, hydroxyprogesterone caproate, medroxyprogesterone acetate, proligestone mifepristone (.11β-[p-(Dimethylamino)phenyl]-1 7 - Γθχν- 7-(1-propynyl)estra-4,9- dien-3- one) and pharmaceutically acceptable salts thereof or their metabolites, analogues and mimic molecules. In another embodiment the antagonist of the nuclear steroid receptor family can be synthesized and administered as a prodrug.

In another embodiment, the antagonist of the mineralocorticoid / aldosterone receptor. Is 7 a-acetylthio-4- pregnene-3,20-dione represented by formula B -

In which Ri is hydrogen, hydroxy, hydroxy), a mineral acid ester such as sulfate, phosphate or nitrate group, or acyloxy-OR 2, 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 R 4 may be substituted or unsubstituted, saturated or unsaturated, straight chain or branched, alicyclic, aryl, heterocyclic or mixed and R3 is methyl.

In one embodiment, is hydroxyl or O R2 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 R is hydroxy, monocarboxylic, straight or branched-chain alkanooyloxy group having up to 12 carbon atoms.

In another embodiment R is hydrogen, hydroxy, acetoxy, propionyloxy, n- butyryloxy, 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- 4- pregnene-3,20-dione; 7a-acetylthio-21-acetoxy-4-pregnene~3,20-dione; 7a- acetylthio-21-propJonyloxy-4-pregnene-3,20-dione; 7a-acetyfthio-21-n-butyryloxy-4- pregnene-3,20-dione; 7a-acetylthio-21 -trimethylacetoxy-4-pregnene-3,20-dione; 7a- acetylthio-21-n-valeroyloxy-4-pregnene-3,20-dione; and 7a-acetylthio-21 - heptanoyloxy-4-pregnene-3,20-dione.

In another embodiment the antagonist of the mineralocorticoid / 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 nuclear steroid receptor family with a suitable carrier is two fold: 1. The antagonist of the nuclear steroid receptor family both inhibits filovirus, Bunyaviridae and Alphavirus and other hemorrhagic viral families replication and eliminates the these viruses ability to cause vascular endothelial instability for filovirus and Bunyaviridae and eliminates muscular pain and rheumatic symptoms for Chikungunya virus infection ( Alphavirus ). 2. The antagonist of the nuclear steroid receptor family 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, Bunyaviridae .Alphavirus or other hemorrhagic viral entities.

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.

The principal discovery of this patentis that the Antagonists of the nuclear steroid family and their metabolites together with certain calcium channel blockers have the ability to directly undermine the pathogenicity of filovirus .Bunyaviridae and Alphavirus and other hemorrhagic viral families and 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 antagonist of the nuclear steroid receptor family compounds outlined herein that is clinically effective at inhibiting filovirus, flavivirus, Bunyaviridae, Alphavirus and other hemorrhagic viruses replication and also as a consequence of lowering circulating viral load treating or preventing vascular endothelial instability, and muscle pain and long term rheumatic pain which the subject suffers or is at risk of due to Ebola viral ,CCHF ,or Chikungunya viral infection.

Pursuant to a preferred embodiment of the invention, an effective amount of antagonist of the nuclear steroid receptor family, or calcium channel blocker such compounds thus administered are such as to produce a circulating concentration of the Nuclear receptor antagonist compounds and their metabolites sufficient to prevent filoviral .Bunyaviridae .Alphavirus viral replication completely and consequently have an effect on the vascular endothelial system and immune effector cells such as macrophages / dendritic cells. This effect of the presented direct antiviral compounds on these viruses may also be utilized in combination with vaccine candidates for these viruses to availe of the enhanced immune stimulation occurring in the patient and to direct a viral specific immune clearance.

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 antagonist compounds to the nuclear steroid receptor family 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, topical, 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 ingredient.

In the preferred embodiment of the invention, the antagonist compound to the nuclear receptor family is micronized. In accordance with the present invention, the expression "micronized" means that the compound has been micronized in accordance with any process for 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 Antagonists of the nuclear steroid receptor family outlined in 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, non-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 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(lactic-acid) polyester, cellulose acetate phthalate, hydroxypropyl-methyl cellulose phthalate poly(butyl methacrylate), (2-dimethyl aminoethyl) methacrylate, and methyl methacrylate. 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 Antagonists to the nuclear receptor family 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 S- adenosylhomocysteine hydrolase (SAH), but are not limited to: Carbocyclic 3-deazaadenosine (C-c Ado), 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 neplanocin A

9-(hydroxyalkenyl)purines (adenines and 3-deazaadenines), which analogues of neplanocin A , 3-deazaneplanocin A, Bromine Epiandrosterone

• 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'-f?-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 · 1'-, 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~ (SIBA) • (S)-9-(2,3-dihydroxypropyl)adenine • ribavirin · vidarabine • pyrazofurin • tubercidin • 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 or any of the pharmaceutical formulations disclosed herein can be administered simultaneously (in a combination formulation), 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. For example, a compound of the present invention, at least one antagonist to the nuclear steroid receptor family of compounds 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 these currently prescription compounds to be incorporated into the pharmaceutical composition of the antiviral 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 g/ml to about 5000 g/ml of plasma, typically about 3 to about 50 g/ml or about 5 to about 25 gg/ml. However, when liposomes targeted to Ebola viral infected cells are used to administer the antagonists to the nuclear receptor family compounds, high doses of 25 mg/ml are used. The effective amount is optionally administered in a dosage ranging between 10 g/kg and about 20,000 pg/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 antagonists compounds to the nuclear receptor often about 5 to 500 mg/day, ideally 400 mg/day with an optimal blood plasma concentration of 60 µΜ in the blood all day. Preferable paediatric doses range from 0.001 to 100 mg/kg/day, with optimal doses in the range of 3 mg/kg/day.

Administration of the antagonists to the nuclear steroid receptor family .these compounds have a direct effect, as reported herein to maintain vascular endothelial stability and prevent hypovolemic shock and improve immune cell viability in a patient infected by filovirus.

The Nuclear steroid receptor family, mediates its biological response by binding with ligands which cross the plasma membranes of cells and interacting with Ligand

Receptor proteins in the cytosol or nucleus, to form complexes. The nuclear steroid Receptor family has been shown to form a heterooligomeric complex that includes a 90 kDa heat shock protein (HSP90).

The Nuclear steroid Receptor family form complexes that 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 a component of the nuclear steroid receptor family have been shown to be functionally present on lymphocytes (Leukemia (2000) 14, 1097-1 104, 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 as component parts of the nuclear receptor

(aldosterone antagonist and/or molecules which mimic aldosterone structure) in particular compete with the Ebola viral glycoprotein GP1 / GP2 coat proteins involved in Nuclear Receptor family attachment and reduce viral cell entry, vascular endothelial instability and apoptosis of macrophages and dendritic cells. In another embodiment the anti-sense mineralocorticoid Steroid Receptor or any other receptor from the nuclear receptor family and the DNA are delivered to viral infected cells by incorporating the anti-sense mineralocorticoid Steroid Receptor RNA and /or the DNA into liposomes or carbohydrate vehicles targeted to infected viral cells by placing Ebola or other coat proteins from Bunyaviridae and Alphavirus or other hemorrhagic viral directed antibodies on the surface of these liposomes or vesicles.

In another embodiment of the invention, liposomes are provided carrying the the anti- sense mineralocorticoid Steroid Receptor RNA and the DNA targeted to Ebola infected cells by putting antibodies to the Ebola coat protein GP1 or GP2 on its surface or antibodies targeted to the other hemorrhagic viral targets. The advantage of this is that the liposome can selectively target viral 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 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, flavivirus, Bunyaviridae and Alphavirus and other hemorrhagic viruses 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 these viruses to the lipophilic binding site on the Nuclear steroid Receptor. The 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 or any other lipophilic ligand binding site on the nuclear receptor.

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, progesterone, estrogen and glucocorticoid.

The term steroid hormone receptors are defined herein as nuclear receptors that are phosphoproteins that include receptors for mineralocorticoid and other steroid or lipophilic ligands of the nuclear receptor 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 in both construction and detail within the scope of the claims.

In-vitro Example 1. ( Figure 1)

The effects of aldosterone, Ebola Glycoprotein GP1 and the combination of aldosterone and eplerenone, and Ebola Glycoprotein GP1 and eplerenone on NO (Nitrouse Oxide) production in human macrophages (Figure )

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 separated from blood samples by histopaque density gradient centrifugation.

Monocytes 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, 100 Mg/ml streptomycin and 10 g/ml fungizone (Gibco, Grand Island, NY). The cells were counted and the number of monocytes was adjusted to

1 x 10 /ml. Equal numbers of cells ( 1 x 106 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% C02 in air. After a 24 h incubation, the supernatant was carefully removed and replaced with medium supplemented with aldosterone (10 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). TT conversion

The viability of monocytes treated with the studied compound and Ebola GP1 was evaluated using the MTT conversion method. MTT (2.5 g/ml) was added to the medium 3 h before the scheduled end of the experiment, and then the cultures were incubated at 37 °C in 5% C02/95% air. Then, the monocytes were lysed in 100 L DMSO, 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% C02/95% air). After 24 h, the cells were resuspended in DMSO containing NBT ( 1 mg/ml), (Sigma- 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, epierenone, and the combination of aldosterone with epierenone plus Ebola GP1 with epierenone 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 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 epierenone, and Ebola GP1 and epierenone on NO production in human macrophages (Figure 1)

Treatment with 10' M aldosterone or the 20 micro grams of Ebola GP1 significantly increased the production of NO. In contrast, treatment with 10 M Aldosterone in combination with 0 epierenone, and the treatment of Ebola GP1 in combination with 10 5 M epierenone, had a significant effect in reducing the NO production by the Aldosterone and most importantly demonstrates the ability of epierenone to inhibit the aldosterone mimic activity of Ebola GP1.

The effects of aldosterone, Ebola GP1 and the combination of aldosterone and epierenone, and Ebola GP1 and epierenone on ROS production in human macrophages (Figure 2)

Treatment with 0 7 M aldosterone or the 20 micro grams of Ebola GP1 significantly increased ROS production.

In contrast, treatment with 10 7 M Aldosterone in combination with 10 5 M epierenone, had a significant effect on reducing ROS production. Treatment of the 20 micro grams of Ebola GP1 in combination with 0 5 M epierenone, this combination had a significant effect on reducing ROS production and most importantly again demonstrates the ability of epierenone to inhibit the aldosterone mimic activity of Ebola GP1.

Experiment .

1. Name and address of the sub-contract facility: 1.1. Department of Virology, Texas Biomedical Research Institute, 7620 NW Loop 410, San Antonio (TX) 78227 1.2. Date started: 30 November 2014 1.3. Date completed: 5 December 2014

2. Objectives To test Spironolactone (CB-008) for inhibitory activity against Ebolavirus at 16 dilutions in HeLa and Vera cell types

3. Safety Procedures: BSL-2 and BSL-4 safety training is required. Proper PPE required as per the Texas Biomed Biohazards and Safety Manual.

4. Procedure: 1.1. Verified the current expiration date status, if applicable, of all reagents to be used in the performance of this assay and documented on the appropriate laboratory worksheet. 1.2. In the BSL-2 tissue culture room, HeLa and Vero cells were plated on 384 well plates with High Glucose DMEM + 10% fetal bovine serum (complete medium). 4000 cells/well for 384 well plates were dispensed. The volume of medium per well was kept as 25µ . Prior to infection, cells were grown to ≥75%, but <100% confluence in a humidified incubator at 37°C ± 2°C with 5% C02. 1.3. At BSL-2, diluted compound at the final concentration of 1:100 (as specified in SOW) in complete medium. 1.4. To a 384 well plate, added 25 L of diluted compound, to the first column of each dilution series (indicated by T in Figure 7). 1.5. Serially diluted test/control samples by removing 25µ from the first column and transferring to column two, mixing several times with a pipette tip. Then removing 25pL from column two and transferred to column three, again mixing by pipette up and down 3 times. Repeated through column 16 of each series. 1.6. After the last dilution was mixed in sixteenth column of every dilution series, removed 25µ from that well and discarded. 1.7. In rows 1 and 2 columns 17 through 24 added 25pL of 40nM Bafilomycin A 1 diluted in complete medium (indicated by B in Fig.7). This concentration became 20nM upon mixing with 25µ medium already

present in the well. After mixing thoroughly 25µί was removed from these wells and discarded to keep the final volume in the well at 25µ . The final concentration of Bafilomycin A 1 became 10nM upon further addition of 25µΙ virus. 1.8. In rows 11-14 columns 17 through 24 added complete medium alone (indicated by UT, Fig 7.) 1.9. Assay plates treated with compounds were incubated at 37°C for 1 hour. 0 . During this time transferred materials to the BSL-4. Plates were taken in to the BSL-4 at the same time the personnel was entering the lab to perform the assay.

1. Retrieved and thawed an appropriate amount of stock virus (Zaire Ebolavirus Mayinga) from the freezer. Since for this assay the right viral load to be used was not predetermined. 4 different concentrations were tested - 1:500, 1:1000, 1:2000 and 1:4000 (depicted by yellow, green, blue and orange respectively in Figure 7).

2 . Dilution of virus in complete medium that gave approximately 5% for untreated cells/well infected was considered for analysis. 3. Added 25 L of the diluted virus to each well at the concentrations indicated in step 4.2 of the 384 well plate and incubate for 24 hours at 37°C ± 2°C. . After the incubation period, removed the plates from incubator and inactivate by immersing in 10% neutral buffered formalin. Packaged the plates in a heat sealed bag, filling the container with enough 10% formalin to cover the plate. It was ensured that all the wells of the plate were completely filled with formalin upon immersion. The sealed containers were stored in the 4°C ± 5°C refrigerator overnight in BSL-4 5. Passed the sealed containers of plates out of the BSL-4 via the chemical dunk tank.

6. Disposed of the formalin in a properly labeled hazardous waste container and washed each plate by dipping 3 times in 1X PBS.

7. Permeabilized the fixed cells by incubating at room temperature with

25pL/well of 0 .1% TritonX-1 00 in 1X PBS for 20 minutes. 8 . After incubation discarded the triton-X and blocked the plates by incubating with 5% goat serum in 1XPBS for 1 hour at room temperature. 9. Treated the cells for 2 hours at room temperature with primary antibody diluted in 5% final concentration of goat serum in 1XPBS-anti Ebolavirus glycoprotein antibody at concentration of 1:2000. 0. After primary antibody treatment, discarded the antibody from the plates and washed each plate by immersing in 1X PBS and leaving the plates with PBS for 5-10 minutes and then repeating the wash once more.

1. Stained the cells for 1 hour with secondary antibody diluted in 5% final concentration of goat serum in 1XPBS. Alexa fluor 488 conjugated antiMouse antibody (1:1000 dilution). 2. After secondary antibody treatment, discarded the antibody from the plates and washed each plate by immersing in 1X PBS and leaving for 0 minutes and then repeating the wash two to three more times. 3. Discarded the PBS and add 25µ Ι_ΛινθΙΙ of Hoechst (1 :50,000 inlXPBS) to the plate. Incubated at room temperature with Hoechst for 30 minutes. 4. Imaged the plates on the microscope using blue and green fluorescence channels. 5. Analyzed images using Cell Profiler using HTS pipeline. Pipeline is stored with data folder. al methods used: . Raw data about nuclei number and infected cell number was plotted in Microsoft Excel. . Rate of infection was determined by dividing number of infected cells with total number of cells . Infection rates were entered in Graphpad Prism software. . A linear and non-linear regression analysis of the data was done. . Infection rates were plotted with standard deviations versus doses used, indicated as dilution factor. . A hyperbolic curve was fitted to the data from Spironolactone (CB- 008) . R-squared regression factors were calculated for all data . EC-50 (Effective concentration that inhibits 50% infection rate) was estimated. d control articles: . Spironolactone (CB-008) . Bafilomycin A1 (stock concentration^ OmM in DMSO) . Untreated (Complete medium with 0.1% v/v DMSO) that may affect assay outcome: . Cell density . Cell passage history . Virus passage history nel: Study director: Dr. Robert A. Davey Technician: Dr. anu Anantpadma

9. Results: 1.1. R-Squared values for Spironolactone (CB-008): 1.1.1. HeLa-0.7644 1.1.2. Vero-0.1947 1.2. EC-50 1.1.1. HeLa- 1:731.9 ± 146.1 1.1.2. Vera - could not be calculated from current data

STANDARD OPERATING PROCEDURE

1. Scope: This Standard Operating Procedure provides instructions on how to perform drug screens in 384 well format.

2. Safety Procedures: BSL-2 and BSL-4 safety training is required. Proper PPE required as per the Texas Biomed Biohazards and Safety Manual. Refer to the BSL-4 Operations & Safety Manual for detailed safety procedures.

3. Specimen Requirements: Samples for use in this assay include: Drugs, compounds, chemicals. These are supplied by the client. On receipt of plates, each is logged using inventory software by bar code reader and stored at 4°C or -80°C as indicated by the client. If barcodes are not present, data will be entered manually.

4 . Procedure: 1.1. Verify the current calibration status, if applicable, of all equipment to be used in the performance of this assay. 1.2. Verify the current expiration date status, if applicable of all reagents to be used in the performance of this assay and document on the appropriate laboratory worksheet. 1.3. In the BSL-2 tissue culture room, HeLA/Vero cells will be plated on 384 well plates with High Glucose DMEM + 10% fetal bovine serum (complete medium). Dispense 3500-4500 cells/well for 384 well plates.

The volume of medium per well will be kept at 25µΙ . Prior to infection, cells will be grown to ≥75%, but <100% confluence in a humidified incubator at

37°C ± 2°C with 5% C0 2. Cells will not be used at >24 hours after plating ( 1 1/30/14). 1.4. At BSL-2, dilute compounds at 4 times the final concentration determined by the client in complete medium (Concentration used 1:100 - starting) 1.5. To a 384 well plate, add 25µΙ_ of diluted compound, in quadruplicates, to the first column of each dilution series (indicated by T in Fig.7). 1.6. Serially dilute test/control samples by removing 25µ _ from the first column and transferring to column two, mixing several times with the pipette tip. Then remove 25µ from column two and transfer to column three, again mixing by pipette up and down 3 times. Repeat through column 16 of each series. 1.7. After the last dilution is mixed in sixteenth column of every dilution series, remove 25µΙ from that well and discard. 1.8. In rows 1 and 2 columns 17 through 24 add 25µ of 20nM Bafilomycin A 1 diluted in complete medium. 1.9. In rows 11-14 column 17 through 24 add complete medium alone 1.10. Assay plates treated with compounds will be incubated at 37°C for a

minimum of 1 hour and up to 2 hours ( 1 hour 10 mins)

1.1 1. During this time transfer materials to the BSL-4. Plates will be taken in to the BSL-4 at the same time the personnel is entering the lab to perform the assay. 1.12. Retrieve and thaw an appropriate amount of stock virus from the freezer. The amount of virus needed will depend upon the number of samples being tested and the predetermined titer of the virus. Since for this assay the right viral load to be used was not predetermined. I used 4 different concentrations - 1:500, 1:100, 1:2000 and 1:4000 (depicted by different colours in plate layout). 1.13. Dilution of thawed stock virus in complete medium that will give 5-20% for untreated cell/well infected will be considered for analysis 1.14. Add 25µ of the diluted virus to each test well (fig 7) of the 384 well plate and incubate for 24 hours at 37°C ± 2°C. 1.15. After the incubation period, remove the plate/plates from incubator

and inactivate by immersing in 10% neutral buffered formalin. Package the

plates in a sealed container (either a heat seal bag or a sealable plastic container) filling the container with enough 10% formulation to cover the

plate. It must be ensured that all the wells of a plate are completely filled

with formalin upon immersion. Allow the sealed containers to sit in the 4°C ± 5°C refrigerator overnight (12 hours at minimum). Prolonged exposure

(periods longer than overnight or 12 hours) to formalin will not adversely affect the results of the assay. 1.16. Pass the sealed containers of plates out of the BSL-4 via the chemical dunk tank.

1.17. Dispose of the formalin in a properly labeled hazardous waste

container and wash each plate by dipping 3-4 times in 1XPBS. 1.18. Permeabilize the fixed cells by incubating at room temperature with

25pl Jwell of 0.1% TritonX-1 00 in 1X PBS for 15-20 minutes. 1.19. After incubation discard the Triton-X and block the plates by

incubating with 5% goat serum in 1XPBS for 1 to 2 hours at room temperature or overnight (12-20 hours at 4°C. Prolonged blocking times of more than 20 hours will not adversely affect the outcome of the experiment. 1.20. Treat the cells for 2 hours at room temperature or overnight at 4°C

with primary antibody diluted in 5% final concentration of goat serum in 1XPBS - anti Ebolavirus glycoprotein antibody at concentrations of 1:1000 to 1:2000 (1:2000). 1.21. After primary antibody treatment, discard the antibody from the plates

and wash each plate by immersing in IX PBS and leaving the plates with PBS for 5-10 minutes and then repeating the wash once more.

1.22. Stain the cells for 1-2 hours with secondary antibody diluted in 5%

final concentration of goat serum in 1XPBS. 1.23. After secondary antibody treatment, discard the antibody from the

plates and wash each plate by immersing in 1X PBS and leaving for 10-15 minutes and then repeating the wash two to three more times.

1.24. Discard the PBS and add 25pUwell of Hoechst (1:50,000 in 1XPBS) to the plate. Incubate at room temperature with Hoechst for at least 30 minutes). 1.25. Image the plates on the microscope using blue and green fluorescence channels. 1.26. Analyze images using Cell Profiler using HTS pipeline. Pipeline is stored with data folder.

5. Documentation: • Written documentation of the assay as it is performed will be done by crossing the boxes at marked steps in the protocol. • Test compound source plate code will by carried through on all file names • Raw data (images) will be saved onto a file Server that is backed up daily and one other storage media and will be labeled appropriately. • Final data will be provided as total cell nuclei, infected cell numbers and % infected relative to test compound concentration in Microsoft Excel Spreadsheets. • The laboratory worksheet and associated raw and final data will be reviewed by a second personnel/PI within one week of completion and before the data summary is provided and documented on the print out and/or worksheet. Additionally, the laboratory worksheet and raw and final data will be stored on the server as well as the printed copy will be filed in the appropriate file and archived. • Recording, reviewing and correction of data will be in accordance with SOP205.

Experiment 2 .

• Name and address of the sub-contract facility: Department of Virology, Texas Biomedical Research Institute, Loop 410, San Antonio (TX) 78227 Date started: 22 nd January 2015 Date completed: 28 th January 201 5 • Objectives T o test Spironolactone combined with Mifepristone (CB-008+) for inhibitory activity against Ebolavirus at 16 dilutions in HeLa and Vera cell types

• Safety Procedures:

BSL-2 and BSL-4 safety training is required. Proper PPE required as per the Texas Biomed Biohazards and Safety Manual.

• Procedure:

1.1. Verified the current expiration date status, if applicable, of all reagents

to be used in the performance of this assay and documented on the appropriate laboratory worksheet.

1.2. In the BSL-2 tissue culture room, HeLa and Vero cells were plated on 384 well plates with High Glucose DMEM + 10% fetal bovine serum (complete medium). 4000 cells/well for 384 well plates were dispensed. The volume of medium per well was kept as 25 L. Prior to infection, cells

were grown to ≥75%, but <100% confluence in a humidified incubator at 37°C ± 2°C with 5% C02. 1.3. At BSL-2, diluted compound at a concentration of 1:25, this concentration became 1:50 upon mixing with 25µ medium already

present in the well. The final concentration become 1:100 upon further

addition of 25 L virus (as specified in SOW) in complete medium. 1.4. To a 384 well plate, added 25pL of diluted compound, to the first

column of each dilution series (indicated by T in Figure 8). 1.5. Serially diluted test/control samples by removing 25µ from the first column and transferring to column two, mixing several times with a pipette

tip. Then removed 25µ from column two and transferred to column three, again mixing by pipette up and down 3 times. Repeated through column 16 of each series.

1.6. After the last dilution was mixed in sixteenth column of every dilution series, removed 25 L from that well and discarded.

1.7. In row 16 and 17 through 24 added 25µ of 40nM Bafilomycin A 1

diluted in complete medium (indicated by B in Fig.8). This concentration

became 20nM upon mixing with 25µ medium already present in the well. After mixing thoroughly 25pL was removed from these wells and discarded to keep the final volume in the well at 25µΙ_. The final concentration of Bafilomycin A 1 became 10nM upon further addition of 25µ Ι virus. . In rows 1-15 columns 7 through 24 added complete medium alone (indicated by UT, Fig 8.) . Assay plates treated with compounds were incubated at 37°C for 1 hour. 0. During this time transferred materials to the BSL-4. Plates were taken in to the BSL-4 at the same time the personnel was entering the lab to perform the assay.

1. Retrieved and thawed an appropriate amount of stock virus (Zaire Ebolavirus Mayinga) from the freezer. HeLa cells were treated with :500 dilution of virus, while Vero cells were treated with 1:2000 dilution. (Figure 8 is the plate layout for HeLa cells. Figure 9 is the plate layout for Vero cells) 2 . Added 25 L of the diluted virus to each well at the concentrations indicated in step 4.1 1 of the 384 well plate and incubate for 24 hours at 37°C ± 2°C. 3. After the incubation period, removed the plates from incubator and inactivate by immersing in 10% neutral buffered formalin. Packaged the plates in a heat sealed bag, filling the container with enough 10% formalin to cover the plate. It was ensured that all the wells of the plate were completely filled with formalin upon immersion. The sealed containers were stored in the 4°C ± 5°C refrigerator overnight in BSL-4 4. Passed the sealed containers of plates out of the BSL-4 via the chemical dunk tank. 5. Disposed of the formalin in a properly labeled hazardous waste container and washed each plate by dipping 3 times in 1X PBS. 6 . Permeabilized the fixed cells by incubating at room temperature with 25MlJwell of 0.1% TritonX-100 in 1X PBS for 20 minutes. 7 . After incubation discarded the triton-X and blocked the plates by incubating with 5% goat serum in 1XPBS for 1 hour at room temperature. 8. Treated the cells for 2 hours at room temperature with primary antibody diluted in 5% final concentration of goat serum in 1XPBS-anti Ebolavirus glycoprotein antibody at concentration of :2000. 1.19. After primary antibody treatment, discarded the antibody from the

plates and washed each plate by immersing in 1X PBS and leaving the plates with PBS for 5-10 minutes and then repeating the wash once more.

1.20. Stained the cells for 1 hour with secondary antibody diluted in 5% final

concentration of goat serum in 1XPBS. Alexa fluor 488 conjugated antiMouse antibody (1:1000 dilution). 1.21. After secondary antibody treatment, discarded the antibody from the

plates and washed each plate by immersing in 1X PBS and leaving for 10 minutes and then repeating the wash two to three more times.

1.22. Discarded the PBS and add 25pl Jwell of Hoechst ( 1:50,000 inlXPBS) to the plate. Incubated at room temperature with Hoechst for 30 minutes. 1.23. Imaged the plates on the microscope using blue and green fluorescence channels.

1.24. Analyzed images using Cell Profiler using HTS pipeline. Pipeline is stored with data folder.

• Statistical methods used:

1.1. Raw data about nuclei number and infected cell number was plotted in Microsoft Excel. .2. Rate of infection was determined by dividing number of infected cells with total number of cells

1.3. Infection rates were entered in Graphpad Prism software. 1.4. A linear and non-linear regression analysis of the data was done. 1.5. Infection rates were plotted with standard deviations versus doses used, indicated as dilution factor. 1.6. A hyperbolic curve was fitted to the data from Spironolactone (CB- 008+) 1.7. R-squared regression factors were calculated for all data 1.8. EC-50 (Effective concentration that inhibits 50% infection rate) was estimated.

• Test and control articles: 1.1. Spironolactone (CB-008+)

1.2. Bafilomycin A 1 (stock concentration^ OmM in DMSO) 1.3. Untreated (Complete medium with 0 .1% v/v DMSO) • Factors that may affect assay outcome: 1.1 . Cell density 1.2. Cell passage history .3. Virus passage history

Personnel: 1.1. Study director: Dr. Robert A. Davey 1.2. Technician: Dr. Manu Anantpadma

· Results: o At dilutions of 1:100, 1:200 and 1:400 cell loss was observed. This generally is a result of cytotoxicity of the test compound o CB008+ Activity against WT Ebola in HeLa cells (Figure 10) o CB008+ Activity against WT Ebola in Vera cells (Figure 11) o R-Squared values for Spironolactone (CB-008+): 1.1.1. HeLa-0.7490 1.1.2. Vero-0. 1476 o EC-50

■ HeLa- 1:2787 ± 536

■ Vero - could not be calculated from current data

EXPERIMENT 3

Test Antiviral Results from USAMRIID / Canopus 5/5/15 Compounds tested : for antiviral activity against Ebola virus and Chikungunya virus. Amlodipine Bicalutamide Canrenoate Cyproterone Mifepristone Nimodipine Spironolactone EXPERIMENT 4

Results for compounds tested in EBOV Synergy Assay Overviews of the Test

• Mifepristone was tested in combination with Amlodipine, Drospiredone, Spironolactone and Cyproterone acetate in EBOV infection assay with HeLa cell line. • Stock solutions were prepared at 10mM in 100% DMSO. • Compounds were dispensed 2h before infection by HP D300 directly from the 100% DMSO stock into assay wells with cells as depicted on the (plate map next slide). The highest concentrations were limited by the total amount of DMSO in assay wells that should not exceed 1%. Concentration of DMSO in all wells was normalised to 1%. • Mifepristone was tested at 7 point dose response with 3 fold step dilution . 4 other compounds in - 9 point dose response with 2-fold step dilution. Test repeated on two plates (n=2). • 16 wells were treated with 1% DMSO to be used as a neutral control. Additionally, 16 wells were not infected and were used as a low signal controls. Infection was stopped after 48h by fixing cells with a formalin solution.

• Synergy effect was assessed by comparison to the additive effect that was calculated as sum of the observed value for selected dose of compound added to value for each doses of mifepristone. o If observed value (%lnhibition) is close to calculated additive value- there was NO synergy o If observed values (%lnhibition) exceeding the calculate additive value - there is potential synergy observed.

Summary of results • 4 compounds were tested in EBOV/Hela assay synergy teste with mefiprestone. • EC50 values for all 5 compounds were similar to preciously reported. • There was synergy effect observed for Mifreprestone in combination with 6.25uM and 3.1 uM Amplodipine. Higher doses resulted in >60% results of cell number. • Mifepristone in combination with Cyproterone, Drosperinone and Spironolactone did not show any synergistic effect at doses that did not cause more then 60% reduction of cell number

EXPERIMENT 5

Results for cmds tested in EBOV/Μφ Assays Overviews of experiment

• Amiodipine, Drosperinon, Mifepriston and Spiranolactone were tested in EBOV assay with PBMC-derived macrophage cells • Cells were purified using ficol columns from 50 ml of blood and criopreserved at 2xE6 cells/vial • 7 days before assay cells were put in culture dishes for differentiation in the presence of 5ng/ml GM-CSF and 50uM BME. Media was changed about every 2 days and in 5 to 7 days attached cells were harvested with EDTA7PBS and scrapping and plated 40,000 cells/ well in 96 well for assay. • Cells were Incubated on the plate for 24h before infection • 2 h prior infection compounds were dispensed from 100% DMSO stock of 10mM using HP-6000 digital dispenser. DMSO were normalised to 1% in all wells using HP dispenser. • Compounds were tittered in 6 points from 50 or 100 uM in 3 fold step dilution. Each dose were tested 3 times on the plate n=3 Cells were infected with EBOV(Zaire) at MOI=7 and kept on the cells for 1h. Then media was replaced with fresh containing same concentration of compounds as before and incubated for 24h. Infection was stopped by fixing cells in formalin solution. Immunostaining was done to detect GP-protein in cells Images were taken by PE Opera confocal platform with 10x objective, analysed using Acapella software. Data was analysed using GeneData software

Summary of results • Amiodipine, Drosperinon, Mifepriston and Spiranolactone were tested in EBOV assay with PBMC -derived macrophage cells. o Only Amiodipine gave full dose response with EC50 =9uM but Sl=6 due to the toxicity of 50uM dose o Mifepriston and Spiranolactone reach efficacy only of 50% inhibition o The value shown as Ec50 for Mifepriston is 50% of total response and =20% inhibition

EXPERIMENT 6 - Dengue 1, 2, 3 and 4 strains Inhibition using compound Amiodipine

STANDARD OPERATING PROCEDURE

TITLE: Viral Reduction Antiviral Assay

Viral Reduction Antiviral Assay

1.0. PURPOSE: To test antiviral agent against four Dengue viruses.

2.0. DEFINITIONS: CPE: Cytopathic Effect. Changes to virus-infected cells, which include altered shape, detachment from the substrate, lysis, membrane fusion, altered membrane permeability, inclusion bodies. EC50: Concentration of antiviral compound that reduces the viral titer by 50% as compared to the untreated control (virus + DMSO).

3.0. EQUIPMENT AND MATERIALS:

3.1 Equipment: a . Biological safety cabinet Type If b. Incubator (37°C, 5% C02) c. Inverted light microscope with phase contrast optics d . Standard Laboratory Equipment

3.2 Materials: a. Cells at a concentration of 2x1 05 c/ml, A549 (CCL-185 from ATCC Human Lung Carcinoma) cells b. Antiviral compound. c. EMEM-2% FBS: Eagles minimum essential medium containing 100 units per ml penicillin, 100 ug/ml streptomycin sulfate. Store at 2-8° C, expiration 3 weeks d. Ethanol (100%) 1% Ethanol - EMEM-2% FBS 2% Ethanol - EMEM-2% FBS e. Standard laboratory materials

4.0. SETUP:

4.1 Twenty four hours prior to infection seed plates as indicated below, and incubate at 37°C w/5% C02. 1ml/well for a concentration of 2x1 0s c/well Prepare antiviral and viral dilutions on day of infection 4.2 Dilute antiviral compounds to the desired concentrations. Dilute in 2% Ethanol+ 2%EMEM for 2X concentration used for inoculation and 1% Ethanol+ 2%EMEM for 1X concentration used in overlay. Formula ul of antiviral = ( desired cone. X volume needed) / cone of stock 4.3 Dilute each virus to yield a MOI of 0.1 PFU/cell in the inoculation volume. 24 well plates: 8x105 PFU/ml, inoculate with 25ul

5.0. PROCEDURE:

5.1 Remove media from wells

5.2. Each antiviral concentration is done in triplicate. Add inoculum, 50 ul of antiviral, to each well. Incubate 1 hour at 37°C. 5.3 Remove antiviral. 5.3. Add 25ul of virus to each well and 25ul of antiviral at 2x the concentration desired. Controls: For each virus include 3 wells that have virus plus 1% Ethanol, 3 wells with virus plus media, and 3 wells that are mock infected with media only. 5.4 Incubate at 37°C, 5% C02 for 60 to 80 minutes, rocking plates every 15 to 20 min. 5.5 At the end of the infection period, remove the inoculum, and add 0.5 ml of cell maintenance medium with appropriate concentration of antiviral agent (eg., EMEM-2% FBS) to each well. 5.6 Incubate at 37°C, 5% C02, harvest at 42-46 hours. 5.7 Examine plates each day under the phase microscope. The presence of virus often gives rise to CPE. Using following scale, rate the CPE: 1+ (0- 25% CPE), 2+ (26-50% CPE), 3+ (51-75% CPE) and 4+ (76-100% CPE). Examinations may reveal damage to the cell sheet that was caused by toxicity from the antiviral compound of the inoculum.

5.8 Harvest samples in duplicate and Store at -80°C. Add 100ul of FBS (heat inactivated) to each well to yield a concentration of 20% FBS. Divide into two labeled cryo-tubes.

5.9 Samples will then be titered on Vero cells according to SOP VI-001.1

Plaque Titration in 6 well plates. The concentration of antiviral that reduces

the viral titer by 50% as compared to the titer of the virus + Ethanol wells is considered the EC50.

.0. REFERENCES:

6.1. Biosafety in Microbiological and Biomedical Laboratories (BMBL) 4 edition CDC/NIH 1999. 6.2. Virology Methods Manual. 1996, Brian WJ Mahy and Hillar O Kangro, ed. Academic Press, London.

SAFETY PRECAUTIONS: 7.1. Follow the safety guidelines for working with infectious agents in the BLS-3 laboratory, found in the most recent version of the Arbovirus Laboratory Manual, and in Biosafety in Microbiological and Biomedical Laboratories (BMBL) 4th edition CDC/NIH 1999. 7.2. Handle all specimens as if infectious. Take great care in handling 6- well plates when transferring from incubator to hood to microscope. Claims

A pharmaceutical formulation for inhibiting viral replication of all families of Arenaviridae, Filoviridae, Alphavirus ( Chikungunya ) Bunyaviridae, Flaviviridae, and Rhabdoviridae in a human comprising administering to a patient in need thereof a therapeutically effective amount of the drugs tested and listed herein in compositions either alone or in combination comprising at least one known antagonist of the human nuclear steroid receptor family as a direct antiviral.

2. A pharmaceutical formulation for inhibiting the replication of Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, Alphavirus ( Chikungunya )and Rhabdoviridae viruses and prevents apoptosis of immune cells particularly macrophage and dendritic cells in an infected patient.comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising at least one of the direct antiviral compounds outlined in this patent

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- methylene- spirorenone, eplerenone, Drospirenone, potassium canrenoate, canrenoate, canrenone and pharmaceutically acceptable salts thereof or their metabolites.

The pharmaceutical formulation as claimed in claim 3, wherein the Antagonist of the mineralocorticoid / aldosterone receptor is specifically administrated in combination with the progesterone receptor antagonist such as Mifepristone as direct antiviral combination to inhibit Arenaviridae, Filoviridae, Alphavirus ( Chikungunya) Bunyaviridae, Flaviviridae. 5. The pharmaceutical formulation as claimed in any preceding claim wherein the antagonist to the mineralocorticoid receptor and the progesterone receptor are anti-idiotypic humanized monoclonal antibodies to the aldosterone binding site and the progesterone binding site.

6 . A pharmaceutical formulation as claimed in any preceding claim wherein, the antagonist of the mineralocorticoid / aldosterone receptor is a 7 a-acetylthio-

4-pregnene-3,20-dione represented by formula B in which R is hydrogen, hydroxy, hydroxyl, 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 R may be substituted or unsubstituted, saturated or unsaturated, straight

chain or branched, alicyclic, aryl, heterocyclic or mixed and R3 is methyl.

7. A pharmaceutical formulation as claimed in claim 5 wherein, R is hydroxyl or

OR2 where R2 is derived from a carboxylic acid of the formula R OOH which

may have up to 12 carbon atoms, and in which R 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 R is hydroxy, monocarboxylic, straight or branched-chain alkanooyloxy group having up to 12 carbon atoms.

9 . A pharmaceutical formulation as claimed in claim 6 or 7 wherein R is hydrogen, hydroxy, acetoxy, propionyloxy, n-butyryloxy, trimethylacetoxy, n- valeroyloxy or n-heptanoyloxy.

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: 7a- acetylthio-4-pregnene-3,20-dione; 7a-acetylthio-21- hydroxy-4-pregnene-3,20- dione; 7a-acetylthio-21 -acetoxy-4-pregnene-3,20- dione; 7a-acetylthio-21- propionyloxy-4-pregnene-3,20-dione; 7a-acetylthio- 21-n-butyryloxy-4-pregnene- 3,20-dione; 7a-acetylthio-21-trimethylacetoxy-4- pregnene-3,20-dione; 7a-acetylthio-21-n-valeroyloxy-4-pregnene-3,20-dione; 7a-acetylthio-21 - heptanoyloxy-4-pregnene-3,20-dione; 7a-Acetylthio-3-oxo-4, 15-androstadiene- [17$-1')-spiro-5']perhydrofuran-2'-one, 3-Oxo-7a- propionylthio-4, 15- androstadiene-[17 (3-1')- spiro-5']perhydrofuran-2'-one,

6P,7p-Methylene-3-oxo- 4 ,15-androstadiene-[1 7(β- 1')- spiro-5']perhydrofuran- Z-one, 15a, 16a- Methylene-3-oxo-7a-propionylthio-4-androstene-[17$-1')- spiro- 5']perhydrofuran-2'-one, 6β,7β , 15a, 16a-Dimethylene-3-oxo-4- androstene- [17( β-1')- spiro-5']perhydrofuran-2'-one, 7a-Acetylthio-15a, 16a- methylene-3- oxo-4-androstene-[17^-1')- spiro-5']perhydrofuran-2'-one, 7a-

Acetylthio- 15β,16β-methylene-3-oxo-4-androstene-[1 7(β- 1')- spiro- 5']perhydrofuran-2'- one, propionylthio-4-

androstene-[17( β- 1,)- spiro- 5']perhydrofuran-2'-one, 6β,7β,15 β,16 β-

Dimethylene-3-oxo-4-androstene- [17(β- 1')- spiro-5']perhydrofuran-2'-one.

11. A pharmaceutical formulation as claimed in any preceding claim wherein the

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

12. The pharmaceutical formulation as claimed in any proceeding claim wherein the composition further includes at least one other nuclear steroid receptor antagonist to create an antiviral combination to inhibit replication of Arenaviridae, Filoviridae, Bunyaviridae, Alphavirus ( Chikungunya ) Flaviviridae, and Rhabdoviridae which viruses cause VHF ( viral haemorrhagic fever.

13. The pharmaceutical formulation as claimed in claim 11 wherein the anti-viral agents are selected from the group comprising:Carbocyclic 3- deazaadenosine (C-c Ado), 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 &-R- alkyl (i.e., 6'-f?-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, 1'-, 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- (SIBA), (S)-9-(2,3-dihydroxypropyl)adenine, ribavirin, vidarabine, pyrazofurin, tubercidin, 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.

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.

16. The pharmaceutical formulation as claimed in claim 14, wherein the liposomes or carbohydrate vehicles are targeted to the Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and Rhabdoviridae and Alphavirus ( Chikungunya ) by attaching viral directed antibodies to the delivery vehicle .

17. The pharmaceutical formulations as claimed in any preceding claim, wherein

the composition is administered prophylactically to prevent Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and Rhabdoviridae and Alphavirus (

Chikungunya) viral infection taking hold in the patient. 18. The pharmaceutical formulation as claimed in any preceding claim, wherein the formulation is a unit dose that comprises 5-500 mg of any antagonist of the nuclear 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 nuclear receptor family is manufactured and administrated as a Pro-Drug entity.

21. A pharmaceutical formulation as outlined in previous claims a combination antiviral formulation containing any combination of the documented antiviral compounds of this patent for use in treating or preventing a future infection and/or minimising the effects of a future infection by a Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and Rhabdoviridae and Alphavirus ( Chikungunya )

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 ,2a-methylene- 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.

24. The pharmaceutical formulation as claimed in claim 20, 2 1 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 β- hydroxyprogesterone, 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-4-pregnene-3,20-dione represented by formula B as defined in

claim 5 in which is hydrogen, hydroxy, hydroxyl, a mineral acid ester such

as sulfate, phosphate or nitrate group, or acyloxy-OR 2, the acyl group R2 being derived from a carboxylic acid of the formula R OOH which may have

up to 12 carbon atoms, and in which R may be substituted or unsubstituted, saturated or unsaturated, straight chain or branched, alicyclic, aryl,

heterocyclic or mixed and R3 is methyl.

27. A pharmaceutical formulation as claimed in claim 25 wherein, 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.

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 1 is hydrogen, hydroxy, acetoxy, propionyloxy, n-butyryloxy, trimethylacetoxy, n- valeroyloxy 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; 7a- acetylthio-21-hydroxy-4-pregnene- 3,20-dione; 7a-acetylthio-21 -acetoxy-4- pregnene-3,20-dione; 7a-acetylthio-21 - propionyloxy-4-pregnene-3,20-dione; 7a-acetylthio-21-n-butyryloxy-4-pregnene- 3,20-dione; 7a-acetylthio-21 - trimethyiacetoxy-4-pregnene-3,20-dione; 7a- acetylthio-21 -n-valeroyloxy-4- pregnene-3,20-dione; 7a-acetylthio-21 - heptanoy!oxy-4-pregnene-3,20- dione; 7a-Acetylthio-3-oxo-4, 15- androstadiene-[f7(P-1 ')-spiro- 5']perhydrofuran-2'-one, 3-Oxo-7a- propionylthio-4, 15-androstadiene-[170-i ')- spiro-5']perhydrofuran-2'- one, 6 ,7p-Methylene-3-oxo-4,15-androstadiene- [17(β-1')- spiro- 5']perhydrofuran-2'-one, 15a,16a-Methylene-3-oxo-7a- propionylthio-4- androstene-[17(P-1')- spiro-5']perhydrofuran-2'-one, 6p,73,15a,16a- Dimethylene-3-oxo-4-androstene-[17(3-1')- spiro- 5']perhydrofuran-2'- one, 7a-Acetylthio-15a, 16a-methylene-3-oxo-4- androstene-[1 7(β-1')- spiro-5']perhydrofuran-2'-one, 7a-Acetylthio-153, 16β- methylene-3-oxo-4- androstene-[17(P-1> spiro-5']perhydrofuran-2'-one, 15β, Ι β-Methylene- 3-οχο-7β- propionylthio-4- androstene-[17(3-1')- spiro- 5']perhydrofuran- 2J-one, 6β,7β, 15β, ^-Dimethylene-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 9,1 1-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 3 1, wherein the halogen is selected from the group consisting of chlorine, bromine, fluorine and iodine.

34. The pharmaceutical formulation as claimed in claim 33, wherein the co¬ administered anti-viral agents are selected from the group comprising: 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 &-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, 1'-, 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~ (SIBA), (S)-9-(2,3-dihydroxypropyl)adenine, ribavirin, vidarabine, pyrazofurin, tubercidin, 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.

35. The pharmaceutical formulation as claimed in claim 33 or 34 wherein the anti¬ viral 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; Famotrrre 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.Bromine Epiandrosterone . 36. 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.

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

38. The pharmceutical formulation as claimed in claim 37, wherein the liposomes or carbohydrate vehicles are targeted toArenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and Rhabdoviridae and Alphavirus ( Chikungunya virus)which viruses cause VHF ( viral haemorrhagic feverby putting viral directed antibodies on its surface.

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

40. 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 human nuclear steroid receptor or calcium channel blockers as specified in this patent.

41. 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.

42. 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.

43. A method of rendering all families of Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and Rhabdoviridae and Alphavirus ( Chikungunya )which viruses cause VHF ( viral haemorrhagic fever)and are characterized by fever and bleeding disorders , not capable of endothelial cell damage causing haemorrhaging but viable for immune clearance and immune memory cell development by vaccination comprising deleting from the above viral genomes the code directed to the hydrophobic amino acid sequences listed that bind the virus to the binding site on the Nuclear steroid Receptor family including but not limited to the following sequences:

44. A method of claim 43 wherein the hydrophobic amino acid sequences listed that bind the virus are any one or combination of those outlined herein

Hydrophobic Amino Acid Sequences • DWYEDIIQAYREY • WIDSPFIWDNVMF • LTHWYAVDFNMWT • PWWYWEMRAFDAE • DWNIWDGWYREIY • RTHFEREFDDWFL

45. A method for the treatment and/or prophylaxis to inhibit replication ofArenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, Alphavirus and Rhabdoviridae which viruses cause VHF ( viral haemorrhagic fever.) 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 nuclear receptor antagonist composition to a subject in need of treatment.

46. A method as claimed in claim 44 wherein the filoviridae virus is selected from the group comprising: Ebola and Marburg.-and the Alphavirus is present as the Chikungunya virus.

47. 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 / 2 binding 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 48. Use of an antagonist of the mineralocorticoid / aldosterone receptor in the preparation of a medicament for the treatment of vascular endothelial cell instability resulting from a filoviridae infection.

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

50. The preferred embodiment is where the antagonist of the mineralocorticoid / aldosterone receptor is micronized and delivered to the patient by IV administration either for prophylactic or therapeutic protection from vascular instability resultant from the actions of filoviral coat glycoproteins GP1 /. 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.

51. A method of rendering all families of Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and Rhabdoviridae and Alphavirus ( Chikungunya) which viruses cause VHF (viral haemorrhagic fever) and are characterized by fever and bleeding disorders not capable of endothelial cell damage causing haemorrhaging but viable for immune clearance and immune memory cell development as vaccination antigen comprising deleting from the viral genome the codes directed to the provision of binding ability for these viruses to the Lipophilic binding site on the Human nuclear Receptor.

52. A pharmaceutical formulation for treating or preventing vascular endothelial cell instability resultant from the actions of filoviral virus infection 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 / aldosterone receptor combined with a selective estrogen receptor modulator or antagonist.

53. 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 / aldosterone receptor in combined with a selective estrogen receptor modulators or antagonists

54. The pharmaceutical formulation as claimed in this patent, wherein the antagonist of the mineralocorticoid receptor compound is selected from the group comprising spironolactone, spirorenone, 1 ,2-dihydro-spirorenone, 1 ,2a-methylene- spirorenone, eplerenone, Drospirenone, potassium canrenoate, canrenoate, canrenone and pharmaceutically acceptable salts thereof or their metabolites combined with selective estrogen receptor modulators or antagonists seated from clomifene .femarelle .ormeloxifene .raloxifene .tamoxifen .toremifene Jasofoxifene, ospemifene.afimoxifene, arzoxifene.and bazedoxifene and pharmaceutically aceptable salts thereof or their metabolites.

55. The pharmaceutical formulation as claimed in claim 3, wherein the Antagonist of the mineralocorticoid / aldosterone receptor is spironolactone combined with the selective estrogen receptor antagonist toremifene.

56. 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 combined with a anti-idiotypic humanised monoclonal to the estrogen receptor site.

57. The pharmaceutical formulation combining Spironolactone in combination with the Progesterone receptor antagonist compound is selected from the group comprising Mifepristone (abortifacient).

58. The pharmaceutical formulation as claimed in this patent, wherein the selective progesterone receptor modulator is selected from the group comprising Aglepristone.

59. The pharmaceutical formulation as claimed in this patent wherein the estrogen receptor antagonist compound is selected from the group comprising of Clomiphene and Toremifene. 60. The pharmaceutical formulation as claimed in this patent, wherein the selective estrogen receptor modulator compound is selected from the group comprising Tamoxifen, Clomifene, Femarelle, Ormeloxifene, Raloxifene, Toremifene, Lasofaxifene, Fulvestrant and Ospemifene.

6 . The pharmaceutical formulation as claimed in claimed in this patent wherein the selective Androgen receptor compound is selected from the group comprising Flutamide, Nilutamide and Bicalutamide

62. The pharmaceutical formulation as claimed in this patent, wherein the following selected compounds shown in experiment 3 demonstrated effective direct antiviral activity against Ebola and Chikungunya virus. Amlodipine ,Besylate .Bicalutamide .Casodex .potassium Canrenoate .Cyproterone acetate , Mifepristone .Nimodipine, comprising Tamoxifen, Clomifene, Femarelle, Ormeloxifene, Raloxifene, Toremifene, Lasofaxifene, Fulvestrant and Ospemifene.

63. A pharmaceutical formulation for inhibiting the replication of Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, Alphavirus (Chikungunya) and Rhabdoviridae viruses and preventing apoptosis of immune cells particularly macrophage and dendritic cells in an infected patient, comprising administering to a patient in need thereof a synergistically effective amount of a composition comprising Mifepristone and Amlodipine as direct antiviral compounds against the above viruses.

A . CLASSIFICATION O F SUBJECT MATTER INV. A61K31/4422 A61K31/56 A61P31/12 ADD.

According to International Patent Classification (IPC) o r to both national classification and IPC

B . FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) A61K

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)

EPO-Internal , WPI Data, MEDLINE, EMBASE, BIOSIS

C . DOCUMENTS CONSIDERED TO B E RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

US 2007/259844 Al (KIM JONG J [US] ) 1, 2 , 14, 8 November 2007 (2007-11-08) 15 , 17 , 2 1 , 36-38,47 paragraphs [0014] , [0016] [0024] , [0034] , [0040]

US 2007/259014 Al (KIM JONG J [US] ) 1-52 8 November 2007 (2007-11-08) paragraphs [0019] , [0020] , [0038] , [0044] , [0099] -/-

X| Further documents are listed in the continuation of Box C . See patent family annex.

* Special categories of cited documents : "T" later document published after the international filing date o r priority date and not in conflict with the application but cited to understand "A" document defining the general state of the art which is not considered the principle o r theory underlying the invention to be of particular relevance "E" earlier application o r patent but published o n o r after the international "X" document of particular relevance; the claimed invention cannot be filing date considered novel o r cannot b e considered to involve a n inventive "L" documentwhich may throw doubts o n priority claim(s) orwhich is step when the document is taken alone cited to establish the publication date of another citation o r other "Y" document of particular relevance; the claimed invention cannot be special reason (as specified) considered to involve a n inventive step when the document is "O" document referring to a n oral disclosure, use, exhibition o r other combined with one o r more other such documents, such combination means being obvious to a person skilled in the art "P" document published prior to the international filing date but later than the priority date claimed "&" document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report

18 March 2016 08/04/2016

Name and mailing address of the ISA/ Authorized officer European Patent Office, P.B. 5818 Patentlaan 2 N L - 2280 HV Rijswijk Tel. (+31-70) 340-2040, Fax: (+31-70) 340-3016 Engl , Bri gi tte C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

US 2009/053294 Al (PRENDERGAST PATRICK T 1-3 , [AU] ) 26 February 2009 (2009-02-26) 6-15 , 17-33 , 35-42 , 45-49 ,52 paragraphs [0022] - [0049] , [0056] , [0057] , [0059] , [0062] , [0077] , [0082]

SALIM Y S ET AL: "Anti -corti costeroi 1-52 anti bodi es i n AIDS pati ents" , APMIS : ACTA PATHOLOGICA, MICROBIOLOGICA, ET IMMUNOLOGICA SCANDINAVICA OCT 1988, vol . 96, no. 10, October 1988 (1988-10) , pages 889-894, XP002755387 , ISSN : 0903-4641 the whol e document

KASUMI ISHIDA ET AL: " Effect of the 1-63 steroi d receptor antagoni st RU486 (mi fepri stone) on an I FN-gamma-i nduced persi stent Chi amydophi l a pneumoni ae i nfecti on model i n epi thel i al HEp-2 eel I s " , JOURNAL OF INFECTION AND CHEMOTHERAPY ; OFFICIAL JOURNAL OF THE JAPANESE SOCI ETY OF CHEMOTHERAPY AND THE JAPANESE ASSOCIATION FORI NFECTI 0US DISEASES, SPRINGER-VERLAG, TO, vol . 18, no. 1, 9 July 2011 (2011-07-09) , pages 22-29 , XP035014262 , ISSN : 1437-7780, D0I : 10. 1007/S10156-011-0274-6 the whol e document Patent document Publication Patent family Publication cited in search report date member(s) date

US 2007259844 A l 08-11-2007 AU 2004249280 A l 29-12-2004 CA 2529847 A l 29-12-2004 CN 101014349 A 08-08-2007 EP 1643981 A2 12-04-2006 P 2007537131 A 20-12-2007 KR 20060052709 A 19-05-2006 US 2007259844 A l 08-11-2007 O 2004112720 A2 29-12-2004

US 2007259014 A l 08- 1 1 -2007 AU 2004249295 A l 29-12-2004 CA 2529852 A l 29-12-2004 EP 1643946 A2 12-04-2006 P 2007524616 A 30-08-2007 KR 20060039867 A 09-05-2006 US 2007259014 A l 08-11-2007 O 2004112724 A2 29-12-2004

US 2009053294 A l 26- 02 -2009 CA 2627463 A l 03-05-2007 CN 101277702 A 01-10-2008 EP 1940414 A2 09-07-2008 IE 20050723 A l 30-05-2007 US 2009053294 A l 26-02-2009 WO 2007049265 A2 03-05-2007