TREATMENT OF DNA VIRAL INFECTIONS OF THE EYE

This invention relates to the prevention and treatment of DNA viral infections and their sequellae.

DNA viruses have a central core of DNA within a proteinaceous structure. The DNA carries the genetic code to reproduce the virus which must infect a living cell to reproduce. There are numerous viral proteins that are well characterised, including important enzymes, which act as ideal targets for anti-viral activity. These include DNA polymerase and thymidine kinase which are required for DNA replication. The replication of viral DNA is essential for virus infectivity.

It is known that infecting viruses can alter the natural ionic balances of a living cell in the course of their replication.

In a previously filed PCT application, published as WO 01/49242, we disclose and claim the use of a synergistic combination of a loop diuretic and a cardiac glycoside for the treatment of DNA viral infections.

In a further previously filed PCT application, published as WO 02/24207, we disclose the use of loop diuretics, thiazide diuretics and sulphonylureas as single active ingredients for the treatment of DNA viral infections.

Loop diuretics are substances which act on the ascending loop of Henle in the kidney. They are typically sulphonamides but may be other substances too. Typical examples include:

acetazolamide mefruside ambuside methazolamide azosemide piretanide bumetanide torsemide butazolamide tripamide chloraminophenamide xipamide clofenamide clopamide ethacrynic acid clorexolone etozolin disulfamide ticrynafen ethoxzolamide furosemide

Furosemide is an anthrilic acid derivative, chemically 4-chloro-N-furfuryl-5- sulfamoylanthranilic acid. It is practically insoluble in water at neutral pH, however is freely soluble in alkali. Furosemide exerts its physiological effect by inhibition of the transport of chloride and potassium ions across cell members. Furosemide is a loop diuretic with a short duration of action. It is used for treating oedema due to hepatic, renal, or cardiac failure and treating hypertension. The bioavailability of furosemide is between 60% to 70% and it is primarily excreted by filtration and secretion as unchanged drug. Furosemide acts on the Na ⁺ /K ⁺ /2CI ^" cotransporter. For its diuretic effect, its predominant action is in the ascending limb of the loop of Henle in the kidney. Loop diuretics markedly promote K ⁺ excretion. This may lead to the most significant complication of long term systemic furosemide usage namely a lowered serum potassium. We postulate that it is this action however which makes furosemide useful as an agent against DNA viral infections.

Recent evidence suggests that the major biotransformation product of furosemide is a glucuronide. Furosemide is extensively bound to plasma proteins, mainly albumin. Plasma concentrations ranging from 1 to 400 mcg/ml are 91-99% bound in healthy individuals. The unbound fraction ranges between 2.3-4.1% at therapeutic concentrations. The terminal half life of furosemide is approximately 2 hours, and it is predominantly excreted in the urine.

Thiazide diuretics include the benzothiadriazines derivatives, also known as thiazides. Typical examples are: althiazide hydrobenzthiazide bemetizide hydrochlorothiazide bendroflumethiazide hydrofluoromethiazide benzthiazide indapamide benzylhydrochlorothiazide mebutizide buthiazide methylcyclothiazide chlorothiazide meticane chlorothalidone metalazone cyclopenthiazide paraflutizide cyclothiazide polythiazide epithiazide quinethazone ethiazide teclothiazide fenquizone trichlormethiazide

Sulphonylureas are anti-diabetic drugs which influence ion transport across cell membranes. They are instanced by: acetohexamide glyburide

1 -butyl-3-metanilylurea glybuthiazole carbutamide glybuzole chlorpropamide glycycloamide glibenclamide glyclopyramide glibornuride glyhexamide gliclazide glymidine glimepiride glypinamide glipizide phenbutamide gliquidone tolazamide glisentide tolbutamide glisolamide tolcylamide glisoxepid

The cardiac glycosides include digoxin, digitoxin, medigoxin, lanatoside C, proscillaridin, k strophanthin, peruvoside and ouabain. Plants of the digitalis species (e.g. digitalis purpura, digitalis lanata) contain cardiac glycosides such as digoxin and digitoxin which are known collectively as digitalis. Other plants contain cardiac glycosides which are chemically related to the digitalis glycosides and these are often also referred to as digitalis. Thus the term digitalis is used to designate the whole group

of glycosides; the glycosides are composed of two components a sugar and a cardenolide.

Ouabain is derived from an African plant Strophanthus gratus (also known as strophanthin G) and is available in intravenous form (it is not absorbed orally) and is used for many laboratory experiments in the study of glycosides, because of its greater solubility. It is known to have a virtually identical mode of action as digoxin.

Digoxin is described chemically as (3b, 5b, 12b)-3- hexopyranosyl-( I "4)-O-2,6- dideoxy-b-D-ribo-hexopyranosyl-( I "4)-2,6-dideoxy-b-D-ribo-hexopyranosyl) oxy]- 12,1 4-dihydroxy-card-.20-22)-enolide. Its molecular formula is C and its molecular weight is 780.95. Dixogin exists as odourless white crystals that melt with decomposition above 230 ⁰ C. The drug is practically insoluble in water and in ether; slightly soluble in diluted (50%) alcohol and in chloroform; and freely soluble in pyridine.

Because some patients may be particularly susceptible to side effects with digoxin, the dosage of the drug should always be selected carefully and adjusted as the clinical condition of the patient warrants.

At the cellular level, digitalis exerts it main effect by the inhibition of the sodium and potassium transport enzyme sodium potassium adenosine triphosphatase (Na/K ATPase); this is directly responsible for the electrophysiological effects of heart muscle and according to our understanding also its activity against DNA viruses. This activity also has an effect on the efficiency of myocardial contractility due to secondary changes in intracellular calcium. At very low intracellular concentrations of digitalis, the

opposite effects can be seen with a reduced efficiency of cardiac contractions as the digitalis stimulates the Na/K ATPase.

As has been previously stated, studies have demonstrated the anti-viral DNA effects of a composition of the invention are dependent on a depletion of intracellular potassium ions. Briefly these studies demonstrate:

• replacement of potassium will restore DNA synthesis;

• use of frusemide and digoxin in combination have comparable effects on potassium depletion; • the level of potassium depletion is sufficient to allow normal cell function;

• the potassium depletion has no cytotoxic effects.

Thus, by altering the cellular concentrations of ions, cellular ionic balances, cellular ionic milieu and cellular electrical potentials by the application of a loop diuretic and a cardiac glycoside it is possible to change the metabolism of the cell without detriment to the cell but so that virus replication is inhibited. Accordingly, we are confirmed in the view that the use of a loop diuretic and a cardiac glycoside is of benefit in preventing or controlling virus replication by inhibiting the replication of viral DNA. Anti-viral efficacy has been demonstrated against the DNA viruses HSV1 and HSV2, CMV, VZV, papillomaviruses and adenoviruses. We believe that efficacy against parvoviruses, pseudorabies, apoviruses, hepadnoviruses and poxviruses will also be found.

It has now been surprisingly found that the anti-viral efficacy of previously disclosed anti-viral DNA agents can be enhanced by controlling the extra cellular ionic concentration when administering anti-viral DNA agents.

In particular, it has been surprisingly found that altering the extra cellular ionic concentration of monovalent ions can enhance efficacy of anti-viral DNA agents.

A first aspect of the invention provides a composition for topical application for the treatment of DNA viral infections; the composition comprises a diuretic and/or a cardiac glycoside and one of a source of potassium ions and a medium depleted in or free from sodium.

There is further provided, in a second aspect of the invention, there is provided a composition for the treatment of DNA viral infections comprising a diuretic and a cardiac glycoside together with a source of potassium ions.

In a third aspect of the invention, there is provided a topical viral treatment composition comprising a diuretic and/or a cardiac glycoside and a source of potassium ions.

In a fourth aspect of the invention, a method of treating DNA viral infections, the method comprising topically administering one or both of a diuretic and a cardiac glycoside together with a source of potassium ions.

A fifth aspect of the invention provides the use of one or both of a diuretic and a cardiac glycoside together with a source of potassium ions for the manufacture of a medicament for topical application for the treatment of DNA viral infections in subjects.

A sixth aspect of the invention provides the use of a diuretic and a cardiac glycoside together with a source of potassium ions for the manufacture of a medicament for the treatment of DNA viral infections in subjects.

The diuretic may be one or more selected from loop diuretics, thiazide diuretics and/or sulphonylyureas.

Preferably the loop diuretic is one or more of frusemide, bumetamide, ethacyrnic acid or torasemide.

Preferably the thiazide diuretic is one or more of chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, trichlormethazide, benzthiazide, bendroflumethazide, bendrofluazide, polythiazide or cyclothiazide.

Preferably the sulphonylurea is one or more of tolbutamide, tolazamide, tolcyclamide, glibornuridum, acetohexamide, chlorpropamide, carbutamide, glyburide or glipizide

The cardiac glycoside may be one or more of digoxin, digitoxin, medigoxin, lanatoside C, proscillaridin, k strophanthin, peruvoside and ouabain.

Preferably, the composition further comprises a medium depleted in or free from sodium.

In this specification the term 'treatment ¹ is intended to include the prevention of infection and/or the application of a composition to prevent DNA viral infections. It is also intended that 'treatment of DNA viral infections' encompasses treatment of the sequellae of DNA viral infections.

In this specification the terms or phrases "sodium-depleted" and "depleted in sodium", together with similar phrases, are intended to mean a sodium concentration of less than 1200 mM, preferably less than 600 mM, preferably less than 400 mM, preferably

less than 300 mM, and most preferably between 300 mM and 150 mM. The terms "sodium-free" or "free of sodium" together with similar phrases, are intended to mean a sodium concentration of less than 150 mM, preferably less than 125 mM and even more preferably less than 100 mM, more preferably less than 75 mM, and most preferably less than 50 mM, e.g. less than 1 mM.

The term 'medium' is intended to mean a suitable carrier.

The medium may be a liquid, gel, or solid. Preferably, the medium is suitable for topical application. The medium may be, or may comprise, or may be dissolved in, an adhesive. The medium may be dissolved in, or may comprise, a solvent.

In order that the invention may be more fully understood, it will now be described by way of example only with reference to the following drawings, in which:

Figure 1 is a graph showing the effects of NaCI on Herpes Simplex Virus (HSV) plaque formation

Figure 2 is a graph showing the effects of KCI on HSV plaque formation

Figure 3 is a graph showing the effect of NaCI depletion on Furosemide mediated ionic centra viral therapy (ICVT)

Figure 4 is a graph showing the effect of NaCI depletion on Digoxin mediated

ICVT

Figure 5 is a graph showing the effect of KCI depletion on Furosemide mediated

ICVT Figure 6 is a graph showing the effect of KCI depletion on Digoxin mediated ICVT

Figure 7 is a graph showing the effect of NaCI supplementation on Furosemide mediated ICVT

Figure 8 is a graph showing the effect of NaCI supplementation on Digoxin mediated ICVT

Figure 9 is a graph showing the effect of KCI supplementation on Furosemide mediated ICVT

Figure 10 is a graph showing the effect of KCI supplementation on Digoxin mediated ICVT Figure 11 is a graph showing the combined effects of NaCI and KCI on

Furosemide mediated ICVT

Figure 12 is a graph showing the combined effects of NaCI depletion and KCI supplementation on Furosemide and Digoxin in combination.

Figure 13 is a graph showing the effect of KCI supplementation on Digoxin mediated ICVT.

Figure 14 is a graph showing the effect of KCI supplementation on Furosemide mediated ICVT 2.3. HSV replication (A549 cells)

Figure 15 is a graph showing the effect of KCI on Furosemide mediated ICVT against HSV in A549 cells Figure 16 is a graph showing the effect of KCI on Digoxin mediated ICVT against

HSV in A549 cells.

Figure 17 is a graph showing the effects of KCI on ICVT mediated by Furosemide and Digoxin, against HSV in A549 cells.

The displacement of potassium ions from an intracellular site essential to viral DNA synthesis is fundamental to Ionic Contraviral Therapy (ICVT) as disclosed, for example, in WO 01/49242. When using a cardiac glycoside (e.g. Digoxin) and a loop diuretic (e.g. Furosemide), potassium displacement is affected specifically through NaVK ^+" ATPase and the Cl ^" cotransporter respectively.

However, the rates of activity of NaVK ⁺ ATPase and the Cl ^" cotransporter are dependent upon 'ambient' concentrations of Na ⁺ and K ⁺ , as indeed are the rates of diffusion of these cations across electrochemical gradients. It is important, therefore, to determine the influence of the 'ambient' concentrations of Na ⁺ and K ⁺ upon ICVT. Mindful of the complexities of cellular electrochemistry, we also examined the influence of the divalent cations Mg ²⁺ and Ca ²⁺ on ICVT.

The effects of the cations of calcium and magnesium and of sodium and potassium on virus replication and on cardiac glycoside and/or diuretic mediated ICVT have been determined.

Comparative Example 1

Tissue culture media (TCM) were specially formulated; each constituent inorganic salt, i.e. the source of each cation under investigation, was excluded as required:

^• NaCI-Free TCM for Na ⁺

• KCI-Free TCM for K ^{+ *} MgSO4-Free TCM for Mg ²⁺

• CaCI2-Free TCM for Ca ²⁺

Confluent cell monolayers (Vero calls and A549 cells) were incubated in the required salt-free medium for three hours to enable intracellular cation depletion and then lower and upper limits of tissue culture tolerance were established by the stepwise replacement of each salt in replicate cultures. Tissue culture tolerance was initially adjudged by the presentation of normal cell morphology and the continuing ability of cells to replicate. As a second stage, MTT assays were undertaken to measure the effect on the rate of cell metabolism.

Uninfected tissue culture cells were surprisingly tolerant of calcium chloride and magnesium sulphate depletion and cells replicated apparently normally in both calcium-free and magnesium-free media. Cells did not replicate in media totally free of sodium chloride or potassium chloride though partial depletion was tolerated.

Cells were tolerant of potassium supplementation but relatively intolerant of sodium chloride, magnesium sulphate or calcium chloride supplementation, as adjudged by microscopic observation of cell morphology.

MTT assays were undertaken in culture media containing a range of concentrations of inorganic salts within the limits of tolerance established above; ranging from partial salt depletion to an excess. Ranges of concentrations that inhibited the rate of cell metabolism by only twenty per cent were selected for subsequent studies on virus replication.

Comparative Example 2

Within the limits of tissue culture tolerance established above, neither calcium chloride nor magnesium sulphate had an effect on virus replication as adjudged by HSV plaque formation. In addition, neither calcium chloride nor magnesium sulphate had any effect on inhibition of HSV plaque formation by Digoxin or Furosemide. Calcium and magnesium were excluded from further consideration in this context.

Comparative Example 3

As shown in Figure 1, NaCI depletion was inhibitory to HSV plaque formation in Vero cells and the stepwise replenishment of NaCI recovered virus levels until a maximum was reached at the 'normal' (120OmM) tissue culture NaCI concentration. Cells were intolerant of NaCI supplementation and virus plaque numbers decreased as a consequence; we attributed this to cytotoxicity since cell morphology was adversely affected and the rate of cell metabolism, as adjudged by MTT assays, was severely decreased (>75%).

Comparative Example 4

As shown in Figure 2, KCI depletion was inhibitory to HSV plaque formation. The stepwise replenishment of KCI 'recovered' virus replication until a maximum was reached at the 'normal ¹ (5mM) KCI concentration. Cells were, however, tolerant of supplemental KCI; morpholgy was normal and the rate of cell metabolism was decreased by less than twenty per cent while virus replication was unaffected.

Example 1

The effect of sodium depletion, together with either Furosemide or Digoxin, on virus replication was greater than the effect of either drug with the normal sodium concentration (Figures 3 and 4). The graphs of Figures 3 and 4 illustrate the effects of Furosemide and Digoxin on HSV plaque formation in media with 'normal' concentrations of sodium (lines A and C respectively) and depleted sodium (lines B and D respectively).

Example 2

The effect of KCI depletion together with either Furosemide or Digoxin was greater than the effect of either drug with the normal potassium concentration (Figures 5 and 6). The graphs of Figures 5 and 6 illustrate the effects of Furosemide and Digoxin on HSV plaque formation in media with 'normal' concentrations of KCI (lines E and G respectively) and reduced KCI (lines F and H respectively).

Example 3

NaCI supplementation had no effect on the contraviral activities of Furosemide and Digoxin (Figures 7 and 8). The dose dependent effects of Furosemide and Digoxin, on virus replication, were identical in both the 'normal' sodium (as NaCI) concentration

(lines I and K respectively) and with supplemented sodium (as NaCI) (lines J and L respectively).

Example 4 It was surprising, however, that KCI supplementation augmented the activity of Furosemide (Figure 9). There were fewer plaques when there was supplemental potassium (line M) than the 'normal' concentration (line N).

Example 5 Conversely however, and as shown in Figure 10, KCI supplementation countered the antiviral effect of Digoxin on HSV replication (line 0) when compared to 'normal' potassium concentrations (line P).

Example 6 The effects of Furosemide on HSV replication under normal concentrations of KCI and KCI supplementation, in both isotonic and sub-isotonic concentrations of NaCI were compared. In each case, normal osmolarity was maintained by the substitution of mannitol for missing salt (NaCI).

As shown in Figure 11A, with the 'normal' (i.e. 5mM) KCI concentration (line Q-1) HSV replication was less inhibited by Furosemide at a concentration of 400ug/m€ than it was with supplemented (i.e. 2OmM) KCI (line R-1). As shown with supplemental KCI, HSV replication was inhibited by 50% by Furosemide at a concentration of only 200 μg/mC.

As shown in Figure 11 B, with the 'normal' KCI concentration (line Q-2) HSV replication was inhibited by almost 50% by Furosemide at a concentration of 400ug/ml. However,

with supplemented KCI (line R-2) HSV replication was inhibited by more than 50% by Furosemide at a concentration of only 200 μg/mC.

As shown in Figure 11 C, the 'normal' KCI concentration (line Q-3) HSV replication was inhibited by 50% by Furosemide at a concentration of 400ug/ml. However, with supplemented KCI (line R-3) HSV replication was inhibited by more than 75% by Furosemide at a concentration of only 200 ug/ml.

In conclusion, Furosemide was twice as effective against HSV under conditions of KCI supplementation (2OmM) and the antiviral effect was further augmented by NaCI depletion. Normal osmolarity was maintained by inclusion of Mannitol in the culture medium without detriment to Ionic Contra-Viral activity.

Example 7 It was important to exclude unexpected interactions between the two drugs, so the effects of potassium supplementation and sodium depletion, on ICVT mediated by Furosemide and Digoxin in combination were examined using HSV. In each case, normal osmolarity was maintained by the substitution of mannitol for missing salt (NaCI/KCI).

Using Furosemide at its ID50 concentration (1000 μg per mC) together with a 'sub- ID50' concentration of Digoxin (20 ng per m£), the ID50 effect was achieved under the 'normal' concentration (5mM) of KCI (Fig. 12, first datum of line S). The combined drug antiviral activity remained much the same with increasing KCI concentration (>5mM); thus, potassium-mediated enhancement of Furosemide activity was maintained while digoxin activity was countered by KCI supplementation.

Using Digoxin at its ID50 concentration (60 ng per ml) together with a 'sub-ID50' concentration of Furosemide (500 μg per ml) the ID50 effect was achieved under the 'normal' concentration (5mM) of KCI only (Fig. 12, first datum of line T). The combined antiviral activity was lost with increasing KCI concentration (>5μM); thus, potassium- mediated enhancement of Furosemide activity was effectively countered by potassium induced neutralisation of Digoxin activity.

With both drug combinations, the effect of KCI on the combined ICV activity was in accordance with the effect of KCI on the predominant drug in the combination. When the predominant drug in the combination was Digoxin, combined activity was lost by potassium supplementation. When the predominant drug in the combination was Furosemide, combined activity was maintained by potassium supplementation. There were, therefore, no unexpected interactions between the two drugs under the optimum cationic conditions defined earlier for each drug used separately.

Further Studies

As has been previously demonstrated, ICVT uniquely has a broad spectrum of antiviral activity and it was important to establish the optimum Ionic Contra-Viral potential for other target human pathogens. We provide the following results against another pathogen, namely, adenovirus. This necessitated the use of a different cell line (A549) which might possibly require different cationic conditions for optimal Ionic Contra-Viral effect.

Example 8 As shown in Figure 13, KCI supplementation (2OmM) countered the antiviral effect of Digoxin on Adenovirus replication in A549 as shown in line U compared with its efficacy

at 'normal' KCI concentrations (line V). As stated above, HSV was similarly affected in Vero cells (Figure 10).

Example 9 As shown in Figure 14, KCI supplementation (2OmM) augmented the antiviral activity of Furosemide against Adenovirus as shown in line W compared with its efficacy at 'normal' KCI concentrations (line X) (Figure 14). HSV was similarly affected in Vero cells (Figure 9).

ICVT elicits antiviral effect through change in the electrochemistry of the cell. The cationic requirements for optimum Ionic Contra-Viral activity against HSV in Vero cells and against AV in A549 cells have here been established and they are the same.

Example 10 KCI supplementation (2OmM) augmented the antiviral activity of Furosemide against HSV in A549 cells (Fig.15). AV was similarly affected in A549 cells (Fig. 14).

Example 11

KCI supplementation (2OmM) countered the antiviral effect of Digoxin on HSV replication in A549. (Fig.16). AV was similarly affected in A549 cells (Fig. 13).

Example 12

Using Furosemide at its ID50 concentration (1000 μg per m£) together with a 'sub-

ID50' concentration of Digoxin (20 ng per mC), the ID50 effect was achieved under the 'normal' concentration (5mM) of KCI (Fig. 17, first datum line 9). The combined drug antiviral activity as shown in line 9 remained much the same with increasing KCI

concentration (>5mM); thus, potassium-mediated enhancement of Furosemide activity was maintained while Digoxin activity was countered by KCI supplementation.

Using Digoxin at its ID50 concentration (60 ng per mC) together with a 'sub-ID50' concentration of Furosemide (500 mg per m£) the ID50 effect was achieved under the 'normal' concentration (5mM) of KCI only (Fig. 17, first datum line Z). The combined antiviral activity was lost with as shown in line Z increasing KCI concentration (>5mM) ; thus, potassium-mediated enhancement of Furosemide activity was effectively countered by potassium induced neutralisation of Digoxin activity.

With both drug combinations, the effect of KCI on the combined Ionic Contra-Viral activities was in accordance with the effect of KCI on the predominant drug in the combination. When the predominant drug in the combination was Digoxin, combined activity was lost by potassium supplementation. When the predominant drug in the combination was Furosemide, combined activity was maintained by potassium supplementation. There were, therefore, no unexpected interactions between the two drugs under the optimum cationic conditions defined earlier for each drug used separately.

This is consistent with the effects observed using HSV in Vero cells (Fig. 12).

The IC50 values for Digoxin and Furosemide have been established against a number of DNA viral infections as shown below:

Also, at the IC50 value, Feline Herpes Virus replication is almost completely inhibited on application of Digoxin and Furosemide separately (99.5 and 98.5% inhibition at the IC50 concentration respectively). When applied together, virus inhibition is complete (99.99999%).

A similar picture is found for other DNA viruses.

Digitoxin can be shown to be twice as active as Digoxin with an IC50 of 30 ng/m£ compared to 60 ng/m€ against HSV in Vero cells.

It is further postulated that similar increases in the efficacy of other loop diuretics and cardiac glycosides will be seen with potassium supplementation and/or sodium depletion extra cellular.

In conclusion, neither calcium ions nor magnesium ions have any effect on virus replication and no affect on either digoxin or furosemide mediated ICVT. Sodium and potassium ions, however, have significant bearing on both virus replication and on ICVT.

Tissue culture cells were intolerant of total NaCI depletion and of total KCI depletion but fairly tolerant of partial depletion of either NaCI or KCI. Under conditions of partial

depletion of NaCI or KCI, that is at concentrations which inhibited the rate of cell metabolism by less than twenty per cent, virus replication was inhibited. These effects are consistent with ICVT, albeit non-drug mediated.

Whilst we do not wish to be bound by any particular theory we understand that exclusion of KCI from the medium was inhibitory to virus replication because once potassium ions have leached from the cell there is no source of potassium ions for cellular import. We believe that exclusion of NaCI down-regulates Na/K-ATPase activity which in turn brings about a reduction in intracellular potassium ion concentrations and consequently inhibited virus replication.

The gross inhibitory effect on virus replication of partial NaCI depletion and the addition of either Digoxin or Furosemide was greater than the effect of either drug alone. Similarly, the gross inhibitory effect on virus replication of KCI depletion and the addition of either Digoxin or Furosemide was greater than the effect of either drug alone.

NaCI supplementation has no effect on ICVT mediated by either Digoxin or Furosemide.

Supplemental potassium, however, affected the Contra-Viral activities of both Digoxin and Furosemide: Supplemental potassium antagonised the antiviral effect of Digoxin on HSV replication (Fig. 10) due, we believe, to increased potassium ion import through channels other than Na-K ATPase, the site of action of Digoxin.

It was surprising, however, that supplemental KCI enhanced the antiviral activity of Furosemide but the clear conclusion is that Furosemide is twice as effective when

formulated with 2OmM potassium chloride and the antiviral effect is further enhanced by the exclusion of sodium.

The same effects were observed using herpes simplex virus in vero cells, adenovirus in A549 cells and HSV in A549 cells.

In conclusion, cardiac glycosides and diuretics (e.g. loop diuretics), for example Digoxin and Furosemide, are synergistic and their combined contraviral activity is optimised by the exclusion of sodium chloride from the formulation. Lowered osmolarity, as a consequence of sodium chloride exclusion, can successfully be restored by the inclusion of mannitol without detriment to Contra-Viral activity.

Ionic Contra-Viral activity will be optimised both by sodium exclusion and by the addition of supplemental potassium chloride (2OmM). Normal osmolarity is restored by the addition of mannitol without detriment to Contra- Viral Activity.