GB2376628A

GB2376628A - Treatment of DNA viral infection

Info

Publication number: GB2376628A
Application number: GB0114947A
Authority: GB
Inventors: Christoper Edward Hartley
Original assignee: Henderson Morley Research and Development Ltd
Current assignee: Henderson Morley Research and Development Ltd
Priority date: 2001-06-19
Filing date: 2001-06-19
Publication date: 2002-12-24
Anticipated expiration: 2021-06-19
Also published as: GB0114947D0; GB2376628B

Abstract

The synergistic combination of a diuretic and a cardiac glycoside is useful in the treatment of DNA viral infections in animals.

Description

TREATMENT OF DNA VIRAL INFECTIONS IN ANIMALS The invention relates to anti-viral treatment for veterinary use, and in particular to prophylactic and therapeutic treatments of DNA viral infections in animals.

Herpes viruses are DNA viruses, having a central core of DNA within a proteinaceous structure. DNA carries the genetic code to reproduce the virus. Viruses 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 antiviral chemotherapy.

These include DNA polymerase and thymidine kinase which are needed 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.

According to the invention in one aspect there is provided the use of a loop diuretic and a cardiac glycoside to exert a synergistic effect in the treatment of DNA viral infections in animals.

In another aspect the invention provides a therapeutic composition for veterinary use, useful in the treatment of DNA viral infections in animals, the composition comprising a synergistic combination of a loop diuretic and a cardiac glycoside.

The diuretic may be selected from a range of loop diuretics, sulphonylureas and thiazides.

Loop diuretics are substances which act on the ascending loop of Henlé in the kidney.

They are 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 Sulphonylureas are anti-diabetic drugs which influence ion transport across cell membranes.. They are instanced by: acetohexamide glyburide I-butyl-3-metanilylurea glybuthiazole carbutamide glybuzole chlorpropamide glycycloamide glibenclamide glyclopyramide glibomuride glyhexamide gliclazide glymidine glimepiride glypinamide glipizide phenbutamide gliquidone tolazamide glisentide tolbutamide glisolamide tolcylamide glisoxepid 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 Preferably the diuretic is loop diuretic and is any one or more of frusemide, bumetanide, ethacrynic acid or torasemide. According to our studies loop diuretics mediate their antiviral effects through alteration to the cellular concentration of ions, cellular ionic balances, cellular ionic milieu and electrical potentials.

The preferred loop diuretic for this use is frusemide which is an anthrilic acid derivative, chemically 4-chloro-N-furfuryl-5-sulfamoylanthranilic acid. It is practically insoluble in water at neutral pH, but is freely soluble in alkali. Frusemide exerts its physiological effect by inhibition of the transport of chloride ions across cell membranes. Frusemide is a loop diuretic with a short duration of action. It is used for treating oedema due to hepatic, renal, or cardiac failure and for treating hypertension.

The bioavailability of frusemide is between 60% to 70% and it is primarily excreted by

filtration and secretion as an unchanged drug. Frusemide acts on the Na+/K +/2CIcotransfonner. For its diuretic effect, its predominant action is in the ascending limb of the loop of Henlé in the kidney. Loop diuretics markedly promote K+ excretion, leaving cells depleted in intracellular potassium. This may lead to the most significant complication of long term systemic frusemide usage, namely a lowered serum potassium. We postulate that it is this action which makes loop diuretics useful as an agent against DNA viruses.

Recent evidence suggests that the major biotransfonnation product of frusemide is a glucuronide. Frusemide 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 frusemide is approximately 2 hours, and it is predominantly excreted in the urine.

Cardiac glycosides exhibit positive inotropic activity which is mediated by inhibition of sodium-potassium adenosine triphosphate (Na/K-ATPase). Substances which fall into this category include: acetyldigitoxon gitalin acetyldigoxin gitoxin cymarin Lanatoside C deslanoside medigoxin Digitalin meproscillarin Digitalis Lanata leaf oubain Digitalis Leaf peruvoside Digitoxin proscillaridin Digoxin strophanthin-K Preferably the cardiac glycosides may be any one or more of digoxin, digitoxin, 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 strophanthidin 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 has a virtually identical mode of action as digoxin. The preferred cardiac glycoside for this use is digoxin.

Digoxin is described chemically as (3b, 5b, 12b) -3-[O-2, 6-dideoxy-b-D-riobhexopyranosyl- (l"4)-0-2, 6-dideoxy-b-D-o-hexopyranosyI- (l"4)-2, 6-dideoxy-b-Dribo-hexopyranosyl) oxy]-12, 14-dihydroxy-card-20-22)-enolide. Its molecular formula is C41H64Ü14, and its molecular weight is 780. 95. Dixogin exists as odourless white crystals that melt with decomposition above 230oC. 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 animals 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 particular animal warrants.

At the cellular level digitalis exerts it main effect by the inhibition of the sodium 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.

A preferred combination is the loop diuretic frusemide and the cardiac glycoside digoxin. It is preferred that concentrations are frusemide 300/- ! g/ml and digoxin 0. 12/- ! g/ml. It is within the scope of the invention to separate the application of the two active ingredients by a short time period.

Studies (including X-ray microanalysis) 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 to 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, and Pseudorabies and Adenoviruses.

The compositions of the invention may be adapted for external or internal administration. Topical and systemic applications are likely to be the most useful. The formulations may be adapted for slow release. It is a much preferred feature of the invention that the compositions are formulated for topical application. Other ingredients may be present, provided that they do not compromise the anti-viral activity; an example is a preservative. Preferably the invention provides a combination of frusemide and digoxin as a topical application in a buffered saline formulation for the treatment of corneal eye infections. So far as we are aware, the combination of existing, licensed compounds, for the treatment of viral infections in animals is without precedent.

One feature of this invention is the use of local concentrations of loop diuretic and cardiac glycoside for the highly effective treatment of virus infections of the eye.

A depot application of a loop diuretic and cardiac glycoside applied intra-occularly would be a suitable method for the treatment of cytomegalovirus retinitis.

The invention is intended for veterinary use and is thus of value in treating DNA viral infections in cats, dogs; pigs, cattle, sheep, horses; poultry; fish; wild animals; and the like.

The invention will now be described by way of illustration only with reference to the following examples.

EXAMPLE 1

In vitro bioassays with Felid herpes virus 1 (FHV1) were undertaken to follow the antiviral activity of the administration of frusemide (600tg/ml).

FHV1 strains used: FHV1 strain B927, was obtained from the Department of Veterinary Pathology, Faculty of Veterinary Science, The University of Liverpool.

Cell cultures: Feline kidney cells (FK) were obtained from the Department of Veterinary Pathology, The Faculty of Veterinary Science, The University of Liverpool, and were used as the only cell line for all experiments in the examples.

Method: (a) FHV plaque inhibition assay FHV plaque inhibition assays were performed by inoculation of preformed cell monolayers with 1 ml of an appropriate virus dilution in a suitable culture medium to give rise to between 50 and 100 plaques per 5cm petri dish. After adsorption (lhour) at

37 C the inoculum was removed and the cell sheets overlaid with culture medium containing 0. 8% v/v carboxymethylcellulose and 6001lg/ml frusemide. Cultures were incubated at 37 C for 48 hours and fixed and stained with carbol fuchsin.

(b) FHV growth curve Confluent FK cell monolayers were infected at a multiplicity of infection of 10 plaque forming units per cell by incubation for 1 hour at 37 C with 1ml of virus diluted to

achieve 10 pfu/cell. The virus inoculum was removed and the infected cell monolayers washed three times with pre-warmed culture medium to remove unadsorbed virus. Cultures were harvested at selected times. Cells were scraped from the culture surface using a silicone-tipped'policeman'and harvested together with the incubation medium and stored at-80 C until assayed. After thawing and before assay, infected cell suspensions were disrupted by ultrasonic vibration (Megason bath) and centrifuged at 2000G for 10 minutes to remove cellular debris. Virus dilution at which plaques were counted were always of sufficient magnitude to exclude inhibition of virus replication by residual frusemide during assay.

Results: Figure 1 shows the infection of FK cells at low, intermediate and high multiplicities of

infection by FHV I in the presence of600g/ml of frusemide.

While frusemide (600/J. g/ml) induced a slight morphological change in uninfected FK cells compare Figures la and I b] uninfected FK cells appeared to replicate at a normal rate and to a normal cell density in the presence of frusemide at this concentration [Figure 2]. This demonstrates that the administration of frusemide at this concentration would not be harmful to healthy cells, and therefore side-effects are minimal.

In the absence of frusemide, FHVI cytopathology at low, intermediate and high multiplicities of infection (MOI) [see Figures Ic, Ie, and lg.]

FK cells infected with FHV1 at low, intermediate and high MOI, cultured in the presence of frusmeide (600 g/ml) were morphologically indistinguishable from uninfected FK cells compare figure la with Figures Id, If and Ih) EXAMPLE 2 In vitro bioassays with Felid herpes virus 1 (FHV1) were undertaken to follow the antiviral activity of the administration of digoxin (0.12 g/ml).

The FHV1 strains, cell cultures, and methodology used in this experiment followed those used in Example 1.

Results: Figure 3 shows the infection of FK cells at low, intermediate and high multiplicities of infection by FHV I in the presence of0. 12) ng/ml ofdigoxin.

Inhibition of FHV1 replication by digoxin (0. 12g/ml) was clear at low, intermediate and high MOI compare Figure 3a to Figures 3d, If and Ih]. Digoxin at this concentration had no adverse effect on the host cell compare Figures la and lb]. There was no adverse effect of digoxin on uninfected cell morphology.

Uninfected FK cells replicated in the presence of digoxin at a highly virus inhibitory concentration, albeit at a reduced rate [see Figure 4].

EXAMPLE 3

In vitro bioassays with Felid herpes virus 1 (FHV1) were undertaken to follow the antiviral activity of the simultaneous administration of frusemide (300ug/ml) and digoxin (0. 24g/ml).

Results: Figure 5 shows the infection of FK cells at an intermediate multiplicity of infection by

FHV1 in the presence of 300Mg/ml of frusemide andO. 24Mg/ml of digoxin, individually and in combination.

Figure 6 shows the infection of FK cells at a high multiplicity of infection by FHV 1 in the presence of 300Mg/ml frusemide and 0. 24ug/ml of digoxin, individually and in combination.

At certain antivirally effective doses of digoxin, there were visible, minute foci (microplaques) of infected cells [see Figures 5a, 5b, 6a and 6b]. By the application of individually and incompletely effective doses of digoxin and frusemide, in combination, it was possible to completely inhibit virus replication whilst maintaining sub-cytotoxic doses of chemotherapy. Figures 5 and 6 illustrate this effect at intermediate and high MOI.

For example, frusemide alone, at a concentration of 300fJ. g/ml, had only a moderate antiviral effect [see Figures 5c and 6c].

However, when frusemide (300ug/ml) and digoxin (0. 24ug/ml) were applied together, virus replication was completely inhibited [see Figures 5d and 6d].

The synergistic activities of frusemide and digoxin were clear when their effects on infectious viral yield were measured. While individually, there was partial inhibition of virus replication, there was complete inhibition of virus replication when both were used in combination. This is shown in the following table:

Sample Virus yield (plaque forming units per cell) Time 0 : input virus 0.01 1. Time 24 hours: Normal virus yield 50 2. Time 24 hours: Frusemide 300 g/ml 0.25 3. Time 24 hours: Digoxin 0.12 g/ml 0.75 4. Time 24 hours: Frusemide and digoxin 0.00001 [300Ilg/ml and 0. 12Ilg/ml] 1. In the absence of inhibitors there was a five thousand-fold increase in virus titre. 2. In the presence of frusemide (300 g/ml) there was only a twenty five-fold increase in virus titre.

3. In the presence of digoxin (0. 12Ilg/ml) there was only a seventy five-fold increase in virus titre.

4. In the presence of frusemide (300ug/ml) and digoxin (0. 12/-lg/ml) there was a one thousand-fold decrease in virus titre.

CONCLUSION Both frusemide and digoxin have potent anti-feline herpes viral activities at concentrations which permit cell replication. Antiviral activity was enhanced, virus replication completely inhibited, and sub-cytotoxic doses maintained when frusemide and digoxin were applied in combination. Our evaluations suggest that this effect will be exerted with any combination of diuretic, especially loop diuretic and cardiac glycoside as disclosed herein. The relative proportions will be determinable by routine experimentation.

Claims

CLAIMS 1. Use of a diuretic and a cardiac glycoside to exert a synergistic effect in the treatment of DNA viral infections in cells of animals.
2. Use according to Claim 1, wherein the diuretic is a loop diuretic.
3. Use according to Claim 2, wherein the loop diuretic is frusemide.
4. Use according to Claim 1,2 or 3, wherein the cardiac glycoside is digoxin.
5. A therapeutic composition useful in the treatment of DNA virus infections in cells of animals, the composition comprising a synergistic combination of a diuretic and a cardiac glycoside, in a concentration which is sufficient to lower the level of the potassium ion to a concentration at which the DNA virus cannot replicate and above the level at which cell function is substantially unimpaired.
6. A composition according to Claim 5, wherein the diuretic is a loop diuretic.
7. A composition according to Claim 5 adapted for topical application.
8. A composition according to Claim 5 adapted for systemic application.

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9. A composition according to Claim 5, comprising 3001lg/ml of loop diuretic and 0.12 g/ml of cardiac glycoside.
10. A composition according to Claim 5, adapted for intra-occular depot application.
11. A composition according to any of Claims 5 to 10, wherein the loop diuretic is frusemide.
12. A composition according to any of Claims 5 to 10, wherein the cardiac glycoside is digoxin.