WO1993018776A1

WO1993018776A1 - Combinations of compounds having antiviral activity

Info

Publication number: WO1993018776A1
Application number: PCT/EP1993/000541
Authority: WO
Inventors: Paolo Chiesi; Carlo De Giuli Morghen
Original assignee: Chiesi Farmaceutici S.P.A.
Priority date: 1992-03-16
Filing date: 1993-03-10
Publication date: 1993-09-30
Also published as: AU3632093A; ITMI920605A0; IT1254519B; ITMI920605A1

Abstract

Drug combinations consisting of a purine or pyrimidine nucleoside analogue and a folic acid competitive antagonist, exert an effective antiviral activity, particularly against Herpes viruses. Such an activity is also shown against mutant viruses that developed drug resistance.

Description

COMBINATIONS OF COMPOUNDS HAVING ANTIVIRAL ACTIVITY

The present invention refers to combinations of antiviral drugs useful for treating Herpes virus infections.

Notwithstanding the remarkable progress in the control of infections diseases, while in the field of antibacterial therapy good results have been obtained thanks to very effective drugs, major problems are found in the antiviral field.

Only a few antiviral drugs proved to be effective: for the treatment of DNA virus infections, particular interest has been paid for drugs able to inhibit the DNA synthesis.

These are mostly purine or pyrimidine bases analogues generally used in the treatment of Herpes viruses.

5~Iodo-2 '-deoxyuridine is the parent pyrimidine nucleoside effective in the topical treatment of Herpetic keratitis. Similar biological properties are shared by 5-iodo-2 '-deoxycytidine (IDC), characterized, iⁿ comparison with IDϋ, by an higher thermal stability and by a slower catabolism.

More recently, acyclovir has been successfully introduced in the clinical practice.

It is known that drug may exert its antiviral activity by interacting with different stages of virus replication process.

The main mechanism of the inhibition of the viral replication by said compounds is the de novo DNA synthesis. During the normal DNA synthesis, free nucleoside triphosphates bind to the end of a growing DNA chain. The reaction is catalysed by the enzyme DNA-polymerase.

A suitably modified nucleoside can interfere with the process by incorporating into the DNA, changing its properties and interrupting the replication. In fact, the DNA-polymerase binds to the activated nucleoside drug (i.e. phosphorylated as hereinafter explained) and connects this unit to the growing single-strand DNA. The drug bound to the DNA strand, differently from a true nucleoside, has no binding sites for a subsequent bond, interrupting thereby the elongation, once incorporated into the DNA.

The nucleoside drug, however, must be phosphorylated to interact with the viral DNA synthesis.

The phosphorylation process requires, as a first step, an enzyme named thymidine kinase which is selectively induced in the cells infected by the virus and not in healthy cells. The drug can therefore exert its action selectively against infected cells.

Another important factor which concurs to the selective inhibition of viral DNA is due to the fact that the phosphorylated nucleoside drug binds irreversibly to the viral DNA polymerase which is therefore unavailable for subsequent reactions.

The nucleoside antivirals can therefore inhibit the viral replication acting at several levels: - at the enzymatic level, on one hand by an activation of the drug through virus-induced enzymes and, on the other hand, by irreversibly binding the viral DNA-polymerase; at the level of nucleic acid, by incorporation of the drug into the viral genome after phosphorylation by cellular enzymes. However, these drugs are limited in their use due to their ability to select resistant viral strains.

The resistant strains have functional impairments in one or both the virus-induced enzymes, thymidine kinase and DNA polymerase. A rational approach to improve the efficacy of antiviral agents should be therefore the use of combination of drugs, each acting against a different viral function.

By using drugs with different mechanisms of action, the possibility of developing drug resistance should be considerably decreased: in fact the virus should develop means to neutralize contemporaneously different drugs.

Indeed, while there is an abundant literature on the use of combinations of antibacterial drugs, some of which found wide applicability in the therapeutic practice, the combinations of antiviral drugs have not been thorougly studied.

The development of a synergistic combination of antiviral agents actually asks for a particular attention to the molecular mechanisms of the drugs.

It has been shown that the genetic mutations responsible for the resistance of Herpes viruses to antiviral purine and pyrimidine nucleoside analogues concern the coding of thymidine kinase.

This enzyme, in fact, is absent or inactive in resistant viruses, which can therefore no longer phosphorylate either thymidine or its analogues, but, although endowed with a lower infective capacity, can survive in a latent condition, so that they are presumed to be able to develop metabolic pathways alternative to thymidine kinase.

The present knowledges on the Herpes virus replication have shown that an alternative pathway to the thymidine triphosphate synthesis may be the synthesis of thymidine monophosphate starting from deoxyuridine monophosphate by means of the thymidilate synthetase enzyme-..

If the use of nucleoside antiviral drugs causes the onset of mutants relying on the thymidilate synthetase pathway, it may be useful to combine other drugs which can inhibit this alternative metabolic pathway.

However, as stated above, not all the drug combinations can synergistically contribute to the inhibition of viral replication.

In certain instances, some compounds used in combination have even shown antagonistic effects.

It has now been found that nucleoside analogues such as particularly 5-iodo-2'-deoxycytidine (IDC), 5- iodo-2 '-deoxyuridine (IDU) and acyclovir, used in combination with particular inhibitors of the DNA synthesis interacting with alternative metabolic pathways, exert a synergistic antiviral effect.

In the past some efforts have been made to find drugs combinations effective against Herpes virus.

Wigand R. and Hassinger M. , Medical Microbiology and Immunology, 1980, 168, 179-190, tested the effects of the combination of different substances against Herpes virus type 1 (HSVl) in _in vitro cell cultures, but this study had no further development. In their study, the authors used among the DNA inhibitors also methotrexate or aminopterine, folate antagonist antimetabolite used in therapy as antitumoral chemotherapeutic and, more recently, in the treatment of psoriasis and of rheumatoid arthritis. Similarly to other folate analogues, methotrexate inhibits the dihydrofolate reductase enzyme, blocking in this way the production of tetrahydrofolate, essential cofactor for the DNA synthesis.

In the study of igand et al. , methotrexate turns out to be, together with 5-fluoro-2 '-deoxyuridine (FUdr) the only compound not interacting with the others, and in fact the authors conclude with the statement that it seems not suited for the use in Herpes virus infections. It has now surprisingly been found that antiviral nucleosides such as 5-iodo-2 ^■-deoxycytidine and acyclovir exert synergistic effects in combination with methotrexate against both type 1 and type 2 Herpes virus (HSVl and HSV2). This synergism has been shown both iri vitro in the "plaque reduction test" and iτ\ vivo in the "yield reduction test" in guinea-pigs infected with the two viral strains. Example 1 Plaque reduction test This test allows to evaluate the capability of the virus to lyse the infected cells forming areas of death cells (plaques) and it measures the virus diffusion from the infected cells to other cells. The inhibition of the virus diffusion, determined according to the plaques number, represents an indirect measure of the inhibition of the viral multiplication.

Iododeoxycytidine (IDC), acyclovir and methotrexate (MTX) were used.

In an initial step of the study hydroxyurea (HU) , a reversible inhibitor of the DNA synthesis, able to prevent the DNA viral replications, was also used. Hydroxyurea is one of the substances that, in the study of Wigand and Hassinger, previously cited, gave good results, showing synergistic effects in combination with two antiviral AraC (Arabinofuranosylcytosine) and AraA (Arabinofuranosyladenine) .

The drugs were used in the following concentrations and combinations: Test for HSV-1 dissolved in phosphate buffer dissolved in distilled water dissolved in distilled water

dissolved in saline solution buffered with Earle salts IDC 1 μg/ml + MTX 0.1 μg/ml IDC 0.5 μg/ml + MTX 0.05 μg/ml IDC 1 μg/ml + HU 10 μg/ml Acyclovir 1 μg/ml + MTX 0.1 μg/ml Test for HSV-2 IDC 10 μg/ml MTX 0.1 μg/ml HU 20 μg/ml Acyclovir 1 μg/ml

IDC 10 μg/ml + MTX 0.1 μg/ml IDC 5 μg/ml + MTX 0.05 μg/ml IDC 10 μg/ml + HU 20 μg/ml Acyclovir 1 μg/ml + MTX 0.1 μg/ml Cell lines

Confluent VERO cell monolayers grown in 5 cm diameter Petri dishes were used. The cells were cultivated at 37°C, in incubator with controlled atmosphere containing 5% CO., in Dulbecco's Modified Minimal Essential Medium (DMEM) added with 10% fetal calf serum. —

Each test was performed in quadruplicate and 4 plates inoculated with the virus, without the addition of drugs, were used as a positive control. Viral strains

HSVl and HSV2 were cultured in Hela cells until a p titer of about 10 PFU/ml (PFU = Plaque Forming Units) in VERO cells was obtained and they were stored in aliguots at -80°C.

For the performance of the test, each VERO cell plate was inoculated with an aliquot sufficient to give from 200 to 400 plaques per dish. Test procedure Dishes of confluent VERO cells were incubated for 2h at 37°C, in atmosphere containing 5% C0₂, in DMEM containing 2.5% calf serum and the tested drugs at the desired final concentrations. 4 Petri dishes were used for each drug solution, whereas 4 dishes without drug were used as a control.

The cells were then inoculated with virus aliquots so as to give from 200 to 400 PFU and incubated at 37°C for lh to allow the virus adsorption.

The virus inoculation was then removed and the cultures were washed twice with 2 ml of Earle's saline medium containing 1% calf serum.

The monolayers cells were then covered with 5 ml of DMEM added with 2.5% calf serum, 1% agarose and the different drugs.

After agarose solidification, the dishes were incubated at 37°C for 2-3 days, until the onset of plaques in the controls. For a better evaluation of the results, 2 ml of a solution of neutral red (DMEM containing 0.03% neutral red and 2.5% calf serum) were added to each dish and after 4 hours the plaques were examined and counted.

The results obtained respectively with HSVl and HSV2 in different tests, each carried out in quadruplicate, are reported in Tables 1 and 2.

Table 1. Inhibition of HSVl multiplication, determined by the plaque reduction test in VERO cell lines

IDC = 5-iodo-2 '-deoxycytidine MTX = methotrexate HU = hydroxyurea ACV = acyclovir PFU = Plaque Forming Unit Table 2. Inhibition of HSVl multiplication , determined by the plaque reduction test in VERO cell lines

Example 2 Yield reduction test

This test directly measures the viral replication in a one-step multiplication cycle.

The test was carried out in two steps : the virus was first grown in VERO cells with the different drugs under examination and the produced virus was then assayed. The first step was carried out substantially as in Example 1. After removal of the culture medium and addition of 1 ml of Earle's medium, the cultures were sonicated at 0°C for 5 min at 15 seconds interval to release the virus from the cells. The viral suspensions obtained by cells incubated with the same drug were pooled and different virus dilutions were prepared. Second step

Confluent VERO cells in 5 cm Petri dishes, prepared as previously described, were inoculated with aliquots (0.2 or 0.25 ml/Petri) of the viral suspensions obtained in the presence or absence of the drugs as above described and diluted from 10 -5 to 10-7

6 Petri dishes inoculated in duplicate were used for each suspension.

Test procedure:

After removal of the culture medium, the VERO cells were inoculated with the different virus dilutions and incubated for lh at 37°C to allow adsorption.

The not absorbed viral inoculum was removed by washing twice with 2 ml of Earle's saline solution and immediately after a layer of DMEM added with 2.5% calf serum and 1% agarose was added to each dish. After solidification of this layer, the dishes were incubated at 37°C for 2-3 days, until plaques appeared in the controls.

For a better evaluation of the results, 2 ml of a solution of neutral red (DMEM containing 0.03% neutral red and 2.5% calf serum) were added to each dish and after 4 hours the plaques were examined and counted. The results obtained respectively with HSVl and HSV2 are reported in Tables 3 and 4. As it is evident from the results reported in Tables 1-4, the two combinations IDC+MTX and ACV+MTX cause a very high inhibition of the viral multiplication. The data show the high anti-herpetic activity of the two combinations and their efficacy in preventing the viral replication.

Table 3. Inhibition of the HSVl replication determined by the yield reduction test in VERO cell lines

Table 4. Inhibition of the HSV2 replication determined by the yield reduction test in VERO cell lines

Drugs and concen¬ PFU/ml of viral suspension trations (μg/ml) Test N.l N.2 N.3 IDC 10 1.48X10"' 1.57X10-' 2.10X10-' MTX 0.1 0.74X10^! 5.94X10^! 7.00X10¹ HU 20 4.30X10^f 5.00X10⁽ 7.94X10

IDC 10 + MTX 0.1 1.21X10^: 2.20X10' 3.88X10- IDC 5 + MTX 0.05 1.30X10^" 9.81X10- 4.31X10- IDC 10 + HU 10 6.41X10 2.87X10* 8.90X10* CONTROLS 2.72X10 1.33X10 1.99X10

The results obtained in the plaque reduction test show in fact that the combinations IDC 1 μg/ml + MTX 0.1 μg/ml; IDC 0.5 μg/ml + MTX 0.05 μg/ml (even though to a slightly lower extent) and ACV 1 μg/ml + MTX 0.1 μg/ml can inhibit the primary infection by Herpes virus and its spread out from the infected cells to the adjacent ones.

The yield reduction test shows, on the other hand, that the virus produced in the presence of these drugs combinations has a very decreased infectivity. The effect is even more evident if compared with the low inhibitory activity shown by hydroxyurea at the concentration of 10 μg/ml, both alone and in combination with IDC. Example 3 In vivo tests Male Charles River guinea-pigs weighing 700-900 g housed in individual cages were inoculated with HSVl 7 and HSV2 suspensions respectively containing 6x10

PFU/ml and 2.9x10 PFU/ml, prepared and assayed as stated above. Before inoculation, guinea-pigs were anaesthetized with diethyl ether and a six-square grid was drawn with permanent ink on the carefully shaved and epilated back. After one day, so as to allow the restoration of the skin, the viral suspensions were applied at each square by multiple intradermal injections. Five guinea-pigs were inoculated with HSVl and five with HSV2. The animals were examined daily to evaluate the development of lesions.

After 17 hours from the virus inoculation, about

1 g of gel containing the drugs under examination was applied on the six squares on the animals back and the treatment was repeated twice a day for 10 days.

The following drugs were used:

0.5% 5'-iododeoxycytidine gel (0.5% IDC)

5% Acyclovir cream (5% ACV) - 1% 5'-iododeoxycytidine + 0.1% methotrexate gel

(1% IDC + 0.1% MTX)

0 .5% 5 ' -iododeoxycytidine + 0 . 05% methotrexate gel

( 0 . 5% IDC + 0 . 05% MTX) .

Since DMSO was used as penetration enhancer in some compositions, one square was treated with 10% dimethylsulfoxide gel to exclude any contribution to the antiviral action.

Each compound was tested on a different square of the same animal, varying in each animal the rostral- caudal level of drugs application.

The antiviral activity of the drugs under examination was evaluated according to the treatment period necessary to achieve recovery and to the capability to prevent the infection development, comprising the steps of papula, vesicle, pustule, ulcer and scab, and to induce cicatrization as soon as possible.

In fact, the more a drug is considered to have a good efficacy, the more it favours a rapid cicatrization, avoiding ulcer and scab. HSV-1 Infections

Both the 1% IDC + 0.1% MTX and 0.5% IDC + 0.05%

MTX combinations and 5% acyclovir were able to block the progression of the infection, the cicatrization final stage being reached in all the animals with no passage through an ulcer intermediate stage.

However, the best results were obtained with the 1% IDC + 0.1% MTX combination, which allowed to reach cicatrization on the fifth day after inoculation in 4 out 5 tested animals, in any case always 1-2 days before than that obtained with 5% ACV alone, in one case even skipping the pustule stage.

The results obtained with the 0.5% IDC + 0.05% MTX combination were also satisfactory, this combination in most cases showing a progress almost superimposable to that of the combination at higher concentrations.

10% DMSO proved to be definitely inactive and the progression of the lesion in the DMSO treated area turned out to be very similar to the one observed in the control area. The results obtained with the various medicaments in a guinea-pig are reported in Figure 1, by way of examples. HSV-2 Infections

Also in this case, the 1% IDC + 0.1% MTX combination proved to be the most effective treatment, allowing to reach the cicatrization stage within the fifth day after inoculation in 4 out 5 tested animals and allowing to skip in all the animals at least two intermediate stages of the infection.

The results obtained with the 0.5% IDC + 0.05% MTX combination at lower concentrations were also satisfactory, with an intermediate progression between that of the higher concentration and the one of 5% acyclovir.

5% Acyclovir, though proving to be an effective medicament, lead to cicatrization within longer times, comprised from 5 to 10 days on the average.

Example 4 Evaluation of the antiviral activity of the combinations against resistant Herpes virus 1- and 2- types.

In vitro tests - Plaque reduction test To further prove the synergistic antiviral effect of the combinations of the invention, which effect derives from the ability to inhibit both the possible metabolic pathways that can be used by virus to multiplicate, the activity of the above combinations against HSV-1 and HSV-2 virus, made acyclovir- resistant, i.e. which could multiplicate in the presence of acyclovir concentrations up to 100 μg/ml, was tested.

The plaque reduction test was carried out according to the procedures described in Example 1.

The medicaments were used in the followinσ concentrations and combinations: μg/ml μg/ml μg/ml + MTX 0.1 μg/ml μg/ml + MTX 0.1 μg/ml + ACV 5.6 μg/ml μg/ml + ACV 5.6 μg/ml μg/ml + ACV 5.6 μg/ml

μg/ml

Six different tests were performed, each of them being carried out in quadruplicate. Four plates in which ACV at a 5.6 μg/ml concentration had previously been incorporated., inoculated with the ACV-resitant virus, were used as the controls.

The summarized results, expressed as percent mean inhibition against virus multiplication, calculated on the basis of the reduction percentages obtained in each test, are reported in Table 5.

Table 5. Percent inhibition of the multiplication of a HSV-2 strain made resistant to ACV 100 μg/ml, evaluated by the plaque reduction test on VERO cell lines. Mean of 6 different tests, each carried out in quadruplicate

Drugs and coneen- % decrease trations (μg/ml)

IDC 1 - 15.05 MTX 0.1 33.28

IDC 1 + MTX 0.1 61.73

IDC 1 + MTX 0 . 1 + ACV 5 . 6 67 . 93

IDC 1 + ACV 5. 6 - 8 . 4

MTX 0.1 + ACV 5.6 66 ACV 5,6 (controls) 0

IDC = 5-iodo-2 '-deoxycytidine

MTX = methotrexate

ACV = acyclovir % reduction = percent reduction of the plaques compared with dishes containing ACV alone, used as controls.

From the above Table, it is evident that acyclovir alone is completely ineffective in inhibiting the growth of the resistant virus.

The same applies to IDC and the IDC + ACV combination, as expected as a confirmation that the two medicaments act with the same action mechanism. Some inhibition is exerted by methotrexate, which blocks an alternative synthetic pathway of viral DNA. The highest decrease in viral multiplication, by 60-70%, was obtained with the IDC+MTX e ACV+MTX combinations, whereas the combination of the three medicaments IDC+MTX+ACV gave no supplementary advantages. Even more evident is the inhibition exerted on a HSV-1 acyclovir-resistant strain.

In this case, in two different experiments, each carried out in quadruplicate, a 100% plaque reduction was obtained with the tested combinations. The confirmation of the antiviral activity of the combinations of the invention also against resistant viruses was obtained in the in vivo test, performed according to the procedures described in Example 3.

Male guinea-pigs inoculated with HSV-1 virus resistant to ACV 100 μg/ml were treated in different dorsal areas with the combinations 1% IDC + 0.1% MTX, 0.5% IDC + 0.05% MTX, 1% ACV + 0.1% MTX, 5% ACV + 0.5% MTX compared with the single medicaments 5% ACV and 1% IDC. On the fifth day of treatment, the areas treated with the IDC+MTX e ACV+MTX combinations, at both the tested concentrations, showed abortive lesions that quickly healed, leaving only a slight topical irritation. Conversely, the areas treated with the single medicaments 5% ACV and 1% IDC were swollen and reddened and showed lesions at the stage of serous pustule.

The above data further corroborate the effective anti-herpes action of combinations consisting of a nucleoside antiviral compound and methotrexate.

The combinations of the invention can be administered, depending on the type of pathology to be treated, by any route, and precisely by the oral, rectal, parenteral, topical, vaginal, nasal routes and they can be formulated in any forms: tablets, capsules, powders or granules, solutions, suspensions or emulsions, suppositories, vials, ointments, creams or gel, eye-drops, foams or sprays.

Therefore, another object of the invention is provided by pharmaceutical compositions containing therapeutically effective amounts of an antiviral active ingredient consisting of a purine or pyrimidine derivative in combination with a folic acid competitive antagonist and with pharmaceutically and biologically compatible excipients. The amount of the active ingredients can vary depending on the type of composition, the prescriptions and the severity of the condition to treat.

The two active components, respectively consisting of a purine or pyrimidine nucleoside analogue and of a folate antagonist, will be present in ratios from 1:0.01 to 1:0.5.

Depending on the formulation, the nucleoside compound will be either in unit form from 100 to 500 mg or in a concentration from 0.1 to 50%. Example 5

Gel compositions of IDC + MTX

COMPONENTS (in %)

5-Iodo-2 '-deoxycytidine 1.00 0.50 active principle

Methotrexate 0.10 0.05 active principle

Dimethylsulfoxide 10.00 10.00 penetration enhancer p-Hydroxybenzoic acid methyl and propyl esters 0.21 0.21 preservants

Polymerized acrylic acid 0.7 0.7 gelling agent

Purified water q.s. to 100 100 carrier

Example 6

Ready to use or extemporaneous solutions of IDC + MTX

COMPONENTS (in %)

5-Iodo-2 '-deoxycytidine 1.00 3.00 active principle

Methotrexate 0.10 0.30 active principle

Dimethylsulfoxide 70.00 70.00 penetration enhancer

Propylene glycol q.s. to 100 100 carrier

Example 7

Ophthalmic ointments of IDC + MTX

COMPONENTS (in %)

5-Iodo-2 '-deoxycytidine 1.00 0.50 active principle Methotrexate 0.10 0.05 active principle

White vaseline q.s. to 100 100 carrier Example 8

Gel compositions of ACV + MTX

COMPONENTS ( in %)

Acyclovir 5.00 1. 00 active principle Methotrexate 1.00 0 . 10 active principle Dimethyl sulf oxide 10.00 10 .00 penetration enhancer p-Hydroxybenzoic acid methyl and propyl esters 0 .21 0 .21 preserving agent

Polymerized acrylic acid 0 .7 0 . 7 gelling agent

Purified water q. s . to 100 100 carrier

Example 9

Ready to use or extemporaneous solutions of ACV + MTX

COMPONENTS (in %)

Acyclovir 3.00 10.00 active principle Me hotrexate 0.30 1.00 active principle Dimethylsulfoxide 70.00 65.00 penetration enhancer

Propylene glycol q.s. to 100 100 carrier

Example 10

Ophthalmic ointments of ACV + MTX

COMPONENTS (in %)

Acyclovir 5.00 1.00 active principle

Methotrexate 0.50 0.10 active principle

White vaseline q.s. to 100 100 carrier Example 11

Parenteral compositions of IDC + MTX

COMPONENTS (in mg) lyophilized bottles

5-Iodo-2 '-deoxycytidine 50.000 25.000 active principle

Methotrexate sodium salt 5.484 2 . 924 active principle Sodium chloride 17.200 17.200 isotonic agent

Sodium hydroxide 45.000 45.000 pH regula¬ tion agent

Example 12

Enteral compositions of IDC + MTX

COMPONENTS (in mg) tablets 5-Iodo-2 ' -deoxycytidine 50 .00 25.00 active principle

Methotrexate 5.00 2.50 active principle

Lactose 97.00 48.50 diluent

Microcrystalline cellulose 50.00 25.00 binder Crospovidone 10.00 5.00 disaggregei tion agent

Polyvinylpyrrolidone 5.00 2.50 binder Magnesium stearate 3.00 1.50 lubricant Example 13

Parenteral compositions of ACV + MTX

COMPONENTS (in mg) lyophilized bottles

Acyclovir 50.000 25.000 active principle

Methotrexate sodium salt 5.484 2.924 active principle Sodium chloride 17.200 17.200 isotonic agent

Sodium hydroxide 45.000 45.000 pH regula¬ tion agent

Example 14

Enteral compositions of ACV + MTX

COMPONENTS (in mg) tablets Acyclovir

Methotrexate

Lactose 9

Microcrystalline cellulose 5 Crospovidone

PolyvinyIpyrrolidone Magnesium stearate

Claims

1. Compositions having antiviral activity, containing purine or pyrimidine nucleoside analogues in combination with a folic acid antagonist.

2. Compositions according to claim 1, wherein the purine or pyrimidine nucleoside analogue is selected from 5-iodo-2 ' -deoxyuridine, 5-iodo-2 'deoxycytidine and acyclovir.

3. Compositions according to claim 1, wherein the folic acid antagonist is methotrexate.

4. Compositions according to claims 1-3, in combination with at least one pharmaceutically or biologically compatible excipient.

5. Compositions according to claim 4, for the oral, parenteral, rectal, topical, nasal or vaginal administrations.

6. Compositions according to claims 4 and 5, wherein the active ingredients, respectively purine or pyrimidine nucleoside analogues and folic acid antagonist, are present in ratios from 1:0.01 to 1:0.5.

7. Compositions according to claims 4 to 6 , wherein the purine or pyrimidine nucleoside is present in ratios from 100 to 500 mg per unit dose or in a concentration from 0.1 to 50%.

8. The combined use of a purine or pyrimidine nucleoside analogue and a folic acid antagonist for the treatment of viral infections caused by a virus of the Herpes group.