WO2022013813A1 - Compound and pharmaceutical composition for use in treating human cytomegalovirus infections - Google Patents

Abstract

A triazole antifungal compound or a pharmaceutically acceptable salt thereof or a prodrug thereof for use as an antiviral in the method for treating and/or preventing human cytomegalovirus infections.

Description

COMPOUND AND PHARMACEUTICAL COMPOSITION FOR USE IN TREATING HUMAN CYTOMEGALOVIRUS INFECTIONS

DESCRIPTION

The present invention relates to a triazole antifungal compound and a pharmaceutical composition comprising said compound for use as an antiviral in the method for treating human cytomegalovirus infections.

BACKGROUND

Human cytomegalovirus (HCMV) is a ubiquitous beta-herpesvirus that infects 60% to 80% of the human population worldwide.

Although HCMV rarely causes symptomatic disease onset in immunocompetent individuals, it is still responsible for a variety of serious diseases in immunocompromised patients, and furthermore, once HCMV has been contracted, it remains latent within the body throughout life and can reactivate when the immune system is weakened, even in healthy patients (Griffiths P et al., J Pathol 2015).

HCMV is also an opportunistic pathogen, responsible for diseases such as pneumonia, gastrointestinal diseases and retinitis, in patients with primary or secondary immunodepression.

In addition to this, HCMV is the leading cause of birth defects, including severe ones, in newborns, such as deafness and central nervous system disorders (Britt WJ et al., Viruses 2018).

To date, there are no vaccines available to prevent HCMV infections, nor are there any drugs approved for the treatment of congenital infections (Manicklal S et al., Clin Microbiol Rev 2013).

Disadvantageously, moreover, the treatment of HCMV infections has been further complicated in recent years by the emergence of HCMV strains that are resistant to the drugs normally in use (Meesing A et al., Drugs 2018).

To date, only a few antiviral agents are approved for use against HCMV: ganciclovir (GCV), the prodrug thereof valganciclovir, foscarnet (FOS), acyclovir, the prodrug thereof valacyclovir, cidofovir (CDV), and the more recent letermovir (Mercorelli B et al., Rev Med Virol 2008).

The targets of the aforesaid antiviral agents are essentially the proteins encoded by the virus, in particular DNA polymerase and viral terminase (Britt WJ et al., Antiv Res 2018).

Specifically, GCV, the most widely used anti-HCMV drug, and CDV are nucleoside analogues that function as terminators of DNA synthesis, while FOS inhibits HCMV DNA polymerase by mimicking the structure of pyrophosphate, a by-product of the polymerisation reaction.

The discovery of such antiviral agents has contributed to improving the management of the anti-HCMV therapy; however, the aforesaid compounds have some limitations such as, for example, low bioavailability, non-negligible toxicity and limited efficacy (Mercorelli B et al. , Rev Med Virol 2008).

Moreover, as these anti-HCMV drugs share the same mechanism of action, the emergence of drug-resistant viral strains, mentioned above, is an increasing problem in the management of HCMV diseases.

Still disadvantageously, strains of mutant viruses resistant to a drug often also show cross-resistance towards other drugs (Villarreal EC et al., Prog Drug Res 2003).

There is therefore a need to identify new anti-HCMV compounds that have a good safety profile, a better pharmacokinetic profile than the known drugs in use and, in addition, that are directed against targets other than viral DNA polymerase.

In particular, there is a need for new pharmaceutical compositions with one or more improved characteristics compared to the currently available compositions.

It is therefore an object of the present invention to provide a new compound with antiviral action for use in the treatment and/or prevention of infections caused by HCMV.

It is further an object of the present invention that such a compound interacts with a different target than the targets of known antiviral compounds, so as to provide a drug with an alternative mechanism of action to those known to treat and/or prevent HCMV infections.

It is further an object of the present invention that the use of such a compound by a subject for the treatment and/or prevention of HCMV infections does not result in the emergence of HCMV strains that are resistant to said compound. Furthermore, it is an object of the present invention that the use of such a compound or a pharmaceutical composition comprising such a compound exhibits fewer side effects than known antiviral drugs.

Said objects are achieved by the use of a triazole antifungal compound as an antiviral in the method for treating and/or preventing human cytomegalovirus infections, in accordance with independent claim 1.

The objects are also achieved by a pharmaceutical composition comprising a therapeutically effective amount of said compound and at least one pharmaceutically acceptable excipient for use as an antiviral in the method for treating and/or preventing HCMV infections, in accordance with claim 11.

The objects are further achieved by a pharmaceutical composition comprising a therapeutically effective amount of a combination of the aforesaid triazole compound and one or more antiviral agents and at least one pharmaceutically acceptable excipient and, further, by the use of said pharmaceutical composition in the method for treating and/or preventing human cytomegalovirus infections in accordance with claims 20 and 12, respectively. Further features of the invention are described in the dependent claims.

The features and advantages of the present invention will be apparent from the detailed description given below, and from the embodiments provided by way of non-limiting examples.

DESCRIPTION OF THE INVENTION

The inventors of the present invention carried out a massive screening on the action of known drugs towards FICMV and several follow-up studies in case of established antiviral action, in order to identify new compounds and targets for the treatment of FICMV viral infections.

Surprisingly, some azole compounds with known antifungal activity were also found to have antiviral action towards FICMV.

The term “azoles” refers to those organic compounds having at least one N-heterocyclic pentatomic ring containing two nitrogen atoms, the imidazoles, or three nitrogen atoms, the triazoles.

These azole antifungal compounds comprise in particular the triazole compounds posaconazole (PCZ), isavuconazole (ICZ), itraconazole (ITZ), fluconazole (FCZ) and voriconazole (VCZ), and the imidazole compounds miconazole (MCZ), ketoconazole (KTZ), clotrimazole (CTZ) and econazole (ECZ).

The antiviral activity of these compounds was assessed by means of plaque reduction assays (PRA), as reported in Examples 2.1, 2.2.1 and 2.2.2.

The aforesaid compounds are known to possess antifungal activity towards a wide spectrum of pathogenic fungi such as for example C. albicans, C. tropicalis, C. parapsilosis, C. neoformans, Coccidioides, Histoplasma capsulatum, Aspergillus and others.

In particular, some triazole-structured antifungal compounds were found to possess a high antiviral activity, as shown by the data in Tables 1, 2 and 3. Preferably, such triazole compounds are selected from the group comprising posaconazole, isavuconazole and itraconazole.

Among these triazole compounds, PCZ and ICZ have proved to have the greatest antiviral activity towards HCMV.

These compounds were therefore analysed in more detail in order to identify their mechanism of action towards the virus.

The antiviral activity of the triazole antifungal compounds has been found to involve the inhibition of the human cytochrome enzyme P450 51 (also called hCYP51 or lanosterol-14a-demethylase).

The fungal enzyme CYP51 represents the known target of PCZ and ICZ during the treatment of fungal infections.

Surprisingly, the inventors of the present invention discovered that the triazole compounds interfere with the human enzyme CYP51 by inhibiting HCMV replication.

Additionally, the inhibition of hCYP51 by the triazole compounds PCZ and ICZ was found to cause a decrease in the viral load of HCMV, supporting the hypothesis that the virus exploits this human enzyme for its replication.

The triazole compounds of the present invention thus represent a new and alternative therapeutic instrument for the treatment and prevention of infections caused by HCMV.

Additionally, as the target of these compounds is a human enzyme, i.e. an enzyme of the host organism, the use of these compounds in the method for treating HCMV infections allows to overcome the problems of viral resistance of the antiviral agents of known type.

A triazole antifungal compound or a pharmaceutically acceptable salt thereof or a prodrug thereof for use as an antiviral in the method for treating and/or preventing HCMV infections is therefore an object of the present invention.

The term “pharmaceutically acceptable salt” means a salt of the aforesaid triazole antifungal compound prepared with pharmaceutically acceptable non toxic organic or inorganic acids or bases.

The term “prodrug” is used to define a precursor compound of the aforesaid triazole compound which does not have its own intrinsic pharmacological activity, or whose pharmacological activity is insufficient but which, once introduced into the body, undergoes one or more biotransformations of chemical or enzymatic type which transform it into the pharmacologically active compound.

An example of this prodrug not to be considered as limiting is the compound isavuconazonium sulphate, a prodrug of the azole compound ICZ.

Specifically, according to the invention, the triazole compound interferes with the hCYP51 enzyme to inhibit HCMV replication.

Preferably, the aforesaid compound is selected from the group comprising PCZ, ICZ and ITZ.

From the analyses reported subsequently, the compounds that proved to have the greatest anti-HCMV action were the PCZ and ICZ compounds, with an EC_5O equal to about 3.3 mM and 7 pM, respectively, as shown in Tables 1 and 3.

According to one aspect of the invention, the triazole antifungal compound for use in the method for treating and/or preventing HCMV infections is chosen from PCZ and ICZ.

According to another aspect of the present invention, the triazole antifungal compound for use in the method for treating and/or preventing HCMV infections is PCZ.

As shown by the graph in Fig. 1A, PCZ advantageously possesses a dose- dependent inhibitory action on HCMV viral replication.

In order to assess whether the antiviral activity of PCZ is due to its cytotoxicity, the cytotoxicity of PCZ towards HFF cells was also determined, as reported in Example 1.3.

Advantageously, the antiviral activity of PCZ is not due to a cytotoxic action, as reported by the results shown in Table 1. In fact, no toxic effects of PCZ on cell viability were found at concentrations up to 250 pM.

In order to assess the antiviral activity spectrum of PCZ, this compound was also tested on three different HCMV viral strains isolated from clinical samples (TB40-UL32-EGFP, VR1814 and 388438U), as reported in Example 2.2.1. Advantageously, the antiviral activity of PCZ is independent of the type of target HCMV strain, in fact the calculated EC₅₀ value was comparable between the different strains, as shown in Table 2.

Still advantageously, PCZ possesses an antiviral activity also towards HCMV strains that are resistant to currently used antiviral drugs as shown by the results of Example 2.2.1.

In particular, PCZ is active against viral strains with mutations in the UL54 gene, which are known to be HCMV strains resistant to ganciclovir (strain 759^rD100), to ganciclovir and cidofovir (strain GDG^rP53) and to foscarnet and acyclovir (strain PFA^rD100).

PCZ thus represents an alternative therapeutic instrument for use in the treatment of viral infections caused by known drug-resistant strains of HCMV. Still advantageously, the antiviral action of PCZ towards HCMV is not cell lineage dependent; in fact, as highlighted by the results shown in Table 2, the EC_5O values measured in epithelial cells (4.2 mM), in endothelial cells (4.8 pM) and in fibroblasts (3.7 pM) are comparable between them.

Additionally, these EC₅₀ values were determined with an HCMV strain which is naturally resistant to the antiviral drugs ganciclovir and cidofovir (TR strain), further confirming the antiviral activity of PCZ towards HCMV strains resistant to DNA polymerase inhibitors.

Again advantageously, the treatment of the cells with PCZ significantly reduces the infectivity of HCMV viral progeny, with an increase in the viral particle/PFU ratio by about 7-10 fold compared to HCMV-infected control cells treated with DMSO, as shown in Figure 5B.

According to another aspect of the present invention, the triazole antifungal compound for use in the method for treating and/or preventing HCMV infections is ICZ.

Advantageously, ICZ has been shown to have an inhibitory action against the replication of different HCMV strains in HFF cells, including clinical isolates and strains resistant to antiviral drugs currently used in the therapy.

As can be seen from the results of the tests shown below in Table 3, ICZ has an EC50 of about 7 pM calculated in HFF cells infected with HCMV AD169.

In order to assess the antiviral activity spectrum of ICZ, the compound was also tested against different HCMV viral strains, two of which were isolated from clinical samples (TB40-UL32-EGFP and VR1814), as reported in Example 2.2.2.

Advantageously, the antiviral activity of ICZ is independent of the HCMV strain used; in fact, the calculated EC₅₀ value was comparable between the different strains, as shown in Table 3. In particular, ICZ has an antiviral activity towards HCMV strains isolated from clinical samples (TB40-UL32-EGFP and VR1814).

Still advantageously, ICZ is active against viral strains resistant to ganciclovir and cidofovir (strain GDG^rP53) and to foscarnet and acyclovir (strain PFA^rD100).

ICZ thus represents an alternative drug for use in the treatment of viral infections caused by FICMV strains, particularly caused by known drug- resistant strains.

According to the invention, the triazole antifungal compound is used in the method for treating and/or preventing FICMV infections.

According to one aspect of the invention, such a method of treatment and/or prevention comprises administering to a subject a therapeutically effective amount of the aforesaid compound.

The term “administering” means introducing the triazole antifungal compound of the present invention into the body by any known route of administration.

This route of administration comprises, by way of example to be considered as non-limiting, the oral, sublingual, buccal, rectal, vaginal, ocular, auricular, nasal, topical or systemic dermal route and the transdermal route, or the injective route which involves the administration by intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intranodal or intrasplenic injection, or the inhalation or nebulisation route.

Preferably, this route of administration is selected from the oral, sublingual, rectal or injective route.

More preferably, this route of administration is the oral route.

The term “subject” means an individual. It is specified that “subject” may include, for example, domestic pets, such as cats, dogs, etc., livestock (e.g. cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g. mice, rabbits, rats, guinea pigs, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish and any other animal.

The subject is preferably a mammal, such as a primate or man, more preferably the subject is a human being.

According to one aspect of the invention, the subject is a paediatric subject.

The term “paediatric subject” means an individual aged between birth and eighteen years, as defined in Regulation (EC) No. 1901/2006.

The term “therapeutically effective amount” means the amount of the triazole compound that is sufficient to treat the infection herein intended of a subject, but low enough to avoid serious side effects, in a reasonable risk/benefit ratio, according to the intent of the medical criteria.

This therapeutically effective amount may depend on different factors such as, by way of non-limiting examples, the route of administration chosen, the severity of the disease to be treated, the age, height, weight and the physical condition of the subject to be treated, the medical history of the subject to be treated, the duration of the treatment, the nature of concomitant therapies, and the desired therapeutic effects.

According to one aspect of the invention, the triazole antifungal compound of the invention is in the form of a pharmaceutical composition comprising a therapeutically effective amount of said triazole compound or a pharmaceutically acceptable salt thereof or a prodrug thereof as defined above, and at least one pharmaceutically acceptable excipient.

The term “pharmaceutically acceptable excipient” means a compound or a mixture thereof that is optimal for use in a formulation designed for the treatment and/or prevention of a disease, in particular of HCMV infections. Suitable excipients for this use are sweeteners, diluents, disaggregants, glidants, colourants, binders, lubricants, stabilisers, adsorbents, preservatives, surfactants, humectants, flavourings, softeners, film-forming substances, emulsifiers, wetting agents, release retardants, colloids, non-permeant compounds, preservatives and mixtures thereof, and others per se known from the pharmaceutical industry.

The expert in the field is able to determine whether and to what extent particular pharmaceutical excipients may serve one or more functions in relation to how much they are present in the composition, what other excipients are present and the route of administration of the composition.

More specifically, the pharmaceutical composition of the present invention may be formulated in a form suitable for administration by oral, sublingual, buccal, rectal, vaginal, ocular, auricular, nasal, topical or systemic dermal route and by transdermal route, or by injection route such as intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intranodal or intrasplenic injection, or by inhalation or by nebulisation.

Preferably, the pharmaceutical composition of the present invention is formulated in a form suitable for oral, sublingual, rectal or injective administration.

Non-limiting examples of a form suitable for oral administration are tablets, capsules, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets and cachets or formulations suitable for inhalation such as aerosols, solutions or powders.

Non-limiting examples of a form suitable for administration by injection route are aqueous buffer solution and oil suspension.

It is specified that “administration by injection route” means administration by intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intranodal or intrasplenic route.

Preferably, the aforesaid composition of the invention is in a form suitable for oral administration.

It is not ruled out that, according to variant embodiments of the aforesaid composition, it is in a form suitable for topical administration.

Non-limiting examples of a form suitable for topical administration are cream, ointment, pomade, solution, suspension, eye drops, ovule, aerosol, spray, powder and gel.

Advantageously, both PCZ and ICZ are compounds approved for clinical therapy (as antifungals) for systemic administration, particularly for oral administration.

Still advantageously, PCZ and ICZ are triazole compounds used in therapy also for use in paediatric subjects.

PCZ and ICZ therefore represent anti-HCMV drugs alternative to the known ones that can be used for the clinical treatment of infections caused by HCMV in paediatric subjects.

The use of such a composition in the method for treating and/or preventing HCMV infections is therefore also part of the present invention.

Advantageously, the triazole compounds of the present invention are drugs already approved for use in humans as antifungals, supporting the fact that their use as antivirals in the form of pharmaceutical compositions shows a minimal in vivo toxicity.

Furthermore, the use of this composition in the method for treating and/or preventing HCMV infections in paediatric subjects is part of the present invention.

The inventors have also surprisingly found that the combination of the triazole compound of the invention, or a pharmaceutically acceptable salt thereof or a prodrug thereof, and one or more antiviral agents produces a synergistic effect in inhibiting HCMV replication in infected cells.

The term “antiviral agent” refers to any molecule or composition in which the molecule or composition is useful in treating viral infections.

The term “synergistic effect” means that the therapeutic effect of a combination comprising two or more agents is greater than the therapeutic effect of a treatment in which a single agent is used alone. In addition, the synergistic effect of a combination of one or more agents allows the use of a lower dosage of one or more of the agents and/or a less frequent administration of the aforesaid agents to the subject. Furthermore, a synergistic effect may result in an improved effectiveness of the agents in preventing, managing or treating the disease or pathological conditions and may avoid or reduce adverse or undesirable side effects associated with the use of one of the agents alone.

In addition, the synergistic effect may result in a better ability to reduce the emergence of drug-resistant virus strains.

According to one aspect of the invention, therefore, the triazole compound is used in a method for treating and/or preventing HCMV infections which comprises administering to a subject a therapeutically effective amount of a combination of the aforesaid triazole antifungal compound or of a pharmaceutical composition as described above and of an antiviral agent. According to one aspect of the invention, such an antiviral agent is selected from the group comprising ganciclovir, foscarnet (FOS), cidofovir, valganciclovir, acyclovir, valacyclovir, penciclovir, famciclovir, fomivirsen, maribavir, alkoxyalkyl derivatives of cidofovir such as hexadecyloxypropyl- cidofovir (CMX001) and analogues, alkoxyalkyl derivatives of 9-(S)-[3-hydroxy- 2-(phosphonomethoxy)propyl]adenine (HPMPA) as octadecyloxyethyl- HPMPA, hexadecyloxypropyl-HPMPA and analogues, alkoxyalkyl derivatives of 1-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine (HPMPC) as octadecyloxyethyl-HPMPC, hexadecyloxypropyl-HPMPC and analogues, alkoxyalkyl derivatives of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)- propyljguanine (HPMPG) as octadecyloxyethyl-HPMPG, hexadecyloxypropyl- HPMPG and analogues, alkoxyalkyl derivatives of 9-(S)-[3-hydroxy-2- (phosphonomethoxy)-propyl]-2,6-diaminopurine (HPMPDAP) such as octadecyloxyethyl-HPMPDAP, hexadecyloxypropyl-HPMPDAP and analogues, and alkoxyalkyl derivatives of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]- 2-amino-6-cyclopropylaminopurine (HPMP-cPrDAP) such as octadecyloxyethyl-HPMP-cPrDAP hexadecyloxypropyl-HPMP-cPrDAP and analogues, and letermovir (LMV).

Preferably, the aforesaid antiviral agent is a viral DNA polymerase or viral terminase inhibitor agent.

Or, such an antiviral agent may be an antisense oligonucleotide against viral genes, such as for example fomivirsen.

According to one aspect of the present invention, the aforesaid antiviral agent is ganciclovir (GCV).

It was surprisingly found that in the presence of the triazole antifungal compound, in particular in the presence of PCZ, the potency of GCV towards HCMV is about 10 times greater than a treatment with the administration of GCV alone (EC₅₀ of GCV in the presence of PCZ = 0.13 mM versus EC₅₀ of GCV in the presence of DMSO = 1.44 pM, fig. 6).

Advantageously, PCZ acts synergistically with GCV in inhibiting HCMV replication at concentrations that are below their respective EC₅₀ as single drugs and that are achievable in the plasma of patients undergoing treatment with such drugs.

Further, in order to assess the combined inhibitory effect of PCZ and GCV on HCMV replication, plaque reduction assays were performed with combinations of different doses of the two compounds.

As shown by the results in Table 5, the combination of PCZ and GCV results in a synergistic antiviral action in all combinations tested.

In fact, the Combination Index (Cl) value calculated using the Chou & Talalay’s method (Chou TC, Pharmacol Rev 2006) was in all cases <0.9. Advantageously, together with the synergistic effect of the combination of the azole compound and GCV, no evident cytotoxicity was found, supporting the hypothesis that the reduction in the number of viral plaques is due to the combination of the antiviral action of two compounds with different target and mechanism of action.

Still advantageously, in addition to decreasing the number of viral plaques, the combined use of PCZ with GCV has been shown to also allow the size of the aforesaid plaques to decrease.

Advantageously, also with regard to the triazole compound ICZ, the experiments treating HCMV-infected cells with GCV, FOS or LMV alone and in combination with ICZ have shown that the combination of each of these antiviral agents with ICZ has a certain synergistic effect against viral replication, as shown in the results in Table 7.

Additionally, no statistically evident cytotoxic effect was observed in the combined treatment of these antiviral agents with ICZ.

Indeed, thanks to the calculation of the Dose Reduction Index (DRI), it was possible to identify that, by way of example, when GCV, FOS or LMV are used in combination with ICZ in vitro, 95% inhibition of HCMV replication can be achieved reducing the dose of ICZ by 7, 5 and 3 times, respectively, compared to using ICZ alone.

Therefore, the pharmaceutical composition defined above comprising a therapeutically effective amount of a combination of a triazole antifungal compound or a pharmaceutically acceptable salt thereof or a prodrug thereof, as defined above, including variants, and one or more antiviral agents and at least one pharmaceutically acceptable excipient is also part of the present invention.

Preferably, this triazole compound is selected from PCZ and ICZ.

According to one aspect of the pharmaceutical composition of the invention, the triazole compound is PCZ.

According to another aspect of the pharmaceutical composition of the invention, the aforesaid triazole compound is ICZ.

According to a further aspect of the pharmaceutical composition of the invention, it comprises a combination of PCZ and ICZ and one or more antiviral agents and at least one pharmaceutically acceptable excipient.

Also such a pharmaceutical composition comprising a therapeutically effective amount of the combination of the triazole compound, an antiviral agent and at least one excipient of the invention is preferably in a form suitable for oral, sublingual, rectal or injective administration.

More preferably, this composition is in a form suitable for oral administration.

In fact, the triazole compounds of the invention are approved for systemic use, particularly for oral use, ensuring antiviral efficacy by this route of administration, which is the route that provides a greater therapeutic compliance by the treated subject.

According to one aspect of the composition of the invention, said one or more antiviral agents are selected from the group comprising ganciclovir, foscarnet, cidofovir, valganciclovir, acyclovir, valacyclovir, penciclovir, famciclovir, fomivirsen, maribavir, alkoxyalkyl derivatives of cidofovir such as hexadecyloxypropyl-cidofovir (CMX001) and analogues, alkoxyalkyl derivatives of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) such as octadecyloxyethyl-HPMPA, hexadecyloxypropyl-HPMPA and analogues, alkoxyalkyl derivatives of 1-(S)-[3-hydroxy-2-(phosphonomethoxy)- propyl]cytosine (HPMPC) such as octadecyloxyethyl-HPMPC, hexadecyloxypropyl-HPMPC and analogues, alkoxyalkyl derivatives of 9-(S)- [3-hydroxy-2-(phosphonomethoxy)-propyl]guanine (HPMPG) such as octadecyloxyethyl-HPMPG, hexadecyloxypropyl-HPMPG and analogues, alkoxyalkyl derivatives of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]-2,6- diaminopurine (HPMPDAP) such as octadecyloxyethyl-HPMPDAP, hexadecyloxypropyl-HPMPDAP and analogues and alkoxyalkyl derivatives of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2-amino-6- cyclopropylaminopurine (HPMP-cPrDAP) such as octadecyloxyethyl-HPMP- cPrDAP, hexadecyloxypropyl-HPMP-cPrDAP and analogues, and letermovir. Preferably, the aforesaid antiviral agent is a viral DNA polymerase or viral terminase inhibitor agent.

Or, such an antiviral agent may be an antisense oligonucleotide against viral genes, such as for example fomivirsen.

According to one aspect of the composition of the invention, such one or more antiviral agents comprise ganciclovir.

According to a further aspect of the composition of the invention, such one or more antiviral agents comprise foscarnet.

According to another aspect of the composition of the invention, such one or more antiviral agents comprise letermovir.

The use of such a pharmaceutical composition as a medicinal product is also an object of the present invention.

In particular, such a pharmaceutical composition is used as an antiviral in the method for treating and/or preventing HCMV infections so as to achieve the above-mentioned advantages.

Furthermore, the use of this composition in the method for treating and/or preventing HCMV infections in paediatric subjects is part of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

- Figures 1A and 1B show the susceptibility of FICMV to the treatment with the azole antifungal compounds PCZ, KTZ, FCZ, ITZ and VCZ. Plaque reduction assays were performed in HFF cells infected with FICMV and treated with different doses of the indicated compounds (in concentrations from 0.1 to 25 mM) or infected and treated with GCV as a control. The treatments with PCZ and KTZ show an inhibitory effect on the replication of the dose-dependent type FICMV AD169 virus (Figure 1A).

The treatment with FCZ, ITZ and VCZ did not show a significant inhibitory effect on the viral replication (Figure 1B). ITZ was only tested up to a concentration of 10 pM due to its low solubility. In the graphs, the data represent the means ± standard deviations (error bars) of at least three independent experiments in duplicate.

- Figure 2 shows that PCZ dose-dependently inhibits the replication of different FICMV strains, shown in the figure. Plaque reduction assays were performed in HFF cells infected with the HCMV strains shown in the figure and treated with PCZ at different doses (in concentrations from 0.1 to 25 pM). In the graphs, the data represent the means ± standard deviations (error bars) of three independent experiments in duplicate.

- Figures 3A, 3B and 3C show that the inhibition of the hCYP51 enzyme of the host blocks the HCMV replication. (R)-N-(1-(3,4’-difluoro-[1 ,T-biphenyl]- 4-yl)-2-(1 H-imidazol-1 -yl)ethyl)-4-(5-phenyl-1 ,3,4-oxadiazol-2-yl)benzamide (VFV), PCZ and VCZ inhibit the in vitro enzymatic activity of the purified hCYP51 enzyme (2 min reaction). In the graph, the data represent the means ± standard deviations (error bars) of three independent experiments in duplicate (figure 3A). Plaque reduction assays were also performed in HFF cells infected with HCMV AD169 strains and treated with VFV inhibitor of the hCYP51 enzyme at different doses (in concentrations from 0.1 to 25 pM). In the graph, the data represent the means ± standard deviations (error bars) of at least three independent experiments in duplicate (figure 3B). In addition, viral progeny reduction assays were performed showing that VFV treatment dose-dependently inhibits viral progeny in HCMV-infected HFF cells. In the graph, the data represent the means ± standard deviations (error bars) of four independent experiments in duplicate (figure 3C).

- Figures 4A, 4B and 4C show that hCYP51 expression is activated by HCMV infection. Figure 4A shows the activation of the hCYP51 enzyme promoter in uninfected control (mock) U-373 MG cells or cells infected with HCMV AD169 or infected with HCMV inactivated by UV radiations. Data are expressed as Relative Luciferase Units (LU/FU, RLU), i.e. as luciferase activity units normalised for fluorescence units obtained from the expression of the co-transfected eGFP reporter gene. In the graph, the data represent the means ± standard deviations (error bars) of three independent experiments in duplicate. The values were statistically analysed by one-way ANOVA analysis followed by Tukey’s test for the multiple comparison. ***p<0.0001; ****p<0.0001. Figure 4B shows data relating to the mRNA of hCYP51 in HFF cells infected with HCMV, obtained by qPCR analysis at the times indicated in the figure. UL54 mRNA was analysed and used as a control of the infection progression at 24 and 48h p.i. The mRNA values were normalised with respect to cellular GAPDH values and the gene expression was reported as relative quantification (RQ) with respect to the control sample (mock cells for hCYP51 and HCMV-infected cells at 24h p.i. for UL54). In the graph, the data represent the means ± standard deviations (error bars) of three independent experiments in duplicate. Figure 4C shows the protein expression analysis of hCYP51 and of the viral protein IEA during virus replication. The host hCYP51 protein and the IE viral antigen (IEA) were analysed by Western Blot in uninfected HFF cells (mock, M) and in HFF cells infected with HCMV at MOI = 0.5 PFU/cell at the indicated post infection times (h p.i.). b-actin was used as a loading control protein. The molecular weights are expressed in kDaltons on the left.

- Figures 5A and 5B show the effects of hCYP51 enzyme inhibition on HCMV replication and on viral infectivity. Figure 5A shows how the pharmacological inhibition of the hCYP51 enzyme results in a reduction in the number of viral genomes in HFF cells infected with HCMV at MOI of 0.5 PFU/cell and treated with 10 mM PCZ or VFV, or DMSO 0.1% as a control, for 120 h. The HCMV genomic copies in the supernatant of each sample were quantified by qPCR. In the graph, the data represent the means ± standard deviations (error bars) of three independent experiments in quadruplicate. Data were analysed by one-way ANOVA analysis followed by Dunnett’s test for multiple comparisons. ***p<0.001; **p<0.005 compared to the control (infected and DMSO-treated sample). Figure 5B shows that the pharmacological inhibition of hCYP51 reduces the infectivity of viral particles. The particle/PFU ratio was obtained by dividing the number of HCMV genomes determined in supernatants derived from HFF cells infected with FICMV at MOI 0.5 PFU/cell and treated with the test compounds (determined by qPCR) with the viral particle titre obtained from the same sample volume (determined by titration in new HFF cells). In the graph, the data represent the means ± standard deviations (error bars) of three independent experiments in quadruplicate. Data were analysed by one-way ANOVA analysis followed by Dunnett’s test for multiple comparisons. *p<0.05; **p<0.005 compared to the control (infected and DMSO-treated sample).

- Figure 6 shows that the treatment with a therapeutic dose of PCZ increases the anti-FICMV activity of GCV. The antiviral activity of GCV against FICMV strain AD169 in the absence (+ DMSO) or in the presence (+ PCZ) of 3 mM PCZ, as determined by plaque reduction assay, is shown. In the graph, the data represent the means ± standard deviations (error bars) of at least three independent experiments in duplicate.

- Figure 7 shows the quantification of the hCYP51 induction in HFF cells infected with FICMV. The overexpression of the host hCYP51 protein in HFF cells infected with FICMV AD169 was analysed by Western Blot and densitometric analysis conducted with the ImageJ software. The hCYP51 signal was normalised with respect to the b-actin protein signal and plotted against the untransfected sample. In the graph, the data represent the means ± standard deviations (error bars) of four independent experiments. Data were analysed by one-way ANOVA analysis followed by Dunnett’s test for multiple comparisons. ***p=0.0001; ****p<0.0001 , compared to the control (mock cells).

- Figure 8 shows the immunofluorescence analysis of hCYP51 and of viral proteins during FICMV infection in live cells. The host hCYP51 protein and the IE viral antigen (IEA) were detected by immunofluorescence on uninfected HFF cells (Nl) and HFF cells infected with FICMV at MOI = 0.25 PFU/cell at the indicated post-infection times (h p.i.). Draq5 was used for nuclei staining. The white arrows in the panel of the images at 24 and 48h p.i. indicate the nuclei of uninfected HFF cells that are in the same field as the HCMV-infected cells. The progression of the FICMV replicative cycle is confirmed by the reniform appearance of the nuclei of HCMV-infected cells at 48h p.i.

- Figures 9A, 9B and 9C show that ICZ inhibits viral replication in a dose- dependent manner. Plaque reduction assays were performed in HFF cells infected with the FICMV strains shown in the figure and treated with ICZ at different doses (in concentrations from 0.1 to 25 mM). The cell viability curve in the presence of ICZ was obtained by means of MTT assay at 120 h (Figure 9A). The treatment with ICZ in HFF cells infected with FICMV dose- dependently decreases the production of viral progeny (Figure 9B). The treatment with ICZ also inhibits the viral replication of FICMV strains that are resistant to the inhibitor compounds of the viral DNA polymerase (Figure 9C). In the graphs, the data represent the means ± standard deviations (error bars) of at least three independent experiments in duplicate.

- Figure 10 shows that ICZ inhibits the enzyme activity of the hCYP51 enzyme in vitro. The graph shows the inhibition of the activity of the purified hCYP51 enzyme in the presence of VCZ, ICZ and PCZ (2-minute reaction). In the graph, the data represent the means ± standard deviations (error bars) of three independent experiments in duplicate.

EXAMPLES

The following examples are given in order to provide the person skilled in the art with a complete disclosure and description of how the compounds and compositions of the present invention have been evaluated. These examples are to be intended purely as non-limiting examples.

Example 1. Materials and methods Example 1.1. Materials, cells and viruses

Ganciclovir (GCV), foscarnet (FOS) and all the azole antifungal compounds used were from the company Sigma-Aldrich. Cidofovir (CDV, Vistide) was from the company Gilead Sciences. VFV ((R)-N-(1-(3,4’-difluoro-[1 ,T-biphenyl]-4- yl)-2-(1 H-imidazol-1 -yl)ethyl)-4-(5-phenyl-1 ,3,4-oxadiazol-2-yl)benzamide) was synthesized at Vanderbilt University, according to the method reported in Lepesheva G et al. , Tetrahedron Lett 2017. For the cellular assays, a 100X stock of VFV was prepared in 25% DMSO/34% 2-hydroxypropyl-p-cyclodextrin (Sigma) in water (v/v).

Human Foreskin Fibroblast (HFF), ARPE-19, and U-373 MG cells were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 100 U/ml penicillin and 100 pg/ml streptomycin sulfate (P/S, Life Technologies).

The human dermal microvascular endothelial cells (HMVECs) (CC-2543) were purchased from Clonetics and cultured in endothelial growth medium (EGM) (Clonetics). The cell cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.

HCMV (strain AD169) was purchased from ATCC. HCMV strain TB40E-UL32- EGFP (courtesy of C. Sinzger, University of Ulm, Germany) has been previously described (Sampaio KL et al. , J Virol 2005). HCMV strain VR1814 (courtesy of G. Gerna, IRCCS Policlinico San Matteo, Pavia) was obtained from a clinical sample of a congenital infection, as described in Revello MG et al., J Infect Dis 2001. HCMV strain 388438U was isolated from a clinical urine sample at the Microbiology and Virology Operating Unit of the Hospital of the University of Padua. The antiviral drug-resistant HCMV strains were obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, MD, USA) as described previously (Mercorelli B et al., Antimicrob Agents Chemother 2009). HCMV TR was reconstituted by transfection in HFF cells with the corresponding TR-BAC prepared from the GCV- and CDV-resistant TR clinical strain isolated from an ocular specimen (Murphy E et al., Proc Natl Acad Sci USA 2003). The reconstitution of the BAC-derived TR strain in fibroblasts allows the generation of infectious viral particles that retain the ability to infect both epithelial and endothelial cells (Cavaletto N et al., J Virol 2015).

Example 1.2 Plaque reduction assay

Plaque reduction assays (PRA) with HCMV have been performed according to what has been previously described (Loregian A et al., Antimicrob Agents Chemother 2010). Briefly, HFF, ARPE-19 and HMVEC cells were seeded at a density of 1.5x10^s cells per well in 24-well plates. On the following day, the cells from each well were infected with 80 Plaque Forming Units (PFU) of virus in DMEM without serum at 37°C. After 2h p.i., the inocula were removed, the cells were washed and the media containing various concentrations of each compound, 2% FBS and 0.6% methylcellulose were added. After 10 days of incubation at 37°C, the cell monolayers were fixed, stained with crystal violet and the viral plaques were counted. Example 1.3 Cytotoxicity assay

The cytotoxicity of the tested compounds was determined by a method with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT; Sigma- Aldrich) as described previously (Loregian A et al. , Chem Biol 2006).

Example 1.4 Viral progeny reduction assays

To perform the viral progeny reduction assays, HFF cells were seeded at a density of 2x10⁴ cells per well in 96-well plates, incubated overnight, and infected on the following day with FICMV AD169 at MOI = 0.1-0.05 PFU/cell. After viral adsorption for 2h at 37°C, the cells were washed and incubated with 0.2 ml of medium containing 5% FBS in the absence or in the presence of the tested compounds. The plates were then incubated for 5 days at 37°C and subjected to a freeze/thaw cycle. The viral titres were determined by transferring a 0.1 ml aliquot from each well to a monolayer of HFF cells in 96- well plates, followed by serial dilution 1:5 in the plate. The cells were incubated for 7 days and, at the end of the incubation, they were fixed, stained and the number of plaques was determined.

Example 1.5 In vitro enzyme assays

The recombinant human protein CYP51 and its redox correspondent NADPFI- cytochrome P450 reductase (CPR) were expressed in Escherichia coli and purified as previously reported (Flargrove TY et al, J Lipid Res 2016). The reaction mixture contained 0.5 mM hCYP51 and 1.0 pM CPR, 100 pM L-a-1,2- dilauroyl-snglycerophosphocholine, 0.4 mg/ml isocitrate dehydrogenase, and 25 mM sodium isocitrate in 50 mM potassium phosphate buffer (pH 7.2) with 10% glycerol (v/v). After addition of radiolabelled ([3-3FI])lanosterol (about 4000 dpm/nmol; dissolved in 45% FIPCD, w/v, final concentration of 50 pM) and inhibitors (in concentrations from 0.1 to 250 pM), the resulting mixture was pre-incubated for 30 sec at 37°C in a vibrating bath. The reaction was initiated by the addition of 100 pM NADPFI and terminated by the extraction of the sterols with 5 ml ethyl acetate. The extracted sterols were dried, dissolved in methanol and analysed by means of a reversed-phase FIPLC system (Waters) provided with a b-RAM detector (INUS Systems) using a NovaPak octadecylsilane (C18) column and a linear gradient with water/acetonitrile/methanol (10:4.5:4.5, v/v/v) (solvent A) and CFI₃OFI (solvent B), increasing from 0 to 100% B for 30 min at a flow rate of 1.0 ml/min. IC₅₀ values were calculated using GraphPad Prism 6 and plotted with the percentage of lanosterol converted against the inhibitor concentration and curve interpolation with non-linear regression (log(inhibitor) vs. normalised response - variable slope).

Example 1.6 Plasmids

The pCYP51-luc plasmid, containing the region of the -314/+343 promoter of the human gene CYP51 ( hCYP51 ) located upstream of the luciferase reporter gene, was kindly provided by D. Rozman (Centre for Functional Genomics and Bio-Chips Institute of Biochemistry, Faculty of Medicine University of Ljubljana, Slovenia) and has been previously described (Haider SK et al. , Mol Endocrinol 2002). pGAPDH-eGFP plasmids containing the promoter region of the cellular gene GAPDH upstream of the enhanced gene Green Fluorescent Protein {· eGFP ), have been previously described (Comely OA et al., J Antimicrob Chemother 2017) and used as a transfection control.

Example 1.7 Cell transfection and infection of HCMV

For HCMV transfection/infection experiments, U-373 MG cells were grown in 24-well plates and were co-transfected using calcium phosphate (Calcium Phosphate Transfection Kit, Sigma) with 1 pg of pCYP51-luc plasmid and 0.2 pg of pGAPDH-eGFP plasmid as a control for the normalisation of the transfection efficiency. On the following day, the transfected cells were either uninfected (mock) or infected with HCMV AD169 at MOI = 0.5 PFU/cell for 2h and incubated with medium with 5% FBS. After 48h, the luciferase activity and the eGFP expression were measured. In all experiments, the values were normalised by dividing the values of luciferase activity units (LU) by the fluorescence units (FU) obtained from the expression of eGFP and expressed as relative luciferase units (LU/FU, RLU).

For UV inactivation, the procedure reported previously was followed (Chaumorcel M et al., J Virol 2012). Briefly, HCMV diluted in DMEM without serum was exposed for 8 min to ultraviolet light (VL-6MC, 254 nM, 6w) at a distance of 4 cm. The virus inactivation was assessed by immunofluorescence. Example 1.8 Quantification of gene expression

HFF cells were seeded in 6-well plates at a density of 6x10⁵ cells/well and incubated overnight at 37°C.

In case of infection, cells were infected on the following day with HCMV AD169 at MOI = 1 PFU/cell for 2h and subsequently incubated with 5% FBS medium. The total RNA was extracted from the samples collected at different times using the total RNA Purification Plus Kit (Norgen Biotek) according to the protocol provided by the manufacturer. The cDNA was generated from RNA (2 pg) using random primers (Applied Biosystems) and M-MLV reverse transcriptase (Applied Biosystems). The qPCR reaction was carried out with SYBR green reagent (Applied Biosystems), according to the manufacturer’s instructions, using the 7900 HT Fast Real-Time PCR system (Applied Biosystems). The primers used were designed according to known knowledge in the field on the basis of the gene sequences to be sought for GAPDH, HCMV UL54 and hCYP51. The primers for hCYP51 are reported in Fink M et al, Endocrinology, 2005.

The quantification of the gene expression was determined using the “comparative ACT method” (AACT) using the GAPDH gene expression for the normalisation of the values.

Example 1.9 Western Blot

For the Western Blot analysis of the hCYP51 induction during infection with FICMV, sub-confluent HFF cells seeded in 6-well plates were infected with FICMV AD169 at MOI = 0.5 PFU/cell. The whole cellular protein extract was obtained at different post-infection times as described previously (Mercorelli B et al., Cell Chem Biol 2016) and analysed by Western Blot with the primary antibodies anti-IE1/IE2 (Argene-Biosoft), anti-hCYP51 (Sigma) and anti-Actin (Sigma).

The immunocomplexes were visualised by means of conjugated secondary antibodies FIRP-Rabbit IgG (H+L) (Santa Cruz), HRP-Mouse IgG (H+L) (Santa Cruz).

The densitometric analysis was performed with ImageJ software (https://imagej.nih.gov/ij/).

Example 1.10 Immunofluorescence and confocal microscopy For the laser scanning confocal microscopy analysis, HFF cells were infected with HCMV AD169 at MOI = 0.25 PFU/cell. At different times, the cells were fixed with 4% paraformaldehyde in PBS 1X for 15 min at room temperature and permeabilised with 0.1% Triton X-100 in PBS for 20 min at room temperature. After washing with PBS, the cells were incubated with 4% FBS in PBS for 1h at room temperature and then incubated with the primary antibodies shown in the previous example, diluted in 4% FBS in PBS 1X for 1h at 37°C under stirring. The cells were then washed with 4% FBS in PBS 1X and incubated with secondary antibodies Alexa FluorTM 488 Rabbit IgG (H+L) (Invitrogen), Alexa FluorTM 546 Mouse IgG (H+L) (Invitrogen) for 1h at 37°C. The nuclei were visualised by incubation for 20 min with Draq5 (1:8000 in PBS 1X). The cells were visualised using a Nikon Eclipse Ti-E microscope.

Example 1.11 Determination of the viral particle/PFU ratio For the determination of the FICMV particle/PFU ratio produced in the presence of the tested compounds, HFF cells were seeded at a density of 2x10⁴ cells per well in 96-well plates, incubated overnight, and infected on the following day with FICMV AD169 at MOI = 0.5 PFU/cell. After adsorption of the virus for 2h at 37°C, the cells were washed and incubated with 0.2 ml of culture medium containing 5% FBS in the presence or in the absence of the tested compounds. The plates were then incubated for 5 days at 37°C. At the end of the incubation, 0.05 ml of the supernatant were used to determine the number of viral particles produced under the various conditions of the experiment, while 0.05 ml were used to determine the viral titre on new HFF cells in monolayer as described previously, in order to determine the number of PFU present in an equal volume of supernatant.

For the determination of the viral particles, 0.05 ml of supernatant were incubated with 0.2% SDS and proteinase K for 1h at 56°C and then for 15 min at 95°C in order to inactivate proteinase K. Subsequently, viral DNA was extracted using the DNA purification kit (Promega) and quantified by qPCR as described previously. The particle/PFU ratio was determined by dividing the number of FICMV genomes by the number of PFU determined in the same volume of supernatant derived from the same sample.

Example 1.12 Quantification of the viral genome

For the quantification of FICMV genomes in 0.05 ml of supernatant obtained from different samples harvested at 120h p.i., the quantitative Real-Time PCR (qPCR) analysis was performed as described previously (Mercorelli B et al. , Antimicrob Agents Chemother 2009). The number of viral genomes was normalised with respect to the gene copies of cellular b-globin.

The primer sequences used for b-globin are reported in Mengoli C et al, J Med Virol, 2004.

Example 1.13 Drug combination studies

To evaluate the combined effect of PCZ and GCV and the combined effect of ICZ and known anti-HCMV drugs on HCMV AD169 replication, plaque reduction assays were performed as reported previously in Example 1.2 using 0.125X, 0.25X, 0.50X, 1X, 2X and 4X EC₅₀ for each combination of PCZ and GCV or ICZ and anti-HCMV drug, in equipotent ratio. The effects of the combination of the two drugs were assessed using the Chou-Talalay method (Chou TC, Pharmacol Rev 2006) with CalcuSyn software, version 2.0 (Biosoft, Cambridge).

Example 1.14 Statistical analysis

All statistical analyses were carried out using GraphPad Prism software version 6.0.

Example 2. Results

Example 2.1 Anti-HCMV activity of azole antifungal compounds The azole antifungal compounds of the invention were analysed by PRA assay according to the methods reported in Example 1.2, for assessing their anti- HCMV activity.

The results obtained are shown in Tables 1 and 3 and Figures 1A and 9A. It can be noted that in HFF cells, PCZ, KTZ and ICZ have a dose-dependent inhibitory effect on the replication of HCMV virus strain AD169.

To rule out the fact that this antiviral activity was due to a cytotoxic effect, the effects of the treatment with PCZ or ICZ on the viability of uninfected HFF cells were evaluated by means of the MTT assay as reported in Example 1.3.

From the results obtained, shown in Table 1 and in Figure 9A, it can be noted that the antiviral activity of PCZ and ICZ is not due to a corresponding cytotoxic activity. The cytotoxicity assays for ICZ were also repeated at higher ICZ concentrations, up to 250 mM. A tendency for ICZ to precipitate over time (at concentrations > 125 pM and at concentrations > 62.5 pM, after 5 and 10 days of treatment, respectively) emerged. This phenomenon was already known for the marketed pharmaceutical formulation of ICZ (Cresemba®). Thus, the concentration of compound that produced 50% in vitro cytotoxicity

(CC50), after 5 days of treatment, was found to be about 100 pM, and a calculated Selectivity Index of 14.

Table 1

^a 50% Effective Concentration, the concentration of compound that inhibits viral plaque formation by 50%, as determined by plaque reduction assays against HCMV AD169 in HFF cells. The values shown represent the means ± SD of data from at least three independent experiments in duplicate. GCV was included as a positive control. ^b Concentration of compound producing 50% cytotoxicity, determined with MTT assay in HFF cells. The values shown represent the means ± SD of the data from two independent experiments in duplicate ^c SI, Selectivity Index, determined as the ratio of CC₅₀ to EC_50.

N.D., Not Determined.

Example 2.2.1 Broad-spectrum anti-FICMV activity of PCZ and other azole antifungal compounds

In order to assess the spectrum of the anti-FICMV activity of PCZ and other azole antifungal compounds, PRA assays were performed, according to the methods reported in Example 1.2, on different strains of FICMV virus, including three clinical isolates (TB40-UL32-EGFP, VR1814 and 388438U).

The results, shown in figure 2 and Table 2, show that the antiviral activity of PCZ does not depend on the FICMV strain, in fact the EC50 values obtained from the treatment of PCZ against different strains are comparable between them.

In addition, PRA assays were also repeated with FICMV strains resistant to viral DNA polymerase inhibitors (759^rD100, PFA^rD100, GDG^rP53 and TR). Advantageously, PCZ completely inhibits the replication of viral strains mutated in the UL54 gene, with mutations conferring on the viral strain cross-resistance to the drugs GCV and cidofovir or to the drugs foscarnet and acyclovir (Table 2, strains 759^rD100 and PFA^rD100 respectively).

Furthermore, the antiviral activity of PCZ does not depend on the type of cell that is infected by FICMV, in fact the EC₅₀ values of PCZ measured against the TR strain in epithelial, endothelial and fibroblast cells are comparable between them (Table 2).

Table 2

^a 50% Effective Concentration, the concentration of compound that inhibits viral plaque formation by 50%, as determined by plaque reduction assays against

HCMV in HFF cells and against FICMV strain TR in epithelial cells (ARPE-19) and endothelial cells (FIMVECs). The values shown represent the means ± SD of data from at least three independent experiments in duplicate ^b GCV was included as a positive control in all strains, except for the PFA^rD100 strain, for which foscarnet was used.

N.D., Not determined.

Example 2.2.2 Broad-spectrum anti-HCMV activity of ICZ

In order to assess the spectrum of the anti-HCMV activity of ICZ, PRA assays were performed according to the methods reported in Example 1.2 on different strains of HCMV virus, including three clinically isolated strains (TB40-UL32-

EGFP, VR1814 and 388438U).

The results, shown in Table 3, show that the antiviral activity of ICZ does not depend on the HCMV strain, in fact the EC50 values obtained from the treatment of ICZ against different strains are comparable between them.

In addition, PRA assays were also repeated with HCMV strains resistant to viral DNA polymerase inhibitors (PFA^rD100 and GDG^rP53).

Advantageously, ICZ inhibits the replication of the viral strains mutated in the UL54 gene, with mutations conferring on the viral strain cross-resistance to the drugs foscarnet and acyclovir (Table 3, figure 9C, strain PFA^rD100), or cross resistance to the drugs ganciclovir and cidofovir (Table 3, figure 9C, strain GDG^rP53).

Table 3

^a50% Effective Concentration, the concentration of compound that inhibits viral plaque formation by 50%, as determined by plaque reduction assays against HCMV in HFF cells. The values shown represent the means of the data from at least three independent experiments in duplicate ^b Concentration of compound producing 50% cytotoxicity, determined with MTT assay in HFF cells. The values shown represent the means of the data from three independent experiments in duplicate.

N.D., Not determined.

Example 2.3 Effect of the treatment with PCZ and ICZ on viral progeny and correlation of anti-FICMV activity with inhibition of host hCYP51 by PCZ and ICZ

The effect of the treatment with PCZ and the treatment with ICZ on viral progeny production was evaluated.

Table 4 and Figure 9B show the results obtained. The treatment with PCZ inhibits the production of viral progeny in a dose- dependent manner, in other words it decreases the replication of the virus. The treatment with ICZ also inhibits viral progeny production in a dose-dependent manner, with an EC₅₀ equal to 3.16 mM.

The effect of hCYP51 enzyme inhibition on FICMV viral replication was also analysed using in vitro enzyme inhibition assays. The results obtained are shown in Table 4 and in Figures 3A and 10.

Both VFV, a known inhibitor of hCYP51, and PCZ and ICZ inhibit the initial conversion rate of lanosterol, presenting an IC50 of 0.5 mM, 8 pM and 50.5 pM, respectively. This inhibitory effect was not found after incubation with VCZ (IC5o>100 pM), which was used as a negative control. VFV was also tested by PRA assay and the results show that VFV has a dose-dependent inhibitory activity on FICMV replication (Figure 3B), although it has a higher EC₅₀ value than that obtained with PCZ (13.3 pM versus 3.3 pM, values obtained by PRA assay). Flowever, considering the high hydrophobic nature of VFV (LogP 5.4), it is reasonable to assume that VFV was not fully solubilised in the methylcellulose medium used during the PRA assays. Therefore, viral progeny reduction assays were also performed by VFV treatment (Figure 3C). VFV was found to have a high inhibitory effect on viral replication, with an EC₅₀ comprised in the low micromolar range, confirming that hCYP51 is an enzyme necessary for the production of FICMV viral progeny.

Table 4.

a50% Effective Concentration, the concentration of compound that inhibits viral plaque formation by 50%, as determined by viral titre assays in HFF cells. The values shown represent the means ± SD of the data from four independent experiments in duplicate. b 90% Effective Concentration, the concentration of compound that inhibits 90% of viral plaque formation, as determined by viral titre assays in HFF cells. The values shown represent the means ± SD of the data from four independent experiments in duplicate. c 50% Inhibitory Concentration, the concentration of compound causing the 50% decrease in lanosterol conversion rate, determined by reconstituting the hCYP51 activity in vitro, with a 2 min reaction. The values shown represent the means ± SD of the data from three independent experiments in duplicate and calculated using GraphPad Prism 6.0 (dose-response-inhibition). N.D., Not Determined.

Example 2.4 Infection by HCMV activates the expression of hCYP51 In order to assess the effects of HCMV infection on the modulation of the hCYP51 promoter in the host, U-373MG cells were transfected with a plasmid containing a reporter gene under control of the hCYP51 promoter and subsequently infected with HCMV as shown in Examples 1.6 and 1.7.

As can be noted from the graph in Figure 4A, HCMV infection activated the hCYP51 promoter by about 40-fold.

However, this activation is not present in the cells transfected with HCMV inactivated by UV radiations, which is able to enter the aforesaid cells but not to express its genes, supporting the hypothesis that de novo synthesised virus proteins are required for the activation of the hCYP51 promoter (Figure 4A).

It was also observed that during the early stages of HCMV viral replication, there is an about 2-fold increase in hCYP51 mRNA levels (Figure 4B) and an accumulation of the corresponding protein (Figures 4C, 7 and 8). Together, these results support the hypothesis that HCMV infection activates the hCYP51 gene expression.

Example 2.5 Inhibition of the hCYP51 enzyme during HCMV replication reduces the infectivitv of viral progeny

The viral particle/PFU ratio of free HCMV viral particles released from HCMV- infected HFF cells treated with the compounds PCZ, VFV or DMSO as controls was determined according to Example 1.11 and assuming that each viral particle (regardless of the infectivity thereof) contained a genome.

As shown by the graphs in Figures 5A and 5B, the treatment of HCMV-infected HFF cells for 120h with PCZ or VFV significantly reduces both the number of HCMV genomes and the level of infectivity of the viral progeny, with an about 7-10-fold increase in the particle/PFU ratio compared to infected cells treated with DMSO alone.

It is therefore believed that the enzymatic activity of hCYP51 is necessary for a productive viral replication purpose and may contribute to the production of infectious viral particles.

Example 2.6 GCV and PCZ act synerqisticallv against HCMV replication in the infected cells

The antiviral efficacy of the antiviral agent GCV in the absence and in the presence of PCZ at a concentration of 3 mM, which represents a dose approximately equivalent to the average PCZ concentration that can be found in the plasma of PCZ-treated subjects, was tested using the PRA assay and according to the methods reported in Example 1.13 (Clark NM et al. , Sem Resp Critical Care Med 2015). Advantageously, in the presence of PCZ, GCV was about 10 times more potent towards HCMV than the treatment in the absence of PCZ and with DMSO solvent alone (EC50 0.13 mM for the combination GCV + PCZ versus 1.44 pM for GCV + DMSO, Figure 6).

The results, also summarised in Table 5, show that GCV and PCZ have an antiviral effect that is synergistic with all the combinations tested. In fact, the value of the Combination Index (Cl) calculated by Chou’s method (Chou TC, Pharmacol Rev 2006) was in all cases <0.9 (Table 5).

Additionally, no evident cytotoxic effect was observed in the combined treatment of these two compounds. Table 5

^a Multiples or submultiples of the EC50 for GCV and PCZ resulting in an equipotent concentration ratio between the two drugs in combination.

The EC50 values were determined by PRA assay for HCMV AD169 in HFF cells for each compound alone or in combination at concentrations comprised between 0.25 and 4 times the equipotent ratio of the compounds based on the 1 : 1.33 ratio approximated by the values in Table 1. ^b Combination Index, obtained from the computational analysis with Calcusyn software. The values shown represent the means ± SD of the data from three independent experiments in triplicate. ^c Effect of the combination of the compounds defined as: very strong synergy for Cl<0.1; strong synergy for 0.1<CI<0.3; synergy for 0.3<CI<0.7; moderate synergy for 0.7<CI<0.9, according to Chou TC, Pharmacol Rev 2006.

In addition to a reduction in the absolute number of plaques formed, a reduction in viral plaque size was also observed when GCV was used in combination with PCZ.

The calculated dose reduction indices are shown in Table 6. Table 6

^a Concentration of compound required for the inhibition of viral replication to the extent indicated, determined with PRA assay in HFF infected cells. The values shown represent the means ± SD of the data from three independent experiments in triplicate. ^b Dose reduction index, i.e. the simulated value of the dose reduction required for each compound used in combination with respect to the dose required to achieve the same inhibitory effect with the compound used alone.

Thus, PCZ and GCV demonstrate to have potent synergistic antiviral activity without a corresponding increase in toxicity. The therapy in combination with GCV thus provides a new therapeutic strategy with which to treat and/or prevent diseases caused by FICMV infection, while at the same time such therapy reduces the toxicity of the compound and the emergence of drug- resistant mutant strains.

Example 2.7 Combinations between GCV and ICZ, FOS and ICZ, LMV and ICZ act synergisticallv against FICMV replication in infected cells The antiviral efficacy of the antiviral agents GCV, FOS and LMV in the absence and in the presence of ICZ was tested by PRA assay and according to the methods reported in Example 1.13. As can be seen from the results in Table 7, there is a synergistic effect between ICZ and all three antiviral compounds tested.

Additionally, no statistically evident cytotoxic effect was observed in the combined treatment of these compounds. From the experiments made, it was also possible to extrapolate the Dose Reduction Index (DRI), an index that provides a measure of how many times the dose of a drug can be reduced when used in combination with another drug, with the same efficacy, compared to the doses of that drug used alone.

In the present case, by way of example, when GCV, FOS or LMV are used in combination with ICZ in vitro, 95% inhibition of FICMV replication can be achieved by reducing the dose of ICZ by 7, 5 and 3 times, respectively, compared to using ICZ alone.

Table 7

a EC_5O for FICMV AD169 in HFF cells for each compound obtained by PRA assay. b EC₅O of the compound-_|/EC₅o of the compound₂ obtaining an equipotent concentration ratio between the two compounds combined. c Combination Index (Cl), at the indicated % value of inhibitory effect obtained from the computational analysis with Calcusyn software. The values shown represent the means ± SD of the data from three independent experiments in triplicate or duplicate. d Weighted average of the Combination Index (C ), calculated as Clwt= (CI₅₀ + 2XCI₇₅ ⁺ 3xClgo + 4XCI₉₅)/10. e Effect of the combination of the compounds defined as: strong synergy for 0.1 <Clwt<0,3 and synergy for 0.3<Clwt<0,7, in accordance with Chou TC, Pharmacol Rev 2006.

Claims

1) Triazole antifungal compound or a pharmaceutically acceptable salt thereof or a prodrug thereof for use as an antiviral in the method for treating and/or preventing human cytomegalovirus infections.

2) Compound for use according to the preceding claim, characterized in that said compound interferes with the human cytochrome enzyme P45051 to inhibit the replication of human cytomegalovirus.

3) Compound for use according to the preceding claim, characterized in that said compound is chosen from posaconazole and isavuconazole.

4) Compound for use according to the preceding claim, characterized in that said compound is posaconazole.

5) Compound for use according to claim 3, characterized in that said compound is isavuconazole.

6) Compound for use according to any one of the preceding claims, characterized in that said method of treatment comprises administering to a subject a therapeutically effective amount of said compound, said subject being preferably a mammal.

7) Compound for use according to any one of the preceding claims, characterized in that said method of treatment comprises administering to a subject a therapeutically effective amount of a combination of said compound and of an antiviral agent.

8) Compound for use according to the preceding claim, characterized in that said antiviral agent is selected from the group comprising ganciclovir, foscarnet, cidofovir, valganciclovir, acyclovir, valacyclovir, penciclovir, famciclovir, fomivirsen, maribavir, alkoxyalkyl derivatives of cidofovir, alkoxyalkyl derivatives of 9-(S)-[3-hydroxy-2-

(phosphonomethoxy)propyl]adenine (HPMPA), alkoxyalkyl derivatives of 1-(S)- [3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine (HPMPC), alkoxyalkyl derivatives of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]guanine (HPMPG), alkoxyalkyl derivatives of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)- propyl]-2,6-diaminopurine (HPMPDAP), alkoxyalkyl derivatives of 9-(S)-[3- hydroxy-2-(phosphonomethoxy)propyl]-2-amino-6-cyclopropylaminopurine (HPMP-cPrDAP) and letermovir.

9) Compound for use according to the preceding claim, characterized in that said antiviral agent is ganciclovir. 10) Compound for use according to any one of claims 6 to 9, characterized in that said subject is a paediatric subject.

11) Pharmaceutical composition comprising a therapeutically effective amount of a triazole antifungal compound or a pharmaceutically acceptable salt thereof or a prodrug thereof as defined in any one of claims 1 to 10, and at least one pharmaceutically acceptable excipient for use in the method for treating and/or preventing human cytomegalovirus infections.

12) Pharmaceutical composition for use according to the preceding claim, characterized by comprising a therapeutically effective amount of a combination of said triazole antifungal compound or a pharmaceutically acceptable salt thereof or a prodrug thereof, and one or more antiviral agents and at least one pharmaceutically acceptable excipient.

13) Pharmaceutical composition for use according to the preceding claim, characterized by being in a form suitable for oral, sublingual, rectal or injective administration.

14) Pharmaceutical composition for use according to the preceding claim, characterized by being in a form suitable for oral administration.

15) Pharmaceutical composition for use according to any one of claims 12 to 14, characterized in that said one or more antiviral agents are selected from the group comprising ganciclovir, foscarnet, cidofovir, valganciclovir, acyclovir, valacyclovir, penciclovir, famciclovir, fomivirsen, maribavir, alkoxyalkyl derivatives of cidofovir, alkoxyalkyl derivatives of 9-(S)-[3-hydroxy- 2-(phosphonomethoxy)propyl]adenine (HPMPA), alkoxyalkyl derivatives of 1 -(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine (HPMPC), alkoxyalkyl derivatives of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]guanine (HPMPG), alkoxyalkyl derivatives of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)- propyl]-2,6-diaminopurine (HPMPDAP), alkoxyalkyl derivatives of 9-(S)-[3- hydroxy-2-(phosphonomethoxy)propyl]-2-amino-6-cyclopropylaminopurine (HPMP-cPrDAP) and letermovir.

16) Pharmaceutical composition for use according to any one of claims 12 to 15, characterized in that said one or more antiviral agents are inhibitors of viral DNA polymerase or viral terminase or antisense oligonucleotides against viral genes.

17) Pharmaceutical composition for use according to any one of claims 12 to 16, characterized in that said one or more antiviral agents comprise ganciclovir.

18) Pharmaceutical composition for use according to any one of claims 12 to 17, characterized in that said one or more antiviral agents comprise foscarnet.

19) Pharmaceutical composition for use according to any one of claims 12 to 18, characterized in that said one or more antiviral agents comprise letermovir.

20) Pharmaceutical composition comprising a therapeutically effective amount of a combination of a triazole antifungal compound or a pharmaceutically acceptable salt thereof or a prodrug thereof, and one or more antiviral agents and at least one pharmaceutically acceptable excipient, as defined in any one of claims 12 to 19.

21) Pharmaceutical composition according to the preceding claim, characterized in that said triazole antifungal compound is isavuconazole.

22) Pharmaceutical composition according to claim 20, characterized in that said triazole antifungal compound is posaconazole.

23) Pharmaceutical composition as defined in any one of claims 20 to 22, in a form suitable for oral, sublingual, rectal or injective administration.

24) Pharmaceutical composition according to the preceding claim, characterized by being in a form suitable for oral administration.