VETERINARY PREVENTION AND/OR TREATMENT OF VIRAL

INFECTIONS"

DESCRIPTION

The present invention concerns the use of methotrexate in the pharmaceutical or veterinary treatment of viral infections.

BACKGROUND OF THE INVENTION Viruses are smaller and simpler in construction than unicellular microorganisms, and they contain as genetic material only one type of nucleic acid, either DNA or RNA. They replicate only within cells of the host that they infect. Many relevant human diseases are caused by viruses. Animal virology developed largely from the need to control viral diseases in livestock farming. Viruses, like other infectious agents, enter the animal body through one of its surfaces. They then spread either locally on one of the body surfaces or through lymphatic and blood vessels to produce systemic infections.

When a virus enters the body, it triggers the body's immune defences. These defences begin with white blood cells, such as lymphocytes, which attack and destroy the virus or the cells it has infected. If the body survives the viral infection, the lymphocytes "remember" the invader and can respond more quickly and effectively to a later infection with the same virus. This is the basis of adaptive immunity. Adaptive immunity can also be produced by vaccination .

Drugs that fight viral infections are called antiviral drugs. Antiviral drugs work by interfering with viral replication. Most of the antiviral drugs, now available, target specific viral proteins involved in the viral replication (RNA polymerase, RNA primase) whose structures are generally deduced by sequencing the viral genome. Since new viral epidemics are often generated by spill-over, it is needed first to determine the sequence of genetic material before being able to start to construct the specific antiviral drug. Hence, it takes years after the breakthrough of a new viral epidemic to obtain a drug able to block the replication of the new specific virus. Therefore, antiviral drugs are much more difficult to develop than antibiotics. In addition, viruses can develop resistance to antiviral drugs. The antiviral drugs themselves can also be toxic to animals and humans. Antibiotics are not effective against viral infections, but if an animal has a bacterial infection in addition to a viral infection, an antibiotic is generally needed.

Respiratory tract infections are common infections of the upper respiratory tract (e.g., nose, ears, sinuses, and throat) and lower respiratory tract (e.g., trachea, bronchial tubes, and lungs) . Symptoms of upper respiratory tract infection include runny or stuffy nose, irritability, restlessness, poor appetite, decreased activity level, coughing, and fever.

Current therapies for respiratory tract infections involve the administration of antiviral drugs (when available) , anti-bacterial, and antifungal agents for the treatment, prevention, or amelioration of viral, bacterial, and fungal respiratory tract infections, respectively. Unfortunately, in regard to certain infections, there are no therapies available, infections have been proven to be refractory to therapies, or the occurrence of side effects outweighs the benefits of the administration of a therapy to a subject. The use of anti-bacterial agents for treatment of bacterial respiratory tract infections may also produce side effects or result in resistant bacterial strains. The administration of antifungal agents may cause renal failure or bone marrow dysfunction and may not be effective against fungal infection in subjects with suppressed immune systems. Additionally, the infection causing microorganism (e.g., virus, bacterium, or fungus) may be resistant or develop resistance to the administered therapeutic agent or combination of therapeutic agents. In fact, microorganisms that develop resistance to administered therapeutic agents often develop pleiotropic drug or multidrug resistance, that is, resistance to therapeutic agents that act by mechanisms different from the mechanisms of the administered agents. Thus, as a result of drug resistance, many infections prove refractory to a wide array of standard treatment protocols.

Therefore, it is felt the need of a new effective and quickly available antiviral drugs to be used as soon as an epidemic insurges.

SUMMARY OF THE INVENTION

The inventor of the present patent application has surprisingly found that methotrexate, an inhibitor of DNA and RNA synthesis in human and animal cells, can be usefully used for the inhibition of the viral replication and therefore for the treatment of viral diseases in human and animals. Therefore, methotrexate has been found as a medicament having a pan-antiviral potential application .

OBJECT OF THE INVENTION

In a first object, the present invention discloses methotrexate as a medicament for the prevention and/or the treatment of viral infections in humans or in animals, with the proviso that the treatment in humans is excluded for viral infections caused by SARS-CoV2 and ZIKA viruses.

In a particular embodiment of the invention, methotrexate is disclosed as a medicament for the treatment and/or the prevention of viral infections caused by RNA viruses.

In a particular embodiment, the present invention discloses methotrexate as a medicament for the prevention and/or the treatment of viral infections in humans or in animals, with the proviso that the treatment in humans is excluded for viral infections caused by SARS-CoV2 and ZIKA viruses, together with additional metabolic cofactors. In a second object, the present invention discloses a pharmaceutical or veterinary composition for the prevention and/or the treatment of viral infections in humans or in animals, with the proviso that the treatment in humans is excluded for viral infections caused by SARS-CoV2 and ZIKA viruses.

In a third object, the present invention discloses a method for the prevention and/or the treatment of viral infections in humans or in animals, with the proviso that the treatment in humans is excluded for viral infections caused by SARS-CoV2 and ZIKA viruses.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the inhibition mechanism of methotrexate on RNA synthesis.

Figure 2 shows the results of the assay on the antiviral activity of methotrexate against CVA3.

Figure 3 shows the results of the assay on the antiviral activity of methotrexate against BHV-1.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Methotrexate is a known chemotherapy agent and immune system suppressant, also known as amethopterin, 4-amino-10-methyl folic acid and also briefly referred to as MTX in the following description.

For the purposes of the present invention, the term "prevention" shall be intended as the prevention of the onset of the disease.

Prevention shall also include a prophylaxis treatment, wherein a subject at risk of being contaged with a virus can prevent the development of the disease.

For the purposes of the present invention, the term "treatment" shall be intended as the curing the disease caused by a virus.

For the purposes of the present invention, "viral infections" shall be intended as diseases caused by a virus.

Viral infections are particularly, but not exclusively, represented by respiratory tract infections.

The term "respiratory tract infection" has its general meaning in the art and is intended to designate infections of the upper respiratory tract (e.g., nose, ears, sinuses, and throat) and lower respiratory tract (e.g., trachea, bronchial tubes, and lungs). Other viral infections can be treated with methotrexate according to the present invention; in fact, other tissues, such as the liver, comprise high expression of the folate transported (FOLR1) or of the dihydrofolate reductase, and therefore have high sensibility towards the treatment with methotrexate.

For the purposes of the present invention "virus" shall include both DNA virus and RNA virus.

As shown in Figure 1 and demonstrated in the Experimental Section, methotrexate is also active against DNA virus, as it blocks the mRNA synthesis and DNA synthesis.

For the purposes of the present invention a "RNA virus" is meant to comprise a negative single-strand RNA virus, a positive single-strand RNA virus and a double-strand RNA virus.

For the purposes of the present invention, "virus" shall include enveloped and non-enveloped viruses.

Viruses - human infections

Viruses are the predominant cause of respiratory tract illnesses and include DNA and RNA viruses.

RNA viruses include: such as respiratory syncytial virus, influenza virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus.

Human respiratory syncytial virus

Human respiratory syncytial virus (also known as human orthopneumovirus, or HRSV, or just RSV) causes respiratory tract infections, with the infected cells of the mucosa fusing together to form a syncytium. It is a major cause of lower respiratory tract infections and hospital visits during infancy and childhood. A prophylactic medication, palivizumab, can be employed to prevent HRSV in preterm (under 35 weeks gestation) infants, infants with certain congenital heart defects (CHD) or bronchopulmonary dysplasia (BPD), and infants with congenital malformations of the airway. Treatment is limited to supportive care, including oxygen therapy and more advanced breathing support with CPAP or nasal high flow oxygen, as required.

In temperate climates, annual epidemic episode during the winter months occurs; in tropical climates, infection is most common during the rainy season.

Severe HRSV infections have increasingly been found among elderly patients. Young adults can be re infected every five to seven years, with symptoms looking like a sinus infection or a cold (infections can also be asymptomatic).

To date, treatment has been limited to supportive measures.

Influenza virus

Influenza virus that infect humans are

Alphainfluenzavirus, Betainf luenzavirus and

Gammainfluenzavirus and can be identified by antigenic differences in their nucleoprotein and matrix protein.

Vaccines and drugs are available for the prophylaxis and treatment of influenza virus infections. Vaccines are composed of either inactivated or live attenuated virions of the H1N1 and H3N2 human influenza A viruses, as well as those of influenza B viruses. Because the antigenicities of the wild viruses evolve, vaccines are reformulated annually by updating the seed strains.

When the antigenicities of the seed strains and wild viruses do not match, vaccines fail to protect the vaccinees. In addition, even when they do match, escape mutants are often generated.

Parainfluenza virus

Human parainfluenza viruses (HPIVs) are the viruses that cause human parainfluenza. HPIVs are a paraphyletic group of four distinct single-stranded RNA viruses belonging to the Paramyxoviridae family.

HPIVs remain the second main cause of hospitalisation in children under 5 years of age suffering from a respiratory illness (only Human orthopneumovirus causes more respiratory hospitalisations for this age group).

Human parainfluenza viruses include:

Human parainfluenza virus type 1 (HPIV-1), which is the most common cause of croup

Human parainfluenza virus type 2 (HPIV-2), which causes croup and other upper and lower respiratory tract illnesses,

Human parainfluenza virus type 3 (HPIV-3), which is associated with bronchiolitis and pneumonia Human parainfluenza virus type 4 (HPIV-4), which is includes subtypes 4a and 4b.

HPIVs belong to two genera: Respirovirus (HPIV-1 & HPIV-3) and Rubulavirus (HPIV-2 & HPIV-4).

Despite decades of research, no vaccines currently exist.

Recombinant technology has however been used to target the formation of vaccines for HPIV-1, -2 and - 3 and has taken the form of several live-attenuated intranasal vaccines. Two vaccines in particular were found to be immunogenic and well tolerated against HPIV-3 in phase I trials. HPIV-1 and -2 vaccine candidates remain less advanced.

Human metapneumovirus (HMPV)

Human metapneumovirus (HMPV) is a negative-sense single-stranded RNA virus of the family Pneumoviridae and is the second most common cause after Human orthopneumovirus (RSV) of lower respiratory infection in young children.

The peak age of hospitalization for infants with HMPV occurs between 6-12 months of age, slightly older than the peak of RSV, which is around 2-3 months. The clinical features and severity of HMPV are similar to those of RSV. HMPV is also an important cause of disease in older adults.

The virus is distributed worldwide and, in temperate regions, has a seasonal distribution generally following that of RSV and influenza virus during late winter and spring.

No treatment is yet known, only ribavirin has shown effectiveness in an animal model.

Rhinovirus Rhinovirus is the most common viral infectious agent in humans and is the predominant cause of the common cold. Rhinovirus infection proliferates in temperatures of 33-35 °C (91-95 °F), the temperatures found in the nose. Rhinoviruses belong to the genus Enterovirus in the family Picornaviridae.

The three species of rhinovirus (A, B, and C) include around 160 recognized types of human rhinovirus that differ according to their surface proteins (serotypes).

In asthma, human rhinoviruses have been recently associated with the majority of asthma exacerbations for which current therapy is inadequate. Intercellular adhesion molecule 1 (ICAM-1) has a central role in airway inflammation in asthma, and it is the receptor for 90% of Human rhinoviruses. Human rhinovirus infection of airway epithelium induces ICAM-1.

There are no vaccines against these viruses as there is little-to-no cross-protection between serotypes. At least 99 serotypes of human rhinoviruses affecting humans have been sequenced. However, a study of the VP4 protein has shown it to be highly conserved among many serotypes of human rhinovirus, opening up the potential for a future pan-serotype human rhinovirus vaccine.

Enterovirus are picornaviruses (pico, or small RNA viruses). All enteroviruses are antigenically heterogeneous and have a wide geographic distribution. Enteroviruses include:

• Coxsackieviruses A1 to A21, A24 and B1 to B6

• Echoviruses (human orphan cytopathic enteric viruses) 1 to 7, 9, 11 to 21, 24 to 27 and 29 to 33

• Enteroviruses 68 to 71, 73 to 91 and 100 to

101

• Type 1 to 3 polioviruses

Enteroviruses are found in respiratory secretions and feces and are sometimes present in the blood and cerebrospinal fluid of infected patients.

Infection typically spreads through direct contact with respiratory secretions or stool, but can be transmitted from environmental contaminated sources (eg, water). Infection transmitted from a mother during childbirth can cause severe disseminated neonatal infection, which can include hepatitis or liver necrosis, meningoencephalitis, myocarditis, or a combination of these, and can lead to sepsis or death. Enteroviruses can cause various diseases: epidemic pleurodynia, hand-foot-mouth disease, herpangin, poliomyelitis.

An exemplary enterovirus is represented by Coxsackievirus (A3): CV-A3.

Coronaviruses

Coronaviruses are a group of related RNA viruses that cause diseases in mammals and birds. In humans, these viruses cause respiratory tract infections that can range from mild to lethal. Mild illnesses include some cases of the common cold (which is also caused by other viruses, predominantly rhinoviruses), while more lethal varieties can cause SARS, and MERS.

Coronaviruses constitute the subfamily Orthocoronavirinae, in the family Coronaviridae, order Nidovirales, and realm Riboviria. They are enveloped viruses with a positive-sense single- stranded RNA genome and a nucleocapsid of helical symmetry.

Coronaviruses vary significantly in risk factor.

Some can kill more than 30% of those infected, such as MERS-CoV, and some are relatively harmless, such as the common cold. Coronaviruses can cause colds with major symptoms, such as fever, and a sore throat from swollen adenoids. Coronaviruses can cause pneumonia (either direct viral pneumonia or secondary bacterial pneumonia) and bronchitis (either direct viral bronchitis or secondary bacterial bronchitis). The human coronavirus discovered in 2003, SARS-CoV, which causes severe acute respiratory syndrome (SARS), has a unique pathogenesis because it causes both upper and lower respiratory tract infections.

Six species of human coronaviruses are known, with one species subdivided into two different strains, making seven strains of human coronaviruses altogether.

Four human coronaviruses produce symptoms that are generally mild:

Human coronavirus OC43 (HCoV-OC43), b-CoV,

- Human coronavirus HKU1 (HCoV-HKUl), b-CoV,

Human coronavirus 229E (HCoV-229E), -CoV,

Human coronavirus NL63 (HCoV-NL63), a-CoV.

Three human coronaviruses produce symptoms that are potentially severe:

Middle East respiratory syndrome-related coronavirus (MERS-CoV), b-CoV

Severe acute respiratory syndrome coronavirus (SARS-CoV), b-CoV

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), b-CoV.

There are as yet no vaccines or antiviral drugs to prevent or treat human coronavirus infections.

Viruses - veterinary infections

Veterinary viral infections are caused by both DNA viruses and RNa viruses.

RNA virus causing viral infections is selected from the group comprising: Arteriviridae, Roniviridae, Tobaniviridae, Toroviridae, Picornaviridae, Caliciviridae, Reoviridae, Togaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Retroviridae, Bunyaviridae, Arenaviridae, Coronaviridae, and Birnaviridae.

Preferably, the veterinary treatment according to the present invention is particularly suitable against viral infections in pigs.

In this regard, it should be reminded that Coronaviridae (including the genera Alphacoronavirus, Betacoronavirus and Gammacoronavirus) and Toroviridae including the genera Torovirus and Bafinivirus) may cause in pigs different diseases, such as transmissible gastroenteritis (TGE), haemagglutinating encephalomyelitis (HEV), porcine epidemic diarrhoea (PED), porcine respiratory coronavirus (PRCV), deltacoronavirus (PDCoV).

Three porcine coronaviruses are associated with digestive disorders (TGE, PED and PDCoV).

Porcine respiratory coronavirus (PRCV) is associated with respiratory problems, and the HEV virus gives rise to two different syndromes, vomiting and wasting disease and encephalomyelitis.

The TGE virus is a coronavirus that has rarely been found since the late 20th century. First reported in 1946, it reached its highest prevalence in the 1970, 80s, and 90s in both Europe and the USA, with over 95% of European farms seropositive in the 80s. It is a highly infectious virus that causes diarrhoea, dehydration, and occasionally vomiting as a distinctive symptom, and causes high mortality in young pigs. The TGE virus has been completely sequenced and only one serotype is known. Homologies exist between TGE virus and bovine coronavirus, as well as with porcine respiratory coronavirus. From an epidemiological point of view, there are both epidemic and endemic cases. It is a relative thermostable virus, and is resistant to low pH and to many disinfectants. At 37°C, it is inactivated in less than two hours. It is very sensitive to light and highly resistant to freezing. The virus survives for long periods of time in frozen carcasses. This means that most outbreaks of the disease occur during cold months.

The most characteristic presentation in TGE-free farms becoming infected is the onset of peracute and acute cases, showing a sudden and dramatic diarrhoea affecting all ages in a matter of days. The diarrhoea is severe, watery, yellowish green and sometimes foul-smelling. In nursing piglets, vomiting is common accompanied by a lack of appetite, without fever or nervous signs. Dehydration with high mortality occurs in 24-48 hours. Mortality can reach 100% in piglets less than one week old, 50% in the second week of lactation, and up to 25% in the third week. The piglets die dehydrated, with a distended stomach, milk and haemorrhagic petechiae in the small intestine, hypertrophied mesenteric nodes (characteristic lesion), atrophy of intestinal microvilli (duodenum, jejunum and ileum) with thinner walls, and necrosis of enterocytes in the jejunum with reduced enzymatic activity and yellowish content. In piglets, co-infections with gastrointestinal bacteria such as E. coli and Clostridium spp are frequent, worsening and prolonging the clinical picture.

To date, the treatment has been only symptomatic. No commercial vaccines are available. There used to be one in the US, but it is no longer commercially available today. Hydration is the main treatment measure and controlling concomitant infections helps us to reduce the impact, also reinforcing biosecurity measures, down time of the facilities and strict control of the environmental conditions, which are so critical for young piglets. The primary objective is to infect all the animals on the farm as quickly as possible to acquire active immunity. To control the process, the feedback of feces is common practice.

Porcine epidemic diarrhoea is a highly contagious disease. The virus has a genomic structure and replication very similar to those affecting other animals. The virulence of the viruses isolated in different countries during clinical cases was similar, with minimum genetic dispersion. Only one serotype is known. The epidemic spread throughout

Europe in the 1970s, causing severe diarrhoea in nursing piglets and recurrent diarrhoea in weaned piglets and fattening pigs. New epidemic outbreaks in both Europe and Asia occurred in the 1980s, 1990s, and 2000s until 2009 (Italy, Korea, Thailand), with intermittent occurrences in most pig producing countries worldwide.

Morbidity at any stage of production can reach 100%, and is more variable in breeding sows. Mortality in nursing piglets can be as high as 100%, with rates normally between 30 and 50%. The main clinical sign is watery diarrhoea leading to death from dehydration within 2-4 days in nursing piglets, in addition to vomiting and poor appetite. Weaned piglets and fattening pigs can recover from diarrhoea after one week, showing anorexia, lethargy, and considerable growth retardation. After a clinical infection in breeding sows, they acquire a strong immunity, which they pass on to their piglets via colostrum. The course of the disease on a breeding farm usually lasts a maximum of one month, varying according to farm size and production system. This period can be prolonged if secondary complications arise from gastrointestinal infectious agents such as

Escherichia coli, Clostridium perfringens, Rotavirus, Isospora suis, Salmonella spp, Lawsonia intracellularis or Brachispira spp. It should be noted that the lesions are found exclusively in the small intestine, which appears distended with yellowish watery contents, and the stomach is found empty. There is up to 70% atrophy of intestinal villi and vacuolization of enterocytes with reduced enzymatic activity.

To date, the treatment has been only through supportive care, with the priority being hydrating nursing and weaned piglets using saline, electrolyte agents, and milk replacers. To establish strong immunity, the transmission of specific IgA via colostrum from the sow to the piglets is critical. Therefore, the rapid exposure of the virus to gestating sows, infecting them with piglet feces to stimulate rapid lactogenic immunity (depending on the production phase where the infection started), allows us to shorten the duration of the clinical picture and lessen the spread to the different production areas as much as possible.

Porcine respiratory coronavirus is a variant of the TGE virus family that infects the respiratory tract, and is not excreted via feces. Porcine respiratory coronavirus produces antibodies that neutralize the TGE virus. The virus infects animals of all ages, either by direct contact or by aerosol transmission, being more prevalent in areas with a high pig density.

Clinical signs include coughing, dyspnea, abdominal breathing, lethargy, anorexia and slight growth retardation; symptoms similar to most problems within the porcine respiratory disease complex (PRDC). Worsening of the symptoms occurs in cases combined with PRRS virus or bacterial infectious agents, which in these cases cause pneumonia that can be severe. The respiratory coronavirus can be located in both the upper and lower respiratory tract. The most characteristic lesions, which are not pathognomonic, are lung consolidation, broncho- interstitial and broncho-catarrhal pneumonia, bronchiolar epithelial hyperplasia with loss of epithelial cells, infiltration of leukocytes, lymphocytes and macrophages into the alveolar septum. Early infection in nursing and weaned piglets by respiratory coronavirus results in immunity to PRCV, but also creates partial immunity against gastrointestinal problems from the TGE coronavirus. To date, there are no antibiotic or antiviral treatments, only treatments against respiratory symptoms and secondary aggravating agents. The first preventative measure is to prevent the entry of the virus via the replacement animals, through discussion with the genetic supplier and testing during quarantine. Internal and external biosecurity measures are one of the greatest safeguards to keep free of the disease. Standards for downtime and all- in-all-out systems are equally recommended. There are currently no commercial vaccines available for porcine respiratory coronavirus.

Bovine herpes virus 1 (BHV-1)

Bovine herpes virus 1 (BHV-1) has a tropism for the mucous membranes of the upper respiratory tract and genitals and causes infectious rhinotracheitis, conjunctivitis and vulvovaginitis, pustular balanoposthitis and less frequently, also abortion, systemic neonatal infection, encephalitis, infertility. An important feature of the virus is the ability to cause latent infection. It is responsible for respiratory forms (IBR) and infections of the genital system: vulvovaginitis (IPV) and pustular balanoposthitis (BPH); in addition: abortion, systemic neonatal infection, encephalitis and infertility. Incubation of 2-5 days is followed by an acute phase of 5-10 days, respiratory symptoms (cough, nasal discharge, conjunctivitis) are related to a sharp drop in production lactea, possible bacterial complications (pneumonia) may occur.

The infection is widespread throughout Italy and many European countries. The economic and health damage caused by BHV 1 in cattle farms due to the action of the virus on the respiratory and reproductive system have been known for some time. To these is added the damage of the exclusion of infected subjects from commercial exchanges.

From the above, it is clear that the present invention concerns a medicament having a pan antiviral potential application.

In a first object, the present invention discloses methotrexate for use as a medicament for the prevention and/or for the treatment of viral infections in humans or in animals, with the proviso that the treatment in humans is excluded for viral infections caused by SARS-CoV2 and ZIKA viruses.

These exclusions have been decided by the

Applicant for ethical reasons, so that the treatment of the respiratory tract infections caused by SARS- CoV-2 RNA virus and ZIKA RNA virus is explicitly outside the scope of protection of the present patent application .

For the purposes of the present invention, methotrexate is disclosed for use as a medicament for the prevention and/or for the treatment of viral infections in humans, wherein said virus is a DNA virus or an RNA virus.

In particular, said RNA virus is selected from the group comprising RNA virus causing viral respiratory tract infections is selected from respiratory syncytial virus, influenza virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus .

In a particular embodiment, said RNA virus is represented by Coxsackie A3 virus (CVA3).

For the purposes of the present invention, methotrexate is disclosed for use as a medicament for the prevention and/or for the treatment of viral infections in animals, wherein said virus is a DNA or an RNA virus. In particular, said DNA virus is represented by Herpesvirus and more in particular by Bovine herpesvirus 1 (BHV-1).

In particular, said RNA virus is selected form the group comprising Arteriviridae, Roniviridae, Tobaniviridae, Picornaviridae, Caliciviridae, Reoviridae, Togaviridae, Toroviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Retroviridae, Bunyaviridae, Arenaviridae, Coronaviridae, and Birnaviridae.

According to a preferred embodiment, said RNA virus is represented by Coronaviridae and Toroviridae .

For the purposes of the present invention, animals include livestock animals.

In particular, livestock animals comprise: poultry, horse, donkey, cattle, zebu, yak, buffalo, gayal, mink, sheep, goat, reindeer, camel, llama, alpaca, pig, rabbit, or deer.

In a preferred embodiment of the invention, animals are represented by pigs.

For the purposes of the present invention, methotrexate is disclosed for use as a medicament for the pharmaceutical or veterinary prevention and/or treatment of viral infections wherein the dosage is about 150-300 pg/week per kg of weight (human body or animal weight).

In particular, said weekly dose is administered in fractioned amounts.

In an embodiment of the invention, the prevention and/or treatment dosage regimen is according to the known "metronomic" regimen.

In an embodiment of the present invention, methotrexate is administered to a human or animal in need thereof at the earliest appearance of symptoms.

In an embodiment of the invention, methotrexate is disclosed for use as a medicament for the prevention and/or for the treatment of viral infections in humans or in animals, together with one or more additional pharmaceutical or veterinary compounds.

For the purposes of the present invention, said one or more additional pharmaceutical or veterinary compounds may be selected from the group comprising: anti-inflammatory compounds (steroidal or FANS).

For the purposes of the present invention, said additional pharmaceutical or veterinary compounds may be administered before, together or after the administration of methotrexate.

In another embodiment of the present invention, methotrexate is disclosed for use as a medicament for the prevention and/or for the treatment of viral infections in humans or in animals, with the proviso that the treatment in humans is excluded for viral infections caused by SARS-CoV2 and ZIKA viruses, together with a coadjuvant compound.

For the purposes of the present invention, said coadjuvant compound is represented by a metabolic cofactor.

In particular, said metabolic cofactor may be selected from the group comprising: carnitine, N- acetylcysteine, nicotinamide, biotin, serine, vitamin B complex, nicotinic acid, or derivatives thereof.

A carnitine derivative is represented by L- carnitine tartrate.

A nicotinamide derivative is represented by nicotinamide riboside.

As for the dosage regimen, the above compounds may be administered with the following indicative dosage: Vitamin B complex may be administered according to the below dosage per day:

For the purposes of the present invention, said metabolic cofactor may be administered before, together or after the administration of methotrexate.

For the purposes of the present invention, methotrexate is administered via nasal, oral, subcutaneous or intramuscular administration.

In a preferred embodiment, the pharmaceutical composition is administered via nasal administration. In a preferred embodiment, the veterinary composition is administered via oral administration.

In a second object, the present invention discloses a pharmaceutical or veterinary composition for the prevention and/or for the treatment of viral infections in humans or in animals, with the proviso that the prevention and/or treatment in humans is excluded for viral infections caused by SARS-CoV2 and ZIKA viruses.

In an embodiment of the present invention, said pharmaceutical or veterinary composition may include one or more additional pharmaceutical or veterinary compounds may be selected from the group comprising anti-inflammatory compounds (steroidal or FANS).

For the purposes of the present invention, said pharmaceutical composition is a nasal, oral, subcutaneous or intramuscular composition.

According to a preferred embodiment, the pharmaceutical composition of the invention is represented by a nasal composition.

For the purposes of the present invention, said pharmaceutical composition is in the form of sealed in soft-gel capsules or in a solid form, such as a tablet, mini-tablet, micro-tablet, granule, micro- granule, pellet, multiparticulate, or micronised particulate, or powder or in the form of a solution, emulsion, gel, vial, drops, aerosol or spray.

According to a preferred embodiment, the pharmaceutical composition of the invention is represented by an aerosol.

The term "aerosol" refers to the delivery of a drug to the body, for either topical or systemic effect, via the airways by delivering it in an aerosolised form. The greatest advantage of this pharmaceutical form is the use of smaller doses and thus, minimal systemic adverse effects, besides giving a rapid response.

The particle size (expressed as mass median aerodynamic diameter) is of critical importance as the drug delivery depends on the same to a major extent. Several devices such as metered dose inhaler (MDI), dry powder inhaler (DPI) and nebulisers are available.

Physical characteristics of aerosol particles including the size (diameter), density, electrical charge, hygroscopy, shape and the velocity of the aerosol have an impact on the deposition of the aerosol. These characteristics are dependent on several factors. For example, a solution based formulation of an aerosol generates much smaller sized particles (~2 pm) as compared to suspension based formulations where particles size is in the range of 4 pm are generated. These characteristics become pertinent since among the various physical characteristics, droplet size of the aerosol is the most important factor in the delivery of the drug to the lungs. Size of the aerosol droplets is generally characterised by mass median aerodynamic diameter (MMAD). This measure determines the particle size (in pm) above and below which 50% of the mass of the particles is contained. This is the particle size that evenly divides the mass, or amount of the drug in the particle size distribution. The higher the MMAD, the more particle sizes are of larger diameters. Indeed, larger particles between 10-15 pm deposit mostly in the upper airways, particles within the 5-10 pm range reach the large bronchi, and particles of 1-5 pm penetrate to the lower airways and lung periphery.

Another characteristic that may modify the penetration of the drug is the shape of aerosol where a more aerodynamically shaped droplet is likely to be associated with greater penetration. Finally, the velocity at which the aerosol is generated also affects the fraction delivered to the lower airways. Those aerosols that are generated at a very high velocity tend to get deposited in the upper airways and consequently the delivery to the lower airways is compromised. An MDI is a typical example of a generator that produces aerosols at a high velocity, which are in the range of 10-100 m/s. On the other hand, dry powder and nebulizers produce aerosols with relatively low velocities. Slower flow minimises oropharyngeal and upper airway deposition and enhances distal delivery and depositing.

The advantages connected to an aerosol administration are well-know and in particular are represented by the facts that: i) the aerosol doses are generally smaller than systemic doses, ii) the onset of effect with inhaled drugs is faster than with oral dosing, iii) the drug is delivered directly to the airways, with minimal systemic exposure, iv) the systemic side effects are less frequent and severe with inhalation when compared to systemic delivery, v) the spray and powder forms may inadvertently deposit in the eyes and result in eye irritation. Conversely, inhaled aerosol delivered with a face mask allows to avoid these problems and increases the amount of drug delivered to the distal airways, and vi) the inhaled drug therapy is less painful than injection and is relatively comfortable.

For the purposes of the present invention, said veterinary composition is a nasal, oral, subcutaneous or intramuscular composition.

According to a preferred embodiment, the pharmaceutical composition of the invention is represented by an oral composition.

According to a preferred embodiment, the pharmaceutical composition of the invention is represented by a tablet.

For the purposes of the present invention, the pharmaceutical or veterinary composition of the invention comprises one or more suitable pharmaceutical or veterinary excipients according to the route of administration.

Suitable excipients include: acidifiers, acidity regulators, anti-caking agents, antioxidants, bulking agents, firming agents, gelling agents, coating agents, modified starches, sequestrants, thickeners, sweeteners, diluents, disintegrants, glidants, colourings, binders, lubricants, stabilisers, adsorbents, preservatives, humectants, flavourings, filmogenic substances, emulsifiers, wetting agents, release retardants and mixtures thereof.

Preferably, said excipients are potassium sorbate, sodium benzoate, e-polylysine, sucralose, maltodextrin, citric acid, sodium carbonate, calcium carbonate, magnesium carbonate, magnesium stearate, stearic acid, polyethylene glycol, natural starch, partially hydrolysed starch, modified starch, maize starch, potato starch, lactose, lactose monohydrate, calcium phosphate, calcium carbonate, calcium sulphate, polyvinylpyrrolidone, silica, colloidal silica, precipitated silica, magnesium silicates, aluminium silicates, sodium lauryl sulphate, magnesium lauryl sulphate, methacrylate copolymers, sodium dehydroacetate, xanthan gum, guar gum, tara gum, locust bean gum, fenugreek gum, gum arabic, alginic acid, sodium alginate, propylene glycol alginate, sodium croscarmellose, polyvinylpolypyrrolidone, polysorbate, glyceryl behenate, titanium dioxide, indigo carmine, cellulose, modified cellulose, calcium carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, ethylcellulose, gelatine, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, polydextrose, carrageenan, methylcellulose, saccharose, saccharose esters, sorbitol, xylitol, dextrose, fructose, maltitol, tragacanth gum, pectin, agar-agar, carboxypolymethylene, hydroxypropylmethylcellulose, tragacanth, mannitol, or a mixture thereof.

The pharmaceutical and veterinary composition of the present invention may be prepared by using methods known to a person skilled in the art.

In a third object, the present invention discloses a method for the prevention and/or for the treatment of viral infections in humans or in animals comprising the step of administering to a human or to an animal in need thereof of a pharmaceutically or veterinary effective dose of methotrexate according to one or more of the embodiment of the above description, with the proviso that the prevention and/or treatment in humans is excluded for viral infections caused by SARS-CoV2 and ZIKA viruses.

According to the above description, the method for the prevention and/or treatment of viral infections comprises the administration of a dose of methotrexate of about 150-300 pg/week per kg of weight (human body or animal weight).

In particular, said weekly dose is administered in fractioned amounts.

According to the above description, the method for the prevention and/or treatment of viral infections may further comprise the administration of one or more additional pharmaceutical or veterinary compounds.

More in particular, said one or more additional pharmaceutical or veterinary compounds may be selected from the group comprising anti-inflammatory compounds (steroidal or FANS).

According to the above description, the method for the prevention and/or treatment of viral infections may further comprise the administration of a coadjuvant compound.

For the purposes of the present invention, said coadjuvant compound is represented by a metabolic cofactor.

In particular, said metabolic cofactor may be selected from the group comprising: carnitine, N- acetylcysteine, nicotinamide, biotin, serine, vitamin B complex, nicotinic acid, or derivatives thereof.

A carnitine derivative is represented by L- carnitine tartrate.

A nicotinamide derivative is represented by nicotinamide riboside.

As for the dosage regimen, the above compounds may be administered with the following indicative dosage:

Vitamin B complex may be administered according to the below dosage per day: For the purposes of the present invention, said metabolic cofactor may be administered together with methotrexate during the prevention and/or treatment regimen.

Alternatively, said metabolic cofactor may be administered according to a different posology regimen.

The present invention will be further disclosed in the following Experimental Section. Experimental Section

Experiments have been carried out to show the efficacy of methotrexate against viruses.

The results of the assay are shown in the enclosed

Figures. In particular, figure 2 shows the results of the assay performed on CVA3 virus.

Coxsackie A3 (CVA3) is a Picornaviridae, single- stranded positive-sense RNA viruses and it is nonenveloped . The plateau of efficacy is reached at about 25 mM methotrexate and IC50 is 6.25 mM. In particular, Figure 3 shows the results of the assay performed on BHV-1 virus.

Bovine herpesvirus 1 (BHV-1) is a Herpesviridae double-stranded DNA genome virus and it is enveloped. The plateau of efficacy is reached at about 1.5 mM methotrexate and IC50 is 0.19 mM.

The assays carried out show that methotrexate is effective both against DNA and RNA viruses.

From the description above reported, there will be evident the surprising effects and advantages related to the invention above described.

First of all, methotrexate is a well-known compound used in the chemiotherapic treatment of neoplasia. Nothing in the art had ever suggested that methotrexate could find application for the prevention and/or for the treatment of viral infections .

Moreover, MTX has shown high efficacy in inhibiting viral reproduction, with negligible effects on host cell viability.

Interestingly, the inhibition of viral replication occurs at a concentration of mM, which is significantly lower, reduced by about one to two orders of magnitude, compared to the cytotoxic dose in the host cell.

According to the present invention, methotrexate has shown to provide an antiviral effect both in the pharmaceutical and in the veterinary field. Thus, it can be administered both to a human or to an animal in need thereof at the earliest appearance of symptoms .

Also, methotrexate can be administered in a prophylaxis treatment when an epidemic is diffusing and widespreading, so to effectively prevent viral replication and diffusion of the infection.

Having shown that methotrexate is effective towards both DNA and RNA viruses, it is expected that it is also effective against most of the DNA and RNA viruses.

Methotrexate is commercially largely available and it is not expensive, thereby it may represent a socially convenient pan-viral medicament. k k k