WO2011121162A1 - Use of valproic acid as an antiviral agent against enveloped viruses - Google Patents

Abstract

The invention relates to the use of valproic acid or the salts or solvates thereof for the treatment of diseases caused by enveloped viruses, such as, for example, West Nile fever virus or Usutu virus.

Description

 Use of valproic acid as an antiviral against enveloped virus

The present invention describes the use of valproic acid or its salts or solvates for the treatment of diseases caused by West Nile virus, Usutu virus and other enveloped viruses.

STATE OF THE PREVIOUS TECHNIQUE

Viruses with lipid envelope (also called envelope) acquire this from the host cell by a process in which the newly formed viral particles are coated with an outer layer from cell membranes. There are different families of viruses with envelopes that include among their members viruses that cause important diseases in animals and smokers, among which we can highlight the families: Arenaviridae, Arteriviridae, Bunyaviridae, Hantaviridae, Nairoviridae, Plebhoviridae, Coronaviridae, Toroviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Alphaviridae, Rubiviridae and Rhabdoviridae West Nile virus (WNV) is classified within the Flaviviridae family. It is a virus with a lipid envelope, whose virions have icosahedral symmetry and an approximate diameter of 50 nm (Mukhopadhyay et al. Science, 302: 248, 2003). As a genetic material, WNV has a single single-stranded and positive polarity RNA molecule of about 1,1000 nucleotides in length. RNA encodes by a single open reading pattern, flanked by two non-coding regions at its 5 ^' and 3 ^' ends, which results in a polyprotein that is processed by viral and cellular proteases to originate 10 mature proteins: 3 structural proteins ( of the capsid, C, of the pre-membrane, pM, and membrane, M, and the envelope glycoprotein, E) and 7 non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) responsible for the viral replication, the assembly of virions and the evasion of the host's immune response (Brinton, M. Annu. Rev. Micobiol. 56: 371-402, 2002).

WNV is a clear example of re-emerging zoonosis, which is currently a serious problem for human and animal health since it causes fatal meningoencephalitis in humans, equidae and birds, in which it generally produces subclinical infections, but in which occasionally it causes a high mortality (Granwehr, BP, et al. Lancet Infecí. Dis. 4: 547-556, 2004). The main vectors of WNV are mosquitoes, mostly of the Culex genus, although many other species of mosquitoes that also act as vectors have been described, with birds being the main hosts.

WNV was first isolated in Uganda in 1937 (Smithburn, K.C., et al. Am. J. Trop. Med. Hyg., 20: 471-492, 1940). Sporadic outbreaks of WNV have been reported in horses, birds and serious infections in humans.

At present there is no treatment against the disease and as tools for its control there is only a commercial vaccine for equidae based on the use of inactivated virus and in the case of humans, interferon-based therapies (IFN) or antibody administration (Ng et al. Dev. Biol., 1 14: 221-227, 2003; Davis et al. Ann. Neurol., 60: 286-300, 2006). Therefore, one of the priority aspects in the research on WNV is the determination of possible antiviral agents. This patent application describes the antiviral effect of valproic acid (VPA), a compound used for the treatment of epilepsy and bipolar disorder (Kostrouchova, et al. Folia Biol., 53: 37-49, 2007; Shaltiel et al., Biol. Psychiatry, 456: 868-874, 2004), on the infection of WNV in cell cultures and in vivo, as well as on the infection of the Usutu virus, another flavivirus genetically closely related to WNV (Weissenbóck et al. , Emerg. Infecí. Dis., 8: 652-656, 2002), which has already been detected in Spain (Busquéis et al., Emerg. Infecí. Dis., 14: 861-863, 2008) and for which has described, very recently, the first case of neuroinvasive infection in the world in an Italian patient (Pecorari et al. Euro Surveill., 14: 19446, 2009).

DESCRIPTION OF THE INVENTION

The present invention describes the use of valproic acid as an antiviral against enveloped viruses such as those of the Flaviviridiae family, especially against West Nile virus and Usutu virus. Valproic acid is a chemical compound (2-propylvaleric acid) that has clinical use as an anticonvulsant and mood stabilizer, so it is used in the treatment of epilepsy, bipolar disorders, depression, migraines and schizophrenia. In a first aspect, the present invention relates to the use of valproic acid or its pharmaceutically acceptable salts or pharmaceutically acceptable solvates for the manufacture of a medicament for the treatment of a disease caused by a virus with a lipid envelope. In other words, the present invention relates to valproic acid or its pharmaceutically acceptable salts or its pharmaceutically acceptable solvates for use in the treatment of a disease caused by a virus with lipid envelope. In a preferred embodiment, the disease is selected from dengue, Japanese encephalitis, Kyasanur disease, Murray Valley encephalitis, St. Louis encephalitis, tick meningoencephalitis, Nile encephalitis, Usutu virus encephalitis, yellow fever, hepatitis C Ebola, equine arteritis, La Crosse encephalitis, Hanta virus disease, Crimea-Congo disease, Rift Valley fever, severe acute respiratory syndrome (SARS), hepatitis B, influenza, mumps, Newcastle disease, rabies , rubella, Lassa fever, Argentine hemorrhagic fevers, Bolivian and Venezuelan, and other diseases caused by enveloped viruses belonging to the families to which these viruses belong.

In another preferred embodiment, the virus of the Flaviviridae family is selected from the West Nile virus or the Usutu virus.

The use of valproic acid as an antiviral according to the present invention can be carried out together with other active ingredients that enhance this viral activity to provide a combination therapy, for administration at the same time or at a different time. Preferably, this other active ingredient is an antiviral such as, but not limited to, IFN or Ribavirin.

The term "pharmaceutically acceptable salts or solvates" refers to any pharmaceutically acceptable salt or solvate, or any other compound that, when administered to a receptor, is capable of providing (directly or indirectly) a compound as described herein. However, it will be appreciated that pharmaceutically acceptable salts are also within the scope of the invention since these may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts and solvates can be carried out by methods known in the art.

For example, pharmaceutically acceptable salts of compounds provided herein are synthesized by conventional chemical methods from an original compound containing an acidic moiety. Generally, such salts are prepared, for example, by reacting the free acid forms of the compounds with a stoichiometric amount of the appropriate base in water or in an organic solvent or in a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of base addition salts include inorganic salts such as, for example, sodium, potassium, calcium, ammonium, magnesium, aluminum and lithium salts, and salts of organic bases such such as, for example, ethylenediamine, ethanolamine, N, N-dialkylene ethanolamine, triethanolamine, glucamine and salts of basic amino acids such as lysine and arginine. The nature of the pharmaceutically acceptable solvate is not critical as long as it is pharmaceutically acceptable. In a particular embodiment, the solvate is a hydrate. Solvates can be obtained by conventional solvation methods well known to those skilled in the art. The amount of valproic acid, its pharmaceutically acceptable salts, or solvates thereof, therapeutically effective to be administered as well as its dosage to treat a pathological state with said compounds will depend on numerous factors, including age, the patient's condition. , the severity of the disease, the route and frequency of administration, the modulator compound to be used, etc.

Valproic acid can be used alone or together with other drugs to provide a combination therapy. The other drugs may be part of the same composition, or be provided as a separate composition, for administration at the same time or at a different time.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. BRIEF DESCRIPTION OF THE FIGURES Fig. 1. Shows the results obtained for the treatment with valproic acid of cells infected with WNV and Usutu virus.

Fig. 2. Shows the results obtained for the treatment of WNV with valproic acid in vivo

EXAMPLES

Example 1

Valproic acid (VPA) dissolved in the culture medium at a concentration of 50 mM inhibits the production of WNV, strain NY99 (Lanciotti et al. 1999) in the Vero mammalian cell line (Figure 1). Infections were performed at a multiplicity of infection (MOI) of 0.5 plaque forming units (PFU) / cell. Viral production at 24 h p.i was determined by plating assays in semi-solid medium in Vero cells. The inhibitory effect for the multiplication of the observed WNV was total, at least 8 logarithms of inhibition, both when the cells were pretreated for 10 minutes and the drug was maintained until 24 h post-infection (pi), as when the VPA was added 3 h pi and maintained for the rest of the infection. Asterisks indicate statistically significant differences (P <0.005).

Example 2 Valproic acid (VPA) dissolved in the culture medium at a concentration of 50 mM inhibits the production of the Usutu virus (Buckley et al. J. Gen. Virol., 84: 2807-2817, .2003) in the line Vero mammalian cell (figure 1), under the conditions described and following the methodology indicated in example 1. A total inhibitory effect of VPA, of at least 6 logarithms, was observed in the multiplication of the Usutu virus. In the same way as for WNV, the inhibitory effect was observed both by pretreating the cells for 10 minutes and keeping the drug until 24 h pi, and when the VPA was added 3 h pi and maintained for the rest of the infection. Asterisks indicate statistically significant differences (P <0.005).

These results (Fig. 1) indicate the antiviral effect of valproic acid on the multiplication of WNV and the Usutu virus in cell cultures.

Example 3

Groups of 12 Swiss mice 6-8 weeks old were inoculated with two daily doses (4x10 ² mg / kg) of valproic acid administered intraperitoneally with a 12-hour interval. One group was treated from the same day of infection (VPA + 0) and another from two days after it (VPA + 2). The treatment was maintained until the end of the experiment, 15 days after infection. As a control, a group of 8 untreated mice (Non-VPA) inoculated with phosphate buffered saline and infected under the same conditions as those treated with VPA were included. The infection was performed by intraperitoneal injection of 1 x 10 ² plaque forming units (pfu) of West Nile virus (neurovirulent strain NY-99). The animals were observed daily to detect signs of infection with WNV. In those cases in which the disease developed, the animals were anesthetized and slaughtered.

The results (Fig. 2) indicated that 16.7% (2/12) and 25% (3/12) of the animals of the VPA + 0 and VPA + 2 groups, respectively, succumbed to the infection, compared to 75 % (6/8) of mortality recorded in untreated animals.

These preliminary results, performed on a small group of animals, suggest that valproic acid could have an antiviral effect in vivo.

Claims

 Use of valproic acid or its pharmaceutically acceptable salts or pharmaceutically acceptable solvates for the manufacture of a medicament for the treatment of a disease caused by a virus with lipid envelope. Use according to claim 1 wherein the virus is a member of the Flaviviridiae family. Use according to claim 2 wherein the virus of the Flaviviridiae family is selected from the West Nile virus or the Usutu virus. Use according to any of claims 1 to 3 wherein the disease is selected from dengue, Japanese encephalitis, Kyasanur disease, Murray Valley encephalitis, St. Louis encephalitis, tick meningoencephalitis, Nile encephalitis, Usutu virus encephalitis, yellow fever, hepatitis C, Ebola, equine arteritis, La Crosse encephalitis, disease caused by Hanta virus, Crimea-Congo disease, Rift Valley fever, severe acute respiratory syndrome (SARS), hepatitis B, influenza, mumps, Newcastle disease, rabies, rubella, Lassa fever, Argentine, Bolivian and Venezuelan hemorrhagic fevers, and other diseases caused by enveloped viruses belonging to the families of the viruses causing these diseases Use according to any of claims 1 to 4, wherein the medicament further comprises at least one other active ingredient. Use according to claim 5 wherein the active ingredient is an antiviral. Valproic acid or its pharmaceutically acceptable salts or pharmaceutically acceptable solvates for use in the treatment of a disease caused by a virus with lipid envelope.