MEK inhibitors for the treatment of hantavirus infection

Abstract

The present invention relates to MEK inhibitors that can show one or more beneficial therapeutic effects. The MEK inhibitors can be used to prevent and / or treat Hantavirus infection.

Description

Background Art

[0001] Hantavirus (or orthohantavirus) is a single-stranded, enveloped, negative-sense RNA virus in the family Hantaviridae, order Bunyavirales. To date, 28 pathogenic hantavirus species are known. The types of hantavirus vary depending on their geographical location. In Europe, the prevalent hantavirus is Puumala virus, while in the United States, the prevalent ones are Andes virus and SinNombre virus. In Asia, the ones found are Hantavirus and Seoul virus. The natural hosts of Hantavirus are rodents, gophers, and bats, however, no clinical symptoms have been found in the natural hosts. Transmission of Hantavirus occurs primarily through aerosolized rodent excreta (urine, saliva, feces), however, in 2005 and 2019, human-to-human transmission of Andes virus was reported in South America (Martinez VP, Bellomo C, San Juan J, Pinna D, Forlenza R, Elder M, Padula PJ (2005). "Person-to-person transmission of Andes virus". Emerging Infectious Diseases; 11(12): 1848–1853). For Hantavirus, the incubation period in humans lasts up to several weeks.

[0002] Hantavirus infections present a global threat as these infections are associated with high mortality rates and available treatments are very limited. In humans, Hantavirus infections themselves present in two clinical manifestations. The first is Hantavirus hemorrhagic fever with renal syndrome (HFRS), where the mortality rate is 12%, and the second is Hantavirus pulmonary syndrome (HPS), where the mortality rate is 40%. The severity of the disease depends on the species of Hantavirus causing the disease and the viral load. For example, severe courses are known to be caused by Hantaan virus, Dobrava-Belgrad virus, Sin Nombre virus, and Andes virus, while moderate courses are more likely to be caused by, for example, Puumala virus or Saaremaa virus. At least 100,000 cases of HFRS are documented each year. In 2004, there were 40,000-60,000 cases per year in China alone (Zhang et al. 2004, Lee et al. 1996, Lee et al. 1998), compared with approximately 9,000 cases in Europe (Vapalatti et al. 2003, Heyman et al. 2009, Heyman et al. 2011).

[0003] There are currently no FDA or WHO-approved vaccines or antivirals available to treat or prevent hantavirus infection. A vaccine called Hantavax has been in research and used in South Korea since 1990, but it has not been widely approved and has limited effectiveness. Inactivated virus vaccines have not been pursued due to the dangers of large-scale production under high containment and unresolved vaccine efficacy issues. Many laboratories have worked on vaccines that deliver viral antigens via DNA vectors or as recombinant proteins, but none of these have shown effectiveness to date.

[0004] Furthermore, there are currently no approved treatments for HPS or HFRS, other than relieving disease symptoms. The only drug that could potentially be used is ribavirin, but its effectiveness remains unknown. One problem in controlling RNA viruses in particular, such as hantaviruses, is the viral adaptability caused by the high failure rate of the viral polymerase, which makes the production of suitable vaccines as well as the development of antiviral substances very difficult. As a result, most known antiviral drugs are known to cause the development of resistance in individual RNA viruses.

[0005] Due to the very small genome, the necessary functional coding capacity for replication is limited, and all viruses are highly dependent on the function of their host cells. By influencing this cellular function necessary for viral replication, the viral replication in infected cells may be negatively affected. In this case, the virus may not be adapted, particularly by mutation to replace the lacking cellular function, so as to escape selection pressure. This may have been confirmed in influenza A viruses with relatively nonspecific inhibitors of cell kinases and methyltransferases (Scholtissek and Müller, ArchVirol 119, 111-118, 1991).

[0006] It is known in the art that cells have multiple signal transmission pathways, and the signals acting on cells through these pathways are transmitted to the nucleus. Therefore, cells can respond to external stimuli and respond by cell proliferation, cell activation, differentiation or controlled cell death. The common point of these signal transmission pathways is that they contain at least one kinase, which activates at least one protein by phosphorylation and then transmits signals. When observing the cellular processes induced after viral infection, it is found that a large number of DNA and RNA viruses preferentially activate determined signal transmission pathways in infected host cells, i.e., the so-called Raf / MEK / ERK kinase signal transmission pathways (Benn et al., J Virol 70, 4978-4985, 1996; Bruder and Kovesdi, J Virol 71, 398-404, 1997; Popik and Pitha, Virology 252, 210-217, 1998; Rodems and Spector, J Virol 72, 9173-9180, 1998). This signal transmission pathway is one of the most important signal transmission pathways in cells and plays an important role in proliferation and differentiation processes. Growth factor-induced signals are transmitted from serine / threonine kinase Raf to dual specificity kinase MEK (MAP kinase / ERK kinase) and finally to kinase ERK (extracellular signal-regulated kinase) by continuous phosphorylation. As a kinase substrate of Raf, only MEK is known, and ERK subtypes are determined to be the only substrates of MEK, and ERK can phosphorylate all substrates. For example, these belong to transcription factors, and cell gene expression is directly affected (Cohen, Trends in Cell Biol 7, 353-361, 1997; Robinson and Cobb, Curr. Opin. Cell Biol 9, 180-186, 1997; Treisman, Curr. Opin. Cell Biol 8, 205-215, 1996).

[0007] In view of the prior art, it is clear that there is a need for further compounds and compositions that are effective in preventing and treating viral diseases, particularly diseases caused by hantaviruses.

[0008] In this regard, ongoing research on the usefulness of MEK inhibitors in the treatment of other viral diseases, particularly influenza, has revealed that this class of compounds avoids the shortcomings of standard antiviral therapy because it targets cellular components of host cells rather than the virus itself. For this reason, resistance to MEK inhibitors has not yet been observed. WO2001 / 076570 provides the concept of treating or preventing infections caused by (-) RNA viruses, particularly influenza viruses, by MEK inhibitors. WO2014 / 056894 provides specific MEK inhibitors for treating or preventing influenza virus infections, such as AZD-6244, AZD-8330, RDEA-119, GSK-1120212 (trametinib), GDC0973 (cobalaminib), CI-1040, PD-0325901, RO-5126766, MSC1936369 (AS703026). WO2015 / 173788A1 discloses that MEK inhibitors are used in methods for treating influenza virus and bacterial co-infection. In addition, WO 2019 / 076947 discloses a new MEK inhibitor PD-0184264 (also known as ATR-002) for use in methods for preventing and / or treating influenza virus infection.

[0009] However, none of these documents show that MEK inhibitors can be used for hantavirus infection, and there remains an urgent need to provide compositions and compounds for treating and preventing hantavirus infection. Summary of the invention

[0010] In the present invention, it was found that the use of MEK inhibitors in the treatment or prevention of Hantavirus infection resulted in effective treatment of the viral infection. Specifically, when the MEK inhibitors CI-1040 or PD-0184264 were administered to Hantavirus-infected mice, a strong reduction in viral load was observed.

[0011] Therefore, the present invention relates to a MEK inhibitor for use in the treatment or prevention of Hantavirus infection in a mammal, preferably a human or a rodent.

[0012] In the context of the present invention, the MEK inhibitor can be selected from CI-1040, PD-0184264, GSK-1120212, GDC-0973, PLX-4032, AZD6244, AZD8330, AS-703026, RDEA-119, RO-5126766, RO-4987655, PD-0325901, TAK-733, AS703026, PD98059 and PD184352 or a pharmaceutically acceptable salt or metabolite thereof. In a preferred aspect, the MEK inhibitor is CI-1040 or PD-0184264.

[0013] In human patients, MEK inhibitors can be administered to treat Hantavirus infection when the patient exhibits symptoms of Hantavirus hemorrhagic fever with renal syndrome (HFRS) or Hantavirus pulmonary syndrome (HPS). In these cases, the MEK inhibitor is administered up to 12 hours, up to 24 hours, up to 48 hours, up to 72 hours, or between 4 and 10 days after the first symptoms of HFRS or HPS are observed.

[0014] The MEK inhibitors can also be administered to human subjects who have come into contact with rodents or rodent excreta or who are in areas where hantavirus outbreaks are prevalent for the prevention of hantavirus infection.

[0015] Administration of MEK inhibitors to treat or prevent hantavirus infection is particularly desirable when the human subject has been living in or traveling in an area where hantavirus infection that causes HFRS or HPS is known to occur.

[0016] The hantavirus infection can be a hanta or eastern barave virus infection, or a hantavirus infection caused by an American species such as Black Creek Canal virus (BCCV), New York ortho-hantavirus (NYV), Monophyletic Halavirus (MGLV), Norbert ortho-hantavirus (SNV), or Andes virus.

[0017] Preferably, the MEK inhibitor for use according to the present invention can be administered orally or via inhalation.

[0018] In another aspect, it is contemplated to treat rodent populations with MEK inhibitors to prevent infection in humans who come into contact with the rodents. In such uses, the MEK inhibitor may be administered by inhalation, for example, by environmental spraying. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It was shown that in the presence of CI-1040, >2 log was achieved compared to the solvent control group. 10 - Step significant reduction in viral titer. This corresponds to a reduction in viral titer of >99%.

[0020] Figure 2 It was shown that in the presence of ATR-002, >1,5 log was achieved compared to the solvent control group. 10 This corresponds to a reduction in viral titer of >90%.

[0021] Figure 3 It was shown that when the lungs of animals were treated with 75 mg / kg / day of ATR-002, no virus was detected on days 7 and 10 post infection.

[0022] Figure 4It was shown that when the kidneys of animals were treated with 75 mg / kg / day of ATR-002, no virus was detected on days 7 and 10 post infection. DETAILED DESCRIPTION

[0023] The following description includes information that may be useful in understanding the present invention. No admission is made that any of the information provided herein is prior art or relevant to the presently claimed invention, nor is any admission made that any publication specifically or implicitly referenced is prior art.

[0024] As used herein, "MEK inhibitors" inhibit the mitogenic signaling cascade Raf / MEK / ERK in cells or subjects by inhibiting MEK (mitogen-activated protein kinase). This signaling cascade is hijacked by many viruses (especially influenza viruses) to promote viral replication. Therefore, specifically blocking the Raf / MEK / ERK pathway at the MEK bottleneck will impair the growth of viruses (especially influenza viruses). In addition, MEK inhibitors have low toxicity to humans and few adverse side effects. There is also no tendency to induce viral resistance (Ludwig, 2009). A particularly preferred MEK inhibitor is PD-0184264, also known as ATR-002.

[0025] Preferably, the MEK inhibitor is selected from CI-1040, PD-0184264, GSK-1120212, GDC-0973, PLX-4032, AZD6244, AZD8330, AS-703026, RDEA-119, RO-5126766, RO-4987655, PD-0325901, TAK-733, AS703026, PD98059 and PD184352 or a pharmaceutically acceptable salt or metabolite thereof. These MEK inhibitors are known in the art, for example, as described in Table 1 of Fremin and Meloche (2010), J. Hematol. Oncol. 11; 3: 8. The structural formulas of PD-0184264 and CI-1040 are shown below for reference:

[0026]

[0027] The structural formula of PD-0184264

[0028]

[0029] The structural formula of CI-1040

[0030] 2-(2-Chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide

[0031] As used herein, "metabolite" refers to an intermediate end product of the MEK inhibitor metabolism that occurs during degradation of the MEK inhibitor by a subject, for example, in the liver. In a preferred embodiment, the MEK inhibitor is a metabolite of CI-1040, for example, PD0184264 is a metabolite of the MEK inhibitor CI-1040.

[0032] For the purposes of the present invention, MEK inhibitors as defined above also include pharmaceutically acceptable salts thereof. The phrase "pharmaceutically or cosmetically acceptable salts" as used herein refers to salts of the compounds of the present invention that are safe and effective for the desired administration form. Pharmaceutically acceptable salts include salts formed with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0033] As mentioned above, Hantavirus infection is a public health problem worldwide. Currently, there are no vaccines or antiviral drugs approved by the WHO or FDA targeting Hantavirus. However, in the treatment of influenza, the present inventors have earlier demonstrated the antiviral potential of MEK inhibitors, such as CI-1040 and PD0184264 (ATR002) (the active metabolite of CI-1040) against influenza virus at both in vitro and in vivo levels. The following results show that in vitro experiments show that treatment of Vero cells with 40 μM CI-1040 or 40 μM ATR-002 can successfully reduce the reproduction of Hantavirus. In Example 1, a decrease in viral titer of >90% was observed compared to the solvent control group, see Figure 1 and 2 In addition, in vivo experiments showed that treatment of mice with 75 mg / kg / day of ATR-002 for 5 days resulted in a complete reduction in viral titers compared to animals treated with solvent alone, as shown in Examples 2 and Figure 3 and 4 As described in.

[0034] Although hantaviruses replicate in the cytoplasm and are not known to have a nuclear phase, PUUV proliferation was successfully impaired by inhibiting the Raf / MEK / ERK pathway with the MEK inhibitors ATR-002 or CI-1040.

[0035] The viral infection to be prevented or treated by administering the MEK inhibitor of the present invention is an infection caused by a Hantavirus. Known Hantaviruses include Puumala virus, Sin Nombre virus, Seoul virus, Hantaan virus, Dobrava-Belgrad virus, Saaremaa virus, and Andes virus.

[0036] As mentioned above, there are two clinical manifestations of Hantavirus infection itself. The first is Hantavirus Hemorrhagic Fever with Renal Syndrome (HFRS), in which the mortality rate is 12%, and the second is Hantavirus Pulmonary Syndrome (HPS), in which the mortality rate is 40%. The severity of the disease depends on the type of Hantavirus causing the disease and the viral load. For example, severe courses of the disease are known to be caused by Hantavirus, Dobrava-Belgrade virus, Sin Nombre virus and Andes virus, while moderate courses of the disease are more likely to be caused by, for example, Puumala virus or Saaremaa virus.

[0037] Hantavirus hemorrhagic fever with renal syndrome (HFRS) is also known as Korean hemorrhagic fever, epidemic hemorrhagic fever, and epidemic nephropathy. Species that cause HFRS include Hanta virus, Dobrava-Belgrade virus, Saaremaa virus, Seoul virus, Puumala virus, and other Eurasian virus. Symptoms of HFRS usually develop within 1 to 2 weeks after exposure to the infectious agent, but in rare cases, it may take up to 8 weeks to develop. The initial symptoms begin suddenly and include severe headache, back and abdominal pain, fever, chills, nausea, and blurred vision. Individuals may have facial flushing, inflammation or redness of the eyes, or a rash. Later symptoms can include low blood pressure, acute shock, vascular leak, and acute renal failure, all of which can lead to severe fluid overload.

[0038] The severity of illness varies depending on the virus causing the infection. Hantaan and Dobrava virus infections usually cause severe symptoms, while Seoul, Saaremaa, and Puumala virus infections are usually milder.

[0039] The syndrome can also be fatal. In some cases, it has been known to cause permanent renal failure. HFRS is difficult to diagnose clinically alone and serological evidence is often required. The presence of anti-hantavirus antibodies of the IgM type with a four-fold rise in IgG antibody titers within 1 week is good evidence of acute hantavirus infection. HFRS should be suspected in patients with an acute febrile influenza-like illness, renal failure of unknown origin, and sometimes abnormal liver function tests.

[0040] Hantavirus pulmonary syndrome (HPS) is usually caused by American species of hantaviruses. These viruses include Black Creek Canal virus (BCCV), New York ortho-hantavirus (NYV), Monongahela virus (MGLV), Sin Nombre ortho-hantavirus (SNV), and certain other members of the genus Hantavirus native to the United States and Canada. Specific rodents are the primary hosts of hantaviruses, including the bristle-tailed cotton rat (Sigmodon hispidus) in southern Florida, which is the primary host of Black Creek Canal virus, the deer mouse (Peromyscus maniculatus) in Canada and the western United States, which is the primary host of Sin Nombre virus, and the white-footed mouse (Peromyscus leucopus) in the eastern United States, which is the primary host of New York virus. In South America, the long-tailed whistling rat and other species of the genus Sigmodon have been shown to be hosts of Andes virus.

[0041] Symptoms of HPS are flu-like symptoms such as fever, cough, myalgia, headache, lethargy, and shortness of breath, which can rapidly worsen into acute respiratory failure. It is characterized by the sudden onset of shortness of breath and the rapid progression to pulmonary edema; it is often fatal despite mechanical ventilation and potent diuretic intervention. The mortality rate is 36%. HPS is easily overlooked because its early symptoms are very similar to those of the flu. Infected patients experience fatigue, fever, and muscle pain within a few weeks of exposure, often accompanied by headaches, dizziness, and gastrointestinal problems. About a week after the initial symptoms subside, the second stage of the disease begins, and patients experience severe coughing and shortness of breath as the lungs fill with fluid. In the later stages of HPS, the lungs are severely damaged, resulting in a high mortality rate.

[0042] In the therapeutic or preventive uses of the present invention, the patient is preferably a mammal, in a preferred embodiment, a primate, and most preferably a human patient. In an alternative mode of administration, it is planned to treat mammals known to be carriers of hantaviruses, such as rodents and bats, especially rats, mice and deer mice, to prevent human infection through these hosts. In this context, in areas with high rates of hantavirus infection, a broad-spectrum mode of administration via inhalation (environmental spray formulations) may be considered. Since human-to-human transmission is rare in hantavirus infections, such broad application to mammalian non-human hosts such as rodents can be used as a preventive measure.

[0043] In addition, it may be useful to administer a MEK inhibitor to human subjects who have been in contact with rodents or rodent excreta or have been living or traveling in areas where hantavirus outbreaks are common to prevent hantavirus infection, particularly if the human subject has been living or traveling in an area known to have hantavirus infection that causes HFRS or HPS.

[0044] In particular, such treatment or preventive treatment is useful when the human patient has been traveling or living in an area known to have hantavirus or dobrava virus infection or hantavirus infection caused by Black Creek Canal virus (BCCV), New York Hantavirus (NYV), Monongahela virus (MGLV), Sin Noble Hantavirus (SNV), or Andes virus in South Korea, Serbia, or the U.S. Since transmission of hantavirus occurs primarily through aerosolized rodent excreta (urine, saliva, feces), preventive treatment is started immediately after exposure to rodent excreta until there are no symptoms of infection for 10 days after exposure.

[0045] The MEK inhibitor can be administered orally, intravenously, intrapleurally, intramuscularly, topically or via inhalation. Preferably, the MEK inhibitor is administered via inhalation or orally.

[0046] Additionally, the MEK inhibitor may be administered up to 12 hours, up to 24 hours, up to 48 hours, up to 72 hours, or between 4 and 10 days after the first symptoms of HFRS or HPS are observed or the human patient is exposed to rodent excreta.

[0047] In one embodiment of the use of the present invention for treatment or prevention, the compound MEK inhibitor can be administered orally or via inhalation at an effective therapeutic dose. In one embodiment, the therapeutically effective amount of the MEK inhibitor is, for example, 0.1 mg to 2000 mg, 0.1 mg to 1000 mg, 0.1 to 500 mg, 0.1 to 200 mg, 30 to 300 mg, 0.1 to 75 mg, 0.1 to 30 mg.

[0048] As described above, the present invention further provides a pharmaceutical composition comprising a MEK inhibitor or a pharmaceutically acceptable salt or metabolite thereof, which is used as a medicament for preventing and / or treating viral infections, preferably infections caused by Hantavirus.

[0049] The pharmaceutical composition of the present invention can be in the form of a suspension or tablet for oral administration; a nasal spray, for example, a sterile injectable preparation (intravenous, intrapleural, intramuscular) as a sterile injectable aqueous or oily suspension or suppository. When administered orally as a suspension, these compositions are prepared according to the available techniques in the field of pharmaceutical preparations and may contain microcrystalline cellulose for providing volume, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity increasing agent, and sweeteners / flavoring agents known in the art. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and / or other excipients, binders, fillers, disintegrants, diluents and lubricants known in the art. Injectable solutions or suspensions can be formulated according to known techniques using the following substances: suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution; or suitable dispersants or wetting agents and suspending agents, such as sterile, mild, fixed oils including synthetic mono- or diglycerides; and fatty acids including oleic acid. The drug compounds in the methods of the present invention can be administered in any suitable single dosage form. In the context of the pharmaceutical compositions of the present invention, suitable oral formulations can also be in the form of tablets, capsules, suspensions, syrups, chewing gums, wafers, elixirs, and the like. Pharmaceutically acceptable carriers, such as binders, excipients, lubricants, and sweeteners or flavoring agents can be included in oral pharmaceutical compositions. If desired, conventional agents for changing the taste, color, and shape of a particular form may also be included.

[0050] For injectable preparations, the pharmaceutical composition can be a lyophilized powder mixed with a suitable excipient in a suitable vial or tube. Before clinical use, the drug can be reconstituted by dissolving the lyophilized powder in a suitable solvent system to form a composition suitable for intravenous or intramuscular injection.

[0051] In one embodiment, the pharmaceutical composition can be an oral administration form (e.g., tablet or capsule or syrup, etc.) containing a therapeutically effective amount (e.g., 0.1 mg to 2000 mg, 0.1 mg to 1000 mg, 0.1 to 500 mg, 0.1 to 200 mg, 30 to 300 mg, 0.1 to 75 mg, 0.1 to 30 mg) of a MEK inhibitor.

[0052] definition

[0053] Throughout the specification and in the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprise" and "comprising" will be understood to mean the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps. When used herein, the term "comprising" may be replaced with the term "containing" or, at times when used herein, with the term "having".

[0054] When used herein, "consisting of" excludes any element, step or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that have no substantial effect on the basic and novel features of the claim. In various embodiments herein, any one of "comprising", "consisting essentially of" and "consisting of" may be replaced with one of the other two terms.

[0055] As used herein, the conjunction "and / or" between multiple listed elements is understood to include both individual and combined options. For example, if two elements are connected by "and / or", the first option means that only the first element is applicable and the second element is not applicable; the second option means that only the second element is applicable and the first element is not applicable; the third option means that the first element and the second element are applicable together. Any of these options can be understood to fall within this meaning, and therefore meet the requirements of the term "and / or" used herein. The simultaneous application of multiple options can also be understood to fall within this meaning, and therefore meet the requirements of the term "and / or" used herein.

[0056] Example

[0057] Material

[0058] Puumala virus (PUUV) strain Sotkamo

[0059] -The main Hantaviruses in Europe

[0060] -Can be processed in S2 laboratory

[0061] Vero cell lines

[0062] - Cell line derived from African green monkey kidney cells

[0063] - Commonly used for virus growth

[0064] - Type I interferon deficiency

[0065] Cell Culture Medium:

[0066] Basal medium: IMDM, 1% P / S, 1% L-Gln, 10% FCS

[0067] ATR-002 / CI-1040 treatment medium: basal medium w / o FCS

[0068] Virus infection medium: basal medium w / o FCS

[0069] The MEK inhibitor ATR-002 (PD0184264) [2-(2-chloro-4-iodophenylamino)-N-3,4-difluorobenzoic acid, the active metabolite of CI-1040, was synthesized at ChemCon GmbH (Freiburg, Germany).

[0070] The MEK inhibitor CI-1040 [2-(2-chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide] was synthesized at ChemCon GmbH (Freiburg, Germany).

[0071] Example 1 : Virus yield reduction test

[0072] method:

[0073] Vero cells were seeded in 24-well plates (1×10 6 cells / well) and incubated at 37°C, 5% CO2.

[0074] One day after seeding, cells were infected with PUU-virion particles (MOI 0.3).

[0075] One hour after infection, cells were treated with 40 μM Cl-1040, 40 μM ATR-002 or DMSO (solvent control).

[0076] The supernatant was collected 72 h after infection and the TCID 50 The virus titer was determined experimentally.

[0077] TCID 50 Test (SOP-ATR-0119)

[0078] Virus titration was performed using standard operating procedure SOP-ATR-0119. Briefly, 10% homogenates of lung and kidney / supernatant from the VYR assay were diluted in a 1:10 serial dilution. Vero cells were infected with different 10-fold virus dilutions and incubated for 60 min at 37°C in a 5% CO2 atmosphere. After incubation, the cells were rinsed with PBS and supplemented with 200 μl IMDM (Iscove's Modified Dulbecco's medium) / BA (bovine albumin)-medium (0.2% BA, 1 mM MgCl2, 0.9 mM CaCl2, 100 U / ml penicillin, 0.1 mg / ml streptomycin) and incubated at 37°C in 5% CO2 for 7 days. Thereafter, the Vero cells were washed and incubated at 4°C with -Histofix fixation for 30 minutes. After washing with PBS, cells were permeabilized with Triton-X-100 and FCS. Primary antibody (anti-PUUV-NP-AB) was incubated for one hour. After washing, secondary antibody was applied to cells for 30 minutes. Then, cells were washed and stained with substrate TrueBlue for 10 minutes. Analyzed by optical microscopy.

[0079] result:

[0080] from Figure 1 It can be seen that in the presence of CI-1040, compared with the solvent control group, >2log 10 - Step significant reduction in viral titer. This corresponds to a reduction in viral titer of >99%.

[0081] exist Figure 2 Similar results were seen in the presence of ATR-002, achieving >1,5 log 10 -Step reduction in viral titer. This corresponds to a reduction in viral titer of >90%.

[0082] in conclusion:

[0083] Treatment of Vero cells infected with PUUV (MOI 0.3) with 40 μM CI-1040 or 40 μM ATR-002 resulted in a strong reduction in viral titers compared to solvent controls.

[0084] Inhibition of the Raf / MEK / ERK-pathway impairs the propagation of Puumala virus in vitro.

[0085] Example 2: Antiviral effect of ATR-002 against Hantavirus in vivo

[0086] Materials and methods

[0087] Mouse

[0088] There is currently no reliable established animal model for studying Hantavirus infection. Type I interferon-deficient Vero cells have been shown to be a good in vitro model, but type I interferon-deficient mice are not available. Therefore, interferon receptor knockout mice (IFNα / β / γR- / -(AG129) mice) were selected for in vivo studies. AG129 mice were used in the following experiments.

[0089] method

[0090] AG129 mice were intranasally infected with PUUV (5 × 10 5 / pfu, 25 μl inoculated into each nostril).

[0091] Treatment with 75 mg / kg / day ATR-002 (in DMSO / Cremophor EL / PBS) started 5 h after infection and continued for 5 days. Administration route: oral administration, 37.5 mg / kg, twice a day (9am and 6pm). The control group was treated with the solvent only.

[0092] Mice were sacrificed on days 7 and 10 post-infection to determine the viral titers (TCID 50 test).

[0093] result

[0094] After PUUV infection, no animals lost weight, developed clinical symptoms, or died.

[0095] PUUV was detectable in the lungs and kidneys of solvent control animals at 7 and 10 days post infection. Virus titers were higher at day 10.

[0096] In the lungs and kidneys of animals treated with 75 mg / kg / day of ATR-002, no virus was detected at 7 and 10 days post-infection (detection limit: lung: 3.4 log 10 (TCID 50 / g organ) Kidney: 3.2log 10 (TCID 50 / g organs)), respectively Figure 3 and 4 shown.

[0097] The reduction in viral load (comparison between homogenates treated with vehicle control and ATR-002) was highly significant (two-way-ANOVA, P < 0.0001).

[0098] in conclusion

[0099] The study showed that treatment of mice with 75 mg / Kg / day of ATR-002 (in DMSO / CremophorEL / PBS) for five days, starting 5 hours after PUUV infection, significantly reduced viral load in the lungs and kidneys.

Claims

1. Use of a MEK inhibitor in the preparation of a medicament for treating Hantavirus infection in a mammal, wherein the MEK inhibitor is CI-1040 or PD-0184264 or a pharmaceutically acceptable salt thereof.

2. The use according to claim 1, wherein the mammal is a rodent or a human.

3. The use according to claim 2, wherein the mammal is a human who exhibits symptoms of Hantavirus Hemorrhagic Fever with Renal Syndrome (HFRS) or Hantavirus Pulmonary Syndrome (HPS).

4. The use according to claim 3, wherein the MEK inhibitor is administered up to 12 hours, up to 24 hours, up to 48 hours, up to 72 hours or between 4 and 10 days after the first symptoms of HFRS or HPS are observed.

5. The use according to any one of claims 2 to 4, wherein the human has been living or traveling in an area where Hantavirus infection causing HFRS or HPS is known to occur.

6. The use according to claim 5, wherein the hantavirus is a hantavirus or eastern baravovirus infection, or a hantavirus infection caused by an American species.

7. The use according to claim 6, wherein the American species is Black Creek Canal Virus (BCCV), New York Hantavirus (NYV), Monophyletic Halavirus (MGLV), Noboth Hantavirus (SNV) or Anders virus.

8. The use according to any one of claims 1 to 4, wherein the MEK inhibitor is administered orally or via inhalation.

9. The use according to claim 8, wherein the MEK inhibitor is administered by inhalation.

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