Novel MEK-inhibitor for treatment of viral and bacterial infections

Abstract

To provide improved MEK inhibitors for use in a method for the treatment of a viral infection or a viral and bacterial superinfection.SOLUTION: There is provided PD-0184264 or a pharmaceutically acceptable salt thereof for use in a method of treatment of a viral disease, wherein the subject has been symptomatic for the viral infection for at least 24 hours when treatment is started.SELECTED DRAWING: None

Description

[Background technology]

[0001] background Influenza A viruses are causative agents of severe respiratory diseases that result in significant morbidity and mortality. The majority of fatal cases during the course of influenza virus infection are actually the result of secondary pneumonia caused by different bacteria, such as Staphylococcus aureus (S. aureus), Streptococcus pneumoniae, and Haemophilus influenzae (Morens et al., 2008, Chertow et al., 2013). The most prominent problem of bacterial superinfection is the sudden increase in virulence (Iwao et al., 2012, Paddock et al., 2012, Parker et al., 2012) and the limited availability of potent anti-infective drugs against different pathogenic bacteria. The high mutability of influenza viruses and the continuous emergence of new strains (Neumann et al., 2009, Taubenberger et al., 2010, Parry, 2013), the inherent characteristics of bacterial strains (Grundmann et al., 2006, Moran et al., 2006, Gillet et al., 2007, Shilo et al., 2011), and the rapid development of resistance to available drugs / antibiotics, both in influenza viruses (Hayden et al., 1992, Bright et al., 2006, Pinto et al., 2006, De Clercq et al., 2007, Pinto et al., 2007) and bacteria (Grundmann et al., 2006, Moran et al., 2006, Shilo et al., 2011), are major reasons for inadequate treatment options.

[0002] WO2001 / 076570 provides a concept for treating or preventing infection caused by (-)RNA viruses (especially influenza viruses) using MEK inhibitors. WO2014 / 056894 provides specific MEK inhibitors, such as AZD-6244, AZD-8330, RDEA-119, GSK-1120212 (trametinib), GDC-0973 (cobimetinib), CI-1040, PD-0325901, RO-5126766, and MSC1936369 (AS-703026), for use in treating or preventing influenza virus infection. WO2015 / 173788A1 discloses MEK inhibitors for use in methods for treating influenza virus and bacterial co-infection.

[0003] The 2009 H1N1 pandemic clearly demonstrated the strong impact that influenza A viruses (IAVs) have on global healthcare systems (Mackey and Liang, 2012; Monto et al., 2011; Robertson and Inglis, 2011). This has been confirmed by the 2019 / 2020 coronavirus (COVID-19) pandemic. Aside from vaccination, only a few antiviral drugs are approved for influenza, and none are known to date for COVID-19. This highlights the urgent need for additional effective antivirals to better control infection. Particularly in the early stages of a pandemic, when vaccines are unavailable, antivirals are the sole treatment. Furthermore, the emergence of IAVs resistant to currently approved antivirals highlights the urgent need for new and sufficiently available antivirals (Moss et al., 2010).

[0004] However, although a few promising MEK inhibitors are known and provided for use in the treatment or prevention of viral infections, particularly influenza virus infections, there remains a need to provide further, improved MEK inhibitors ideally suited for such uses.

[0005] Furthermore, it is crucial to start antiviral therapy for viral infections as soon as possible after infection. For example, the time frame for initiating influenza treatment with oseltamivir is only 24 hours at most. Very often, patients visit their doctor after this period, failing to undergo standard antiviral treatment. Furthermore, antiviral treatment may be contraindicated due to potential drug interactions, especially in elderly patients, who often must take one or more medications continuously. Very often, stopping such medications is not easy. As a result, the time frame for initiating antiviral therapy is already exceeded before these patients are suitable for antiviral therapy.

[0006] In view of the prior art, it is clear that there is a need for new compounds and compositions that are effective in the treatment of viral diseases, particularly respiratory diseases caused by RNA viruses such as influenza virus (IV) or coronavirus (CoV), especially when treatment initiation is slow. Additionally, there is a need for the treatment of viral infections where the virus is resistant to antiviral therapy, as well as the prevention and / or treatment of bacterial superinfections. Summary of the Invention

[0007] The solution to the technical problem is the provision of PD-0184264, a metabolite of CI-1040, for use in the treatment or prevention of viral infections (e.g., influenza or coronavirus infections), bacterial infections, or co-infections including bacterial and viral infections. This solution is also reflected in the embodiments and claims described below and illustrated in the Examples and Figures. The inventors of the present application surprisingly found that PD-0184264, a metabolite of the MEK inhibitor CI-1040, has greater antiviral and antibacterial activity than CI-1040 itself. Furthermore, PD-0184264 has been shown to be effective even when treatment is initiated more than 24 hours after infection, preferably because the subject has been symptomatic of the viral infection for at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours.

[0008] Preferably, treatment is initiated at least 24 hours and no later than 48 hours after the onset of disease. Preferably, the viral infection is caused by a positive- or negative-strand RNA virus. Preferably, the negative-strand RNA virus is an influenza virus, more preferably, the virus is an influenza A virus (IAV) or an influenza B virus (IBV), even more preferably, the influenza A virus is selected from the group consisting of H1N1, H2N2, H3N2, H5N6, H5N8, H6N1, H7N2, H7N7, H7N9, H9N2, H10N7, N10N8, or H5N1, or the influenza B virus is selected from the group consisting of IBV Yamagata lineage and Victoria lineage. Preferably, the positive-strand RNA virus is a coronavirus, such as severe acute respiratory virus (SARS-CoV and SARS-CoV2) or Middle East respiratory syndrome coronavirus (MERS).

[0009] In some embodiments, the virus is resistant to antiviral treatment, more preferably the antiviral treatment is administration of oseltamivir, zanamivir, peramivir, amantadine, rimantadine, favipiravir, baloxavir marboxil, or pimoxivir.

[0010] In one embodiment, the virus that is resistant to antiviral treatment is the H1N1 H275Y mutant.

[0011] Therefore, the use of PD-0184264 solves the technical problem underlying the present application by providing a more effective treatment option for viral infections (particularly influenza virus infections), as well as viral (particularly influenza virus) and bacterial co-infections, with an extended treatment window that allows treatment initiation later than 24 hours and up to at least 72 hours after the identification of the first symptoms.

[0012] While the MEK inhibitor CI-1040 is already effective in treating or preventing influenza virus infections and influenza virus or bacterial co-infections, the present inventors have discovered that a metabolite of CI-1040 (PD-0184264, Formula 1) is more effective than CI-1040 itself in targeting influenza virus or co-infections including influenza virus and bacterial infections. PD-0184264 is one of several metabolites of CI-1040 (Wabnitz et al., 2004, LoRusso et al., 2005), but it was not known or could have been predicted that the metabolite would be more potent than CI-1040. To the inventors' surprise, as shown in the Examples, they found that one metabolite (PD-0184264, Structure 1 below) is indeed more effective than CI-1040 and has greater therapeutic potential. Indeed, this property of PD-0184264 was unexpected, as PD-0184264 exhibited a weaker inhibitory effect on MEK kinase in vitro than CI-1040 (see Example 9). Furthermore, in vitro assays demonstrated a weaker antiviral effect of PD-0184264 compared to CI-1040 (see Example 10), yet surprisingly, in vivo assays demonstrated a much stronger antiviral effect of PD-0184264 compared to CI-1040 (see Example 11). TIFF2025124703000001.tif68128

[0013] Therefore, the present invention relates to PD-0184264 or a pharmaceutically acceptable salt thereof for use in a method for preventing and / or treating bacterial infections and / or viral diseases. Preferably, the virus causing the viral disease is an RNA virus. If the RNA virus is a minus-strand RNA virus, it is preferably an influenza virus, more preferably an influenza A or B virus. If the RNA virus is a plus-strand RNA virus, it is preferably a coronavirus such as SARS, SARS-CoV2, or MERS.

[0014] The present invention also relates to PD-0184264, or a pharmaceutically acceptable salt thereof, for use in a method for the prevention and / or treatment of a bacterial infection, preferably mediated by a bacterium selected from the group consisting of Staphylococcaceae, Streptococcaceae, Legionellaceae, Pseudomonadaceae, Bacillaceae, Chlamydiaceae, Mycoplasmataceae, Enterobacteriaceae, Pseudomonadales, and / or Pasteurellaceae.

[0015] The present invention further relates to PD-0184264, or a pharmaceutically acceptable salt thereof, for use in a method for the prevention and / or treatment of co-infections, including bacterial infections and viral diseases.

[0016] Preferably, PD-0184264 or a pharmaceutically acceptable salt thereof is for use in a method for the prevention and / or treatment of bacterial infections and co-infections, including viral diseases, wherein the bacterial infection is mediated by a bacterium selected from the group consisting of Staphylococcus, Streptococcus, Legionellaceae, Pseudomonadaceae, Bacillaceae, Chlamydiaceae, Mycoplasmataceae, Enterobacteriaceae, Pseudomonadales, and / or Pasteurellaceae.

[0017] Preferably, PD-0184264 or a pharmaceutically acceptable salt thereof is for use in a method for the prevention and / or treatment of co-infections, including bacterial infections and viral diseases, wherein the virus is a negative-strand RNA virus, preferably an influenza virus, more preferably an influenza A virus or an influenza B virus.

[0018] Preferably, PD-0184264, or a pharmaceutically acceptable salt thereof, is for use in a method for the prevention and / or treatment of a viral disease, wherein PD-0184264, or a pharmaceutically acceptable salt thereof, is administered in combination with a neuraminidase inhibitor, or a pharmaceutically acceptable salt thereof.

[0019] Preferably, PD-0184264, or a pharmaceutically acceptable salt thereof, is for use in a method for the prevention and / or treatment of co-infections, including bacterial infections and viral diseases, wherein PD-0184264, or a pharmaceutically acceptable salt thereof, is administered in combination with a neuraminidase inhibitor, or a pharmaceutically acceptable salt thereof.

[0020] In an alternative embodiment, PD-0184264, or a pharmaceutically acceptable salt thereof, is for use in a method for the prevention and / or treatment of a viral disease, or a method for the prevention and / or treatment of a co-infection, including a bacterial infection and a viral disease, wherein the neuraminidase inhibitor is selected from oseltamivir, oseltamivir phosphate, zanamivir, laninamivir, or peramivir, or a pharmaceutically acceptable salt thereof.

[0021] Also provided by the present invention is a pharmaceutical composition comprising PD-0184264 or a pharmaceutically acceptable salt thereof and a neuraminidase inhibitor or a pharmaceutically acceptable salt thereof.

[0022] Preferably, PD-0184264 is for use in methods for the prevention and / or treatment of viral diseases, and / or in methods for the prevention and / or treatment of bacterial infections, and / or in methods for the prevention and / or treatment of co-infections, including bacterial infections and viral diseases, wherein PD-0184264 is combined with one or more MEK inhibitors.

[0023] Preferably, PD-0184264, or a pharmaceutically acceptable salt thereof, is for use in the prevention and / or treatment of a bacterial infection, viral infection, or superinfection in a subject, preferably a vertebrate, more preferably a bird or mammal, most preferably a human.

[0024] The present disclosure further relates to a method of treating a bacterial infection in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of PD-0184264 or a pharmaceutically acceptable salt thereof.

[0025] Furthermore, the present invention relates to a method for treating a viral disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of PD-0184264 or a pharmaceutically acceptable salt thereof. In a preferred embodiment, PD-0184264 is administered to a subject once daily at an oral dose of 100 mg to 900 mg, preferably 600 mg.

[0026] In a further aspect, the present invention relates to a method of treating a co-infection, including a bacterial infection and a viral disease, in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of PD-0184264 or a pharmaceutically acceptable salt thereof.

[0027] Preferably, the virus is a negative-strand RNA virus, more preferably the virus is an influenza virus, and most preferably the influenza virus is an influenza A virus or an influenza B virus.

[0028] Preferably, the bacterial infection is mediated by a bacterium selected from the group consisting of Staphylococcus, Streptococcus, Legionellaceae, Pseudomonadaceae, Bacillaceae, Chlamydiaceae, Mycoplasmataceae, Enterobacteriaceae, Pseudomonadales, and Pasteurellaceae. [The present invention 1001] PD-0184264 or a pharmaceutically acceptable salt thereof for use in a method for treating a viral disease, wherein the subject has been exhibiting symptoms of the viral infection for at least 24 hours at the time treatment is initiated. [The present invention 1002] PD-0184264 or a pharmaceutically acceptable salt thereof for use in accordance with the present invention, wherein the subject has been showing symptoms of a viral infection for at least 36 hours, at least 48 hours, or at least 72 hours at the time treatment is initiated. [The present invention 1003] Further, PD-0184264 or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of bacterial co-infections according to the present invention 1001 or 1002. [The present invention 1004] PD-0184264 or a pharmaceutically acceptable salt thereof for use in any of inventions 1001 to 1003, wherein the viral infection is a respiratory infection caused by a negative-strand or positive-strand RNA virus. [The present invention 1005] PD-0184264 or a pharmaceutically acceptable salt thereof for use in the present invention 1004, wherein the negative-strand RNA virus is an influenza virus. [The present invention 1006] PD-0184264 or a pharmaceutically acceptable salt thereof for use in the present invention 1005, wherein the virus is an influenza A virus or an influenza B virus. [The present invention 1007] PD-0184264 or a pharmaceutically acceptable salt thereof for use in the present invention 1006, wherein the influenza A virus is selected from the group consisting of H1N1, H2N2, H3N2, H5N6, H5N8, H6N1, H7N2, H7N7, H7N9, H9N2, H10N7, N10N8 or H5N1. [The present invention 1008] PD-0184264 or a pharmaceutically acceptable salt thereof for use in the present invention 1006, wherein the influenza B virus is selected from the group consisting of IBV Yamagata strain or Victoria strain. [The present invention 1009] PD-0184264 or a pharmaceutically acceptable salt thereof for use in any of inventions 1001 to 1004, wherein the viral infection is caused by a positive-strand RNA virus. [The present invention 1010] PD-0184264 or a pharmaceutically acceptable salt thereof for use in the present invention 1009, wherein the virus is a coronavirus. [The present invention 1011] PD-0184264 or a pharmaceutically acceptable salt thereof for use in the present invention 1010, wherein the virus is SARS-CoV, SARS-CoV2, or MERS. [The present invention 1012] PD-0184264 or a pharmaceutically acceptable salt thereof for use in any of inventions 1001 to 1011, wherein the virus is resistant to standard antiviral treatment. [The present invention 1013] PD-0184264 or a pharmaceutically acceptable salt thereof for use in the present invention 1012, wherein the standard antiviral treatment is the administration of oseltamivir, zanamivir, peramivir, amantadine, rimantadine, favipiravir, baloxavir marboxil, and / or pimoxivir. [The present invention 1014] PD-0184264 or a pharmaceutically acceptable salt thereof for use in the present invention 1012, wherein the virus is H1N1 virus A H275Y mutant. [The present invention 1015] PD-0184264 or a pharmaceutically acceptable salt thereof for use in any of inventions 1001 to 1014, wherein the subject has been treated with a standard antiviral agent prior to administration of PD-0184264. [The present invention 1016] PD-0184264 or a pharmaceutically acceptable salt thereof for use in any of claims 1001 to 1015, administered in combination with a neuraminidase inhibitor or a pharmaceutically acceptable salt thereof. [The present invention 1017] PD-0184264 or a pharmaceutically acceptable salt thereof for use in the present invention 1016, wherein the neuraminidase inhibitor is selected from oseltamivir, oseltamivir phosphate, zanamivir, laninamivir, or peramivir, or a pharmaceutically acceptable salt thereof. [The present invention 1018] PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any of the preceding inventions in a subject, preferably a vertebrate, most preferably a human. [The present invention 1019] PD-0184264 or a pharmaceutically acceptable salt thereof for use in any of inventions 1001 to 1018, wherein PD-0184264 is administered to a human subject once daily at a dose of 100 to 900 mg, preferably 600 mg. [The present invention 1020] PD-0184264 or a pharmaceutically acceptable salt thereof for use in accordance with the present invention, wherein PD-0184264 is administered to a human subject for 1 to 21 consecutive days, preferably 7 to 14 consecutive days. [The present invention 1021] PD-0184264 or a pharmaceutically acceptable salt thereof for use in accordance with the present invention 1019 or 1020, wherein PD-0184264 is administered to a human subject in an oral dosage form. [Brief explanation of the drawings]

[0029] The drawings show:

[0030] [Figure 1-1] Treatment of mice infected with influenza A with PD-0184264 or CI-1040. Results from the experiment in Example 1 are presented as virus titer (log10) pfu / ml (left) or virus titer % (right). [Figure 1-2] A continuation of Figure 1-1 is shown. [Figure 2]Graph showing the effect of PD-0184264 and CI-1040 on the growth of MRSA bacteria. At various time points, as indicated on the horizontal axis of the graph, the optical density of the cell-free bacterial culture was measured as an indicator of bacterial growth and is shown on the vertical axis of the graph in % (OD600). Data represent the average of three biological replicates as described in Example 2. [Figure 3] Inhibition of bacterial MRSA growth by various concentrations of PD-0184264. PD-0184264 was administered to overnight cultures of Staphylococcus aureus USA300 (MRSA) at various concentrations (as indicated). After 6 hours, optical density was tested. Data shown in the figure represents one of three biological replicates described in Example 2. [Figure 4] Effect of CI-1040 and PD-0184264 on bacterial growth. (A) Effect of various concentrations of CI-1040 on bacterial growth. (B, C) Effect of various concentrations of PD-0184264 on Staphylococcus aureus strain 6850 (B) or strain USA300 (C). [Figure 5] Administration of PD-0184264 to mono- or co-infected cells protects the cells. Human lung epithelial cells (A549) were pretreated with PD-0184264 (at the indicated concentrations) or vehicle (DMSO) and then infected with the human influenza virus strain A / Puerto Rico / 8 / 34 (H1N1). Given the antiviral and potent antibacterial effects of PD-0184264 (Figures 2–4), we analyzed whether this characteristic of this compound could be macroscopically observed in the cell-destructive cytopathic effect (CPE) induced by IAV (influenza A virus) and / or Staphylococcus aureus infection. Following monoinfection with IAV (H1N1) (middle panel) or S. aureus strain 6850 (top panel), slight destruction of the cell monolayer was observed. This CPE was strongly increased upon co-infection with both pathogens (bottom panel) but was inhibited in the presence of increasing concentrations of PD-0184264. [Figure 6]Comparing the antibacterial activity of PD-0184264 with that of common antibiotics. To compare the antibacterial properties of the MEK inhibitor PD0184264 with that of common antibiotics, we treated bacteria overnight with vehicle, MEK inhibitors U0126 and PD-0184264, or various concentrations of the antibiotic gentamicin. Compared to vehicle-treated bacteria, incubation with the first-generation MEK inhibitor U0126 resulted in only a slight reduction in bacterial titer, whereas treatment with PD-0184264 resulted in a very strong reduction in bacterial load. This was true for both bacterial strains. [Figure 7A] Results of a time-of-addition assay comparing the antibacterial activity of PD-0184264 with other MEK inhibitor compounds or the antibiotic gentamicin. [Figure 7B] Results of testing the resistance of S. aureus to the MEK inhibitor PD0184264 compared with treatment with gentamicin or erythromycin or no treatment. [Figure 8] Domain structure of the bacterial kinase PknB (top) (Rakette et al. 2012), and sequence homology between the autophosphorylation site of PknB and the activation site of ERK, a direct target of the mammalian MAP kinases p38, JNK, and MEK (Miller et al. 2010). [Figure 9] Determination of the effect of inhibitor treatment on the minimum inhibitory concentrations (MICs) of various antibiotics. [Figure 10] Stress resistance of Staphylococcus aureus strain 6850 and MRSA strain USA300 upon treatment with PD-0184264. [Figure 11A] Figure 11: Treatment with PD-0184264 attenuates the growth of various serotypes of Streptococcus pneumoniae. (A) Effect of PD-0184264 on cultures of Streptococcus pneumoniae strains TIGR4 (serotype 4) and D39 wt (serotype 2) as measured by OD600. [Figure 11B] (B) Effect of PD-0184264 on cultures of S. pneumoniae strains TIGR4 (serotype 4) and D39 wt (serotype 2), shown in CFU / ml. [Figure 11C](C) Effect of PD-0184264 on cultures of Bacillus subtilis (expressed in CFU / ml). [Figure 12-1] Table 1: Antibiotics. [Figure 12-2] A continuation of Figure 12-1 is shown. [Figure 13] Photographs showing that treatment of cells with PD-0184264 strongly reduces pathogen-induced CPE (cytopathic effect) during bacterial (Staphylococcus aureus) or viral (influenza IV) mono- and co-infection. [Figure 14A] Figure 14: Graph showing the change in intracellular bacterial load upon treatment with CI-1040 (A) or PD-0184264 (B). Comparable results were obtained when CI-1040 or PD-0184264 was administered at later time points during the progression of infection (C). [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 15] Graphs showing cell viability (A, B) and membrane disruption (C, D). (A, B) Shows the viability of A549 cells in the presence of increasing concentrations of PD-0184264. In (C) and (D), an LDH assay was performed to determine membrane disruption by the inhibitor. [Figure 16] Cell-free kinase assay showing the inhibitory effects of CI-1040 and PD-0184264 on the MEK pathway. Kinase activity is measured by determining the amount of phosphorylated target protein ERK using an ELISA assay. Three independent experimental series were performed with similar results. One representative experiment is presented here. [Figure 17]Antiviral activity of PD-0184264 against influenza virus H1N1pdm09 in an in vitro assay. (A) A549 cells were infected with virus (MOI = 0.001) to determine the reduction in virus titer. (B) A549 cells were infected with virus RBI (MOI = 0.001) and treated with various concentrations of PD-0184264 (100 μM, 50 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, and 0.1 μM) to determine the EC50 value. (C) A549 cells were treated with various concentrations of PD-0184264 (100 μM, 50 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, and 0.1 μM) for 24 hours, followed by WST staining for 4 hours to determine the CC50 value. [Figure 18] PD-0184264 reduces viral titers in mouse lungs in vivo. After H1N1pdm09 virus infection, female C57BL / 6 mice were treated orally with 2.8, 8.4, or 25 mg / kg of PD-0184264 (left panel) or 25, 75, or 150 mg / kg of CI-1400 (right panel). Animals were sacrificed 24 hours post-infection, and viral titers were determined using standard methods. [Figure 19] PD-0184264 has better bioavailability than CI-1040. (A) Male NMRI mice were treated intravenously with 75 mg / kg CI-1040 (dark gray area) or 75 mg / kg PD-0184264. Blood was collected 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours (study day 2) after administration, and plasma was analyzed for the presence of drug. (B) Male NMRI mice were treated orally with 150 mg / kg CI-1040 (dark gray area) or 150 mg / kg PD-0184264 via oral gavage. Blood was collected 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours (study day 2) after administration, and plasma was analyzed for the presence of drug. [Figure 20]Mouse survival after treatment with PD-0184264. Eight female C57BL / 6 mice were infected with H1N1pdm09 and treated with 25 mg / kg PD-0184264 (ATR-002, central gray line) or vehicle alone (light gray line) via the oral route. Five days of treatment began 24, 48, or 72 hours after infection, as indicated on the graph (gray bars). Mice had to be sacrificed when they experienced a 20% weight loss (gray dotted line). Graphpad Prism 8 software was used to interpret both figures. P values ​​were determined using the Logrank (Mantel-Cox) test. DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description The following description contains information that may be useful in understanding the present invention. None of the information provided herein is admitted to be prior art to or relevant to the claimed invention, nor is any publication specifically or implicitly referenced admitted to be prior art.

[0032] As noted above, the present invention relates to PD-0184264 for use in methods of treating viral diseases or co-infections involving bacterial and viral infections. As shown in the Examples, PD-0184264 appears to act on the bacterial kinase PknB, and may thereby exert its bacteriostatic effects, at least in part.

[0033] Furthermore, as demonstrated in the accompanying examples, PD-0184264 exhibits efficacy not only in viral and bacterial infection scenarios, but also in bacterial and viral co-infection scenarios. This effect is surprisingly more potent than that of CI-1040, a MEK inhibitor already known in the prior art for treating bacterial and viral infections. Comparing the inhibitory effects of CI-1040 and PD-0184264 on MEK kinase as shown in Example 9, one would actually expect the opposite result, since MEK inhibitors were used to achieve this effect in the prior art.

[0034] The same holds true when comparing the antiviral effects of PD-0184264 and CI-1040 in in vitro assays. Here, CI-1040 is more effective than PD-0184264, as can be seen from Example 10. Specifically, a 10-fold higher concentration of PD-0184264 is required in in vitro assays to achieve the same inhibitory effect. Surprisingly, despite weaker in vitro inhibition, PD-0184264 was found to be superior to CI-1040 in vivo, as can be seen from Example 11. As shown in Figure 18, PD-0184264 already exhibits a reduction in viral titer at 2.8 mg / kg, and 25 mg / kg of PD-0184264 demonstrates at least a 90% reduction in viral titer in the lungs. In contrast, CI-1040 only exhibits a similar reduction at 150 μM. Furthermore, Example 1 demonstrates the reduction of viral titers in the lungs by PD-0184264. In Example 1, mice were infected with influenza virus and treated with 150 mg / kg, 75 mg / kg, or 25 mg / kg of CI-1040 or PD-0184264, respectively. As shown in Figure 1, 25 mg / kg of PD-0184264 already has the same effect as 150 mg / kg of CI-1040. Thus, to the inventors' surprise, PD-0184264 exhibits potent antiviral effects in vivo, even though previous in vitro data provided no incentive to continue research with PD-0184264. This surprising effect may be due to the greater bioavailability of PD-0184264 compared to CI-1040, as shown in Example 12.

[0035] Examples 2, 4, and 6-8 further demonstrate the potent antibacterial effect of PD-0184264 compared to CI-1040 and other MEK inhibitors. Example 2 analyzes the effect of PD-0184264 on bacterial growth. Figure 2 shows that PD-0184264 inhibits bacterial growth, while CI-1040 has no effect on bacterial growth. Figure 3 shows that bacterial growth is inhibited by PD-0184264 in a concentration-dependent manner, demonstrating nearly complete growth inhibition starting at 50 μM PD-0184264. Similarly, Figure 4 also shows that bacterial growth is inhibited by PD-0184264 in a concentration-dependent manner—showing a significant reduction in growth already at 10 μM PD-0184264—while CI-1040 has no effect on bacterial growth. In Example 4, the antibacterial effect of PD-0184264 was compared with that of the MEK inhibitor U0126 or the antibiotic gentamicin. Figure 6 shows that PD-184264 has an antibiotic effect similar to gentamicin, while the MEK inhibitor U0126, known from the prior art, has no effect on bacterial growth. The same is true in Figure 7A, where the bacterial growth process is monitored. Figure 7B further shows that PD-0184264 does not induce resistance to PD-0184264, but bacteria readily develop resistance to gentamicin and erythromycin. Thus, PD-0184264 does not induce resistance in bacteria. In Example 6, the effect of PD-0184264 on bacterial susceptibility to antibiotics was analyzed. As shown in Figure 9 and Table 2, treatment with PD-0184264 indeed resulted in increased bacterial susceptibility to various antibiotics, most notably penicillin and gentamicin. Furthermore, Figure 10 shows that PD-0184264 reduces bacterial stress tolerance. Example 7 provides evidence that the effects of PD-0184264 are not limited to Staphylococcus aureus, but also exert effects on other bacteria, such as Streptococcus pneumoniae (see Figures 11A and B) and Bacillus subtilis (see Figure 11C).

[0036] Examples 3 and 8 highlight the efficacy of PD-0184264 in bacterial and viral co-infection situations. Example 3 analyzed whether the efficacy of PD-0184264 could also be observed macroscopically with respect to cell-destructive cytopathic effect (CPE) induced by viral and / or bacterial infection. Figure 5 shows that PD-0184264 specifically reduces the cytopathic effect induced by bacterial and viral co-infection in a concentration-dependent manner, demonstrating efficacy even at 15 μM PD-0184264. The same analysis was performed in Example 8, whereby Figure 13 demonstrates the positive effect of PD-0184264 in bacterial and viral co-infection scenarios. Example 8 further analyzed the effect of PD-0184264 on intracellular bacterial titers in co-infection scenarios. Figure 14 shows that PD-0184264 reduces intracellular bacterial titers, while CI-1040 has no effect. Furthermore, Example 8 and Figure 15 show that PD-0184264 does not exert a cytotoxic effect.

[0037] Furthermore, Example 13 showed that treatment of mice infected with the H1N1 influenza strain with PD-0184264 resulted in a significant difference in survival, even when the drug was administered as late as 72 hours after infection (see Figure 20).

[0038] These results are comparable to the data obtained with CI-1040 presented in Haasbach's paper (Haasbach et al. 2017) and in further experiments conducted by the present applicant. In these experiments, CI-1040 was found to have a therapeutic window that was extended compared to known antiviral drugs, particularly Tamiflu (oseltamivir), allowing administration during periods when Tamiflu is ineffective. Using a mouse model, the inventors demonstrated that CI-1040 can reduce influenza virus pulmonary titers in vivo. Surprisingly, the therapeutic window of CI-1040 was found to extend to at least 48 hours postinfection, a time when Tamiflu® treatment is ineffective.

[0039] According to FDA labeling, oseltamivir (Tamiflu®) is currently indicated for the treatment of influenza in patients within two days of the onset of symptoms. This corresponds to previous observations in mouse models that oseltamivir is no longer effective when administered more than 48 hours after influenza virus infection. In contrast, PD-0184264 was effective even 72 hours after infection. Thus, PD-0184264, like CI-1040, has been shown to have a longer therapeutic window than oseltamivir. For this reason, the effects observed in mouse models are likely to be extrapolated to human patients, meaning that PD-0184264 or a pharmaceutically acceptable salt or derivative thereof can be administered to patients more than two days after the onset of symptoms of viral infection. This is also consistent with the comparison of the modes of action of CI-1040 and PD-0184264 with that of oseltamivir. As mentioned above, oseltamivir is thought to block the release of virions from infected cells. For this reason, it is only effective when virions have not yet left the cell. In contrast, CI-1040 and PD-0184264 were found to block the MEK pathway, which is necessary for viral replication, thereby retaining the viral RNP complex in the cell nucleus. Regarding the molecular mechanism of action of MEK inhibition, the inventors previously showed that inhibition of the kinase with the prototype inhibitor U0126 resulted in blocking the nucleocytoplasmic transport of the viral RNP complex (Pleschka et al., 2001). This can be confirmed for CI-1040 and PD-0184264. Based on the data in Example 13 and further preliminary studies, PD-0184264 is a metabolite of CI-1040 and targets the same pathway as CI-1040, and is therefore expected to have the same effect as CI-1040.

[0040] Therefore, the methods and uses of the present invention also relate to the treatment of viral infection in subjects who have been symptomatic for at least 24 hours, at least 36 hours, or at least 48 hours.Of course, these times include all intervening time periods, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47 hours for symptomatic subjects.In a preferred embodiment, the subject has been symptomatic for at least 48 hours before treatment.For example, treatment can be initiated between 24 and 48 hours after the onset of symptoms.In this regard, it should be noted that "symptomatic" and "onset of symptoms" can be used interchangeably.However, treatment can also be initiated at least 60 hours, at least 72 hours, or at least 96 hours after infection. If PD-0184264, or a pharmaceutically acceptable salt or derivative thereof, is administered when a different antiviral agent has already been found to be ineffective, PD-0184264, or a pharmaceutically acceptable salt or derivative thereof, may be administered 24 hours after completion of treatment with the previous antiviral agent.

[0041] Furthermore, like CI-1040, PD-0184264 is highly likely to be effective against influenza viruses that are resistant to antiviral treatment. This feature of CI-1040 and PD-0184264 solves many of the problems of standard antiviral treatments and allows them to be administered after standard antiviral drug treatment has failed. As used herein, standard antiviral treatment is defined as treatment with a drug that is approved for use as an antiviral and is effective in inhibiting the development of viral pathogens at any stage of their life cycle. Examples include entry inhibitors, uncoating inhibitors, reverse transcription inhibitors, polymerase inhibitors, endonuclease inhibitors, protein maturation inhibitors, integrase inhibitors, transcription inhibitors, translation inhibitors, protease inhibitors, virion assembly inhibitors, or virion release inhibitors. In the context of influenza viruses, two different standard antiviral treatment approaches are known: neuraminidase inhibitors and M2 protein inhibitors.

[0042] Therefore, in one embodiment, the virus is resistant to antiviral treatment.As outlined herein, antiviral treatment can be related to the administration of neuraminidase inhibitors such as oseltamivir, zanamivir, laninamivir and peramivir, and / or the administration of M2 inhibitors such as amantadine and rimantadine.Therefore, antiviral treatment can be the administration of oseltamivir, zanamivir, amantadine and / or rimantadine.In another embodiment, antiviral treatment can be related to the administration of oseltamivir, zanamivir, peramivir, amantadine, rimantadine, favipiravir, baloxavir marboxil and / or pimoxivir.

[0043] PD-0184264 can be used in methods for the treatment and / or prevention of the medical conditions described herein. Accordingly, the terms "treating" or "treatment" include administering PD-0184264, preferably in pharmaceutical form, to a subject suffering from a co-infection, including bacterial infections and viral diseases, for the purpose of alleviating or ameliorating symptoms associated with such infections. Similarly, the term "treatment" includes administering PD-0184264, preferably in pharmaceutical form, to a subject suffering from a bacterial infection for the purpose of alleviating or ameliorating symptoms associated with such infections. Furthermore, the term "treatment" includes administering PD-0184264, preferably in pharmaceutical form, to a subject suffering from a viral infection for the purpose of alleviating or ameliorating symptoms associated with such infections. Co-infections, including bacterial infections and viral diseases, viral diseases, and bacterial infections are medical conditions that may be treated or prevented by PD-0184264 or a pharmaceutically acceptable salt thereof.

[0044] Furthermore, the terms "prophylaxis" or "prevention," as used interchangeably herein, refer to any medical or public health treatment aimed at preventing a medical condition described herein. As used herein, the terms "prevent," "prevention," and "preventing" refer to a reduction in the risk of acquiring or developing a given condition, i.e., a superinfection including a viral infection and a bacterial infection, a bacterial infection only, or a viral infection only, as described herein. "Prevention" also refers to a reduction or inhibition of recurrence of a superinfection including an influenza virus infection and a bacterial infection, a bacterial infection only, or a viral infection only in a subject.

[0045] PD-0184264 of the present invention is effective in treating superinfections, as shown in Examples 3 and 8. As used herein, "superinfection" includes viral diseases, preferably influenza virus infections, and bacterial infections. Such superinfections can occur when bacteria and influenza viruses co-infect a host, e.g., a subject and / or a single cell. It can also occur when a host, e.g., a subject and / or cell, is simultaneously infected with one or more viral particles and one or more bacteria. However, such superinfections can also occur sequentially. In such cases, a subject and / or cell is first infected with one or more viral particles, and then, at a later time, the same subject and / or cell is infected with one or more bacteria, or vice versa. The period between two infections can be at most 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 6 hours, 3 hours, 1.5 hours, or as short as 30 minutes. In this regard, prolonged administration is expected to result in more effective prevention of secondary bacterial infections due to the extended therapeutic window of PD-0184264. Such a situation may also be a superinfection, in which a second infection with a different microbial agent of exogenous or endogenous origin that is resistant to the treatment used against the first infection is superimposed on a previous infection. Among the influenza virus infections of superinfection, the influenza virus infection can be mediated by influenza A virus or influenza B virus, and preferably, the influenza A virus is H1N1, H2N2, H3N2, H5N6, H5N8, H6N1, H7N2, H7N7, H7N9, H9N2, H10N7, N10N8, or H5N1. In one embodiment, the influenza A virus is H1N1. In another embodiment, the influenza A virus is H3N2, H5N1, and H7N9. In a further embodiment, the influenza A virus is H3N2, H5N1, H1N1, H5N6, H7N2, and H7N9.

[0046] The present invention also relates to "bacterial infections" that may occur in the context of the above-mentioned superinfections present in a host (e.g., a subject and / or a cell). The bacterial infection can be mediated by any bacteria, preferably It is mediated by bacteria selected from the group consisting of Staphylococcus, Streptococcus, Legionellaceae, Pseudomonadaceae, Bacillaceae, Chlamydiaceae, Mycoplasmataceae, Enterobacteriaceae, Pseudomonadales, and / or Pasteurellaceae.

[0047] The bacterial infection may be caused by Staphylococcus, preferably Staphylococcus aureus, methicillin-susceptible and methicillin-resistant Staphylococcus aureus, Panton-Valentine leukocidin (PVL)-expressing Staphylococcus aureus and / or Streptococcus, preferably Streptococcus mitis, Streptococcus pyogenes or Streptococcus pneumoniae, Legionella, preferably Legionella pneumophila, Pseudomonas, preferably Pseudomonas aeruginosa, Bacillus, preferably Bacillus subtilis, Chlamydophila, preferably Chlamydophila pneumonia, Mycoplasma, preferably Mycoplasma pneumoniae The infection may be mediated by a bacterium selected from the group consisting of Klebsiella pneumoniae, Klebsiella, preferably Klebsiella pneumoniae, Moraxella, preferably Moraxella catarrhalis, and / or Haemophilus, preferably Haemophilus influenzae. Preferably, the bacterium is selected from the group consisting of Staphylococcus aureus, Streptococcus pneumoniae, or Haemophilus influenzae. Most preferably, the bacterium is Staphylococcus aureus.

[0048] The "viral disease" or "viral infection" to which the present invention also relates may occur in the context of the above-mentioned superinfection, or may occur as the only infection present in a host, e.g., a subject and / or cells. The viral disease or infection may be mediated by any virus; preferably, it is mediated by a virus that can cause respiratory infection (e.g., an RNA virus). A preferred virus is an influenza virus or a coronavirus. Preferably, the influenza virus infection is mediated by influenza A or influenza B virus, with influenza A virus being preferred. Influenza A virus subtypes H1N1, H2N2, H3N2, H5N6, H5N8, H6N1, H7N2, H7N7, H7N9, H9N2, H10N7, N10N8, and / or H5N1 are particularly preferred. Preferably, the coronavirus infection is mediated by SARS-CoV, SARS-CoV2, or MERS-CoV.

[0049] In alternative embodiments, PD-0184264 is administered in combination with one or more MEK inhibitors. MEK inhibitors include, for example, U0126, PLX-4032, AZD6244, AZD8330, AS-703026, GSK-1120212, RDEA-119, RO-5126766, RO-4987655, CI-1040, PD-0325901, GDC-0973, TAK-733, PD98059, ARRY-438162, ARRY-162, ARRY-300, PF-3644022, and PD184352. The additional MEK inhibitor may be administered simultaneously with, before, or after PD-0184264.

[0050] Preferably, PD-0184264 is for use in the methods of the invention for preventing and / or treating co-infection, wherein PD-0184264 is combined with one or more inhibitors that target influenza virus or bacteria. PD-0184264 may be administered simultaneously with, before, or after the one or more inhibitors that target influenza virus.

[0051] Generally, an inhibitor targeting influenza virus is any inhibitor or drug effective in treating influenza. Various substances are known to be effective in reducing influenza virus infection. Among them are, for example, inhibitors against viral neuraminidase, compounds targeting viral ion channel proteins (M2), and compounds that target viral polymerase activity or endonuclease activity by interfering with components of the viral polymerase complex: PB1, PB2, PA, or NP. Pharmaceutically acceptable salts of these inhibitors are also contemplated by the present invention. However, preferred inhibitors are MEK inhibitors, as described herein, with PD-0184264 being particularly preferred.

[0052] "MEK inhibitors" inhibit the mitogenic signaling cascade Raf / MEK / ERK in cells or in a subject by inhibiting MEK (mitogen-activated protein kinase kinase). This signaling cascade is hijacked by many viruses, especially influenza viruses, to promote viral replication. Therefore, specific blockade of the Raf / MEK / ERK pathway at the bottleneck MEK attenuates the proliferation of viruses, especially influenza viruses. Furthermore, MEK inhibitors have low toxicity and show few side effects in humans. They also do not tend to induce viral resistance (Ludwig, 2009). A particularly preferred inhibitor is PD-0184264.

[0053] "Neuraminidase inhibitors" are antiviral drugs that target influenza viruses and work by blocking the function of the viral neuraminidase protein, thereby preventing the virus from being released from infected host cells, because newly produced viruses cannot bud from the cells in which they replicate. Also included are pharmaceutically acceptable salts of neuraminidase inhibitors. Preferred neuraminidase inhibitors are oseltamivir, zanamivir, peramivir, laninamivir, or pharmaceutically acceptable salts of any of these substances, such as oseltamivir phosphate and oseltamivir carboxylate. The most preferred neuraminidase inhibitors are oseltamivir phosphate, zanamivir, oseltamivir, or peramivir.

[0054] Compounds that target the viral ion channel protein (M2) are, for example, amantadine and / or rimantadine, while compounds that target polymerase or endonuclease activity by interfering with components PB1, PB2, PA, or NP of the viral polymerase complex are, for example, the NP blocker nucleozin or the polymerase inhibitor T-705 (favipiravir).

[0055] Furthermore, PD-0184264 can be combined with one or more inhibitors that target bacteria. Example 6 demonstrates that PD-0184264 increases bacterial susceptibility to antibiotics. The inhibitor that targets bacteria can be any inhibitor that is effective in reducing bacterial infection. While PD-0184264 is strongly preferred as the inhibitor that targets bacteria in the present invention, other inhibitors that target bacteria known to those skilled in the art are antibiotics. Preferred antibiotics can be obtained from Table 1 (Figure 12). Thus, in one embodiment, the antibiotic is selected from the group consisting of antibiotics listed in Table 1 (Figure 12). In a further embodiment, the antibiotic is selected from the group consisting of antibiotic classes listed in Table 1 (Figure 12). In another embodiment, the antibiotic is selected from the group consisting of generic names of antibiotics listed in Table 1 (Figure 12). More preferred antibiotics are selected from gentamicin, rifampicin, lysostaphin, erythromycin, levofloxacin, vancomycin, teicoplanin, penicillin, and oxacillin.

[0056] A "subject" that can be treated with the inhibitors of the present invention, particularly MEK inhibitors, or a combination of inhibitors, is preferably a vertebrate. In the context of the present invention, the term "subject" includes an individual who requires treatment for only a superinfection or a bacterial or viral infection as described herein. Preferably, the subject is a patient suffering from or at risk of only a superinfection or a bacterial or viral infection. Preferably, the patient is a vertebrate, more preferably a mammal. Mammals include, but are not limited to, livestock, sport animals, pets, primates, mice, and rats. Preferably, the mammal is a human, horse, dog, cat, cow, pig, mouse, rat, etc., and particularly preferably a human. In some embodiments, the subject is a human subject, which may optionally be older than 1 year and younger than 14 years, between 50 and 65 years, between 18 and 50 years, or older than 65 years. In other embodiments, the subject is a human subject selected from the group consisting of subjects who are at least 50 years of age, subjects residing in a long-term care facility, subjects with chronic disorders of the pulmonary or cardiovascular system, subjects who have required regular medical follow-up or hospitalization within the previous year due to chronic metabolic disease, renal dysfunction, hemoglobinopathy or immunosuppression, subjects under the age of 14 years, subjects 6 months to 18 years of age receiving long-term aspirin therapy, and women who would be in their second or third trimester of pregnancy during influenza season. In the methods of the present invention, PD-0184264 can be administered orally, intravenously, intrathoracically, intramuscularly, topically, or by inhalation. Preferably, PD-0184264 is administered by inhalation, topically, or orally. In a preferred embodiment, PD-0184264 is administered orally once daily for 7 to 21 days at a dose of 100 mg to 900 mg, preferably 600 mg.

[0057] Specifically, as described in Example 14, PD-0184264 was administered according to a single-ascending dose / multiple-ascending dose (SAD / MAD) regimen, with a starting dose of 100 mg and seven escalation steps (up to three dose levels). The dosing regimen consisted of a single dose of PD-0184264 (SAD) escalated from 100 mg to 900 mg, followed by seven doses of PD-0184264 (MAD) escalated from 100 mg to 600 mg once daily (QD) over seven days. Each dose cohort was deemed safe by the Safety Review Committee (SRC), allowing release of the next higher dose (up to a maximum SAD of 900 mg and a MAD of 600 mg, respectively). The observed pharmacokinetic profile supports the once-daily regimen for further clinical development.

[0058] Overall, only a few adverse events were observed during the study, and no serious adverse events were observed. Therefore, PD-0184264 is considered safe and well tolerated. Pharmacokinetic exposure and MEK inhibition assessments were validated, confirming the maintenance of clinically relevant blood levels.

[0059] The present invention also contemplates various compositions, preferably pharmaceutical compositions. The present invention relates to compositions comprising PD-0184264 for use in methods for preventing and / or treating superinfections, including bacterial infections and viral diseases. The present invention similarly relates to compositions comprising PD-0184264 for use in methods for preventing and / or treating bacterial infections and / or viral diseases. The present invention also provides compositions comprising PD-0184264 and one or more inhibitors that target viruses, particularly influenza viruses and / or bacteria, for use in methods for preventing and / or treating superinfections, including bacterial infections and viral infections, particularly influenza virus infections. In addition, the present invention relates to compositions comprising PD-0184264 and one or more inhibitors that target bacteria, for use in methods for preventing and / or treating bacterial or viral infections.

[0060] As described above, a composition ultimately comprising PD-0184264 and one or more inhibitors targeting bacteria and / or one or more inhibitors targeting viruses can be a pharmaceutical composition. A preferred embodiment of the pharmaceutical composition comprises PD-0184264 and an inhibitor targeting influenza virus, particularly a neuraminidase inhibitor. In another embodiment, the pharmaceutical composition comprises PD-0184264 and an additional MEK inhibitor. Preferably, such a composition further comprises a carrier, preferably a pharmaceutically acceptable carrier. The composition can be in the form of an orally administrable suspension or tablet, a nasal spray, a preparation for an inhalation device, a sterile injectable preparation (intravenous, intrapleural, intramuscular), such as a sterile injectable aqueous or oily suspension, or a suppository.

[0061] The pharmaceutical composition for use in the present invention, comprising PD-0184264 and, optionally, one or more inhibitors targeting influenza viruses and / or one or more inhibitors targeting bacteria, is administered to a mammalian or avian patient. Examples of suitable mammals include, but are not limited to, mice, rats, cows, goats, sheep, pigs, dogs, cats, horses, guinea pigs, canines, hamsters, mink, seals, whales, camels, chimpanzees, rhesus monkeys, and humans, with humans being preferred. Examples of suitable birds include, but are not limited to, turkeys, chickens, geese, ducks, teals, mallards, starlings, pintails, gulls, swans, guinea fowl, or waterfowl. Human patients are a specific embodiment of the present invention.

[0062] The inhibitor is preferably administered in a therapeutically effective amount. As will be apparent to those skilled in the art, the "therapeutically effective amount" of PD-0184264 or each active compound / inhibitor can vary depending on factors including, but not limited to, the activity of the compound used, the stability of the active compound in the patient's body, the severity of the condition to be alleviated, the total body weight of the patient being treated, the route of administration, the ease with which the compound is absorbed, distributed, and excreted by the body, the age and sensitivity of the patient being treated, any adverse events, etc. The dosage can be adjusted to accommodate changes in various factors over time.

[0063] The inhibitors, methods, and uses described herein are applicable to both human therapy and veterinary use. Compounds described herein, particularly PD-0184264, and optionally one or more inhibitors targeting influenza virus and / or one or more inhibitors targeting bacteria, having the desired therapeutic activity, can be administered to a subject in a physiologically acceptable carrier, as described herein. Depending on the mode of delivery, the compounds can be formulated in various ways, as discussed below. The concentration of the therapeutically active compound in the formulation can vary from about 0.1 to 100% by weight. The agent can be administered alone or in combination with other treatments. Example 1 demonstrates that 25 and 75 mg / kg of PD-0184264 are effective in vivo via oral administration. Accordingly, PD-0184264 can be administered at doses ranging from 10 to 100 mg / kg of PD-0184264, preferably from 25 to 75 mg / kg. A preferred dosage is 100-900 mg once daily, including any dose between 200, 300, 400, 500, 600, 700, and 800 mg. In a preferred embodiment, administration is once daily and the dose is 600 mg administered orally. PD-0184264 can be administered for 1 to 21 or more consecutive days, preferably 7 to 14 days.

[0064] The pharmaceutical compounds in the method of the present invention can be administered in any suitable unit dosage form.Suitable oral formulations can be in the form of tablets, capsules, suspensions, syrups, chewing gum, wafers, elixirs, etc.Pharmaceutically acceptable carriers such as binders, excipients, lubricants, and sweeteners or flavoring agents can be included in the oral pharmaceutical composition.If desired, conventional agents can also be included to modify the taste, color, and shape of a particular dosage form. For injectable formulations, the pharmaceutical composition may be a lyophilized powder that is mixed with a suitable excipient in a suitable vial or tube. Prior to use in the clinic, the drug may be reconstituted by dissolving the lyophilized powder in a suitable solvent system to form a composition suitable for intravenous or intramuscular injection.

[0065] Combining PD-0184264 with an antiviral agent (e.g., a neuraminidase inhibitor, such as oseltamivir) results in a synergistic effect. This synergistic effect can be an increased antiviral effect, for example, resulting in a reduced viral titer or an extended therapeutic window. Thus, the present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of PD-0184264 and a therapeutically effective amount of a neuraminidase inhibitor selected from the group consisting of oseltamivir, oseltamivir phosphate, laninamivir, zanamivir, and peramivir. In one embodiment, the composition may be an orally administrable dosage form (e.g., a tablet or capsule or syrup, etc.) having a therapeutically effective amount of a neuraminidase inhibitor (e.g., 0.1 mg to 2000 mg, 0.1 mg to 1000 mg, 0.1 mg to 500 mg, 0.1 mg to 500 mg, 0.1 mg to 200 mg, 30 mg to 300 mg, 0.1 mg to 75 mg, 0.1 mg to 30 mg), as described above.

[0066] In a further aspect, PD-0184264 is for use in a method of the invention for the prevention and / or treatment of co-infection, comprising administering PD-0184264 to a patient comprising: a) Influenza virus b) bacteria When contacted with an in vitro test system comprising cultured cells infected with a bacterium, PD-0184264 reduces both viral infection and bacterial infection compared to the in vitro test system before contact. In another embodiment, PD-0184264 is for use in a method of preventing and / or treating bacterial infection of the invention, wherein when PD-0184264 is contacted with an in vitro test system comprising cultured cells infected with a bacterium, PD-0184264 reduces bacterial infection compared to the in vitro test system before contact.

[0067] Therefore, the present invention also provides a method for producing a pharmaceutical composition comprising: a) Influenza virus and b) bacteria According to this, the present invention also provides an in vitro test system comprising cultured cells infected with a bacterium.

[0068] Again, when the in vitro test system involves viral and bacterial infections, these infections may occur sequentially or simultaneously.

[0069] "Cultured cells" are cells that do not exist in their natural environment, e.g., in a plant or animal. Rather, cultured cells can be primary cell cultures or cell lines that include cells isolated from their natural environment. Preferably, the cultured cells are human lung epithelial cells. Preferably, the cultured cells are at a concentration of approximately 1 x 10 in 0.5 ml, 1 ml, 1.5 ml, 2 ml, 2.5 ml, 3 ml, 3.5 ml, or 4 ml of medium, such as DMEM. 5 , 2 × 10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 10×10 5 , 11×10 5 , most preferably 8 x 10 5Most preferably, cells are seeded at a density of 8 x 10 in 2 ml of DMEM. 5 is the density of cells.

[0070] Such cultured cells are infected with viruses and bacteria, or in other embodiments, only with bacteria. As already mentioned above, superinfection can occur sequentially or simultaneously. For example, cultured cells can be first infected with influenza virus and then infected with bacteria 30 minutes later. It is also possible to add antibiotics to the culture medium after 3 hours to remove extracellular bacteria. In such a scenario, the antibiotics would then be washed away again. In other embodiments, the cells are only infected with bacteria.

[0071] As used herein, the term "contacting" refers to bringing cells containing influenza virus and bacteria into spatial proximity with PD-0184264. This may mean, for example, applying the inhibitor to the medium in which the cultured cells are placed via a syringe.

[0072] In one embodiment, a reduction in viral infection is a reduction in plaque-forming units (PFU) / ml, and a reduction in bacterial infection is a reduction in colony-forming units (CFU) / ml. "Plaque-forming units" is a measure of the number of particles (e.g., virus particles) capable of forming plaques per unit volume. This is a functional measurement, not an absolute measurement of particle quantity: defective virus particles or virus particles that do not infect their target cells do not produce plaques and are therefore not counted. For example, a solution of influenza virus with a concentration of 1,000 PFU / μl indicates that 1 μl of the solution contains enough virus particles to produce 1,000 infectious plaques in a cell monolayer. In the present invention, cell cultures treated with the inhibitor exhibit a reduced number of plaque-forming units in the culture after treatment with PD-0184264 compared to the culture before treatment. A possible "reduction in plaque-forming units (PFU) / ml" is analyzed in the following manner. First, a culture of cells co-infected with influenza virus and bacteria is analyzed for its ability to generate plaque-forming units (PFU) / ml, for example, by spitting some cells from a Petri dish and plating them to count the bacterial plaques formed. This result is then compared with the number of plaque-forming units (PFU) / ml generated by cells from the same culture after an inhibitor has been applied. A reduction in plaque-forming units is observed if the number of plaque-forming units (PFU) / ml is reduced after treatment with the inhibitor compared to the number generated before application of the inhibitor.

[0073] "Colony-forming units (CFU) / ml" estimates the number of viable bacteria in a sample. Different methods exist. For example, to generate colony-forming units, a sample (e.g., a small amount of cultured cells) is spread on the surface of a nutrient agar plate and allowed to dry before incubation for counting. Viable bacteria are defined as the ability to grow via binary fission under controlled conditions. Significant growth is required for colonies in cell culture to be visible—when counting colonies, it is unclear whether the colony arose from a single cell or 1,000 cells. Therefore, results are reported as CFU / ml (colony-forming units per milliliter) for liquids and CFU / g (colony-forming units per gram) for solids (rather than cells / ml or cells / g) to reflect this uncertainty.

[0074] "Colony forming units (CFU) / ml" can be analyzed in the following manner. First, a culture of cells co-infected with influenza virus and bacteria, or infected only with bacteria, is analyzed for its ability to generate colony forming units (CFU) / ml, for example, by sucking a few cells from a Petri dish and plating them for counting. This result is then compared to the number of colony forming units (CFU) / ml generated by cells from the same culture after an inhibitor has been applied. A reduction is noted if the number of colony forming units (CFU) / ml is reduced to the number generated before the inhibitor was applied.

[0075] Generally, those skilled in the art are aware of these well-known techniques for analyzing bacterial and viral infections. Methods that can measure plaque-forming units (PFU) / ml and colony-forming units (CFU) / ml are further described in the literature (Tuchscherr et al. 2011; Hrincius et al. 2010).

[0076] For the purpose of the present invention, the active compound defined above also includes its pharmaceutically acceptable salt.As used herein, the phrase "pharmaceutical or cosmetically acceptable salt" refers to a salt of the compound of the present invention that is safe and effective for the desired administration form.Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0077] The present invention is also characterized by the following: 1. PD-0184264 or a pharmaceutically acceptable salt thereof for use in a method for the prevention and / or treatment of a viral disease. 2. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to Item 1, wherein the virus is a minus-strand RNA virus. 3. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to item 1 or 2, wherein the virus is an influenza virus. 4. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of items 1 to 3, wherein the influenza virus is an influenza A virus or an influenza B virus. 5. PD-0184264 or a pharmaceutically acceptable salt thereof for use in a method for the prevention and / or treatment of a bacterial infection. 6. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to item 5, wherein the bacterial infection is mediated by a bacterium selected from the group consisting of Staphylococcaceae, Streptococcaceae, Legionellaceae, Pseudomonadaceae, Bacillaceae, Chlamydiaceae, Mycoplasmataceae, Enterobacteriaceae, Pseudomonadales, and / or Pasteurellaceae. 7. PD-0184264 or a pharmaceutically acceptable salt thereof for use in a method for the prevention and / or treatment of co-infections, including bacterial infections and viral diseases. 8. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to item 7, wherein the bacterial infection is mediated by a bacterium selected from the group consisting of Staphylococcaceae, Streptococcaceae, Legionellaceae, Pseudomonadaceae, Bacillaceae, Chlamydiaceae, Mycoplasmataceae, Enterobacteriaceae, Pseudomonadales, and / or Pasteurellaceae. 9. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to item 7 or 8, wherein the virus is a minus-strand RNA virus. 10. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of items 7 to 9, wherein the viral infection is caused by an influenza virus. 11. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of items 7 to 10, wherein the viral infection is caused by influenza A virus or influenza B virus. 12. PD-0184264 or a pharmaceutically acceptable salt thereof for the use according to any one of items 1 to 4 and 7 to 11, administered in combination with a neuraminidase inhibitor or a pharmaceutically acceptable salt thereof. 13. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to paragraph 12, wherein the neuraminidase inhibitor is selected from oseltamivir, oseltamivir phosphate, zanamivir, laninamivir, or peramivir, or a pharmaceutically acceptable salt thereof. 14. A pharmaceutical composition comprising PD-0184264 or a pharmaceutically acceptable salt thereof. 15. A pharmaceutical composition comprising PD-0184264 or a pharmaceutically acceptable salt thereof and a neuraminidase inhibitor or a pharmaceutically acceptable salt thereof. 16. The use of any of the preceding clauses, comprising an additional MEK inhibitor. 17. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of the preceding paragraphs in a subject, preferably a vertebrate.

[0078] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "reagent" includes one or more of such different reagents, and reference to a "method" includes reference to equivalent steps and methods known to those skilled in the art that may be modified or substituted for the methods described herein.

[0079] All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent that the material incorporated by reference contradicts or is inconsistent with this specification, the present specification will supersede any such material.

[0080] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0081] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprise" can be replaced with the term "containing," or, when used herein, can also be replaced with the term "having."

[0082] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0083] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms.

[0084]

[0001] Throughout the text of this specification, several documents are cited. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. [Example]

[0085] The following examples illustrate the present invention. These examples should not be construed as limiting the scope of the invention. The examples are included for illustrative purposes, and the present invention is limited only by the claims.

[0086] Example 1: Treatment of mice with PD-0184264 results in reduced viral titers in the lungs 1.5 × 10 cells were injected into 8-week-old C57BL / 6 mice (5 mice per group). 5 PFU (5× MLD 50 Mice were infected with influenza virus strain A / Regensburg / D6 / 2009 (RB1, H1N1pdm09). Starting 1 hour before infection, mice were treated at 8-hour intervals with either 150 mg / kg CI-1040, 75 mg / kg CI-1040, 25 mg / kg CI-1040, 75 mg / kg PD0184264, 25 mg / kg PD0184264, or vehicle (control): 50 μl DMSO / 150 μl Cremophor / 800 μl PBS. All animals received a 200 μl oral dose. 24 hours after infection, mice were sacrificed, and lungs were weighed and transferred to Lysing Matrix D tubes (MP Bio). A 10-fold lung volume of BSS (buffered salt solution) was applied to the sample. Organs were minced using a FastPrep FP 120 (Savant). To remove cellular debris, the homogenate was centrifuged at 2000 rpm for 15 minutes, and the supernatant was collected. Determination of virus titer in the homogenate was performed using the AVICEL® plaque assay. Results are presented in Figure 1 as virus titer (log10) pfu / ml (left) or virus titer % (right). Virus titers were determined independently by two researchers. The average values ​​of all titrations are presented.

[0087] Example 2: Administration of CI-1040 or PD-0184264 exhibits inhibitory effects on the growth of bacteria, including MRSA, in vitro To generally examine the effects of CI-1040 or PD-0184264 on bacterial growth, cell-free overnight cultures of S. aureus MRSA strain (USA300) were supplemented with 50 μM CI-1040 or PD-0184264 or the same volume (40 μl) of DMSO (serving as the solvent) (Figure 2). Bacterial growth was monitored for 360 minutes. PD-0184264 had a strong effect on bacterial growth, almost completely eliminating it throughout the entire observation period. As can be seen in Figure 2, CI-1040 slightly inhibited MRSA growth beginning 120 minutes after the start of the experiment compared to the solvent control. This indicates that, in addition to blocking intracellular MEK, PD-0184264 as well as CI-1040 block bacterial components involved in bacterial growth.

[0088] To determine the concentration of PD-0184264 required to inhibit bacterial growth, PD-0184264 was administered at various concentrations (as shown in Figure 3) to overnight cultures of Staphylococcus aureus USA300 (MRSA). MRSA bacteria were incubated with various concentrations of the MEK inhibitor ranging from 0 to 100 μM, and bacterial growth was monitored after 6 hours of incubation. The concentration required to inhibit 50% of bacterial growth ranged from 15 to 25 μM.

[0089] These data can be validated in a slightly different experimental setup, where actual bacterial titers can be determined instead of OD at later time points after treatment. Day cultures of S. aureus 6850 were set at 20 CFU / ml and treated overnight with various concentrations of CI-1040 as indicated at 37°C and 5% CO2. Optical density (OD) measurements were then taken. 600The remaining culture was washed with PBS, and serial dilutions were plated on BHI agar plates. Bacterial titers are shown as colony-forming units per ml (CFU / ml). Results represent the mean ± SD of three independent biological experiments with two technical replicates and are shown in Figure 4A. Additionally, daytime cultures of Staphylococcus aureus 6850 (Figure 4B) or MRSA strain USA300 (Figure 4C) were set at 20 CFU / ml and treated overnight at 37°C and 5% CO with various concentrations of PD-0184264, as indicated. In the morning, the optical density (OD600) was measured. The remaining culture was washed once with PBS, and serial dilutions were plated on BHI agar plates. Bacterial titers were determined using a colony counter (protocol 3) and are shown as colony-forming units per ml (CFU / ml) on a logarithmic scale. Results represent the mean ± SD of three independent biological experiments with two technical replicates. Statistical significance was analyzed by one-way ANOVA followed by Dunnett's multiple comparison test. Both CI-1040 (Figure 4A) and PD-0184264 (Figure 4B, C) were effective in these assays against S. aureus strain 6850 (Figure 4A, B) and MRSA strain USA 300 (Figure 4C). At 20 μM PD-0184264, very strong titer reductions of up to 1.5-2 orders of magnitude were detectable (Figure 4B, C).

[0090] Example 3: Administration of PD-0184264 to singly or coinfected cells protects cells from the cytopathic effects of IAV and / or Staphylococcus aureus Given the antiviral and potent antibacterial effects of PD-0184264 (Figures 2-4 in Example 2 above), we analyzed whether this compound's signature could also be observed macroscopically with respect to the cell-destructive cytopathic effect (CPE) induced by IAV (influenza A virus) and / or Staphylococcus aureus infection. Human lung epithelial cells (A549) were pretreated with PD-0184264 (at the indicated concentrations) or vehicle (DMSO) and infected with the human influenza virus strain A / Puerto Rico / 8 / 34 (H1N1) at a multiplicity of infection (MOI) of 0.001 at 37°C. After 30 min, the virus dilutions were removed, and the cells were rinsed with PBS and supplemented with infiltration medium DMEM / INV (containing 1% human serum albumin and 25 nM HEPES) with or without S. aureus 6850 (MOI = 0.1) in the presence of the indicated concentrations of inhibitor or vehicle control. Three hours after bacterial infection, cells were treated with DMEM / FBS containing 10% FBS and 2 μg / ml lysostaphin for 20 minutes to remove extracellular bacteria. After further washing with PBS, cells were supplemented with infection medium DMEM / BA (0.2% BA, 1 mM MgCl2, 0.9 mM CaCl2, 100 U / ml penicillin, 0.1 mg / ml streptomycin) containing inhibitor or vehicle. After a 24-hour incubation period at 37°C, cell morphology was examined by light microscopy. As shown in Figure 5, slight disruption of the cell monolayer was observed after monoinfection with IAV (H1N1) or S. aureus strain 6850. This CPE was strongly enhanced upon coinfection with both pathogens (lower panel). However, with increasing amounts of MEK inhibitor, the cell monolayer remained intact and cells became less rounded, suggesting that this phenotype may be concentration-dependent. This cytoprotective effect of PD-0184264 fully reflects its antiviral and antibacterial properties (Figure 5).

[0091] Example 4: Comparison of the antibacterial effects of PD-0184264 and common antibiotics To compare the antibacterial properties of the MEK inhibitor PD0184164 with those of common antibiotics, we treated bacteria overnight with vehicle, the MEK inhibitors U0126 and PD-0184264, or various concentrations of the antibiotic gentamicin. Daytime cultures of Staphylococcus aureus 6850 or MRSA strain USA300 were set at 20 CFU / ml and treated overnight at 37°C and 5% CO2 with the MEK inhibitors U0126 and PD-0184264 or the antibiotic gentamicin, as indicated. Optical density (OD600) was measured in the morning. The remaining cultures were washed once with PBS, and serial dilutions were then plated on BHI agar plates. Bacterial titers were determined using a colony counter (protocol 3) and are presented as colony-forming units per ml (CFU / ml). The results shown in Figure 6 represent the mean ± SD of three independent biological experiments with two technical replicates. Statistical significance was analyzed by one-way ANOVA followed by Dunnett's multiple comparison test. Compared to vehicle-treated bacteria, incubation with the first-generation MEK inhibitor U0126 resulted in only a slight reduction in bacterial titer, whereas treatment with PD-0184264 resulted in a very strong reduction in bacterial load, similar to the results previously shown in Figure 4. This was true for both bacterial strains. As expected, gentamicin treatment at concentrations higher than 1 μg / ml strongly inhibited the growth of both bacterial strains, with the antibacterial activity of this antibiotic being stronger in the case of S. aureus 6850. Gentamicin at 0.5 μg / ml failed to detectably reduce bacterial titer for either strain. In summary, the effect of the MEK inhibitor PD-0184264 on bacterial growth was nearly as effective as the antibiotic gentamicin at lower concentrations.

[0092] To further compare the antibacterial activity of PD-0184264 with other MEK inhibitor compounds or the antibiotic gentamicin, a time-of-addition assay was performed (Figure 7a). An overnight culture of S. aureus 6850 was divided into six subcultures containing 15 ml of BHI medium with the solvent DMSO alone, one of the MEK inhibitors U0126 and PD-0184264, or the antibiotic gentamicin at two different concentrations (0.5 or 2 μg / ml). Immediately after addition of the various compounds, OD600 was measured, and serial dilutions were plated on BHI agar plates to calculate bacterial titers. The remaining culture was further incubated at 37°C with 5% CO2 in the presence of the compounds or solvent alone. This procedure was repeated twice, at 3 and 6 hours after inoculation. Bacterial titers were determined using a colony counter (protocol 3) and are expressed as colony-forming units per ml (CFU / ml). Treatment with the MEK inhibitor PD-0184264 showed the strongest inhibition of bacterial growth compared to all other compounds. A medium change was then performed, and the cultures were further incubated without the addition of any substances. All cultures reached the turbidity of previously solvent-treated cultures, suggesting that the MEK inhibitor PD-0184264 exerts a bacteriostatic, rather than bactericidal, effect.

[0093] Resistance to various commonly used antibiotics occurs regularly and is a major problem in the clinic. To test whether the MEK inhibitor PD0184264 causes resistance in S. aureus, cultures were treated continuously for nearly 3 weeks in the presence of the inhibitors, gentamicin, and erythromycin, or left untreated. Specifically, cultures were grown for 24 hours in the presence or absence of the substances, and the OD 600 The β-amyloid ... * p < 0.05; ** p < 0.01; ***p < 0.001; **** p < 0.0001). As seen with gentamicin, resistance development occurred during the first week of treatment, in contrast to the macrolide antibiotic erythromycin. Notably, treatment with the MEK inhibitor did not induce resistance (see results shown in Figure 7b).

[0094] Example 5: The bacterial kinase PknB may be a target of PD-0184264 in bacteria The inhibitor PD-0184264 is believed to be specific for the mammalian kinase MEK. Its direct antibacterial effect raises the question of its mechanism of action in prokaryotes: namely, whether there are MEK-like bacterial components that could be similarly specifically targeted by PD-0184264. In this regard, the bacterial serine / threonine kinase PknB became the focus of investigation. This kinase shows high structural and functional similarity to cellular serine / threonine kinases, more precisely, to MAP kinases that are MEK targets in mammalian cells, such as p38, JNK, and ERK (Miller et al., 2010; Rakette et al., 2012) (Figure 8). Interestingly, this kinase has been shown to be activated by autophosphorylation, strongly suggesting that it exhibits MEK-like activity.

[0095] Example 6: PD-0184264 increases the susceptibility of Staphylococcus aureus to antibiotics and reduces bacterial stress tolerance Due to the expression of three penicillin-binding domains (PASTA) (see Figure 8, top panel), PknB is involved in regulating antibiotic susceptibility. It has been shown that the absence of the kinase results in increased susceptibility to various antibiotics, particularly various β-lactams (Tamber et al. 2010). To investigate whether treatment of bacteria with the MEK inhibitor PD-0184264 can affect bacterial kinases and result in a phenotype similar to that of kinase knockout, bacterial cultures were treated overnight with solvent (DMSO) or 20 μM PD-0184264 and then used to determine the minimum inhibitory concentrations (MICs) of various antibiotics. Daytime cultures of S. aureus 6850 were set at 20 CFU / ml and treated overnight with either solvent (DMSO) or the MEK inhibitor PD-0184264 at 37°C and 5% CO2. Optical density (OD600) was measured in the morning. Briefly, solvent- and inhibitor-treated cultures were washed once with PBS, and 1:1 dilutions were plated onto BHI agar plates. Immediately after inoculation, MIC test stripes of various antibiotics (Thermo Fischer Scientific) were placed in the center of the plates, which were then incubated at 37°C for 18–24 hours. After 18 hours of incubation at 37°C, MIC concentrations were determined by visual analysis of the plates. The concentration at which growth inhibition was no longer observed was designated the MIC for each individual antibiotic. Treatment with PD-0184264 indeed resulted in increased bacterial susceptibility to various antibiotics, most notably penicillin and gentamicin (Figure 9, Table 2). This result is in full agreement with previously published data generated with a kinase-deficient mutant strain (Tamber et al. 2010).

[0096] Table 2. MIC determination after overnight treatment with PD0184264 TIFF2025124703000002.tif45162

[0097] The increased antibiotic susceptibility observed upon treatment with PD-0184264 is consistent with the phenotype of bacteria lacking the kinase (Tamber et al. 2010), strongly suggesting that PknB may be directly targeted by the inhibitor. Because kinases are known to play an important role in bacterial stress resistance, we monitored bacterial growth under heat stress after treating bacteria with the inhibitor PD-0184264. Staphylococcus aureus strain 6850 and MRSA strain USA300 were treated overnight with either solvent or 20 μM PD-0184264. The following day, subcultures with equal amounts of bacteria were prepared (confirmed by OD600 and plated on BHI agar) and further incubated at 42°C for 6 hours. Bacterial titers were then calculated by plating serial dilutions on BHI agar plates. As shown in Figure 10, PD-0184264-treated bacteria were strongly attenuated under these conditions compared to solvent-treated pathogens. This is observed in both the methicillin-susceptible strain 6850 (black bars) and the MRSA strain USA300 (gray bars). Impaired stress resistance in the presence of the inhibitor is another indication that PD-0184264 directly targets the kinase PknB, a key mediator of stress resistance. In summary, the data provide strong circumstantial evidence that PD-0184264 elicits its antibacterial action through inhibition of the bacterial kinase PknB.

[0098] Example 7: Administration of PD-0184264 exhibits inhibitory effects on the growth of Streptococcus pneumoniae and Bacillus subtilis In addition to Staphylococcus aureus, other bacteria are known to cause secondary bacterial pneumonia after influenza virus (IV) infection in patients. The most abundant pathogen in this context is Streptococcus pneumoniae. These bacteria are the most common cause of community-acquired pneumonia. In contrast to Staphylococcus aureus, secondary infection with Streptococcus pneumoniae occurs late after IV infection and therefore represents the end stage of post-influenza pneumonia.

[0099] Similar to Staphylococcus aureus, the majority of Streptococcus pneumoniae strains express eukaryotic-like serine / threonine kinases, such as PknB, which are highly conserved across different genera. Furthermore, these kinases share high homology with cellular MAP kinases (e.g., ERK, JNK, and p38). The results presented in Examples 2–6 obtained using various S. aureus strains already demonstrated the inhibitory effect of PD-0184264 treatment on bacterial growth, indicating the involvement of bacterial kinases, such as PknB, in the observed phenotypes. Surprisingly, a homolog of S. aureus PknB is also present in S. pneumoniae, suggesting that these bacteria may also be susceptible to PD-0184264. Therefore, we analyzed the effects of PD-0184264 on various S. pneumoniae strains. S. pneumoniae strains can be divided into various serotypes, which differ in virulence and overall pathogenicity. To test for serotype- or strain-independent effects, we used encapsulated strains D39 and TIGR4, both virulent but belonging to different serotypes. Treatment with PD-0184264 was found to attenuate the growth of different serotypes of S. pneumoniae. Specifically, daytime cultures of S. pneumoniae strains TIGR4 (serotype 4) and D39 wt (serotype 2) were set to an optical density (OD600) of 1, diluted 1:2000 in BHI medium, and treated overnight with either solvent (DMSO) or various concentrations of the specific MEK inhibitor PD0184264 (PD; the active metabolite of CI-1040), as indicated. OD600 was then measured again (results shown in Figure 11A), and serial dilutions were plated on BHI agar plates to determine bacterial titers (results shown in Figure 11B). As a result, we were able to demonstrate that both serotypes were sensitive to PD-0184264 (Fig. 11a, b).

[0100] The same was true for Bacillus subtilis, where a strong reduction in viable cell count was observed in the presence of 10 μM PD-0184264 and was completely abolished at higher concentrations (see results in Figure 11c). Specifically, to test the potential antibacterial effect on B. subtilis, overnight cultures of B. subtilis were incubated with vehicle or various concentrations of the MEK inhibitor PD-0184264 (as indicated) for 18 hours. Bacterial load was then determined by measuring OD600 and plating serial dilutions on BHI agar plates. The data shown in Figure 11 represent the mean + SD of three independent experiments.

[0101] In summary, these data demonstrate the broad applicability of PD-0184264 in antimicrobial treatment.

[0102] Example 8: PD-0184264, but not CI-1040, reduces intracellular bacterial titers Influenza virus (IV) infection leads to enhanced expression of antiviral cytokines, most importantly type I IFNs, which activate important downstream antiviral responses and may enhance subsequent bacterial infection. To determine whether treatment with CI-1040 or PD-0184264 sensitizes cells to secondary S. aureus infection, immortalized human alveolar basal epithelial cell (A549) cell cultures were infected with influenza virus (IV) and S. aureus in the presence or absence of inhibitors. Specifically, A549 cells were pretreated for 1 hour with 10 μM of the specific MEK inhibitor PD-0184264 or DMSO as a solvent control. Cells were then rinsed with PBS and infected with influenza virus (IV) (MOI as indicated) for 30 minutes at 37°C and 5% CO2. Subsequently, cells were washed with PBS and infected with S. aureus 6850 (MOI as indicated) for 3 hours in the presence or absence of inhibitors. To avoid bacterial overgrowth, an antibiotic wash step with lysostaphin (2 μg / mL) was performed for 20 minutes at 37°C to remove non-internalized bacteria. Cells were then washed once and further incubated in the presence of inhibitors or vehicle for up to 24 hours post-infection (pi). At the end of the incubation period, cell monolayers were analyzed by light microscopy. Microscopy revealed that coinfection with both pathogens resulted in greatly increased cytopathic effect (CPE) compared with single infection (Figure 13, upper panel). CPE was completely abolished in the presence of PD-0184264 (Figure 13, lower panel), indicating reduced viral replication.

[0103] To determine whether treatment with PD-0184264 and CI-1040 was comparable, A549 cells were pretreated with 10 μM CI-1040, PD-0184264, or vehicle (DMSO) for 60 minutes and then infected with influenza IV (H7N7) at an MOI of 0.001 at 37°C. The results are shown in Figures 14A and 14B, respectively. Alternatively, cells were left untreated (DMSO) and infected with IV (H1N1) at an MOI of 0.01 at 37°C. After 30 minutes, virus dilutions were removed, and cells were rinsed with PBS and replenished with infiltration medium with or without S. aureus 6850 (6850) (MOI 0.1) in the presence of 10 μM CI-1040, PD-0184264, or vehicle control. Three hours after bacterial infection, cells were treated with (2 μg / mL) for 20 minutes to remove extracellular bacteria. Cells were then washed and replenished with infection medium containing inhibitor or vehicle. After a total incubation period of 24 hours (post-viral infection), intracellular bacterial titers were analyzed. Results represent the mean + SD of three separate experiments. Statistical significance was assessed by one-way ANOVA followed by Tukey's multiple comparison test ( * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). As can be seen in Figure 14A, treatment with CI-1040 did not sensitize cells to secondary infection with S. aureus, as there was no detectable change in intracellular bacterial load. Surprisingly, as can be seen in Figure 14B, administration of PD-0184264 even resulted in a reduction in intracellular bacterial titers. As shown in Figure 14C, comparable results were obtained when CI-1040 or PD-0184264 was administered at later time points during the infection process.

[0104] To rule out that the reduction in viral and intracellular bacterial replication was the result of a cytotoxic effect of PD-0184264 on A549 cells, cell viability was monitored for 24 and 48 hours in the presence of increasing concentrations of PD-0184264. Furthermore, an LDH assay was performed to determine membrane disruption due to inhibitor treatment. Treatment of A549 cells with PD-0184264 was shown not to induce cytotoxicity. A549 cells were treated with increasing concentrations of PD-0184264 (1, 5, 10, 20, 50, or 100 μM) for 24 hours (as shown in Figures 15A and C) or 48 hours (as shown in Figures 15B and D). After the incubation period, supernatants were collected for measurement of LDH release using the CytoSelect LDH Cytotoxicity Assay Kit according to the manufacturer's instructions (shown in Figures 15C and D). Furthermore, viable cells were counted by staining with trypan blue. Cell viability was normalized to DMSO-treated cells and is shown as percent viability. Data represent the mean + SD of three independent experiments. Statistical significance was calculated by one-way ANOVA followed by Dunnett's multiple comparison test ( * p < 0.05; ** p < 0.01; 27 *** p < 0.001).

[0105] Example 9: IC against CI-1040 and PD-0184264 50 Determining Values Aliquots of inhibitor were dissolved in 100% DMSO (master solution 10 mM). To analyze IC50 values, the following serial dilutions were prepared in a microtiter plate: 50 μM, 25 μM, 5 μM, 2.5 μM, 0.5 μM, 0.25 μM, 0.05 μM, 0.025 μM, and 0.005 μM. 1 μl of each dilution was added to 49 μl of kinase reaction mixture to obtain the following test concentrations: 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, and 0.1 nM.

[0106] Three μl of purified protein solution (active c-Raf1, MEK1wt, and ERK2wt) was mixed with kinase buffer and 1 μl of DMSO or DMSO / inhibitor (final volume 45 μl). The mixture was incubated in the dark at room temperature for 30 minutes. After this preincubation, which ensures binding of the inhibitor to the MEK protein, the kinase reaction was initiated by adding 5 μl of 10 mM ATP and mixing with a pipette. The sample was incubated at 500 rpm for 30 minutes in a 26°C thermomixer (Eppendorf). To stop the kinase reaction, 5.5 μl of 20% SDS solution was added, and the mixture was then incubated at 50°C for 10 minutes. Next, each sample was diluted with 190 μl of blocking buffer (1% BSA in TBST). 100 μl of each sample was added to anti-ERK antibody-coated wells of a 96-well microtiter plate.

[0107] Kinase reaction samples (100 μl / well) were incubated in anti-ERK antibody-coated, BSA-blocked wells of a 96-well microtiter plate for 60 minutes at room temperature. The plate was then washed 3 times with 100 μl of TBST wash buffer for 5 minutes. To detect phosphorylated ERK, anti-phospho-ERK (p44 / p42) antibody (1:3000 in blocking buffer, 100 μl / well) was added and incubated overnight at 4°C.

[0108] After three washing steps (3 x 100 μl / well), HRP-conjugated anti-mouse IgG specific antibody (1:1000 in TBST) was added and incubated for 60 min at room temperature. 100 μl / well of the peroxidase substrate ABTS was added after three additional washing steps (3 x 100 μl / well TBST) and incubated for 30 min at 30°C. The substrate reaction was stopped by adding 2.5 μl of 20% SDS. The optical density (OD) of the mixture was measured at a wavelength of 405 nm in an ELISA reader.

[0109] Cell-free kinase assays revealed that 12.5-fold less CI-1040 was required to inhibit 50% of MEK activity compared to PD-0184264, a weaker inhibitor of MEK kinase (Figure 16). Therefore, one would never have predicted the potent antiviral and antibacterial effects of PD-0184264. However, as shown in the previous examples, PD-0184264 exhibits potent antiviral and antibacterial activity in vivo compared to CI-1040.

[0110] Example 10: Antiviral activity of PD-0184264 in in vitro assays drugs CI-1040 [2-(2-chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide; Lot: CC-5395.0-16] and PD-0184264 (PD0184264) [2-(2-chloro-4-iodophenylamino)-N-3,4-difluorobenzoic acid; Lot: CC-5595.4-10] were synthesized at ChemCon GmbH (Freiburg, Germany). For cell culture experiments, 10 mM stock solutions of CI-1040 (M = 478.66 g / mol) and PD-0184264 (M = 409.55 g / mol) were prepared in DMSO (Merck-Millipore; Germany).

[0111] Viruses and cells Virus inhibition experiments were performed with influenza A virus strain RB1 [A / Regensburg / D6 / 09 (H1N1pdm09)] at an MOI of 0.001.

[0112] Progeny virus inhibition assay A549 cells were infected with RB1 for 30 minutes at 37°C in a 5% CO2 atmosphere. After incubation, virus dilutions were aspirated, and cells were rinsed with PBS and incubated for 24 hours at 37°C in 5% CO2 with 500 μl IMDM (Iscove's Modified Dulbecco's Medium) / BA (bovine albumin)-medium supplemented with 0.6 μl of TPCK-treated trypsin (0.2% BA, 1 mM MgCl2, 0.9 mM CaCl2, 100 U / ml penicillin, 0.1 mg / ml streptomycin) in the presence of either 10 μM CI-1040 or various concentrations of PD-0184264 (100 μM, 50 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, and 0.1 μM, final DMSO concentration = 1%). The solvent control was IMDM / BA-medium with 1% DMSO. Cell culture supernatants were collected and progeny virus titers were determined in MDCK II cells using the AVICEL® plaque assay as previously described (Haasbach et al. 2011; Matrosovich et al. 2006).

[0113] WST assay A549 cells were seeded in 96-well flat-bottom tissue plates (Greiner, Germany) and grown overnight. Subsequently, the cells were treated with various concentrations of PD-0184264 (100 μM, 50 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, and 0.1 μM) dissolved in 100 μl of IMDM (ThermoFisher, Germany) supplemented with 5% fetal bovine serum (Sigma-Aldrich; Germany) at a final DMSO concentration of 1%. The cells were cultured for 24 hours at 37°C and 5% CO2. Then, 10 μl of WST-1 reagent (Roche, Germany) was added to the medium and incubated for 4 hours. During this time, metabolically active cells in culture cleaved the stable tetrazolium salt WST-1 into soluble formazan. After this incubation period, the formed formazan dye was quantified at 405 nm using an ELISA reader. The measured absorbance directly correlated with the number of viable cells.

[0114] result The antiviral activity of PD-0184264 against RB1 was examined using a standard virus inhibition assay (Figure 17A). A 98.87 ± 0.03% reduction in virus titer was observed when cells were treated with 100 μM PD-0184264 (P >0.0001). A similar reduction was observed with 50 μM PD-0184264 (91.50 ± 2.08%; P >0.0001). In contrast, only a weak reduction in virus titer was observed with 10 μM PD-0184264 (58.97 ± 4.45%). 1 μM PD-0184264 resulted in little reduction in progeny virus. Thus, compared to the viral reduction achieved by 10 μM CI-1040 (96.78 ± 0.65%; P > 0.0001), a nearly 10-fold higher concentration of PD-0184264 was required to achieve a similar reduction in progeny influenza virus. This is also consistent with the EC50 values ​​of PD-0184264 compared to CI-1040. For PD-0184264, the EC 50 In another study, the EC value of CI-1040 against RB1 was 0.804 μM (Figure 17B). 50 The value could be determined to be 0.026 μM (Haasbach et al. 2017). 50 This value (Figure 17C) is higher than that of CI-1040 (> 312.3 μM; Haasbach et al. 2017). Thus, PD-0184264 has an SI = 1960 (selectivity index).

[0115] Summary / Discussion The results demonstrate that PD-0184264 has lower antiviral activity in cell culture (i.e., in vitro) compared to CI-1040. Nearly 10-fold higher concentrations of PD-0184264 are required to achieve the same viral reduction as CI-1040 in in vitro assays. The EC 50 The difference in values ​​is even more significant. 50 The value is the EC of CI-1040 50The SI of PD-0184264 against RB1 on A549 cells was also lower compared to that of CI-1040.

[0116] Example 11: PD-0184264 reduces viral titers in mouse lungs in vivo (A) Following H1N1pdm09 infection, female C57BL / 6 mice were treated orally with either 2.8, 8.4, or 25 mg / kg of PD-0184264 (left) or 25, 75, or 150 mg / kg of CI-1400 (left). 24 hours after infection, animals were sacrificed, and lungs were harvested and a 10% suspension was prepared. Virus titers were determined using standard methods. Virus titers in mice treated with the two MEK inhibitors were compared to those in mice treated with vehicle alone (control). The virus titer in the lungs of control mice was set at 100% (black bar). Both figures were displayed using Graphpad Prism 7 software.

[0117] drugs CI-1040 [2-(2-chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide; Lot: CC-5395.0-16] and PD-0184264 (PD0184264) [2-(2-chloro-4-iodophenylamino)-N-3,4-difluorobenzoic acid; Lot: CC-5595.4-10] were synthesized at ChemCon GmbH (Freiburg, Germany). For an oral dose of 25 mg / kg, 2.5 mg of PD-0184264 was dissolved in 50 μl of DMSO (Sigma-Aldrich, Germany) and further diluted with 0.15 ml of Cremophor EL (Merck-Millipore, Germany) and 0.8 ml of PBS (Gibco, Germany). For the 8.4 mg / kg and 2.8 mg / kg doses, 0.84 mg or 0.28 mg of PD-0184264 was dissolved in 50 μl of DMSO (Sigma-Aldrich, Germany) and further diluted with 0.15 ml of Cremophor EL / 0.8 ml of PBS. 202.5 mg of CI-1040 was dissolved in 0.5 ml of DMSO / 0.15 ml of Cremophor EL / 0.8 ml of PBS and further diluted with Cremophor EL and PBS.

[0118] animal Eight-week-old female C57Bl / 6 mice (Charles River Laboratories, Germany) weighing 21.0–24.0 g at the time of administration were used for the antiviral test. Animals were fed a normal diet. Drinking water was available ad libitum.

[0119] Drug administration Drugs were administered using a single dose by oral gavage on study day 1. The administration rate was 15 seconds per dose with a dose volume of 200 μl.

[0120] Lung viral titration assay Twenty-four hours after infection, mice were sacrificed, and lungs were weighed and transferred to Lysing Matrix D tubes (MP Bio). BSS was administered at a volume 10 times the lung volume. Organs were minced using a FastPrep FP 120 (Savant). To remove cellular debris, the homogenate was centrifuged at 2000 rpm for 15 minutes, and the supernatant was collected. Viral titers in the homogenate were determined using the AVICEL® plaque assay as previously described (Haasbach et al. 2011; Mastrosovich et al. 2006).

[0121] Summary / Discussion Figure 18 shows the results of the experiment. Compared to the control experiment, only CI-1040 concentrations of 75 mg / kg or higher showed any effect on reducing viral titers. In contrast, PD-0184264 already demonstrated a reduction in lung viral titers of approximately 70% at a concentration of 2.8 mg / kg. At a concentration of 8.4 mg / kg, viral titers were reduced by approximately 20%, and at 25 mg / kg, viral titers were reduced by approximately 10%. Thus, a 6-fold lower concentration of PD-0184264 was required to achieve similar effects as 150 mg / kg of CI-1040, highlighting the potential for PD-0184264's antiviral effects.

[0122] Example 12: PD-0184264 has higher bioavailability compared to CI-1040 (A) Male NMRI mice were treated orally with either 75 mg / kg CI-1040 (dark gray area) or 75 mg / kg PD-0184264. Blood was collected at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours (study day 2) after administration, and plasma was analyzed for the presence of drug. (B) Male NMRI mice were treated orally by gavage with either 150 mg / kg CI-1040 (dark gray area) or 150 mg / kg PD-0184264. Blood was collected at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours (study day 2) after administration, and plasma was analyzed for the presence of drug. Each data point represents the average of three plasma samples. Both figures were displayed using Graphpad Prism 7 software.

[0123] drugs CI-1040 [2-(2-chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide; Lot: CC-5395.0-15] and PD-0184264 (PD0184264) [2-(2-chloro-4-iodophenylamino)-N-3,4-difluorobenzoic acid; Lot: CC-5595.4-10] were synthesized at ChemCon GmbH (Freiburg, Germany). For intravenous administration, 30.65 mg of CI-1040 was dissolved in 0.075 ml of DMSO (Sigma-Aldrich, Switzerland) and further diluted with 0.225 ml of Cremophor EL (Merck-Millipore, Germany) and 2.7 ml of PBS (Gibco, Germany). PD-0184264 (34.88 mg) was dissolved in 0.075 ml DMSO and further diluted with 0.225 ml Cremophor EL / 2.7 ml PBS. For oral administration, CI-1040 (202.5 mg) was dissolved in 0.5 ml DMSO / 1.5 ml Cremophor EL / 8.0 ml PBS. PD-0184264 (81.0 mg) was dissolved in 0.2 ml DMSO / 0.6 ml Cremophor EL / 3.2 ml PBS.

[0124] animal Eight-week-old male NMRI mice (Charles River Laboratories, Germany) weighing 23.9–36.5 g at the time of dosing were used in the pharmacokinetic study. Animals were fed a normal diet. Drinking water was available ad libitum.

[0125] Blood sampling and plasma preparation Experiments were performed at LPT GmbH (Hamburg, Germany). Sufficient whole blood samples were collected under isoflurane anesthesia to obtain at least 2 × 100 μl of Li-heparin plasma from three animals per group at the following time points: 0 (pre-dose), 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h (study day 2) after administration. Whole blood samples were immediately cooled using an Iso-Therm-Rack system (Eppendorf AG, Germany) until centrifugation within 0.5 h of collection. Immediately after centrifugation, samples were stored at -20°C until further analysis. Plasma analysis was performed using standard procedures at Prolytic GmbH (Frankfurt, Germany).

[0126] Drug administration Drugs were administered by oral gavage or by intravenous bolus injection into the tail vein using a single dose on study day 1. The injection rate was 15 seconds / dose with a dose volume of 200 μl.

[0127] result Pharmacokinetic studies revealed higher PD-0184264 exposure in mouse plasma compared to CI-1040 after iv (Figure 19A) and oral (Figure 19B) administration, with the AUC value of PD-0184264 being 1953.68 μg*h / ml, much higher than that of CI-1040. Note that almost no drug was detected in plasma after iv and oral administration of CI-1040 at 8 hours. In contrast, high concentrations were still observed at the 8-hour data point after oral administration of PD-0184264.

[0128] Summary / Discussion The dramatic difference in plasma exposure between PD-0184264 and CI-1040 after a single intravenous dose has already led to the speculation that CI-1040 may be rapidly degraded. We assumed that the drug would decay monoexponentially. This assumption is generally valid. At low concentrations, drugs typically decay monoexponentially. Furthermore, the terminal elimination rate constant does not change over time or with different concentrations of circulating drug. Nevertheless, at this time, we do not know whether other processes, such as the enterohepatic circulation, play an important role in the terminal phase of the pharmacokinetic profile.

[0129] In summary, PD-0184264 exhibited greater antiviral activity than CI-1040 in vivo, which may be due to the drug's greater bioavailability.

[0130] Example 13: PD-0184264 has an extended therapeutic window Drugs and drug administration For oral administration of 25 mg / kg, 10 mg of PD-0184264 was dissolved in 50 μl of DMSO (Sigma-Aldrich, Germany) and further diluted with 0.15 ml of Cremophor EL (Merck-Millipore, Germany) and 0.8 ml of PBS (Gibco, Germany). For vehicle administration, 50 μl of DMSO (Sigma-Aldrich, Germany) was diluted with 0.15 ml of Cremophor EL (Merck-Millipore, Germany) and 0.8 ml of PBS (Gibco, Germany). Drugs were administered twice daily (BID) by oral gavage with an 8-hour time lag. Treatment began at either 24, 48, or 72 hours postinfection. The administration rate was 15 seconds per dose with a 50 μl administration volume.

[0131] animal Eight-week-old female C57Bl / 6 mice (Charles River Laboratories, Germany) weighing 17.0–21.0 g at the time of administration were used for survival studies. Animals were fed a normal diet. Drinking water was available ad libitum.

[0132] Viral infection For infection, animals were anesthetized by intraperitoneal injection of 200 μl of ketamine / Rompun. Equal volumes of 2% Rompun (Bayer) and 10% ketamine (Sanofi) stock solutions were mixed with PBS in a 1:10 ratio. Mice were inoculated with 3 × 10 5 Mice were infected intranasally with pfu of strain A / Regensburg / D6 / 2009 Influenza H1N1pdm09 (5xMLD50). A sufficient virus dose was diluted in 50 μl of BSS, and 25 μl was inoculated into each nostril. All animal experiments were approved by the Institutional Animal Care and Use Committee of Tübingen.

[0133] Clinical observations Animals were observed and weighed daily in accordance with regulatory regulations. Mice were observed twice daily before the onset of clinical symptoms and three times daily after the onset of illness. Mice had to be sacrificed when they lost 20% of their body weight. None of the animals died in their cages, and all were sacrificed by CO2 exposure followed by cervical dislocation.

[0134] result Female C57BL / 6 mice were infected with H1N1pdm09. Treatment with 25 mg / kg PD-0184264 was initiated either 24, 48, or 72 hours postinfection. Mice served as controls and received vehicle only. These animals had to be sacrificed 4–8 days postinfection due to greater than 20% weight loss. When treatment was initiated 24 hours postinfection, 6 of 8 mice were protected, although 2 of 8 animals had to be sacrificed on day 10. There was no difference in weight loss, but mice regained weight on day 10 postinfection (until the end of the observation period). The difference in survival between vehicle- and PD-0184264-treated mice was statistically significant (log-rank Mantel-Cox test, P = 0.0001). When treatment was initiated 48 hours postinfection, 5 of 8 mice were protected, although 3 of 8 animals had to be sacrificed between days 8 and 10. Again, there was no difference in weight loss. Mice regained weight on day 9 postinfection (until the end of the observation period). Again, the difference in survival was significant (log-rank Mantel-Cox test, P = 0.0004). Even when treatment was initiated 72 hours postinfection, the difference in survival was similarly significant (log-rank Mantel-Cox test, P = 0.0002). In this case, 6 of 8 animals had to be sacrificed between days 7 and 10, but 2 of 8 mice were protected. Mice began to gain weight on day 9 postinfection.

[0135] Summary / Discussion Treatment of H1N1pdm09-infected mice with 25 mg / kg PD-0184264 resulted in a significant difference in survival, even when the drug was administered 72 hours post-infection.

[0136] Example 14: Phase I Clinical Trial of PD-0184264 in Healthy Human Subjects A randomized, double-blind, placebo-controlled, dose-escalation study was conducted to demonstrate the safety and tolerability of PD-0184264 in 70 healthy volunteers. It followed a single-ascending / multiple-ascending dose (SAD / MAD) regimen and an adaptive design with seven treatment arms, starting at 100 mg and escalating up to three times. The dosing regimen consisted of one dose of PD-0184264 (SAD) escalated from 100 mg to 900 mg, followed by seven doses of PD-0184264 escalated from 100 mg to 600 mg once daily (QD) over seven days (MAD). Each dose cohort was deemed safe by the Safety Review Committee (SRC), allowing release of the next higher dose (up to a maximum SAD of 900 mg and MAD of 600 mg, respectively). The observed pharmacokinetic profile supports a once-daily regimen for further clinical development.

[0137] Overall, only a few adverse events occurred during the study, and no serious adverse events were observed. Therefore, PD-0184264 is considered safe and well tolerated. Pharmacokinetic exposure and MEK inhibition assessments were validated, confirming the maintenance of clinically relevant blood levels.

[0138] References TIFF2025124703000003.tif46160TIFF2025124703000004.tif237160TIFF2025124703000005.tif230160TIFF2025124703000006.tif18160

Claims

1. PD-0184264 or a pharmaceutically acceptable salt thereof for use in a method for treating a viral disease, wherein the subject has been exhibiting symptoms of the viral infection for at least 24 hours at the time treatment is initiated.

2. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 1, wherein the subject has been exhibiting symptoms of viral infection for at least 36 hours, at least 48 hours, or at least 72 hours at the time treatment is initiated.

3. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 1 or 2, further for use in the prevention or treatment of bacterial superinfection.

4. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 3, wherein the viral infection is a respiratory infection caused by a negative-strand or positive-strand RNA virus.

5. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 4, wherein the negative-strand RNA virus is an influenza virus.

6. 6. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 5, wherein the virus is an influenza A virus or an influenza B virus.

7. 7. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 6, wherein the influenza A virus is selected from the group consisting of H1N1, H2N2, H3N2, H5N6, H5N8, H6N1, H7N2, H7N7, H7N9, H9N2, H10N7, N10N8 or H5N1.

8. 7. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 6, wherein the influenza B virus is selected from the group consisting of IBV Yamagata lineage or Victoria lineage.

9. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 4, wherein the viral infection is caused by a positive-strand RNA virus.

10. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 9, wherein the virus is a coronavirus.

11. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 10, wherein the virus is SARS-CoV, SARS-CoV2, or MERS.

12. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 11, wherein the virus is resistant to standard antiviral treatments.

13. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 12, wherein the standard antiviral treatment is administration of oseltamivir, zanamivir, peramivir, amantadine, rimantadine, favipiravir, baloxavir marboxil, and / or pimoxivir.

14. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 12, wherein the virus is H1N1 virus A H275Y mutant.

15. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 14, wherein the subject has been treated with a standard antiviral drug prior to administration of PD-0184264.

16. PD-0184264, or a pharmaceutically acceptable salt thereof, for use according to any one of claims 1 to 15, administered in combination with a neuraminidase inhibitor, or a pharmaceutically acceptable salt thereof.

17. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 16, wherein the neuraminidase inhibitor is selected from oseltamivir, oseltamivir phosphate, zanamivir, laninamivir, or peramivir, or a pharmaceutically acceptable salt thereof.

18. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of the preceding claims in a subject, preferably a vertebrate, most preferably a human.

19. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 18, wherein PD-0184264 is administered to a human subject once daily at a dose of 100 to 900 mg, preferably 600 mg.

20. 20. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 19, wherein PD-0184264 is administered to a human subject for 1 to 21 consecutive days, preferably for 7 to 14 consecutive days.

21. 21. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 19 or 20, wherein PD-0184264 is administered to the human subject in an oral dosage form.

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