EA042391B1 - A NEW MEK INHIBITOR FOR THE TREATMENT OF VIRAL AND BACTERIAL INFECTIONS - Google Patents

Description

State of the art

Influenza A viruses are the causative agents of severe respiratory illness leading to significant morbidity and mortality. Most deaths during influenza virus infection are actually the result of secondary pneumonia caused by various bacteria such as Staphylococcus aureus (S. aureus), Streptococcus pneumoniae, and Haemophilus influenza (Morens et al., 2008, Chertow et al., 2013). The most significant problems of bacterial coinfection are the sudden increase in pathogenicity (Iwao et al., 2012, Paddock et al., 2012, Parker et al., 2012) and the limited arsenal of powerful anti-infective agents against various pathogens. High variability of influenza viruses and constant emergence of new strains (Neumann et al., 2009, Taubenberger et al., 2010, Parry, 2013), specific characteristics of bacterial strains (Grundmann et al. 2006, Moran et al., 2006, Gillet et al. ., 2007, Shilo et al., 2011), as well as the rapid development of resistance as 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) in relation to available drugs/antibiotics are the main reasons for the lack of treatment options.

WO 2001/076570 proposes the concept of treating or preventing infections caused by (-)RNA viruses, in particular influenza viruses, with MEK inhibitors. WO 2014/056894 proposes specific MEK inhibitors such as AZD-6244, AZD-8330, RDEA-119, GSK-1120212 (trametinib), GDC-0973 (cobimetinib), CI-1040, PD-0325901, RO-5126766, MSC1936369 (AS-703026) for use in the treatment or prevention of influenza virus infection. WO 2015/173788 A1 discloses MEK inhibitors for use in a method for treating influenza virus and bacterial co-infections.

However, although some potential MEK inhibitors are already known and intended for use in the treatment or prevention of viral infections, in particular influenza virus infection, as well as bacterial co-infections accompanying viral infections, in particular influenza infection, there is still a need to provide further ideally improved inhibitors. MEK for such applications, and also, especially, for the treatment of bacterial infections. Therefore, the technical problem of the present application is to satisfy this need.

Brief description of the invention

The solution to the technical problem is to provide PD-0184264, a metabolite of CI-1040, for use in the treatment or prevention of viral diseases such as influenza virus infection, bacterial infections or co-infection, including a bacterial infection and a viral disease. This decision is also reflected in the embodiments described below and in the claims, and illustrated in the examples and figures. The authors of the present application unexpectedly found that the PD-0184264 metabolite of the MEK inhibitor CI-1040 has a higher antiviral and antibacterial activity than CI-1040 itself. Therefore, the use of PD-0184264 solves the technical problem underlying the present application, providing a more effective option for the treatment of viral infections, in particular influenza virus infections, as well as viral, in particular influenza virus, bacterial co-infections and bacterial infections.

Although the MEK inhibitor CI-1040 is already effective in treating or preventing influenza virus infection and coinfections caused by influenza virus or bacterial infection, the inventors found that the metabolite (PD-0184264, formula 1) CI-1040 is more effective in controlling the virus influenza and/or bacterial infections or co-infections involving an influenza virus and a bacterial infection than CI-1040 itself. PD-0184264 is one of several metabolites of CI-1040 (Wabnitz et al., 2004, LoRusso et al., 2005), but it was impossible to know or suggest that the metabolite is more potent than CI-1040. Much to the inventors' surprise, they did indeed find that one metabolite (PD-0184264, Structure 1 below) was more effective and had a higher therapeutic potential than CI-1040 as shown in the examples. This property of PD-0184264 could not be expected, since PD-0184264 actually shows a weaker inhibitory effect on MEK kinases in vitro than CI-1040 (see example 9). In addition, in vitro assays showed a weaker antiviral effect of PD-0184264 compared to CI-1040 (see example 10), while in vivo assays unexpectedly showed a much stronger antiviral effect of PD-0184264 compared to CI-1040 (see example 11).

Accordingly, the present invention 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 and/or a viral disease. Preferably the virus causing the viral disease is

Negative strand RNA virus, preferably an influenza virus, and more preferably an influenza A or influenza B virus.

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

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 coinfection, including a bacterial infection and a viral disease.

Preferably, PD-0184264 or a pharmaceutically acceptable salt thereof is for use in a method for preventing and/or treating coinfection comprising a bacterial infection and a viral disease, 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.

Preferably, PD-0184264 or a pharmaceutically acceptable salt thereof is for use in a method for preventing and/or treating coinfection comprising a bacterial infection and a viral disease, wherein the virus is a negative strand RNA virus, preferably an influenza virus, more preferably an influenza A virus or flu virus B.

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.

Preferably, PD-0184264 or a pharmaceutically acceptable salt thereof is for use in a method for preventing and/or treating coinfection involving a bacterial infection and 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.

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 in a method for the prevention and/or treatment of co-infection involving a bacterial infection and a viral disease, wherein the neuraminidase inhibitor is selected from oseltamivir, phosphate oseltamivir, zanamivir, laninamivir or peramivir or a pharmaceutically acceptable salt thereof.

Also provided in the present invention is a pharmaceutical composition comprising PD0184264 or a pharmaceutically acceptable salt thereof and a neuraminidase inhibitor or a pharmaceutically acceptable salt thereof.

Preferably, PD-0184264 is for use in a method for the prevention and/or treatment of a viral disease and/or in a method for the prevention and/or treatment of a bacterial disease and/or in a method for the prevention and/or treatment of a co-infection comprising a bacterial infection and a viral disease, wherein PD-0184264 is combined with one or more MEK inhibitors.

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 co-infection in a subject, preferably a vertebrate, more preferably a bird or mammal, most preferably a human.

The present invention also provides a method for treating a bacterial infection in a subject, comprising administering a therapeutically effective amount of PD-0184264 or a pharmaceutically acceptable salt thereof to a subject in need thereof.

Furthermore, the present invention relates to a method of treating a viral disease in a subject, comprising administering a therapeutically effective amount of PD-0184264 or a pharmaceutically acceptable salt thereof to a subject in need thereof.

In a further embodiment, the present invention relates to a method for treating coinfection comprising a bacterial infection and a viral disease in a subject, the method comprising administering a therapeutically effective amount of PD-0184264 or a pharmaceutically acceptable salt thereof to a subject in need thereof.

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.

Preferably, the bacterial infection is mediated by a bacterium selected from the group consisting of Staphylococcaceae, Streptococcaceae, Legionellaceae, Pseudomonadaceae, Bacillaceae, Chlamydiaceae, Mycoplasmataceae, Enterobacteriaceae, Pseudomonadales and Pasteurellaceae.

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Brief description of the drawings

The figures show the following.

Fig. 1. Treatment of mice infected with influenza A virus with PD-0184264 or CI-1040.

The results of the experiment from Example 1 are presented as virus titer (log 10) PFU/ml (left) or % virus titer (right).

Fig. 2. Graph showing the effect of PD-0184264 and CI-1040 on MRSA bacterial growth.

At different time points, as indicated on the horizontal axis of the graph, the optical densities of the cell-free bacterial cultures were measured as an index of bacterial growth shown on the vertical axis of the graph in % (OD600). Data are the average of three biological replicates described in Example 2.

Fig. 3. Inhibition of bacterial growth by MRSA with various concentrations of PD-0184264.

PD-0184264 was administered at various concentrations (as indicated) to an overnight culture of S. aureus USA300 (MRSA). After 6 hours, the optical density was measured. The data shown in the figure represents one experiment from three biological replicates described in Example 2.

Fig. 4. Effect of CI-1040 and PD-0184264 on bacterial growth.

(A) Effect of CI-1040 on bacterial growth at various concentrations. (B, C) Effect of PD-0184264 on S. aureus strain 6850 (B) or USA300 strain (C) at different concentrations of PD-184264.

Fig. 5. Administration of PD-0184264 to single-infected or coinfected cells protects the cells.

Human lung epithelial cells (A549) were pre-treated with PD-0184264 (at indicated concentrations) or vehicle (dimethyl sulfoxide (DMSO)) and infected with human influenza A/Puerto Rico/8/34 (H1N1). Considering the antiviral and potent antibacterial effect of PD-0184264 (FIGS. 2-4), it was analyzed whether this compound feature could also be observed macroscopically in relation to cell-destroying cytopathic effects (CPE) caused by IAV (influenza A virus) and/or S infection. aureus. Little disruption of the cell monolayer was observed after a single infection with IAV (H1N1) (middle panel) or with S. aureus 6850 strain (upper panel). This CPE was greatly increased by co-infection with both pathogens (lower panel), but was inhibited in the presence of increasing concentrations of PD-0184264.

Fig. 6. Comparison of the antibacterial action of PD-0184264 with well-known antibiotics.

To compare the antibacterial properties of the MEK inhibitor PD0184164 with that of a well-known antibiotic, bacteria were treated overnight with solvent, MEK inhibitors U0126 and PD-0184264, or various concentrations of the antibiotic gentamicin. Compared to solvent-treated bacteria, incubation with the first generation MEK inhibitor U0126 resulted in only a slight decrease in bacterial titers, while treatment with PD-0184264 resulted in a very large reduction in bacterial load. This happened with both bacterial strains.

Fig. 7. (A) Results of addition time analyses, comparing the antibacterial activity of PD0184264 with other MEK inhibitory compounds or the antibiotic gentamicin.

(B) Results of the MEK inhibitor resistance test PD0184264 in S. aureus compared with gentamicin, erythromycin, or no treatment.

Fig. Fig. 8. Domain structure of the bacterial PknB kinase (top) (Rakette et al. 2012) and sequence homology of the PknB autophosphorylation site with the mammalian MAP kinase activation sites p38, JNK, and the direct MEK target ERK (Miller et al. 2010).

Fig. 9. Determining the effect of inhibitor treatment on the minimum inhibitory concentration (MIC) of various antibiotics.

Fig. 10. Stress tolerance of S. aureus 6850 strain and MRSA strain USA300 when treated with PD0184264.

Fig. 11. Treatment with PD-0184264 impairs the growth of various serotypes of Streptococcus pneumoniae.

(A) Effect of PD-0184264 on cultures of Streptococcus pneumoniae TIGR4 (serotype 4) and D39 wt (serotype 2) strains as measured by OD600; (B) The effect of PD-0184264 on cultures of Streptococcus pneumoniae TIGR4 (serotype 4) and D39 wt (serotype 2) strains is shown in cfu/ml; (C) The effect of PD-0184264 on cultures of Bacillus subtilis is shown in cfu/ml.

Fig. 12. Tab. 1: antibiotics.

Fig. 13. Images demonstrating that treatment of PD-0184264 cells greatly reduces pathogen-induced CPE (cytopathic effect) in single bacterial (S. aureus) or viral (influenza IV) infection and co-infection.

Fig. 14. Graph showing changes 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 times during ongoing infection (c).

Fig. 15. Graph showing cell viability (a, b) and membrane rupture (c, d).

(a, b) demonstrate the viability of 5A549 cells in the presence of increasing concentrations of PD-0184264. In (c) and (d), an LDH assay was performed to determine membrane rupture due to inhibitor treatment.

Fig. 16. Cell free kinase assay demonstrating the inhibitory effect of CI-1040 and PD0184264 on the MEK pathway.

Kinase activity is measured by determining the amount of phosphorylated ERK target protein using an ELISA assay. Three independent experimental series were performed with similar results. One typical experiment is presented in this application.

Fig. 17. Antiviral activity of PD-0184264 against HlN1pdm09 influenza virus 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 RBI virus (MOI=0.001) and treated with various concentrations of PD0184264 (100 μM, 50 μM, 10 μM, 5 μM, 1 μM, 0.5 μM and 0.1 μM) to determine the EC ₅₀ 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 h followed by four hours of WST staining to determine the CC value ₅₀ .

Fig. 18. In vivo reduction of virus titer in mouse lungs with PD-0184264.

After H1N1pdm09 virus infection, female C57BL/6 mice were treated orally with 2.8, 8.4, or 25 mg/kg PD-0184264 (left panel) or 25, 75, or 150 mg/kg CI-1400 (right panel). Animals were sacrificed 24 hours after infection, and the virus titer was determined by the standard method.

Fig. 19. PD-0184264 has better bioavailability than CI-1040.

(A) Male NMRI mice were treated with either 75 mg/kg CI-1040 (dark gray area) or 75 mg/kg PD-0184264 intravenously. Blood was collected at 15 min, 30 min, 1 h, 2 h , 4 h, 6 h, 8 h and 24 h (test day 2) after administration, and the plasma was analyzed for drug. (B) Male NMRI mice were treated with either 150 mg/kg CI-1040 (dark gray area) or 150 mg/kg PD-0184264 orally (per os) using a gastric tube. Blood was collected at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h (test day 2) after administration, and the plasma was analyzed for drug.

Detailed description of the invention

The following description includes information that may be helpful in understanding the present invention. Any information presented in this application is not prior art or is not relevant to the present inventions, or any publication referred to directly or indirectly is not prior art.

As stated above, the present invention relates to PD-0184264 for use in a method for the prevention and/or treatment of a viral disease, a bacterial infection, or a co-infection, including a bacterial infection and a viral infection. As shown in the examples, PD-0184264 appears to act on the bacterial PknB kinase and thus may be at least partially bacteriostatic.

In addition, as shown in the attached examples, PD-0184264 is effective in viral and bacterial infection scenarios, as well as in bacterial and viral coinfection scenarios. This effect is surprisingly stronger than that of CI-1040, a MEK inhibitor already known in the art for the treatment of bacterial and viral infections. When comparing the inhibitory effects of CI-1040 and PD-0184264 on MEK kinase, as shown in Example 9, one would expect the opposite, since MEK inhibitors have been used in the prior art to achieve these effects.

The same is true when comparing the antiviral effect of PD-0184264 and CI-1040 in in vitro assays. In the present application, CI-1040 is more potent than PD-0184264 as seen in Example 10. In particular, a 10-fold higher concentration of PD-0184264 is needed in an in vitro assay to achieve the same inhibitory effect. Surprisingly, despite weak inhibition in vitro, PD-0184264 was found to be superior to CI-1040 in vivo, as seen in Example 11. As shown in FIG. 18, already 2.8 mg/kg PD-0184264 showed a reduction in virus titer, while 25 mg/kg PD-0184264 showed a reduction in lung virus titer of at least 90%. In contrast, CI-1040 shows a similar reduction at only 150 μM. In addition, example 1 demonstrated the reduction of virus titer in the lungs using PD-0184264. in Example 1, mice were challenged with influenza virus and treated with 150 mg/kg, 75 mg/kg, or 25 mg/kg CI-1040 or PD-0184264, respectively. As shown in FIG. 1, already 25mg/kg PD-0184264 has the same effect as 150mg/kg CI-1040. Thus, much to the surprise of the inventors, PD-0184264 shows a strong antiviral effect in vivo, although previous in vitro data did not provide any incentive to continue working with PD0184264. This unexpected effect may be based on the higher bioavailability of PD-0184264 compared to CI-1040 as shown in Example 12.

Examples 2, 4 and 6-8 further demonstrate the strong antibacterial effect of PD0184264, also compared to CI-1040 and other MEK inhibitors. Example 2 analyzes the effect of PD-0184264 on bacterial growth. In FIG. 2 shows that PD-0184264 inhibits bacterial growth while CI-1040 does not affect bacterial growth. In FIG. 3 shows that bacterial growth is inhibited by PD0184264 in a concentration dependent manner, showing almost complete growth inhibition starting at 50 μM PD-0184264. Similarly, also in FIG. 4 shows that bacterial growth is inhibited

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PD-0184264 in a concentration-dependent manner - as early as 10 μM PD-0184264 shows a significant reduction in growth - whereas CI-1040 does not affect bacterial growth. Example 4 compares the antibacterial effect of PD-0184264 to the MEK inhibitor U0126 or the antibiotic gentamicin. In FIG. 6 shows that PD-184264 has antibiotic activity similar to gentamicin, while the prior art MEK inhibitor U0126 does not affect bacterial growth. The same is true for FIG. 7A, which tracks the progress of bacterial growth. In FIG. 7B further shows that PD0184264 does not cause resistance to PD-0184264, while bacteria easily develop resistance to gentamicin and erythromycin. Thus, PD-0184264 does not induce resistance in bacteria. in example 6 analyzed the effect of PD-0184264 on the sensitivity of bacteria to the antibiotic. As shown in FIG. 9 and Table 2, treatment with PD-0184264 did result in an increase in bacterial susceptibility to various antibiotics, most notably with penicillin and gentamicin. In addition, in FIG. 10 shows that PD-0184264 reduces the resistance of bacteria to stress. Example 7 provides evidence that PD-0184264 is not limited to S. aureus but also affects other bacteria such as Streptococcus pneumoniae (see FIGS. 11 A and B) and Bacillus subtilis (see FIG. 11C) .

Examples 3 and 8 highlight the efficacy of PD-0184264 in the context of bacterial and viral coinfection. in Example 3, it was analyzed whether the efficacy of PD-0184264 could also be observed macroscopically against cell-destroying cytopathic effects (CPE) caused by viral and/or bacterial infection. In FIG. 5 shows that PD-0184264 reduces the cytopathic effect caused in particular by bacterial and viral co-infection in a concentration-dependent manner, showing an effect already at 15 μM PD-0184264. The same was also analyzed in Example 8, which again demonstrates in FIG. 13 positive effect of PD-0184264 in the scenario of bacterial and viral coinfection. in example 8, the effect of PD-0184264 on the intracellular titer of bacteria in a coinfection scenario was further analyzed. In FIG. 14 shows that PD-0184264 reduces intracellular bacterial titer while CI-1040 has no effect. In addition, Example 8 and FIG. 15 demonstrate that PD-0184264 has no cytotoxic effect.

PD-0184264 can be used in the method of treatment and/or prevention of diseases described in this application. Thus, the term treatment or treatment includes administering PD-0184264, preferably in the form of a drug, to a subject suffering from coinfection, including a bacterial infection and a viral disease, in order to reduce or improve the symptoms accompanying such infections. Likewise, it comprises administering PD-0184264, preferably in the form of a medicament, to a subject suffering from a bacterial infection, or for the purpose of ameliorating or ameliorating the symptoms accompanying such infections. It further comprises administering PD-0184264, preferably in the form of a medicament, to a subject suffering from a viral infection or for the purpose of ameliorating or ameliorating the symptoms accompanying such infections. A co-infection including a bacterial infection and a viral disease, wherein the viral disease and the bacterial infection are a medical condition that is treated or prevented by PD-0184264 or a pharmaceutically acceptable salt thereof.

In addition, in this application, the term prophylaxis refers to any medical or public health procedure, the purpose of which is to prevent the medical condition described in this application. As used herein, the terms prevent, prevent, and exercise prevention refer to reducing the risk of acquiring or developing a given condition, namely coinfection, including a viral infection and a bacterial infection, a bacterial infection alone, or a viral infection alone, as described herein. The term prophylaxis also means reducing or inhibiting the recurrence of a co-infection involving an influenza virus infection and a bacterial infection, a bacterial infection alone, or a viral infection alone in a subject.

PD-0184264 of the present invention is effective in the treatment of coinfection as shown in examples 3 and 8. In the present application, the term coinfection includes a viral disease, preferably an influenza virus infection, and a bacterial infection. Such co-infection may occur when a host, eg a subject and/or a single cell, is simultaneously infected with a bacterium and an influenza virus. It may also be that the host, eg the subject and/or the cell is simultaneously infected with one or more viral particles and one or more bacteria. However, such co-infection can also occur sequentially. In such a case, the subject and/or cell is first infected with one or more viral particles, and later the same subject and/or cell is infected with one or more bacteria, or vice versa. The time period between two infections may be a time period of 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 at least 30 minutes.

Such a situation may also represent a superinfection in which the second infection is superimposed on an earlier infection, in particular with another microbial agent of exogenous or endogenous origin that is resistant to the treatment used against the first infection.

As part of co-infection, influenza virus infection may be mediated by influenza A virus or

- 5 042391 influenza B virus, preferably 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 other embodiments, influenza A virus is

H3N2, H5N1 and H7N9. In further embodiments, influenza A virus is

H3N2, H5N1, H1N1, H5N6, H7N2 and H7N9.

The present invention also relates to a bacterial infection that may occur under the conditions of coinfection described above, or may occur as a single infection present in the host, eg, subject and/or cell. Bacterial infection can be mediated by any bacterium; preferably mediated by a bacterium selected from the group consisting of Staphylococcaceae, Streptococcaceae, Legionellaceae, Pseudomonadaceae, Bacillaceae, Chlamydiaceae, Mycoplasmataceae, Enterobacteriaceae, Pseudomonadales and/or Pasteurellaceae.

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

A viral disease or viral infection, to which the present invention also pertains, may occur under the conditions of co-infection described above, or may occur as a single infection present in the host, eg, subject and/or cell. The viral disease or viral infection may be mediated by any virus; preferably they are mediated by the influenza virus. More preferably, the influenza virus infection is mediated by influenza A or influenza B virus, with influenza A viruses being preferred. Particularly preferred are influenza A virus subtypes H1N1, H2N2, H3N2, H5N6, H5N8, H6N1, H7N2, H7N7, H7N9, H9N2, H10N7, N10N8 and/or H5N1.

In an alternative embodiment, PD-0184264 is administered in combination with one or more MEK inhibitors. MEK inhibitors include e.g. PD98059, ARRY-438162, ARRY-162, ARRY-300, PF-3644022 and PD184352. An additional MEK inhibitor may be administered simultaneously, before or after PD-0184264.

Preferably, PD-0184264 is for use in the methods of preventing and/or treating coinfection of the present invention, wherein PD-0184264 is combined with one or more inhibitors that target an influenza virus or a bacterium. PD-0184264 may be administered simultaneously, before or after one or more influenza virus-targeting inhibitors.

In general, an influenza virus-targeting inhibitor is any inhibitor or drug effective in the treatment of influenza. Various substances are known to be effective in reducing influenza virus infection. These include, for example, inhibitors of viral neuraminidase, compounds targeting the viral ion channel protein (M2), and compounds targeting viral polymerase or endonuclease activity by interfering with a component of the viral polymerase complex: PB1, PB2, PA or NP. In accordance with the invention, pharmaceutically acceptable salts of the inhibitors are also provided. However, in the present application, the preferred inhibitor is a MEK inhibitor, particularly preferred PD-0184264.

MEK inhibitors inhibit the Raf/MEK/ERK mitogenic signaling cascade in cells or in a subject by inhibiting ME (mitogen-activated protein kinase kinase). This signaling cascade is captured by many viruses, in particular influenza viruses, to enhance viral replication. Therefore, the specific blockade of the Raf/MEK/ERK pathway at the narrow MEK site prevents the growth of viruses, in particular influenza viruses. In addition, MEK inhibitors exhibit low toxicity and few side effects in humans. There is also no trend towards viral resistance (Ludwig, 2009). A particularly preferred inhibitor is PD-0184264.

A neuraminidase inhibitor is an antiviral drug targeting the influenza virus that works by blocking the function of the viral neuraminidase protein, thus preventing the release of the virus from infected host cells, since newly produced viruses cannot be released from the cell in which they replicate. Also included are pharmaceutically acceptable salts of the neuraminidase inhibitor. Preferred neuraminidase inhibitors are oseltamivir, zanamivir, peramivir, laninamivir, or a pharmaceutically acceptable salt of any of these substances, such as oseltamivir phosphate, oseltamivir carboxylate, etc. The most preferred neuraminidase inhibitors are oseltamivir phosphate, zanamivir, oseltamivir or peramivir.

Compounds targeting the viral ion channel protein (M2) are, for example, amantadine and/or rimantadine, while compounds targeting polymerase or endonuclease activity by acting on a component of the viral polymerase complex, PB1, PB2, PA or NP , are, for example, a nucleosin NP blocker or a T-705 polymerase inhibitor (favipiravir).

In addition, PD-0184264 can be combined with one or more inhibitors that target the bacterium. Example 6 shows that PD-0184264 increases the sensitivity of bacteria to antibiotics. The bacterial-targeting inhibitor can be any inhibitor effective in reducing bacterial infection. In the present invention, PD-0184264 is strongly preferred as a bacterium-targeting inhibitor, but another bacterium-targeting inhibitor known to one skilled in the art is an antibiotic. Preferred antibiotics can be selected from Table 1 (FIG. 12). Thus, in one embodiment, the antibiotic is selected from the group consisting of the antibiotics listed in Table 1 (FIG. 12). In another embodiment, the antibiotic is selected from the group consisting of the class of antibiotics listed in Table 1 (FIG. 12). In another embodiment, the antibiotic is selected from the group consisting of the generic name of the antibiotics listed in Table 1 (FIG. 12). More preferred are antibiotics selected from gentamicin, rifampicin, lysostaphin, erythromycin, levofloxacin, vancomycin, teicoplanin, penicillin and oxacillin.

The subject that can be treated with inhibitors, in particular MEK inhibitors or a combination of inhibitors of the present invention, is preferably a vertebrate. In the context of the present invention, the term subject includes an individual in need of treatment for a coinfection as described herein, or for a bacterial or viral infection alone. Preferably, the subject is a patient suffering from co-infection or only a bacterial or viral infection, or is at risk of developing it. Preferably the patient is a vertebrate, more preferably a mammal. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice, and rats. Preferably the mammal is a human, horse, dog, cat, cow, pig, mouse, rat, etc., particularly preferably a human. In some embodiments, the subject is a human, optionally over 1 year old and under 14 years old, between the ages of 50 and 65, between the ages of 18 or 50, or over 65 years of age. In other embodiments, the subject is a human subject that is selected from the group consisting of subjects who are at least 50 years old, subjects who reside in a permanent care facility, subjects with chronic pulmonary or cardiovascular disorders, subjects who require regular medical supervision or hospitalization within the previous year due to chronic metabolic disease, renal dysfunction, hemoglobinopathies, or immunosuppression, subjects under 14 years of age, subjects 6 months to 18 years of age who are receiving long-term aspirin therapy, and women who will be in their second or third trimester of pregnancy during flu season.

In the method according to the invention, PD-0184264 can be administered orally, intravenously, intrapleurally, intramuscularly, topically or by inhalation. Preferably PD-0184264 is administered by inhalation, topically or orally.

The present invention also provides various compositions, preferably pharmaceutical compositions. The present invention relates to a composition containing PD-0184264 for use in a method for the prevention and/or treatment of coinfection, including a bacterial infection and a viral disease. The present invention also relates to a composition containing PD0184264 for use in a method for preventing and/or treating a bacterial infection and/or a viral disease. The present invention also provides a composition comprising PD-0184264 and one or more inhibitors targeting a virus, in particular an influenza virus and/or a bacterium, for use in a method for the prevention and/or treatment of coinfection, comprising a bacterial infection and a viral infection, in particular influenza virus infection. In addition, the present invention relates to a composition containing PD-0184264 and one or more bacterial-targeting inhibitors for use in a method for preventing and/or treating a bacterial infection or a viral infection.

As mentioned above, a composition comprising PD-0184264 and ultimately one or more bacterial-targeting inhibitors and/or one or more virus-targeting inhibitors, in particular influenza virus, may be a pharmaceutical composition. A preferred embodiment of the pharmaceutical composition comprises PD-0184264 and an influenza virus targeting inhibitor, in particular a neuraminidase inhibitor. In another embodiment, the pharmaceutical composition contains PD-0184264 and an additional MEK inhibitor. Preferably, such compositions further comprise a carrier, preferably a pharmaceutically acceptable carrier. The composition may be in the form of oral suspensions or tablets, nasal sprays, preparations for inhalation devices, sterile injectable preparations (intravenously, intrapleurally, intramuscularly), for example, in the form of sterile injectable aqueous or oily suspensions or suppositories.

A pharmaceutical composition for use according to the invention comprising PD-0184264 and optionally one or more influenza virus-targeting inhibitors and/or one or more bacterial-targeting inhibitors is administered to a mammalian or avian patient. Examples of suitable mammals include mouse, rat, cow, goat, sheep, pig, dog, cat, horse, guinea pig, dog, hamster, mink, seal, whale, camel, chimpanzee, macacuresus, and human, preferably human, but not limited to . Examples of suitable birds include, but are not limited to, turkey, chicken, goose, duck, teal, mallard, starling, northern pintail, gull, swan, guinea fowl, or certain waterfowl. Human patients are a specific embodiment of the present invention.

The inhibitor or inhibitors are preferably administered in a therapeutically effective amount. The therapeutically effective amount for PD-0184264 or each active compound/inhibitor may vary depending on factors including potency of the compound used, stability of the active compound in the patient, severity of conditions to be alleviated, total weight of the patient being treated, route of administration, ease absorption, distribution and excretion of the compound by the body, age and sensitivity of the patient to be treated, side effects and the like, as will be apparent to a person skilled in the art. The amount of administration may be adjusted as various factors change over time.

The inhibitors, methods, and uses described herein are applicable to both human therapy and veterinary use. The compounds described herein, in particular PD-0184264 and optionally one or more inhibitors targeting an influenza virus and/or one or more inhibitors targeting a bacterium having the desired therapeutic activity, may be administered to a subject in a physiologically acceptable carrier, as described in this application. Depending on the route of administration, the compounds can be formulated in various ways, as discussed below. The concentration of the therapeutically active compound in the composition may vary from about 0.1% to about 100% by weight. The agents may be administered alone or in combination with other treatments. Example 1 shows that 25 and 75 mg/kg of PD-0184264 are effective in vivo when administered orally. Accordingly, PD-0184264 may be administered at a dosage in the range of 10 to 100 mg/kg of PD-0184264, preferably in the range of 25 to 75 mg/kg of PD-0184264.

The pharmaceutical compounds in the method of the present invention may be administered in any suitable unit dosage form. Suitable oral formulations may be in the form of tablets, capsules, suspension, syrup, chewing gum, cachets, elixir, and the like. Pharmaceutically acceptable carriers such as binders, excipients, lubricants, and sweetening or flavoring agents may be included in oral pharmaceutical compositions. If desired, conventional flavor, color and shape changing agents for specialty molds can also be included.

For injectable formulations, the pharmaceutical compositions may be in a lyophilized powder in admixture with suitable excipients in a suitable vial or tube. Prior to use in a medical facility, the drugs may be reconstituted by dissolving the lyophilized powder in a suitable solvent system to form a composition suitable for intravenous or intramuscular injection.

The combination of PD-0184264 with an antiviral agent such as a neuraminidase inhibitor such as oseltamivir results in a synergistic effect. This synergistic effect may be an increased antiviral effect resulting, for example, in a reduced viral titer or a prolonged therapeutic window. Accordingly, the present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of PD0184264 as well as a therapeutically effective amount of a neuraminidase inhibitor selected from the group of oseltamivir, oseltamivir phosphate, laninamivir, zanamivir and peramivir.

In one embodiment, the composition may be in an oral administration form (e.g., tablet or capsule or syrup, etc.) in a therapeutically effective amount (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 up to 30 mg) of a neuraminidase inhibitor as described above.

In other embodiments, PD-0184264 is for use in methods for the prevention and/or treatment of coinfection of the present invention, wherein PD-0184264 reduces both viral and bacterial infection upon contact with an in vitro test system, wherein the test system comprises cultured cells infected

a) influenza virus

b) bacterium compared to the pre-contact in vitro test system. In another embodiment of PD-0184264

- 8 042391 is intended for use in methods for the prevention and/or treatment of coinfection according to the present invention, while PD-0184264 reduces bacterial infection upon contact with an in vitro test system, while the test system contains cultured cells infected with a bacterium compared to the test -in vitro system before contact.

Thus, the present invention also relates to an in vitro test system, wherein the test system contains cultured cells infected with

a) influenza virus and

b) bacteria.

In addition, the present invention also provides an in vitro test system, wherein the in vitro test system contains cultured cells infected with a bacterium.

In cases where the in vitro test system includes a viral and bacterial infection, these infections may occur sequentially or simultaneously.

A cultured cell or cultured cells is/are cells that do not exist in their natural environment, eg in a plant or animal. Rather, the cultured cell may be a primary cell culture that contains cells isolated from their natural environment, or a cell line. Preferably, the cultured cells are human lung epithelial cells. Preferably cultured cells are seeded at a density of approximately 1x105, 2x105, ^3x105 , 4x105, 5x105, ^6x105 , 7x105, ^8x105 , ^9x105 , ^{10x105 , 11x105} , most preferably ^8x105 cells in 0.5 ml, 1 ml , 1.5 ml, 2 ml, 2.5 ml, 3 ml, 3.5 ml, 4 ml medium such as DMEM. The most preferred density is 8x10 ⁵ in 2 ml of DMEM.

Such cultured cells are infected with a virus and a bacterium, or in other embodiments with only a bacterium. As already described above, coinfection can be sequential or simultaneous. For example, cultured cells may be infected first with influenza virus and then 30 minutes later with bacterium/bacteria. It is also possible to add an additional antibiotic to the culture after 3 hours to remove extracellular bacteria. In this scenario, the antibiotic is then washed off again. In other embodiments, the cells are infected with the bacterium alone.

In the present application, the term bringing into contact refers to the spatial approximation of the cell containing the influenza virus and bacteria, to PD-0184264. This may, for example, mean that the inhibitor is applied via a syringe to the medium in which the cultured cells are located.

In one embodiment, the reduction in viral infection is a reduction in plaque forming units (PFU)/mL and the reduction in bacterial infection is a reduction in colony forming units (CFU)/mL. Plaque forming units is a measure of the number of particles capable of forming plaques per unit volume, such as viral particles. This is more of a functional measurement than an absolute particle count measurement: viral particles that are defective or unable to infect the target cell will not form plaque and thus will not be counted. For example, a 1000 pfu/µl influenza virus solution indicates that 1 µl of the solution contains enough virus particles to produce 1000 infectious plaques in a cell monolayer. In the case of the present invention, the inhibitor-treated cell culture shows a reduced number of plaque-forming units in the culture after treatment compared to the culture before treatment with PD-0184264.

The possible reduction in plaque forming units (PFU)/ml was analyzed as follows. First, cultured cells that are co-infected with influenza virus and bacteria are analyzed for their ability to generate plaque forming units (PFU)/ml, for example, several cells are selected from a petri dish and seeded to count the bacterial plaques that will form. This result is then compared with the amount of plaque forming units (PFU)/ml generated by cells of the same culture after application of the inhibitor. If the amount of plaque-forming units (PFU)/mL decreases after treatment with the inhibitor compared to the amount formed before the application of the inhibitor, a decrease in plaque-forming units occurs.

Colony forming units (CFU)/mL evaluate the number of viable bacteria in a sample. There are different ways. For example, to create colony forming units, a sample (eg, cultured cells in a small volume) is spread over the surface of a nutrient agar plate and allowed to dry before incubation for counting. A viable bacterium is defined as the ability to reproduce by binary fission under controlled conditions. The appearance of a colony in cell culture requires significant growth - when counting colonies, it is not clear whether the colony originated from a single cell or 1000 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 to reflect this uncertainty (rather than cells/ml or cells/g).

Colony forming units (CFU)/ml can be analyzed as follows. First, cultured cells co-infected with an influenza virus and a bacterium or only one bacterium are analyzed for their ability to create colony forming units (CFU)/ml, for example, by selecting

- 9 042391 a few cells from a Petri dish and seeding them for counting. This result is then compared with the number of colony forming units (CFU)/ml generated by cells of the same culture after application of the inhibitor. If the number of colony forming units (CFU)/ml decreases to the amount generated before the use of the inhibitor, a decrease occurs.

In general, one skilled in the art will be aware of these widely known techniques for assaying bacterial and viral infections. How plaque forming units (PFU)/mL and colony forming units (CFU)/mL can be measured is also described in the literature (Tuchscherr et al. 2011, Hrincius et al. 2010).

For the purpose of the invention, the active compound as defined above also includes the pharmaceutically acceptable salt(s) thereof. As used herein, the phrase pharmaceutically or cosmetically acceptable salt(s) means those salts of the compounds of the invention which are safe and effective for the desired form of administration. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc. and salts formed with cations such as those derived from sodium, potassium, ammonium. , calcium, iron hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The invention is also characterized by the following paragraphs.

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 claim 1, characterized in that said virus is a negative strand RNA virus.

3. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 1 or 2, characterized in that said virus is an influenza virus.

4. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 3, characterized in that said influenza virus is influenza A virus or influenza B virus.

5. PD-0184264 or a pharmaceutically acceptable salt thereof for use in a method for preventing and/or treating a bacterial infection.

6. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 5, characterized in that said 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 coinfection, including a bacterial infection and a viral disease.

8. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to claim 7, characterized in that said 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 claim 7 or 8, characterized in that said virus is a negative strand RNA virus.

10. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of claims 7 to 9, characterized in that said viral infection is caused by an influenza virus.

11. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of claims 7-10, characterized in that said viral infection is caused by influenza A virus or influenza B virus.

12. PD-0184264 or a pharmaceutically acceptable salt thereof for use according to any one of claims 1-4 and 7-11, characterized in that PD-0184264 or a pharmaceutically acceptable salt thereof is 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 claim 12, wherein said neuraminidase inhibitor is selected from oseltamivir, oseltamivir phosphate, zanamivir, laninamivir or peramivir, or a pharmaceutically acceptable salt thereof.

14. Pharmaceutical composition containing PD-0184264 or a pharmaceutically acceptable salt thereof.

15. A pharmaceutical composition containing PD-0184264 or a pharmaceutically acceptable salt thereof and a neuraminidase inhibitor or a pharmaceutically acceptable salt thereof.

16. Use according to any one of the preceding claims, comprising an additional MEK inhibitor.

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

It should be noted that the singular forms used in this application and in the appended claims also include the plural forms, unless otherwise indicated. Thus, for example, reference to the term reagent includes one or more different reagents, and reference to the term method includes equivalent steps and methods known to those skilled in the art, which may be modified or replaced by the methods described in

- 10 042391 of this application.

All publications and patents cited in this specification are incorporated by reference in their entirety. To the extent that material incorporated by reference conflicts or is inconsistent with this specification, the description supersedes any such material.

Unless otherwise indicated, the term at least preceding a series of elements is to be understood as referring to each 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 in this application. Such equivalents are intended to be embraced by the present invention.

Throughout the description and in the claims that follow, the word contain and variants such as contains and containing include the indication of an integer or stage or group of integers or stages, but not the exclusion of any other integer or stage or group integers or stages, unless the context otherwise requires. When used in this application, the term containing may be replaced by the term consisting, or sometimes when used in this application, the term having.

When used in this application, the term consisting of excludes any element, step or ingredient not listed in the claim. When used in this application, the expression consisting essentially of does not exclude materials or steps that do not significantly affect the main and new characteristics of the claims.

In the present application, in each particular case, any of the terms containing, essentially consisting of, and consisting of may be replaced by any of the other two terms.

Several documents are cited throughout this specification. Each of the documents cited in this application (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) above or below is incorporated by reference in its entirety. Nothing in this application should be construed as an admission that such publications constitute prior art.

Examples

The present invention is illustrated by the following examples. These examples should not be construed as limiting the scope of the invention. The examples are included for purposes of illustration and the present invention is limited by the claims only.

Example 1 Treatment of mice with PD-0184264 resulted in a decrease in virus titer in the lung.

8-week-old C57BL/6 mice (five per group) were infected with 1.5x105 PFU (5xMLD ₅₀ ) of influenza virus strain A/Regensburg/D6/2009 (RB1, H1N1pdm09). Beginning one hour before infection, mice were treated with either 150 mg/kg CI-1040; All animals received a volume of 200 μl orally (per os). Mice were sacrificed 24 hours post-infection and lungs were weighed, transferred to a Lysing Matrix D (MP Bio) tube and BSS (buffered saline) at 10 times the lung volume used in the samples. Organs were minced using FastPrep FP 120 (Savant). To remove cell debris, the homogenates were centrifuged for 15 min at 2000 rpm and the supernatant was collected. Determination of virus titer in homogenates was performed using the AVICEL® plaque assay. The results are shown in FIG. 1 as virus titer (log10) PFU/ml (left) or % virus titer (right). The virus titer was determined by two investigators independently. Mean values from all titrations were presented.

Example 2 Administration of CI-1040 or PD-0184264 has an inhibitory effect on bacterial growth including MRSA in vitro.

To study the effect of CI-1040 or PD-0184264 on bacterial growth in general, 50 μM of either CI-1040 or PD-0184264, or the same volume (40 μl) of DMSO, was added to a cell-free overnight culture of S. aureus strain MRSA (USA300) serving as a solvent (Fig. 2). Bacterial growth was monitored for 360 min. PD-0184264 had a strong effect on bacterial growth, which was almost completely absent during the entire observation period. CI-1040 did not significantly inhibit the growth of MRSA starting 120 minutes after the start of the experiment compared to the vehicle control, as seen in FIG. 2. This indicates that PD-0184264 as well as CI-1040, in addition to blocking MEK in cells, also blocks the bacterial component responsible for bacterial growth.

To study the concentrations of PD-0184264 required to inhibit bacterial growth, PD0184264 was administered at various concentrations (as shown in Figure 3) to an overnight culture of S. aureus USA300 (MRSA). MRSA bacteria were incubated with various concentrations of MEK inhibitor ranging from 0 to 100 μM, and growth of the bacteria was monitored six hours after culture. The concentration required to inhibit 50% of bacterial growth was in the range of 15-25 μM.

These data can be verified under slightly different experimental conditions by determining actual bacterial titers instead of OD at later time points after treatment. During the day, the culture of S. aureus 6850 was set at 20 CFU/ml and treated with various concentrations of CI-1040 as indicated overnight at 37°C and 5% CO2. Then the optical density (OD ₆₀₀ ) was measured. The remaining culture was washed with PBS and serial dilutions were placed on BHI agar plates. Bacterial titers are shown as colony forming units per ml (CFU/ml). The results are the mean + SD of three independent biological experiments with two technical replicates shown in FIG. 4A. In addition, overnight cultures of S. aureus 6850 (FIG. 4B) or MRSA strain USA300 (FIG. 4C) were set at 20 cfu/mL and treated with various concentrations of PD-0184264 as indicated overnight at 37°C and 5 % CO2. Optical density (OD600) was measured in the morning. The remaining cultures were washed once with PBS and then serial dilutions were placed on BHI agar plates. Bacterial titers were determined using a protocol3 colony counter and displayed as colony forming units per ml (CFU/ml) on a logarithmic scale. Results are 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 (Fig. 4A) and PD-0184264 (Fig. 4B, C) were also effective in these assays against S. aureus 6850 strain (Fig. 4A, B) and MRSA USA 300 strain (Fig. 4C) . A very strong decrease in titers to 1.5-2 orders of magnitude could be detected with 20 μM PD-0184264 (Fig. 4B, C).

Example 3 Administration of PD-0184264 to single-infected or coinfected cells protects the cells from the cytopathic effects of IAV and/or S. aureus.

Considering the antiviral and potent antibacterial effect of PD-0184264 (FIGS. 2-4 in Example 2 above), it was analyzed whether said compound feature could also be macroscopically observed in relation to cell-destroying cytopathic effects (CPE) induced by IAV (influenza A virus) and /or S. aureus infection. Human lung epithelial cells (A549) were pre-treated with PD-0184264 (at indicated concentrations) or solvent (DMSO) and infected with human influenza A/Puerto Rico/8/34 (H1N1) in multiple infection (MOI=0.001) at 37° WITH. After 30 min, the virus dilution was removed, cells were washed with PBS, and invasive DMEM/INV medium (containing 1% human serum albumin, 25 nM HEPES) with or without S. aureus 6850 (MOI = 0.1) was added in the presence of the indicated concentration of inhibitor or diluent control. 3 hours after bacterial infection, cells were treated with DMEM/FBS containing 10% FBS, 2 μg/ml lysostaphin for 20 minutes to remove extracellular bacteria. After additional washing with PBS, DMEM/BA infection medium (0.2% BA, 1 mM MgCl2, 0.9 mM CaCl2, 100 U/mL penicillin, 0.1 mg/mL streptomycin) containing an inhibitor or solvent was added to the cells. After a 24 hour incubation period at 37° C., cell morphology was examined by light microscopy. As shown in FIG. 5, after a single infection with IAV (H1N1) or S. aureus 6850 strain, little disruption of the cell monolayer was observed. This CPE was greatly increased by co-infection with both pathogens (bottom panel). However, with increasing amounts of MEK inhibitor, this phenotype can be reversed in a concentration dependent manner, as the cell monolayer remains intact and the cells are less round. This protective effect of PD-0184264 cells perfectly reflects its antiviral and antibacterial properties (Fig. 5).

Example 4 Comparison of the antibacterial activity of PD-0184264 with well-known antibiotics.

To compare the antibacterial properties of the MEK inhibitor PD0184164 with that of a well-known antibiotic, bacteria were treated overnight with solvent, MEK inhibitors U0126 and PD-0184264, or various concentrations of the antibiotic gentamicin. Overnight cultures of S. aureus 6850 or MRSA strain USA300 were set at 20 cfu/mL and treated as indicated overnight with either MEK U0126 and PD-0184264 inhibitors or the antibiotic gentamicin at 37°C and 5% CO2. Optical density (OD600) was measured in the morning. The remaining cultures were washed once with PBS and then serial dilutions were placed on BHI agar plates. Bacterial titers were determined using a protocol3 colony counter and are presented as colony forming units per ml (cfu/ml). The results shown in FIG. 6 are 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 solvent-treated bacteria, incubation with the first generation MEK inhibitor U0126 resulted in only a slight decrease in bacterial titers, while treatment with PD-0184264 resulted in a very large decrease in bacterial load, as shown previously in FIG. 4. This happened with both bacterial strains. As expected, the growth of both bacterial strains was strongly inhibited due to treatment with gentamicin at concentrations greater than 1 µg/mL, although the antibacterial activity of the antibiotic was higher in the case of S. aureus 6850. At 0.5 µg/mL gentamicin, no reduction was found bacterial titers for both strains. Thus, the effect of the MEK inhibitor PD-0184264 on bacterial growth was almost as effective as low concentrations of the antibiotic gentamicin.

To further compare the antibacterial activity of PD-0184264 with other MEK

- 12 042391 inhibitory compounds or the antibiotic gentamicin, time addition analyzes were performed (FIG. 7a). An overnight culture of S. aureus 6850 was divided into six subcultures containing 15 ml of BHI medium along with DMSO solvent alone or with one of the U0126 and PD-0184264 MEK inhibitors or two different concentrations of the antibiotic gentamicin (0.5 or 2 μg/ml). Immediately after adding the various compounds, OD600 was measured and serial dilutions were placed on BHI agar plates to calculate bacterial titers. The remaining cultures were additionally incubated at 37°C with 5% CO ₂ in the presence of substances or solvent only. This procedure was repeated twice 3 and 6 hours after inoculation. Bacterial titers were determined using a protocol3 colony counter and are presented 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. The medium was then exchanged and the cultures were additionally incubated without adding any substances. All cultures reached the turbidity of the solvent-pretreated culture, indicating that the MEK inhibitor PD-0184264 is bacteriostatic rather than bactericidal.

The development of resistance to various commonly used antibiotics occurs regularly and is a major problem in healthcare settings. To test whether the MEK inhibitor PD0184264 induces resistance in S. aureus, cultures were continuously treated for nearly three weeks in the presence of the inhibitor, gentamicin, erythromycin, or left untreated. In particular, the cultures were grown for 24 hours in the presence or absence of substances, the OD ₆₀₀ was measured, and then the cultures were set to 20 cfu/ml and grown again for 24 hours. This procedure was repeated for 17 days. Data are means + SD of three independent experiments. Statistical significance was analyzed by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05;**p<0.01;***p<0.001;****p<0.0001). As seen for gentamicin, the development of resistance occurred within 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 Fig. 7b).

Example 5 Bacterial PknB kinase can be targeted by PD-0184264 in bacteria.

It is assumed that the inhibitor PD-0184264 is specific for the MEK kinase that exists in mammals. Its direct antibacterial effect raises the question of the mode of action in prokaryotes, i.e. is there a MEK-like bacterial component that could also specifically target PD-0184264. At this stage, bacterial serine/threonine kinase PknB has become the focus of research. This kinase shows high structural and functional similarity to cellular serine/threonine kinases, more specifically MAP kinases such as p38, JNK and ERK (Miller et al., 2010, Rakette et al. 2012) (Fig. 8), which are MEKs. targets in mammalian cells. Interestingly, the kinase has been shown to be activated by autophosphorylation, thus strongly suggesting that it exhibits MEK-like activity.

Example 6 PD-0184264 increases the sensitivity of Staphylococcus aureus to antibiotics and reduces resistance to bacterial stress.

Through the expression of three penicillin-binding domains (PASTA) (see Fig. 8, top panel), PknB is involved in the regulation of antibiotic susceptibility. It can be shown that kinase deficiency leads to increased susceptibility to various antibiotics, especially various β-lactams (Tamber et al. 2010). To investigate whether treatment of bacteria with the MEK inhibitor PD0184264 could affect bacterial kinase and lead to a kinase knockout-like phenotype, bacterial cultures were treated overnight with solvent (DMSO) or 20 μM PD-0184264 and then used to determine the minimum inhibitory concentration ( MIC) of various antibiotics. During the day, the culture of S. aureus 6850 was set at 20 cfu/ml and treated with either solvent (DMSO) or MEK inhibitor PD-0184264 overnight at 37°C and 5% CO2. Optical density (OD ₆₀₀ ) was measured in the morning. Briefly, solvent and inhibitor treated cultures were washed once with PBS and 1:1 dilutions were plated on BHI agar plates. Shortly after MIC inoculation, test strips for various antibiotics (Thermo Fischer Scientific) were placed in the middle of the dish and then incubated for 18-24 hours at 37°C. 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 visible was designated as the MIC for each individual antibiotic. Treatment with PD0184264 did result in an increase in bacterial susceptibility to various antibiotics, most notably with penicillin and gentamicin (FIG. 9, Table 2). This result is in complete agreement with published data obtained using a mutant strain lacking the kinase (Tamber et al. 2010).

- 13 042391

table 2

Determination of MIC after overnight treatment with PD0184264

Minimum inhibitory concentration (MIC) [mg/l] S. aureus 6850 S. aureus US AZ 00 Not processed PD0184264 Not processed PD0184264 Penicillin 16 0.25 - 0.5 >256 8 Meropenem 0.06 0.03 0.12-0.25 0.06 Linezolid 2 1 2 1 Ciprofloxacin 0.25 0.12-0.25 8 4 - 8 Gentamycin 2 0.12 1 0.12

The observed increase in antibiotic susceptibility with PD-0184264 treatment, which is consistent with the bacterial phenotype lacking the kinase (Tamber et al. 2010), strongly suggests that the inhibitor can directly target PknB. Since the kinase is known to play an important role in resistance to bacterial stress, the growth of bacteria under heat stress conditions was controlled after treatment of the bacteria with the PD-0184264 inhibitor. S. aureus 6850 strain and MRSA strain USA300 were treated overnight with either vehicle or 20 μM PD0184264. The next day, subcultures were prepared with the same number of bacteria (confirmed by OD ₆₀₀ and inoculation 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 FIG. 10, PD-0184264-treated bacteria were severely damaged under these conditions compared to solvent-treated pathogens. This is observed for both the methicillin-sensitive 6850 strain (black bars) and the MRSA USA300 strain (grey bars). Impaired stress tolerance in the presence of an inhibitor is another indication that PD-0184264 directly targets PknB kinase, which is an important mediator of stress tolerance. Thus, the data provide strong circumstantial evidence that PD-0184264 exerts its antibacterial activity by inhibiting the bacterial PknB kinase.

Example 7 Administration of PD-0184264 has an inhibitory effect on the growth of Streptococcus pneumoniae and Bacillus subtilis.

In addition to S. aureus, there are other bacteria known to cause secondary bacterial pneumonia after influenza (IV) virus infection in patients. In this context, the most common pathogen is Streptococcus pneumoniae. These bacteria represent the most common cause of community-acquired pneumonia. Unlike S. aureus, secondary infections with Streptococcus pneumoniae occur late after IV and therefore correspond to the end stage of post-influenza pneumonia.

Like S. aureus, most strains of Streptococcus pneumoniae express eukaryotic serine/threonine kinases such as PknB, which are highly conserved between different genera. In addition, these kinases have high homology to cellular MAP kinases (eg, ERK, JNK, p38). The results shown in Examples 2-6, obtained with various strains of S. aureus, have already demonstrated an inhibitory effect of PD-0184264 treatment on bacterial growth, indicating the involvement of bacterial kinases such as PknB in the observed phenotype. Strikingly, a homolog of S. aureus PknB also exists in Streptococcus pneumonia, suggesting that these bacteria may also be susceptible to PD-0184264. Thus, the effect of PD0184264 on different strains of Streptococci pneumonia was analyzed. Strains of Streptococcus pneumoniae can be divided into different serotypes that differ in their virulence and overall pathogenicity. To test for serotype- or strain-independent action, encapsulated strains D39 and TIGR4 were used, which are both virulent but belong to different serotypes. Treatment with PD-0184264 has been found to disrupt the growth of various serotypes of Streptococcus pneumoniae. In particular, during the day cultures of strains of Streptococcus pneumoniae 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 a solvent (DMSO) or various concentrations specific MEK inhibitor PD0184264 (PD; active metabolite CI-1040) as indicated. OD600 was then measured again (results shown in FIG. 11A) and dilution series were placed on BHI agar plates to determine bacterial titers (results shown in FIG. 11B). As a result, both serotypes could be shown to be susceptible to PD-0184264 (Fig. 11 a, b).

- 14 042391

The same is true for Bacillus subtilis, which also showed a strong decrease in the number of viable bacteria in the presence of 10 μm PD-0184264 and a complete abolition at higher concentrations (see results in Fig. 11c). Specifically, to test for potential antibacterial effect on B. subtilis, overnight cultures of B. subtilis were incubated with either vehicle or various concentrations of the MEK inhibitor PD-0184264 (as indicated) for 18 h. Bacterial load was then determined by measuring OD600 and inoculating serial dilutions. on BHI agar plates. The data shown in FIG. 11 are the mean + SD of three independent experiments.

Overall, these data indicate a broad applicability of PD-0184264 in antibacterial treatment.

Example 8 PD-0184264 but not CI-1040 reduces intracellular bacterial titers.

Influenza (IV) virus infection leads to an increase in the expression of antiviral cytokines, most importantly, type I IFNs, which activate critical subsequent antiviral responses, can also enhance subsequent bacterial infections. To see if treatment with CI-1040 or PD-0184264 would sensitize cells to secondary S. aureus infection, cell cultures of immortalized human alveolar basal epithelial cells (A549) were infected with influenza IV and S. aureus in the presence or absence of inhibitors. In particular, A549 cells were pre-treated for 1 h with 10 μM of the specific MEK inhibitor PD-0184264 or DMSO as a solvent control. The cells were then washed with PBS and infected with influenza (IV) virus (MOI as indicated) for 30 min at 37°C, 5% CO2. The cells were then washed with PBS and infected with S. aureus 6850 (MOI as indicated) in the presence or absence of the inhibitor for 3 h. C to remove non-internalized bacteria. Then the cells were washed once and additionally incubated for up to 24 hours a day in the presence of an inhibitor or solvent. At the end of the incubation period, the cell monolayer was analyzed by light microscopy. Microscopic examination showed that superinfection with both pathogens resulted in a significant increase in cytopathic effect (CPE) compared to single infections (Fig. 13, upper panel). CPE disappeared completely in the presence of PD-0184264 (FIG. 13, bottom panel), indicating a decrease in viral replication.

To test if 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 min and then infected with influenza IV (H7N7) at an MOI of 0.001 at 37°C. The results are shown in FIG. 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, the virus dilution was removed, cells were washed with PBS, and inoculation medium with or without S. aureus 6850 (6850) (MOI 0.1) was added in the presence of 10 μM CI-1040, PD-0184264, or solvent control. 3 hours after bacterial infection, cells were treated with lysostaphin (2 μg/ml) for 20 minutes to remove extracellular bacteria. The cells were then washed and an infection medium containing an inhibitor or solvent was added. After a total incubation period of 24 h (after viral infection), titers of intracellular bacteria were analyzed. Results represent means + 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 from FIG. 14A, treatment with C1-1040 did not sensitize the cells to secondary S. aureus infection as no change in intracellular bacterial load was detected. Surprisingly, administration of PD-0184264 even resulted in a decrease in intracellular bacterial titers, as can be seen in FIG. 14V. Comparable results were obtained when CI-1040 or PD-0184264 were administered at later times during ongoing infection, as shown in FIG. 14C.

To rule out that the reduction in viral and intracellular bacterial replication was the result of the cytotoxic effect of PD-0184264 on A549 cells, cell viability in the presence of increasing concentrations was monitored for 24 and 48 hours. In addition, LDH analysis was performed to determine membrane rupture due to inhibitor treatment. Treatment of A549 cells with PD-0184264 has not been shown to cause cell toxicity. A549 cells were treated for 24 hours (as shown in Figures 15A and C) or 48 hours (as shown in Figures 15B and D) with increasing concentrations of PD-0184264 (1, 5, 10, 20, 50, or 100 µM). After the incubation time, supernatants were sampled for LDH release measurement (as shown in FIG. 15C,D) using the CytoSelect LDH Cytotoxicity Assay Kit according to the manufacturer's instructions. In addition, viable cells were counted by trypan blue staining. Cell viability was normalized for DMSO-treated cells and is shown as % viability. Data are 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).

- 15 042391

Example 9 Determination of IC ₅₀ values for CI-1040 and PD-0184264.

Aliquots of the inhibitor were dissolved in 100% DMSO (10 mM stock solution). To analyze the IC ₅₀ values in a microtiter plate, the following serial dilutions were prepared: 50 μM, 25 μM, 5 μM, 2.5 μM, 0.5 μM, 0.25 μM, 0.05 μM, 0.025 μM, 0.005 μM. 1 µl of each dilution was added to 49 µl of the kinase reaction mixture to give the following test concentrations: 1 µM, 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 0.1 nM.

µl active c-Raf1, 2 µl MEKlwt and 3 µl ERK2wt purified protein solutions were mixed with kinase buffer and 1 µl DMSO or DMSO/inhibitor (final volume 45 µl). The mixture was incubated for 30 min at room temperature in the dark. After this pre-incubation, allowing the inhibitor to bind to the MEK protein, the kinase reaction was started by adding 5 μl of 10 mM ATP and mixing with a pipette. Samples were incubated for 30 min at 26°C in a thermomixer (Eppendorf) at 500 rpm. To stop the kinase reaction, 5.5 μl of 20% SDS solution was added and this mixture was then incubated for 10 min at 50°C. Each sample was then diluted with 190 μl blocking buffer (1% BSA in TBST). 100 μl of each sample was added to the wells of a 96-well microtiter plate coated with anti-ERK antibodies.

Kinase reaction samples (100 μl/well) were incubated for 60 minutes at room temperature in wells of a 96-well microtiter plate coated with anti-ERK antibody and blocking BSA. The dishes were then washed 3x5 min with 100 µl TBST wash buffer. To detect phosphorylated ERK, an antibody to phospho-ERK (p44/p42) (1:3000, 100 μl/well in blocking buffer) was added and incubated overnight at 4°C.

After three washing steps (3x100 µl/well), HRP-conjugated anti-mouse IgG (1:1000 in TBST) was added and incubated for 60 min at room temperature. 100 μl/well ABTS peroxidase substrate was added after three additional washing steps (3x100 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 is measured at a wavelength of 405 nm in an ELISA reader.

Cell-free kinase analysis revealed that 12.5 times less CI-1040 (FIG. 16) was needed to inhibit 50% of MEK activity compared to PD-0184264, which is actually a weaker inhibitor of MEK kinase. Thus, no one expected the strong antiviral and antibacterial effects of PD-0184264. However, as shown in the examples above, PD-0184264 exhibits stronger antiviral and antibacterial activity than CI-1040 in vivo.

Example 10 Antiviral activity of PD-0184264 in an in vitro assay.

Medicines.

CI-1040 [2-(2-chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide; Lot:CC5395.0-16] 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, a 10 mM stock solution of CI-1040 (M=478.66 g/mol) and PD-0184264 (M=409.55 g/mol) in DMSO (Merck-Millipore; Germany) was prepared.

Virus and cells.

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

Viral progeny inhibition assay.

A549 cells were infected with RB1 for 30 min at 37° C. in 5% CO ₂ atmosphere. After incubation, the virus dilution was taken and the cells were washed with PBS and 500 μl of IMDM (Iskov's Dulbecco's medium)/BA (bovine albumin) medium (0.2% BA, 1 mM MgCl ₂ , 0.9 mM CaCl ₂ , 100 U/ml penicillin, 0.1 mg/ml streptomycin) and 0.6 µl TPCK-treated trypsin in the presence of 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, the final concentration of DMSO was 1%) for 24 h at 37°C in 5% CO2. The solvent control was IMDM/BA medium with 1% DMSO. Cell culture supernatants were harvested to determine progeny virus titers on MDCK II cells using the AVICEL® plaque assay as previously described (Haasbach et al. 2011, Matrosovich et al. 2006).

WST analysis.

A549 cells were seeded in a 96-well flat bottom plate (Greiner, Germany) and grown overnight. Thereafter, 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 IMDM (ThermoFisher, Germany) supplemented with 5% fetal bovine serum (Sigma-Aldrich; Germany), the final concentration of DMSO in serum was 1%, and cultivation was carried out at 37°C and 5% CO ₂ for 24 h. After that, 10 μl of WST-1 reagent ( Roche, Germany) and incubated for four hours. During this time, the stable tetrazolium salt of WST-1 was degraded to soluble formazan by metabolically active cells in culture. After this incubation period, the formazan dye formed was quantified using an ELISA reader.

- 16 042391 at 405 nm. The measured optical density directly correlates with the number of viable cells.

Results.

The antiviral activity of PD-0184264 against RB1 was examined in a standard virus inhibition assay (FIG. 17A). A decrease in virus titer by 98.87±0.03% was found when cells were treated with 100 μM PD-0184264 (P > 0.0001). A similar decrease was found with 50 μM PD-0184264 (91.50±2.08%; P>0.0001). In contrast, only a slight decrease in virus titer was found with 10 μM PD-0184264 (58.97±4.45%). 1 μM PD-0184264 almost did not lead to a decrease in the number of progeny of the virus. Thus, compared to virus reduction with 10 µM CI-1040 (96.78±0.65%; P>0.0001), nearly 10-fold higher concentration of PD-0184264 is required to achieve a similar reduction in progeny flu virus. This also corresponds to the EC50 value for PD-0184264 compared to CI-1040. For PD-0184264, the EC50 value is 0.804 μM (FIG. 17B). In another study, the EC50 value for CI-1040 against RB1 can be determined as 0.026 µM (Haasbach et al. 2017). The CC ₅₀ value for PD-0184264 is greater than 1576 (Fig. 17C), which is higher compared to CI-1040 (>312.3 μM; Haasbach et al. 2017). Thus, PD0184264 has an SI of 1960 (selectivity index).

Conclusion / discussion.

The results demonstrate reduced antiviral activity of PD-0184264 in cell culture, ie. in vitro compared to CI-1040. An almost 10-fold higher concentration of PD-0184264 is required to achieve the same reduction in virus as CI-1040 in an in vitro assay. The difference in EC ₅₀ values between these two compounds is even more pronounced. The EC ₅₀ value for PD0184264 is 31 times higher than the EC ₅₀ value for CI-1040 (Haasbach et al. 2017). SI PD-0184264 against RB1 on A549 cells is also reduced compared to the development of CI-1040.

Example 11 Virus titer reduction in mouse lung with PD-0184264 in vivo.

(A) After H1N1pdm09 infection, female C57BL/6 mice were treated orally with either 2.8, 8.4 or 25 mg/kg PD-0184264 (left side) or 25, 75 or 150 mg/kg CI-1400 (left side ). Animals were sacrificed 24 hours after infection and a lung was taken to prepare a 10% suspension. The virus titer was determined by the standard method. The virus titer of mice treated with two MEK inhibitors was compared with that of mice treated with vehicle alone (control). The level of virus in the lungs of control mice was set to 100% (black bar). Graphpad Prism 7 software was used to illustrate both figures.

Medicines.

CI-1040 [2-(2-chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide; Lot: CC5395.0-16] 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 oral administration at 25 mg/kg, 2.5 mg PD-0184264 was dissolved in 50 µl DMSO (SigmaAldrich, Germany) and further diluted with 0.15 ml Cremophor EL (Merck-Millipore, Germany) and 0.8 ml PBS (Gibco, Germany ). For 8.4 mg/kg and 2.8 mg/kg administration, 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 PBS. 202.5 mg CI-1040 was dissolved in 0.5 ml DMSO/0.15 ml Cremophor EL/0.8 ml PBS and further diluted with Cremophor EL and PBS.

Animals.

For antiviral studies, eight-week-old female C57Bl/6 mice (Charles River Laboratories, Germany) weighing 21.0-24.0 g at injection were used. The animals were fed as usual. Drinking water was available ad libitum.

The use of medicines.

Drugs were administered as a single administration on test day 1 via a gastric tube. The application rate was 15 seconds per dose at an injection volume of 200 μl.

Lung virus titration assay.

Mice were sacrificed 24 hours post-infection, lungs were weighed, transferred to a Lysing Matrix D tube (MP Bio) and BSS was applied at 10 times the lung volume. Organs were minced using FastPrep FP 120 (Savant). To remove cell debris, the homogenates were centrifuged for 15 min at 2000 rpm and the supernatant was collected. Determination of virus titer in homogenates was performed using the AVICEL® plaque assay as described previously (Haasbach et al. 2011, Mastrosovich et al. 2006).

Conclusion / discussion.

In FIG. 18 shows the results of the experiments. Compared to the control experiment, only concentrations of 75 mg/kg or higher of CI-1040 showed any effect in reducing virus titer. In contrast, PD-0184264 showed a decrease in virus titer in the lungs already at a concentration of 2.8 mg/kg to about 70%. At a concentration of 8.4 mg/kg, the virus titer is reduced to about 20%, while at 25 mg/kg, the virus titer is reduced to about 10%. Thus, a 6-fold lower concentration of PD-0184264 is required to achieve an effect similar to 150 mg/kg CI-1040, highlighting the high potential of PD-0184264 for antiviral effects.

Example 12 PD-0184264 has higher bioavailability than CI-1040.

(A) Male NMRI mice were treated with either 75 mg/kg CI-1040 (dark gray area) or 75 mg/kg PD0184264 intravenously. Blood was collected at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h (test day 2) after administration, and the plasma was analyzed for drug. (B) Male NMRI mice were treated with either 150 mg/kg CI-1040 (dark gray area) or 150 mg/kg PD-0184264 orally (per os) using a gastric tube. Blood was collected at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h (test day 2) after administration, and the plasma was analyzed for drug. Each data point represents the mean of three plasma samples. Graphpad Prism 7 software was used to illustrate both figures.

Medicines.

CI-1040 [2-(2-chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide; Lot: CC5395.0-15] and PD-0184264 (PD0184264) [2-(2-chloro-4-iodophenylamino)-K-3,4-difluorobenzoic acid; Lot: CC-5595.4-10] were synthesized at ChemCon GmbH (Freiburg, Germany). For intravenous (iv) administration, 30.65 mg CI-1040 was dissolved in 0.075 ml DMSO (Sigma-Aldrich, Switzerland) and further diluted with 0.225 ml Cremophor EL (Merck-Millipore, Germany) and 2.7 ml PBS (Gibco , Germany). 34.88 mg of PD-0184264 was dissolved in 0.075 ml of DMSO and further diluted with 0.225 ml of Cremophor EL/2.7 ml of PBS. For oral administration, 202.5 mg CI-1040 was dissolved in 0.5 ml DMSO/1.5 ml Cremophor EL/8.0 ml PBS. 81.0 mg of PD-0184264 was dissolved in 0.2 ml DMSO/0.6 ml Cremophor EL/3.2 ml PBS.

Animals.

For pharmacokinetic studies, eight-week-old male NMRI mice (Charles River Laboratories, Germany) weighing 23.9-36.5 g when administered were used. The animals were fed as usual. Drinking water was available ad libitum.

Blood sampling and plasma preparation.

The experiments were carried out at LPT GmbH (Hamburg, Germany). Sufficient whole blood was collected under isoflurane anesthesia to obtain at least 2x100 μl of plasma with Li-heparin 3 animals per group, and at the following time points: 0 (before administration), 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h (test day 2) after administration. Whole blood samples were flash-cooled using an Iso-Therm-Rack system (Eppendorf AG, Germany) prior to centrifugation within 0.5 h after retrieval. Immediately after centrifugation, the samples were stored at -20°C until further analysis. Plasma analysis was performed using standard procedures at Prolytic GmbH (Frankfurt, Germany).

The use of medicines.

Drugs were administered either as a single administration on test day 1 via a gastric tube or as an intravenous bolus in the tail vein. The injection rate was 15 s/dose with an injection volume of 200 μl.

Results.

Pharmacokinetic experiments showed that a higher exposure of PD-0184264 was found after IV (Fig. 19A) and oral (per os) (Fig. 19B) administration in mouse plasma compared to CI-1040 with AUC values of 1953.68 µg *h/ml PD-0184264, which are much higher than the values for CI-1040. Of note, almost no drug was detected in plasma eight hours after IV administration and after oral (per os) administration of CI-1040. In contrast, after oral (per os) application of PD-0184264 2, a high concentration was still detected at the time point of 8 hours.

Conclusion / discussion.

The dramatic difference in plasma levels between PD-0184264 and CI-1040 after a single intravenous dose already suggests that CI-1040 may be rapidly degraded. The inventors assumed that drug reduction occurs in a mono-exponential manner. In general, this assumption is correct. At low concentrations, the drug usually decreases in a mono-exponential manner. And the terminal elimination rate constant does not change with time or with different concentrations of circulating drug. However, it is currently unknown what role other processes, such as enterohepatic circulation, play in the terminal phase of the pharmacokinetic profile.

Overall, PD-0184264 exhibits higher antiviral activity than CI1040 in vivo, which may be based on higher drug bioavailability.

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   ФОРМУЛА ИЗОБРЕТЕНИЯ

   1. Применение PD-0184264 или его фармацевтически приемлемой соли для профилактики и/или лечения бактериальной инфекции.
2. 2. Применение PD-0184264 или его фармацевтически приемлемой соли для профилактики и/или лечения вирусного заболевания.
3. 3. Применение PD-0184264 или его фармацевтически приемлемой соли для профилактики и/или лечения коинфекции, включающей бактериальную инфекцию и вирусное заболевание.
4. 4. Применение PD-0184264 или его фармацевтически приемлемой соли по п.2 или 3, где указанный вирус представляет собой РНК-вирус с негативной цепью.
5. 5. Применение PD-0184264 или его фармацевтически приемлемой соли по п.4, где указанный вирус представляет собой вирус гриппа.
6. 6. Применение PD-0184264 или его фармацевтически приемлемой соли по п.5, где указанный вирус гриппа представляет собой вирус гриппа А или вирус гриппа В.

   -