Combination of diltiazem and viral polymerase inhibitors

Abstract

The present invention relates to the combination of diltiazem with at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimodivir, RO-7 and CC-42344. In particular, the present invention relates to the therapeutic use of said combination in the prevention and / or treatment of viral infections, especially viral infections of the respiratory and / or intestinal tract of the human or animal body.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to novel combinations of at least two antiviral compounds.

[0002] In particular, the present invention relates to this combination for use in the prevention and / or treatment of viral infections.

[0003] In particular, the present invention relates to this combination for therapeutic use in the prevention and / or treatment of viral infections, especially viral infections affecting epithelial cells of the respiratory and / or intestinal tract. [Background technology]

[0004] State of the technology Acute respiratory infections (ARIs) in humans are one of the leading causes of medical visits, hospitalizations, and deaths worldwide, and are the leading cause of death among young children, accounting for more than 2 million deaths annually.

[0005] Among the pathogens causing acute respiratory infections, viruses occupy a major position. In fact, they account for the majority of pneumonia cases in children and are the etiology of bacterial pneumonia in adults.

[0006] Among the most representative viruses in terms of frequency (seasonal epidemics) and morbidity, influenza A and B viruses are endemic and constitute risk factors for frequent pandemics. Influenza viruses also constitute pathogens that contribute to increased morbidity and mortality in cases of respiratory co-infections, and contribute to the emergence of antibiotic-resistant bacterial strains, which fundamentally threaten the effectiveness of antibiotic treatment in humans and animals.

[0007] In terms of preventative or therapeutic treatment of viruses, especially influenza viruses, the current arsenal is quite limited.

[0008] Currently, vaccination is the primary strategy for preventing influenza. However, the use of vaccines has drawbacks. For example, when a new seasonal virus strain emerges, it takes 6 to 9 months to develop and formulate a new vaccine. Furthermore, when circulating strains mutate, vaccines lose their effectiveness. Finally, traditional vaccine production methods based on embryonated chicken eggs make it difficult or even impossible to produce vaccines against certain avian-derived pandemic strains, such as H5N1 or H7N9.

[0009] The journal (Pizzorno et al., 2019b) presents the main therapeutic compounds used in the context of influenza treatment and their mechanisms of action.

[0010] When therapeutic action is required, the main antiviral compounds currently in use are: oseltamivir / Tamiflu® (ROCHE); zanamivir / Relenza® (GlaxoSmithKline); and baloxavir marboxil / Xofluza® (ROCHE), which is available in several countries, including the United States and Japan.

[0011] Zanamivir and oseltamivir are neuraminidase inhibitors.

[0012] Baloxavir marboxil is an inhibitor of the influenza virus polymerase complex; recently, it has also been characterized as an inhibitor of the polymerases of other negative-strand RNA genome viruses (Wang et al., 2020).

[0013] Although these antiviral therapeutic agents are relatively effective, they also suffer from significant drawbacks: first, the increasing emergence of viruses resistant to the antiviral compounds, and second, the lack of broad-spectrum antiviral activity of these compounds.

[0014] Indeed, these antiviral compounds target viral determinants and not the host cell. Due to the high frequency of mutations in influenza viruses (intrinsic to their polymerase, which is less faithful in terms of replication, and to their segmented genome, which is a source of genetic reassortment between different viruses), conventional antiviral drugs induce frequent viral resistance.

[0015] In the case of baloxavir marboxil, several recent studies have shown that the risk of developing resistance is very high. In one phase 3 clinical study, the incidence of resistant strains was approximately 10%, primarily with type A viruses (H3N2). More importantly, the resistance rate reached approximately 20% in one pediatric study (Omoto et al., 2018). Furthermore, a further recent study on baloxavir marboxil-resistant mutants showed that one of the main resistance mutations (PA I38T) led to a delayed onset of symptom relief and prolonged viral shedding (Uehara et al., 2019).

[0016] Finally, some infected patients not treated with baloxavir marboxil shed resistant strains, demonstrating the high inter-individual transmissibility of baloxavir-resistant viruses and raising concerns about the potential for widespread spread of these mutant viruses (Takashita et al., 2019). Moreover, the clinical impact of resistance is particularly problematic in hospitalized patients and immunocompromised individuals.

[0017] Therefore, new antiviral compounds are actively sought to limit the occurrence of these resistances.In particular, the combination therapy based on the combination of at least two antiviral compounds seems particularly promising.In particular, when the two antiviral compounds that are combined have synergistic activity, this can reduce the dose of active compound that is used, thus ensuring significant antiviral therapeutic effect and limiting the risk of occurrence of resistance.

[0018] Among the new antiviral compounds identified in the past few years, particular mention may be made of diltiazem, an active compound that allows the treatment of various viral infections by acting on the host cells rather than on the virus.

[0019] International Patent Application WO 87 / 07508 describes the use of diltiazem for the treatment of viral infections related to cytomegalovirus or herpes.

[0020] International patent application WO 2011 / 066657 describes the use of diltiazem for the treatment or prevention of viral infections, such as oral herpes, genital herpes and shingles.

[0021] International application WO 2016 / 146836 and an article (Pizzorno et al., 2019a) describe the use of diltiazem, optionally in combination with other antiviral drugs, for treating infections caused by influenza viruses. A combination of diltiazem and oseltamivir is proposed for treating viral influenza infections.

[0022] International application WO 2019 / 224489 discloses in detail the specific biological effect of diltiazem, which induces the expression of genes encoding type III interferons in respiratory epithelial cells. Diltiazem can therefore be used in a variety of therapeutic applications, particularly in the treatment of viral and bacterial respiratory infections in the respiratory and intestinal epithelium.

[0023] Diltiazem is therefore a very promising new therapeutic option for the treatment of viral infections.

[0024] However, many benefits of the therapeutic use of this compound remain to be identified, and in particular its use in combination with other antiviral drugs may have surprising and unexpected benefits. [Prior art documents] [Patent documents]

[0025] [Patent Document 1] WO 87 / 07508 [Patent Document 2] WO 2011 / 066657 [Patent Document 3] WO 2016 / 146836 [Patent Document 4] WO 2019 / 224489 [Patent Document 5] W0 2 / 094238 [Patent Document 6] US 4,605,552 [Patent Document 7] EP 1117408 Summary of the Invention

[0026] DISCLOSURE OF THE INVENTION The present invention relates to the combination of diltiazem with at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimodivir, RO-7 and CC-42344.

[0027] The present invention also relates to this combination for use as a medicine, especially for use in the prevention and / or treatment of viral infections, especially respiratory and / or enteric infections of the human or animal body, in particular infections caused by influenza viruses.

[0028] The present invention also relates to pharmaceutical compositions comprising a combination of diltiazem and at least one viral polymerase inhibitor compound selected from baloxavir marboxil, pimodivir, RO-7 and CC-42344 in a pharmaceutical vehicle.

[0029] The present invention also relates to this pharmaceutical composition for therapeutic use in the prevention and / or treatment of viral infections, in particular viral infections caused by influenza viruses.

[0030] Finally, the present invention relates to a combination product comprising diltiazem and at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimoxivir, RO-7 and CC-42344 for simultaneous, separate or sequential use in the prevention and / or treatment of viral infections, in particular viral infections caused by influenza virus.

[0031] Text description of the illustration image022.gif. For the sake of brevity, the following names and abbreviations shall be used in the figures and their descriptions: - Dil or DIL refers to diltiazem; - FAV indicates favipiravir; - BLX, Bal or baloxavir refers to the active form baloxavir (baloxavir acid). [Brief explanation of the drawings]

[0032] [Figure 1a] Figure 1 shows the antiviral effect of the combination of diltiazem plus baloxavir on cells in culture infected with H1N1nLuc virus.Timeline of the experiment. [Figure 1b] Figure 1 shows the antiviral effect of the diltiazem + baloxavir combination on cells in culture infected with H1N1nLuc virus. Dose-effect curves (EC50 determination) of diltiazem and baloxavir molecules as monotherapy for H1N1nLuc virus. [Figure 1c] Figure 1 shows the antiviral effect of the combination of diltiazem and baloxavir on cells in culture infected with H1N1nLuc virus.Figure 2 shows a graphical representation of the synergistic effect of diltiazem (DIL) and baloxavir (BLX) in combination against H1N1nLuc recombinant virus, depending on their concentrations used. [Figure 2a] Figure 1 shows the antiviral effect of the combination of diltiazem plus favipiravir on cells in culture infected with H1N1nLuc virus. [Figure 2b]Figure 1 shows the antiviral effect of the combination of diltiazem and favipiravir on cells in culture infected with H1N1nLuc virus. Dose-effect curves (EC50 determination) of diltiazem and favipiravir as monotherapy on H1N1nLuc virus. [Figure 2c] Figure 1 shows the antiviral effect of the combination of diltiazem and favipiravir on cells in culture infected with H1N1nLuc virus.Graphical representation of the synergistic or antagonistic effect of diltiazem (DIL) and favipiravir (FAB) in combination against H1N1nLuc recombinant virus depending on their concentrations used. [Figure 3a] Figure 1 shows the effect of treatment with diltiazem and baloxavir and the combination of both compounds on the replication of A / H1N1 and then A / H3N2 viruses in a human respiratory epithelium model.Timeline of the experiment. [Figure 3b] Figure 1 shows the effect of treatment with diltiazem and baloxavir and the combination of both compounds on A / H1N1 and then A / H3N2 virus replication in a human respiratory epithelium model. A / H1N1 virus infection. Comparison of virus titrations at post-infection (pi) times for untreated epithelia, epithelia treated with the solvent DMSO, treatment with diltiazem (90 μM), treatment with baloxavir (10 nM), and treatment with a combination of diltiazem (90 μM) and baloxavir (10 μM). [Figure 3c] Figure 3b shows the effect of treatment with diltiazem and baloxavir and the combination of both compounds on A / H1N1 and then A / H3N2 virus replication in a human respiratory epithelium model. A / H1N1 virus infection. Tracking transepithelial resistance (TEER) measurements at time post infection (pi) for the epithelium as described above in the description of Figure 3b. [Figure 3d]Figure 1 shows the effect of treatment with diltiazem and baloxavir and the combination of both compounds on A / H1N1 and then A / H3N2 virus replication in a human respiratory epithelium model. A / H3N2 virus infection. Comparison of virus titrations at post-infection (pi) times for untreated epithelia / epithelia treated with solvent DMSO / epithelia treated with diltiazem (90 μM) / epithelia treated with baloxavir (10 nM) / epithelia treated with a combination of diltiazem (90 μM) and baloxavir (10 μM). [Figure 3e] Figure 3 shows the effect of treatment with diltiazem and baloxavir and the combination of both compounds on A / H1N1 and then A / H3N2 virus replication in a human respiratory epithelium model. A / H3N2 virus infection. Tracking transepithelial resistance (TEER) measurements at time post infection (pi) for the epithelium as described above in the description of Figure 3d. [Figure 4a] Figure 4 shows the effect of monotherapy treatment with diltiazem and baloxavir on the replication of baloxavir-resistant A / H1N1 I38T virus in a model of human respiratory epithelium (Figures 4d, 4e), compared with their individual effects on A / H1N1 WT virus (Figures 4b, 4c). Experimental timeline. [Figure 4b] Figure 4d and 4e show the effect of monotherapy treatment with diltiazem and baloxavir on the replication of baloxavir-resistant A / H1N1 I38T virus in a model of human respiratory epithelium, compared with their individual effects on A / H1N1 WT virus (Figures 4b and 4c). A / H1N1 viral infection. Comparison of virus titrations at post-infection (pi) times for untreated epithelium / epithelium treated with solvent DMSO / treatment with diltiazem (90 μM) / treatment with baloxavir (10 nM). [Figure 4c]Figure 4 shows the effect of monotherapy treatment with diltiazem and baloxavir on the replication of baloxavir-resistant A / H1N1 I38T virus in a model of human respiratory epithelium (Figures 4d, 4e), compared with their individual effects on A / H1N1 WT virus (Figures 4b, 4c). A / H1N1 viral infection. Tracking transepithelial resistance (TEER) measurements over time post-infection for the epithelium as described above in the description of Figure 4b. [Figure 4d] Figure 4 shows the effect of monotherapy treatment with diltiazem and baloxavir on the replication of baloxavir-resistant A / H1N1 I38T virus in a model of human respiratory epithelium (Figures 4d and 4e), compared with their individual effects on A / H1N1 WT virus (Figures 4b and 4c). Viral infection with A / H1N1 I38T (baloxavir-resistant strain). Comparison of virus titrations at post-infection (pi) times for untreated epithelium, epithelium treated with solvent DMSO, treatment with diltiazem (90 μM), treatment with baloxavir (10 nM), and treatment with baloxavir (100 μM). [Figure 4e] Figures 4d and 4e show the effect of monotherapy treatment with diltiazem and baloxavir on the replication of baloxavir-resistant A / H1N1 I38T virus in a model of human respiratory epithelium (Figures 4d and 4e), compared with their individual effects on A / H1N1 WT virus (Figures 4b and 4c). Viral infection with A / H1N1 I38T (a baloxavir-resistant strain). Tracking transepithelial resistance (TEER) measurements over time post-infection for the epithelium as described above in the legend to Figure 4d. [Figure 5a] Figure 1 shows the effect of treatment with diltiazem and baloxavir and the combination of both compounds on the replication of the baloxavir-resistant virus strain A / H1N1 I38T in a human epithelial model.Timeline of the experiment. [Figure 5b]1 shows the effect of treatment with diltiazem and baloxavir, and the combination of both compounds, on the replication of the baloxavir-resistant virus strain A / H1N1 I38T in a human epithelial model. Viral infection with A / H1N1 I38T (baloxavir-resistant strain). Comparison of virus titrations at post-infection (pi) times for untreated epithelia, epithelia treated with the solvent DMSO, epithelia treated with diltiazem (90 μM), epithelia treated with baloxavir (10 nM), and epithelia treated with a combination of diltiazem (90 μM) and baloxavir (10 μM). [Figure 5c] Figure 5b shows the effect of treatment with diltiazem and baloxavir and the combination of both compounds on the replication of the baloxavir-resistant virus strain A / H1N1 I38T in a human epithelial model. Viral infection with A / H1N1 I38T. Tracking transepithelial resistance (TEER) measurements over time post-infection for the epithelium as described above in the description of Figure 5b. [Figure 5d] 1 shows the effect of treatment with diltiazem and baloxavir, and the combination of both compounds, on the replication of the baloxavir-resistant virus strain A / H1N1 I38T in a human epithelial model. Viral infection with A / H1N1 I38T (baloxavir-resistant strain). Comparison of virus titration at post-infection (pi) times for untreated epithelia, epithelia treated with diltiazem (90 μM), epithelia treated with baloxavir (100 nM), and epithelia treated with a combination of diltiazem (90 μM) and baloxavir (100 μM). [Figure 5e] Figure 5d shows the effect of treatment with diltiazem and baloxavir and the combination of both compounds on the replication of the baloxavir-resistant virus strain A / H1N1 I38T in a human epithelial model. Viral infection with A / H1N1 I38T. Tracking transepithelial resistance (TEER) measurements over time post-infection for the epithelium as described above in the description of Figure 5d. [Figure 6a] Figure 1 shows experiments performed to observe the emergence of baloxavir-resistant strains in a standard test of serial passage of viral cells under antiviral selective pressure. [Figure 6b]6A-6C show experiments performed to observe the emergence of baloxavir-resistant strains in a standard test of serial passage of virus cells under antiviral selective pressure. The graph shows the concentrations of baloxavir and diltiazem used in the different experimental arms (untreated, treated with baloxavir, treated with diltiazem, and treated with a combination of diltiazem and baloxavir) for each cell passage, according to the experimental protocol previously described in FIG. 6A. The table shows limiting dilutions of infected supernatants obtained from each cell passage (where significant cytopathic effects were observed) and used to infect MDCK cells from each subsequent cell passage in the four experimental arms (untreated, treated with baloxavir, treated with diltiazem, and treated with a combination of diltiazem and baloxavir) previously described in FIG. 6A. The following five figures show the determination of the half maximal inhibitory concentration (IC50) of baloxavir against A / H1N1 virus obtained from the last cell passage in each of the experimental arms (untreated, treated with baloxavir, treated with diltiazem, and treated with the combination of diltiazem and baloxavir) previously described in Figure 6a. [Figure 6c]

[0023] Figure 1 shows an experiment performed to observe the emergence of baloxavir-resistant strains in a standard test of serial passage of virus cells under antiviral selective pressure. Determination of the half-maximal inhibitory concentration of baloxavir against A / H1N1 virus obtained from the first cell passage P0 in MDCK cells treated with various increasing concentrations of baloxavir. The initial IC50 determined for the A / H1N1 virus used in this experiment was confirmed to be 0.2 nM. [Figure 6d]

[0023] Figure 1 shows an experiment conducted to observe the emergence of baloxavir-resistant strains in a standard test of serial cell passage of virus under antiviral selective pressure. The half-maximal inhibitory concentration of baloxavir was determined in MDCK cells treated with increasing concentrations of baloxavir against viruses obtained from cell passage P10 of the untreated experimental arm. The calculated IC50 of baloxavir against A / H1N1 virus derived from cell passage P10 without treatment (untreated) is 0.9 nM. This slight increase can be attributed to viral replication of A / H1N1 virus adapted to continuous cell growth in MDCK cells in this experimental arm. [Figure 6e]1 shows an experiment conducted to observe the emergence of baloxavir-resistant strains in a standard test of serial passage of virus cells under antiviral selective pressure. Determination of the half-maximal inhibitory concentration of baloxavir against viruses obtained from cell passage P10 of the diltiazem-treated experimental arm in MDCK cells treated with increasing concentrations of baloxavir. The calculated IC50 of baloxavir against the A / H1N1 virus derived from the 10th cell passage with diltiazem treatment is 0.3 nM. This IC50 is essentially similar to the IC50 originally determined for the A / H1N1 virus used in this experiment. [Figure 6f]

[0023] Figure 1 shows an experiment performed to observe the emergence of baloxavir-resistant strains in a standard test of serial passage of virus cells under antiviral selective pressure. The half-maximal inhibitory concentration of baloxavir was determined for viruses obtained from cell passage P8 of the baloxavir-treated experimental arm in MDCK cells treated with increasing concentrations of baloxavir. The calculated IC50 of baloxavir for A / H1N1 virus derived from cell passage P8 treated with baloxavir (at increasing concentrations) is 7 nM. This IC50 is much higher than the IC50 originally determined for the A / H1N1 virus used in this experiment. This increase, equivalent to a 35-fold increase over the initial IC50, confirms the emergence of viruses with a baloxavir-resistant phenotype. [Figure 6g]6(a) and 6(b) show experiments performed to observe the emergence of baloxavir-resistant strains in a standard test of serial passage of virus cells under antiviral selective pressure. The half-maximal inhibitory concentration of baloxavir was determined for viruses obtained from cell passage P6 of the experimental arm treated with diltiazem and baloxavir in MDCK cells treated with various increasing concentrations of baloxavir. The calculated IC50 of baloxavir for A / H1N1 virus derived from cell passage 6 treated with diltiazem and baloxavir combination treatment (at increasing concentrations) is 0.8 nM. This IC50 is very similar to the IC50 calculated for baloxavir in A / H1N1 virus derived from cell passage 10 in the experimental arm without treatment (0.9 nM, see FIG. 6(d)), and slightly higher than the IC50 initially calculated for A / H1N1 virus obtained from cell passage P0 used in the experiment (IC50 = 0.2 nM, see FIG. 6(c)). DETAILED DESCRIPTION OF THE INVENTION

[0033] Detailed Description of the Invention The present invention relates to the combination of diltiazem with at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimodivir, RO-7 and CC-42344.

[0034] Within the meaning of the present invention, a "combination" means a composition comprising at least two distinct active compounds, both of which have antiviral activity.

[0035] The combinations may contain equal amounts by weight of each antiviral compound, i.e., 50% by weight diltiazem and 50% by weight viral polymerase inhibitor, or unequal doses of each compound, e.g., 90% by weight diltiazem and 10% by weight viral polymerase inhibitor, 80% by weight diltiazem and 20% by weight viral polymerase inhibitor, 70% by weight diltiazem and 30% by weight viral polymerase inhibitor, 60% by weight diltiazem and 40% by weight viral polymerase inhibitor, 40% by weight diltiazem and 60% by weight viral polymerase inhibitor, 30% by weight diltiazem and 70% by weight viral polymerase inhibitor, 20% by weight diltiazem and 80% by weight viral polymerase inhibitor, or even 10% by weight diltiazem and 90% by weight viral polymerase inhibitor, the percentages being expressed by the weight of the compound relative to the total weight of the combination.

[0036] According to a first embodiment of the invention, the combination comprises diltiazem and baloxavir marboqil. In particular, the combination consists of diltiazem and baloxavir marboqil. In one variant of the invention, the combination comprises diltiazem and the active form of baloxavir (baloxavir acid).

[0037] According to a second embodiment of the invention, the combination comprises diltiazem and pimodivir. In particular, the combination consists of diltiazem and pimodivir.

[0038] According to a third embodiment of the invention, the combination comprises diltiazem and RO-7. In particular, the combination consists of diltiazem and RO-7.

[0039] According to a fourth embodiment of the invention, the combination comprises diltiazem and CC-42344. In particular, the combination consists of diltiazem and CC-42344.

[0040] According to another embodiment, the combination is: - diltiazem, baloxavir marboxil and CC-42344, - diltiazem, baloxavir marboxil and pimoxivir, diltiazem, baloxavir marboxil and RO-7, or even - All possible combinations of diltiazem and baloxavir marboxil, pimoxivir, RO-7 and CC-42344 Includes.

[0041] The combination according to the invention therefore comprises at least two active compounds with antiviral activity, which are described in more detail below.

[0042] This combination has a synergistic antiviral effect, one of the advantages of which is that the dose of each antiviral compound can be reduced compared to the doses conventionally used in monotherapy, which on the one hand limits the occurrence of negative side effects in the treated organism and on the other hand limits the occurrence of viral resistance to said antiviral compounds.

[0043] This patent application also describes combinations of diltiazem with at least one viral polymerase inhibitor compound, such as those described below.

[0044] Diltiazem Diltiazem is a molecule that is a member of the benzothiazepine family and is referred to by the CAS number 42399-41-7. This molecule can be in the form of two enantiomers, L-cis and D-cis, or in a racemic mixture.

[0045] The structural chemical formula of diltiazem hydrochloride is shown below as formula (I). [ka]

[0046] Diltiazem has been known for over 30 years and is approved by drug regulatory authorities in Europe and the United States. It can be administered in the form of diltiazem hydrochloride. Cardizem®, Cartia®, Taztia® and Dilacor® are the most common trade names.

[0047] Many formulations are available, especially sustained-release formulations. Diltiazem is available in various dosage forms, for example, in the form of a cream for topical application, in the form of tablets or capsules for oral administration, in the form of a powder for preparing an injectable solution, or in the form of a pharmaceutical formulation for inhalation (WO 02 / 094238, US 4,605,552).

[0048] The standard dosage regimen used in humans is 180-360 mg / day administered in capsules or tablets for therapeutic use as a calcium channel blocker.

[0049] The first physiological property identified for this compound is calcium channel inhibition, thus inhibiting intracellular calcium flux. Diltiazem specifically inhibits transmembrane calcium entry in cardiac muscle fibers and vascular smooth muscle fibers. This allows a reduction in the intracellular calcium concentration that reaches contractile proteins.

[0050] In humans, diltiazem administration is indicated for its vasodilatory properties with the aim of reducing cardiac workload. It is also used in the treatment of cardiac and circulatory disorders such as angina pectoris, arterial hypertension, myocardial ischemia, and tachycardia.

[0051] Diltiazem also acts from a renal and peripheral perspective by reversing the effects of angiotensin II.

[0052] In topical application, diltiazem may be indicated in cases of chronic anal fissures.

[0053] Patent EP 1117408 describes the use of diltiazem as a calcium channel inhibitor for treating pathologies associated with degeneration of retinal photoreceptors.

[0054] The use of diltiazem to treat viral infections as discussed above has already been described in several patent applications, and clinical trials are currently underway with the aim of obtaining marketing approval for this new antiviral therapeutic indication (FLUNEXT PHRC #15-0442 ClinicalTrials.gov Identifier: NCT03212716).

[0055] Viral polymerase complex inhibitors The influenza virus polymerase complex is composed of three subunits, basic protein 1 (PB1), basic protein 2 (PB2), and acidic protein (PA), which enable transcription and replication of the viral genome.

[0056] Antiviral therapeutics under development and / or currently approved for marketing target different subunits of this polymerase complex and act according to different mechanisms of action: - Baloxavir marboxil is an endonuclease inhibitor mediated by the PA subunit, - Pimodivir is an inhibitor of the PB2 subunit, - Favipiravir is a nucleoside analogue that inhibits RNA elongation by the polymerase complex.

[0057] The first inhibitor compound of this polymerase complex to be approved by regulatory authorities and marketed was baloxavir marboxil. Currently, other PB2 inhibitors (pimodivir) and PB1 inhibitors (favipiravir) are either being tested in phase 3 clinical trials or have restricted approval.

[0058] Baloxavir marboxil Baloxavir marboxil (S-033188), also referred to as baloxavir in this application (especially in the figures), is a molecule that inhibits the cap-dependent endonuclease activity of the PA subunit of the influenza virus polymerase complex. This molecule is a prodrug that, after administration to the body, is metabolized to the active form "baloxavir acid," which inhibits the initiation of viral mRNA synthesis by forming a stable bond with two manganese ions in the active site of the PA subunit of the viral polymerase complex. While the metabolism of the prodrug to the active metabolite is rapid, baloxavir acid has a prolonged hepatic excretion period (half-life: 50-90 hours), allowing for single oral administration (Hayden & Shindo, 2019). In this application, the term "baloxavir" refers to the active form "baloxavir acid" with CAS number 1985605-59-1, obtained after metabolism of the prodrug baloxavir marboxil with CAS number 1985606-14-1.

[0059] This active form is used for all in vitro experiments. Initial in vitro studies demonstrated very good efficacy in inhibiting influenza A virus (average EC50: 1.4–3.1 nM) and influenza B virus (average EC50: 4.5–8.9 nM). Baloxavir has also been shown to be effective against viruses resistant to adamantanes and neuraminidase inhibitors (H274Y) as well as against different subtypes of influenza A virus originating from avian reservoirs. The combination of baloxavir with the neuraminidase inhibitor oseltamivir has demonstrated synergistic effects, allowing protection of mice from lethal infection with influenza A virus (H1N1). This combination also significantly reduced viral titers in the lungs 24 hours after treatment initiation (Fukao et al., 2019).

[0060] Baloxavir marboxil has the potential to revolutionize the treatment of influenza infections given its long half-life, allowing for a treatment regimen consisting of a single dose (as opposed to 5 days of treatment with oseltamivir) and increased antiviral activity compared to neuraminidase inhibitors.

[0061] In a recent phase 3 clinical study, the median time to cessation of viral shedding in non-hospitalized influenza-infected adults was 48 hours in those treated with baloxavir marboxil, compared with 72 hours in those treated with oseltamivir and 96 hours in those treated with placebo, although there was no significant difference in symptom duration between the two treatments (Hayden et al., 2018).

[0062] Baloxavir marboxil is very well tolerated, primarily with gastrointestinal side effects (nausea, diarrhea, etc.) occurring in 4-5% of patients receiving the compound.

[0063] In contrast, one of the major drawbacks of this molecule is the rapid emergence of resistance mutations in the PA subunit of the influenza virus polymerase complex. Initial in vitro studies have isolated viral variants with a major substitution at amino acid 138 (I38T / M / or phenylalanine F) in PA, which reduces the virus's susceptibility to baloxavir by 10-100-fold (Noshi et al., 2018). Clinically, these substitutions are currently very rare, but can be observed in the absence of antiviral treatments (Takashita et al., 2019). During clinical trials, the I38T / M substitution in PA was observed in 2-24% of patients treated with baloxavir marboxil. Furthermore, the duration of clinical symptoms was not significantly shortened in patients with mutant viruses treated with baloxavir marboxil.

[0064] Recently, the antiviral effects of baloxavir marboxil have been observed in other types of viruses with negative-strand segmented RNA genomes, such as severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV), tick-borne pathogens (Wang et al., 2020).

[0065] Pimoziville Pimodivir (JNJ63623872 or JNJ-872 or VX-787) is a complex that inhibits the PB2 subunit of the influenza A virus polymerase complex by binding to the cap-binding site, preventing binding of the natural ligand 7-methylGTP and viral mRNA synthesis (Clark et al., 2014).

[0066] Pimodivir is well tolerated, depending on the dose, with mainly gastrointestinal side effects (nausea, diarrhea, etc.).

[0067] Two phase 3 clinical trials were conducted during the 2017-2018 season.

[0068] For both anti-infective drugs, resistance mutations in PB2 were identified in vitro and in vivo. The M431I substitution in PB2 likely accounts for an approximately 60-fold decrease in susceptibility and was observed in 10% of patients treated with pimoxivir monotherapy (Trevejo et al., 2018).

[0069] RO-7 RO-7, an inhibitor of the endonuclease activity of the acidic protein PA of the influenza virus polymerase complex, was discovered in 2016. This compound has good in vitro activity (EC50: 1.1-21.6 nM) against various strains of influenza viruses circulating in humans and present in avian reservoirs. The use of RO-7 protected mice from lethal infection with influenza virus while significantly reducing the viral load in their lungs. Because RO-7 is structurally very similar to baloxavir marboxil, substitution of amino acid I38 in PA also reduces the susceptibility of influenza viruses to this molecule.

[0070] CC-42344 CC-42344 is one of the most recently identified compounds. It acts by inhibiting the activity of the PB2 subunit of the influenza virus polymerase complex. Although preclinical data have not yet been published, this molecule has good antiviral activity and can be administered orally, via IV, or by inhalation.

[0071] Ribavirin Ribavirin (Virazole) is a broad-spectrum antiviral molecule active against many DNA and RNA viruses. It is a guanosine analogue that, in its monophosphate form, inhibits inosine 5'-monophosphate dehydrogenase (IMPDH), reducing available GTP levels. In its triphosphate form, ribavirin is incorporated into transcribed viral RNA, thus blocking RNA elongation and exerting mutagenic effects. Therefore, ribavirin is considered an inhibitor of viral genome replication.

[0072] In vitro studies have shown that ribavirin is effective against all types of influenza viruses. In contrast, clinical studies have concluded that orally administered ribavirin is not effective against influenza viruses. Furthermore, the many observed side effects limit its use. However, several clinical trials based on the combination of ribavirin with other antiviral compounds are underway in humans.

[0073] Favipiravir Favipiravir or T-705 is a compound used as an antiviral against RNA viruses, particularly orthomyxoviruses, including various influenza viruses, West Nile virus, yellow fever virus, foot and mouth disease virus, and other flaviviruses, arenaviruses, bunyaviruses, and alphaviruses.

[0074] This molecule acts by selectively inhibiting the polymerase of these viruses (Yousuke et al., 2009).

[0075] Regarding its effect on influenza viruses, favipiravir has shown efficacy against influenza A, B, and C viruses, including those resistant to neuraminidase inhibitors, in MDCK cell cultures and in a mouse model infected with the A / PutoRico / 8 / 34 strain. Favipiravir acts as a nucleoside analogue that exhibits low cytotoxicity and can specifically inhibit influenza virus polymerase.

[0076] In vivo, favipiravir has been shown to be effective in protecting mice infected with the highly pathogenic virus A / H5N1, which is either sensitive or resistant to oseltamivir.

[0077] The combination of oseltamivir and favipiravir was found to be synergistic at certain concentrations in mice infected with influenza A virus.

[0078] Favipiravir resistance mutations have been identified in influenza virus polymerase ( Goldhill et al., 2018 ).

[0079] The therapeutic use of this compound in humans has had manufacturing approval in Japan since 2014.

[0080] Therapeutic uses of said combinations. According to one of its aspects, the present invention relates to a combination of diltiazem and at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimodivir, RO-7 and CC-42344 for use as a medicine.

[0081] The present invention particularly relates to the combination of diltiazem with at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimodivir, RO-7 and CC-42344 for therapeutic use in the prevention and / or treatment of viral infections, especially viral infections of the respiratory and / or intestinal tract of human or animal organisms.

[0082] The term "prevention" refers to the fact of preventing or at least reducing the likelihood of the appearance of a viral infection in the human or animal body.

[0083] The term "treatment" refers to the fact of combating viral infections in the human or animal body. This means administering treatment with the aim of reducing the viral load in the body. The term "treatment" also refers to the fact of attenuating the symptoms (fever, fatigue) associated with viral infections.

[0084] The treatment is equally applicable to humans and animals, particularly domestic animals such as pigs, horses and poultry.

[0085] According to a first embodiment of the invention, the present invention relates to a combination of diltiazem and at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimodivir, RO-7 and CC-42344 for therapeutic use in the prevention of viral infections. According to a second embodiment of the invention, the present invention relates to a combination of diltiazem and at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimodivir, RO-7 and CC-42344 for therapeutic use in the treatment of viral infections.

[0086] According to a third embodiment of the present invention, the present invention relates to a combination of diltiazem and at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimodivir, RO-7 and CC-42344 for therapeutic use in the prevention and treatment of viral infections.

[0087] According to one preferred embodiment of the invention, the combination comprises diltiazem and baloxavir marboxil.

[0088] The present invention also relates to a method of treating a patient with a viral infection, particularly a respiratory and / or enteric viral infection, comprising administering to said patient a combination of diltiazem and at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimoxivir, RO-7 and CC-42344.

[0089] The present invention also relates to a method for preventing the occurrence of viral infections, particularly respiratory and / or intestinal viral infections, in a susceptible individual, comprising administering to said individual a combination of diltiazem and at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimoxivir, RO-7 and CC-42344.

[0090] The present invention also relates to a method for preventing and / or treating viral infections, particularly respiratory and / or enteric viral infections, in infected or susceptible animals, comprising administering to said animal a combination of diltiazem and at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimodivir, RO-7 and CC-42344.

[0091] Viral infections and related viruses Within the meaning of the present invention, a "viral infection" refers to a disease caused by a virus that has invaded an organism and infected certain cells of said organism, called "target cells" or "host cells." Viral infections are generally diagnosed by medical professionals based on observing the symptoms of infected patients or animals. Confirmation of the diagnosis may require additional laboratory tests (tests of blood and / or sputum and / or bronchoalveolar lavage fluid and / or biological samples from the intestinal tract).

[0092] The combinations according to the invention are intended for the prevention and / or treatment of viral infections, in particular viral infections of the respiratory and / or enteric tract of the human or animal body, ie infections which affect the respiratory epithelial cells.

[0093] "Respiratory infection" means a viral infection that affects the lungs and airways, i.e., the passages through which air passes to breathe. These include the common cold, flu, and bronchiolitis, among others.

[0094] Viruses involved in viral respiratory infections include respiratory syncytial virus (RSV), influenza virus (flu), parainfluenza virus, adenovirus, and rhinovirus, among others.

[0095] In children, the main causes of viral respiratory infections are rhinoviruses, influenza viruses, parainfluenza viruses, respiratory syncytial virus (RSV), enteroviruses, coronaviruses, and some strains of adenovirus.

[0096] In the context of the present invention, this is in particular acute respiratory infections (ARI).

[0097] "Enteric infection" means a viral infection that affects the digestive tract, especially the upper and lower digestive tract: mouth, pharynx (where the digestive tract and respiratory tract intersect), esophagus, stomach, and intestines.

[0098] The most common causes of gastroenteritis are associated with viral infections caused by norovirus or rotavirus.

[0099] According to one particular embodiment, the therapeutic combination is directed to the treatment and / or prevention of infection with influenza viruses.

[0100] Influenza viruses that cause influenza are classified into four types: A, B, C, and D. Two glycoproteins, the hemagglutinin HA and neuraminidase (NA), are found on the surface of the virus and play an important role in infecting the cells of the infected organism. Depending on the nature of their surface HA and NA glycoproteins, different subtypes of influenza A viruses exist, with 16 types of HA and 9 types of NA identified in viruses circulating in the animal kingdom, particularly among migratory seabirds. Thus, influenza viruses can be defined according to the types of glycoproteins they possess on their surface.

[0101] In humans, subtype A viruses have been circulating for decades as subtypes H1N1, H2N2, and H3N2, with occasional interspecies transmission, particularly from animals to humans, of avian viruses H5N1, H7N7, H7N9, H5N2, and H9N2. Influenza A viruses pose a serious public health threat, as highlighted by the recent emergence of a new pandemic influenza virus, H1N1, of swine, avian, and human origin (a virus involving swine, avian, and human reassortment). Influenza pandemics are particularly the result of antigenic shifts in human populations corresponding to the emergence of viruses endowed with new surface glycoproteins (HA and NA). These shifts allow direct transmission of animal viruses, particularly avian viruses, to humans, as was the case with the highly pathogenic avian H5N1 in Asia since 2003, or the H7N7 influenza epidemic in the Netherlands in 2003 and the H7N9 epidemic in Southeast Asia in 2013. Furthermore, seasonal influenza epidemics, which are the result of, among other things, genetic drift (the emergence of mutations in surface glycoproteins), are a major cause of increased morbidity and mortality in the human population, particularly in the very young, the elderly and immunocompromised, and those with cardiopulmonary disease.

[0102] Within the meaning of the present invention, "influenza virus" refers to influenza pathogens of types A, B, C or D, in particular influenza types A and B that have a human or animal host. "Flu virus" and "influenza virus" are used interchangeably in this application and refer to the same virus.

[0103] The present invention relates inter alia to the combination of diltiazem and baloxavir marboxil for their therapeutic use in the prevention and / or treatment of influenza virus infection.In one variant of the invention, the present invention relates inter alia to the combination of diltiazem and baloxavir acid for their therapeutic use in the prevention and / or treatment of influenza virus infection.

[0104] According to a first aspect of the invention, the combination of the invention is used for the prevention and / or treatment of infection with at least one influenza virus type A. Advantageously, the combination of the invention has a broad spectrum of activity against different subtypes of influenza virus type A.

[0105] In one particular embodiment, the influenza virus is a type A virus selected from the subtypes H1N1, H2N2, H3N2, H5N1, H7N7, H7N9, H5N2 and H9N2.

[0106] According to another aspect of the invention, the combination of the invention is used for the prevention and / or treatment of infection with at least one influenza B virus.

[0107] Viral resistance The present invention particularly relates to a combination of diltiazem and at least one viral polymerase inhibitor selected from among baloxavir marboxil, pimodivir, RO-7 and CC-42344 for therapeutic use in the prevention and / or treatment of infections with influenza viruses, wherein said influenza viruses are resistant to the inhibitory effect of the at least one antiviral compound, especially an anti-influenza compound, in particular a viral polymerase inhibitor.

[0108] Indeed, the combinations according to the invention are particularly interesting when the virus causing the viral infection is not or only slightly sensitive to the action of conventionally used viral polymerase inhibitor compounds, in particular diltiazem and viral polymerase inhibitor compounds selected from among baloxavir marboxil, pimodivir, RO-7 and CC-42344.

[0109] As indicated above, a major drawback to the therapeutic use of this family of compounds that target viral polymerases is the emergence of viral strains that become resistant to their action and insensitive to the inhibitory effects of these compounds.

[0110] The advantages of the combination of diltiazem with at least one viral polymerase inhibitor compound selected from baloxavir marboxil, pimoxivir, RO-7 and CC-42344 are as follows: 1) the emergence of viral strains resistant to at least one viral polymerase inhibitor compound is significantly limited or even completely inhibited during co-administration with diltiazem (see Example 6); 2) Diltiazem alone has effective antiviral activity against viral strains resistant to viral polymerase inhibitors (based on baloxavir-resistant viral strains, see Example 4); 3) The combination of diltiazem + baloxavir marboxil has effective antiviral activity against baloxavir-resistant virus strains due to the presence of diltiazem and high concentrations of baloxavir (see Example 5).

[0111] In particular, the combination according to the present invention comprises diltiazem and baloxavir marboxil and is used in the treatment and / or prevention of infections with influenza viruses that are resistant to the inhibitory effects of viral polymerase inhibitors, for example viruses that display resistance mutations in the polymerase PB2 subunit.

[0112] According to another embodiment of the present invention, the combination comprises diltiazem and baloxavir marboxil for use in the treatment and / or prevention of infections caused by influenza viruses resistant to the inhibitory action of baloxavir marboxil. In particular, this is an influenza A / H1N1 virus strain carrying a mutation in the polymerase PA subunit. More precisely, this is an influenza A / H1N1 virus strain carrying the I38T point mutation in the polymerase PA subunit.

[0113] According to another embodiment of the invention, the influenza virus is resistant to the inhibitory action of at least one anti-influenza compound, in particular a neuraminidase inhibitor, such as oseltamivir (Tamiflu®).

[0114] A variant of the invention relates to diltiazem for therapeutic use in the prevention and / or treatment of infections with influenza viruses, wherein said influenza viruses are resistant to the inhibitory action of at least one antiviral compound, in particular an anti-influenza compound, in particular a viral polymerase inhibitor, in particular one of the following compounds: baloxavir marboxil, pimodivir, RO-7, CC-42344 and neuraminidase inhibitors, in particular oseltamivir.

[0115] Other active drugs present in the combination The present invention also relates to the combination of diltiazem with at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimodivir, RO-7 and CC-42344, further comprising another active agent, in particular an antiviral and / or antibiotic agent.

[0116] According to a preferred embodiment, the antiviral drug is selected from antiviral drugs well known to those skilled in the art and conventionally used for the prevention or treatment of influenza. Antiviral drugs active against at least one influenza virus are commercially available and are described in references, for example, in Le Dictionnaire Vidal. Particular mention may be made of oseltamivir. Therefore, the present invention also relates to the combination of diltiazem, baloxavir marboxil and oseltamivir.

[0117] The antibiotic is selected from antibiotics well known to those skilled in the art, in particular antibiotics used in viral infections to prevent secondary bacterial infections, in particular antibiotics from the macrolide family, in particular roxithromycin.

[0118] The present invention also relates to one of the above-named combinations for therapeutic use, especially for therapeutic use in the prevention and / or treatment of viral infections, in particular for the prevention and / or treatment of infections by influenza viruses.

[0119] Pharmaceutical or veterinary compositions The present invention also relates to pharmaceutical compositions comprising a combination of diltiazem and at least one viral polymerase inhibitor compound selected from baloxavir marboxil, pimodivir, RO-7 and CC-42344 in a suitable pharmaceutical vehicle.

[0120] The present invention also relates to veterinary compositions comprising a combination of diltiazem and at least one viral polymerase inhibitor compound selected from baloxavir marboxil, pimodivir, RO-7 and CC-42344 in a suitable pharmaceutical vehicle.

[0121] According to one preferred aspect of the invention, the viral polymerase inhibitor present in the pharmaceutical or veterinary composition is baloxavir marboxil.

[0122] The pharmaceutical or veterinary composition comprises an effective amount of diltiazem and an effective amount of a viral polymerase inhibitor compound, particularly baloxavir marboxil.

[0123] Within the meaning of the present invention, an "effective amount" means an amount of an antiviral compound sufficient to inhibit the growth and / or replication of a virus and / or the development of a viral infection in an organism, which inhibition can be quantified, for example, by measuring virus production as shown in the examples of the present application.

[0124] For example, so-called "effective" amounts in vitro are as follows: for diltiazem, a concentration of 5 to 200 μM is preferred; - For viral polymerase inhibitor compounds, especially baloxavir (active form), a concentration of 5 to 200 nM is preferred.

[0125] According to the present invention, the term "pharmaceutical vehicle" refers to one or more acceptable pharmaceutical vehicles or excipients according to the present invention, i.e. vehicles or excipients well known to those skilled in the art, whose administration to an individual or animal is not accompanied by significant adverse effects.

[0126] Pharmaceutical or veterinary compositions according to the invention are suitable for oral, sublingual, inhalation, subcutaneous, intramuscular, intravenous, transdermal, ocular or rectal administration.

[0127] According to one preferred aspect of the invention, the pharmaceutical composition is characterized in that it is in a dosage form suitable for administration by inhalation.

[0128] Inhalation means absorption by the respiratory tract, which is the method of absorption for certain substances, particularly therapeutic compounds, in the form of gases, fine droplets, or suspended powders.

[0129] The administration of pharmaceutical or veterinary compositions by inhalation, ie, by the nasal and / or oral routes, is well known to those skilled in the art.

[0130] Inhalation administration is divided into two types: - administration by insufflation when the composition is in the form of a powder; and - administration by nebulization, when the composition is in the form of an aerosol (suspension) or a solution under pressure, e.g., an aqueous solution, in which case the use of a nebulizer or sprayer is recommended for administering the pharmaceutical or veterinary composition.

[0131] Thus, the dosage forms contemplated here are selected from among powders, aqueous suspensions of droplets or pressurized solutions.

[0132] The present invention also relates to a pharmaceutical composition for use as a medicine comprising a combination of diltiazem and at least one viral polymerase inhibitor compound selected from baloxavir marboxil, pimodivir, RO-7 and CC-42344 in a suitable pharmaceutical vehicle.

[0133] The present invention also relates to a pharmaceutical composition comprising a combination of diltiazem and at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimodivir, RO-7 and CC-42344 in a pharmaceutically suitable vehicle for therapeutic use in the prophylaxis and / or treatment of viral infections, especially viral infections of the respiratory and / or intestinal tract of the human or animal body, in particular infections caused by influenza viruses.

[0134] Combination Products The present invention also relates to a combination product comprising diltiazem and at least one viral polymerase inhibitor compound selected from among baloxavir marboxil, pimoxivir, RO-7 and CC-42344 for simultaneous, separate or sequential use for therapeutic use in the prevention and / or treatment of viral infections, especially viral infections of the respiratory and / or intestinal tract of humans or animals, in particular infections caused by influenza viruses.

[0135] This combination product can be used in humans or animals.

[0136] This is comprised in particular of diltiazem and baloxavir marboxil.

[0137] This may include other active compounds, in particular oseltamivir. [Example]

[0138] In the following examples, the term baloxavir is used to refer to baloxavir acid, i.e., the activated form of the compound, and not to refer to the form administered to patients, which is a prodrug.

[0139] Example 1 The combination of diltiazem plus baloxavir has synergistic antiviral effects against recombinant H1N1nLuc virus in cell lines. A549 cells infected with A / H1N1nLuc (multiplicity of infection (MOI) 0.01) were treated 1 h postinfection with increasing concentrations of diltiazem (6 nM to 100 μM) and baloxavir acid (0.0625 nM to 1 nM) (sold by Med Chem Tronica, ref. HY-109025A), alone and in combination.

[0140] The luminescence measured in infected cell supernatants (sampled 48 h post-infection) reflects the level of viral replication (the experimental chronogram is shown in Figure 1a). The EC50 values ​​for each of these two molecules were determined (EC50 for diltiazem = 50102 nM; EC50 for baloxavir = 0.144 nM, see Figure 1b).

[0141] The effect of the molecular combinations was characterized by analysis using Combenefit software from the measured luminescence data (shown in tabular form with a chromatic scale, see FIG. 1c).

[0142] The synergistic or antagonistic effect of the combination is shown by a synergy score (analyzed according to the Loewe model) using a chromatic scale to facilitate the understanding of the results. A coefficient of 5 or more (gray to dark gray) indicates a synergistic effect of the molecular combination, while a coefficient of -5 or less (white) indicates an antagonistic effect of the molecular combination. A coefficient of -5 to 5 indicates non-interference of the molecules in the combination, or even an additive effect between the different treatments. Number of experiments = 3.

[0143] As shown in Figure 1c, the combination of diltiazem and baloxavir acid exhibits synergistic effects.

[0144] Two specific conditions are particularly synergistic: (i) Conditions that combine high concentrations of diltiazem (1562 nm) with baloxavir acid concentrations close to the IC50 (0.1 nM); ii) Conditions that combine lower concentrations of diltiazem (less than 25 nM) with very small doses of baloxavir (less than 0.1 nM).

[0145] No antagonistic effects were observed at the combined concentrations under all conditions tested.

[0146] The results show that the combination of diltiazem and active baloxavir allows a very significant, synergistic reduction of viral replication in A549 cells compared to monotherapy treatment with each of these two molecules alone at the same concentrations (especially under conditions using low doses of baloxavir, which is advantageous in terms of reducing the risk of the emergence of baloxavir-resistant mutations).

[0147] Example 2 The combination of diltiazem plus favipiravir is synergistic or antagonistic depending on the respective concentrations of these two molecules in recombinant H1N1nLuc virus in cell lines. A549 cells infected with A / H1N1nLuc (MOI 0.01) were treated with increasing concentrations of diltiazem (6 nM-100 μM) and favipiravir (6 nM-100 μM) alone and in combination 1 hour postinfection.

[0148] The luminescence measured in infected cell supernatants (sampled 48 h postinfection) reflects the level of virus replication (experimental chronogram is shown in Fig. 2a).

[0149] The EC50 for each of these molecules was determined (Diltiazem EC50 = 53791 nM; Favipiravir EC50 = 2494 nM, see Figure 2b).

[0150] The effect of the molecular combinations was characterized by analysis using Combenefit software from the measured luminescence data (shown in tabular form with a chromatic scale, see FIG. 2c).

[0151] The synergistic or antagonistic effect of the combination is indicated by a synergy score using a chromatic scale to facilitate the understanding of the results. A coefficient of 5 or more (gray to dark gray) indicates a synergistic effect of the molecular combination, while a coefficient of -5 or less (white) indicates an antagonistic effect of the molecular combination. A coefficient of -5 to 5 indicates non-interference of the molecules in the combination, or even an additive effect between the different treatments. Number of experiments = 3.

[0152] The results obtained show that the combination of these two molecules (diltiazem + favipiravir) can improve the antiviral effect, but generally does not allow a significant reduction in the treatment dose.

[0153] Depending on the concentrations of the different diltiazem and favipiravir molecules, these combinations are associated with very heterogeneous effects. To obtain a synergistic effect, large doses of favipiravir (above 1580 nM, which is a disadvantage as it favors an increased risk of developing favipiravir resistance) must be combined with various doses of diltiazem (6 nM to 100 μM).

[0154] Moreover, under certain conditions, the combined effects of these two molecules can be antagonistic. These antagonistic effects are observed when high doses of diltiazem (6250 nM) are used.

[0155] The combination of diltiazem plus baloxavir provides consistent results even when varying concentrations are used, and therefore differs from the less preferred combination of diltiazem plus favipiravir.

[0156] Example 3 Synergistic effects of the diltiazem plus baloxavir combination in a human respiratory epithelium model The reduction in A / H1N1 and A / H3N2 viral replication was significantly greater with treatment with the combination of diltiazem plus baloxavir (active) compared to monotherapy treatment with either diltiazem or baloxavir alone.

[0157] Reconstituted human respiratory epithelia (nasal origin) (MucilAir® HAE, Epithelix) cultured at the air-liquid interface (according to the supplier's instructions) were infected with prototype (non-recombinant) influenza viruses of the A / H1N1 pdm09 (A / Lyon / 969 / 2009 H1N1) strain (MOI 0.1) or the A / H3N2 (A / Texas / 50 / 2012 H3N2) strain (MOI 0.01) and then treated with or without (untreated) diltiazem (90 μM), baloxavir (10 nM), or a combination of diltiazem (90 μM) and baloxavir (10 nM) delivered at 5, 24, and 48 hours post-infection (hpi), respectively (the experimental chronogram is shown in Figure 3a).

[0158] Samples from the apical pole of infected epithelia, treated or untreated, were taken at 24, 48 and 72 hpi to measure virus replication in MDCK cells by infection titration in TCID50 / mL.

[0159] Before each sampling at the apical pole, transepithelial resistance (a physiological marker of epithelial integrity) was also measured using an EVOM2 device and STX2 probe (World Precision Instruments) (FIGS. 3c and 3e).

[0160] As shown in Figure 3b, without treatment (untreated), viral infection caused a decrease in the number of cells in the 10- to 48-hour period after infection. 8 It induces the production of A / H1N1 infectious particles with titers of over TCID50 / mL.

[0161] Diltiazem treatment (90 μM) is able to reduce the production of A / H1N1 infectious particles by up to 2 log10 at 48 hours post-infection and by at least 1 log10 at 72 hours post-infection.

[0162] Baloxavir treatment has significant in vitro antiviral activity, reducing A / H1N1 infectious particle production by 4 log10 at 48 hours post-infection, whereas its solvent alone (DMSO) has no effect on infection.

[0163] The combination of diltiazem and baloxavir molecules significantly improved antiviral efficacy compared to monotherapy treatment, with a nearly 6 log10 reduction in A / H1N1 virus production at 48 and 72 hours post-infection.

[0164] Figure 3c shows tracking of transepithelial resistance (TEER) measurements during infection of the epithelia with A / H1N1 described in Figure 3a, demonstrating a significant decrease in the integrity of untreated epithelia or epithelia treated with the control solvent DMSO at 48 and 72 hours post-infection, which correlates with the highly efficient infection shown in Figure 3b.

[0165] In contrast, treatment with diltiazem maintained stable TEER values ​​and maintained the integrity of the infected epithelium, correlating with the antiviral effect demonstrated by infection titration (Fig. 3b).

[0166] Treatment with baloxavir also allows for the maintenance of the integrity of the infected epithelium, with measured TEER values ​​similar to those obtained with diltiazem treatment.

[0167] Similarly, the integrity of the infected epithelium was maintained by treatment with the combination of both molecules, which correlates with their antiviral efficacy (Fig. 3b).

[0168] These TEER measurements also confirm the lack of cytotoxic effects of combined diltiazem and baloxavir treatment.

[0169] As shown in Figure 3d, without treatment (untreated), epithelial A / H3N2 virus infection reached a titer of 10 48 hours after infection. 9 It induces the production of infectious particles with a titer of more than TCID50 / mL.

[0170] Diltiazem treatment (90 μM) reduces the production of A / H3N2 infectious particles by 2 log10 at 48 hours post-infection and by at least 1 log10 at 72 hours post-infection.

[0171] Baloxavir treatment has significant in vitro antiviral activity, reducing A / H3N2 infectious particle production by 3 log10 at 48 hours post-infection, whereas its solvent alone (DMSO) has no effect on infection.

[0172] The combination of diltiazem and baloxavir molecules significantly improved antiviral efficacy compared to monotherapy treatment, with a nearly 5 log10 reduction in A / H3N2 virus production at 48 and 72 hours post-infection.

[0173] Tracking transepithelial resistance (TEER) measurements during infection of epithelia with A / H3N2 as described in Figure 3a, shown in Figure 3e, shows a significant decrease in the integrity of untreated epithelia or epithelia treated with the control solvent DMSO at 48 and 72 hours postinfection, which correlates with the highly efficient infection measured and shown in Figure 3d.

[0174] In contrast, treatment with diltiazem was able to maintain the integrity of the A / H3N2-infected epithelium, with TEER values ​​remaining stable up to 48 h postinfection, correlating with the antiviral effect demonstrated by infection titration (Fig. 3d).

[0175] Treatment with baloxavir also allows for the maintenance of the integrity of the infected epithelium, with measured TEER values ​​similar to those obtained with diltiazem treatment at 48 hours post-infection.

[0176] Similarly, the integrity of the infected epithelium was maintained by treatment with the combination of both molecules, which correlates with their antiviral efficacy (Fig. 3d).

[0177] These TEER measurements also confirm that combined diltiazem and baloxavir treatment does not have a cytotoxic effect on the epithelium.

[0178] In conclusion, Figures 3b and 3d demonstrate a significant antiviral effect of treatment with diltiazem or baloxavir compared to the state of infection without treatment. These results also demonstrate the beneficial / synergistic effect of diltiazem + baloxavir treatment compared to monotherapy treatment with each of these two molecules alone at the same concentration: this combination indeed allows a significantly greater reduction in viral replication compared to monotherapy treatment in a human respiratory epithelium model.

[0179] Example 4 Diltiazem has effective antiviral activity against the I38T recombinant A / H1N1 virus resistant to baloxavir in a reconstituted human respiratory epithelium model, comparable to its antiviral activity against wild-type (WT) recombinant A / H1N1 virus in the same epithelium model. A resistant A / H1N1 virus was generated by reverse genetics, which carries the I38T resistance mutation in the PA subunit of its polymerase.

[0180] Reconstituted human respiratory epithelium (MucilAir® HAE, Epithelix) of nasal origin, maintained in culture at the air-liquid interface according to the supplier's instructions, was infected with wild-type A / H1N1 pdm09 recombinant influenza virus (WT, MOI 0.1) or with a baloxavir-resistant A / H1N1 pdm09 recombinant influenza virus (I38T mutation in the PA subunit of the viral polymerase) (I38T, MOI 0.1).

[0181] These epithelia were then treated or not (untreated) with three consecutive doses of diltiazem (90 μM) alone, baloxavir alone at concentrations of 10 nM or 100 nM, or the combination of both molecules (diltiazem 90 μM and baloxavir 10 nM or 100 nM), delivered at 5, 24, and 48 hours postinfection (hpi), respectively (experimental chromograms are shown in Figure 4a).

[0182] Samples from the apical pole of infected epithelia, treated or untreated, were taken at 24, 48 and 72 hpi to measure virus replication in MDCK cells by infection titration in TCID50 / mL.

[0183] Before each sampling at the apical pole, transepithelial resistance (a physiological marker of epithelial integrity) was also measured using an EVOM2 device and STX2 probe (World Precision Instruments).

[0184] Each experimental condition was performed in duplicate (n=2). Graphs show mean and standard deviation values ​​generated by GraphPad software.

[0185] Figure 4b shows that without treatment (untreated), virus infection resulted in a titer of 10 8 This indicates that production of more than TCID50 / mL of A / H1N1 WT infectious particles is induced.

[0186] Diltiazem treatment (90 μM) is able to reduce the production of A / H1N1 infectious particles by 2 log10 at 48 hours post-infection and by 0.5 log10 at 72 hours post-infection.

[0187] Baloxavir treatment has significant in vitro antiviral activity, reducing A / H1N1 infectious particle production by 3 log10 at 48 hours post-infection, whereas its solvent alone (DMSO) has no effect on infection.

[0188] Tracking transepithelial resistance (TEER) measurements during infection, shown in Figure 4c, showed a significant decrease in the integrity of untreated epithelia or epithelia treated with the control solvent DMSO at 48 and 72 h postinfection, which correlates with the highly efficient infection shown in Figure 4b.

[0189] In contrast, treatment with diltiazem was able to maintain the integrity of the A / H1N1-infected epithelium, with TEER values ​​remaining stable up to 48 h postinfection, correlating with the antiviral effect demonstrated by infection titration (Fig. 4b).

[0190] Treatment with baloxavir also allows for the maintenance of epithelial integrity infected with A / H1N1, with stable TEER measurements at 48 and 42 hours post-infection.

[0191] Figure 4d shows that without treatment (untreated), epithelial infection with baloxavir-resistant A / H1N1 I38T virus resulted in a 10 8 This indicates that production of infectious particles with a titer close to TCID50 / mL was induced (see FIG. 4d).

[0192] Treatment with 10 nM baloxavir had no antiviral effect against this resistant A / H1N1 I38T virus in an epithelial model of infection, but a significantly increased baloxavir treatment dose (100 nM) was required to achieve still-limited antiviral activity against this resistant A / H1N1 I38T virus (a 1 log10 reduction in infectious particle production at 48 hours post-infection and a 1.5 log10 reduction at 72 hours post-infection).

[0193] In contrast, treatment with diltiazem (90 μM) alone can significantly reduce the production of A / H1N1 I38T infectious particles by approximately 2 log 10 at 48 hours post-infection and by approximately 2 log 10 at 72 hours post-infection.

[0194] Tracking transepithelial resistance (TEER) measurements during infection with A / H1N1 I38T, shown in Figure 4e, demonstrates a significant decrease in the integrity of the intact epithelium at 48 and 72 h postinfection, which correlates with the highly efficient infection shown in Figure 4d.

[0195] In contrast, treatment with diltiazem was able to maintain the integrity of epithelia infected with A / H1N1 I38T virus, with TEER values ​​remaining stable at 48 and 72 h postinfection, correlating with the antiviral effect demonstrated by infection titration (Fig. 4d).

[0196] In contrast, treatment with 10 nM baloxavir failed to maintain the integrity of epithelia infected with A / H1N1 I38T at 48 and 72 hours postinfection, consistent with the lack of antiviral activity reported in (Figure 4d). Only treatment with a high dose of baloxavir (100 nM), for which significant antiviral activity against the resistant A / H1N1 I38T virus is reported in Figure 4d, is able to maintain epithelial integrity, as reported by measurements of stable values ​​of TEER at 48 and 72 hours postinfection.

[0197] Example 5 The diltiazem plus baloxavir combination induces a significantly greater reduction in baloxavir-resistant A / H1N1 I38T virus replication compared with monotherapy treatment, but only when high doses of baloxavir are used. Reconstituted human respiratory epithelium (MucilAir® HAE, Epithelix) of nasal origin, maintained in culture at the air-liquid interface according to the supplier's instructions, was infected with a recombinant influenza virus of type A H1N1 pdm09 resistant to baloxavir (I38T mutation in the PA subunit of the viral polymerase) (I38T, MOI 0.1).

[0198] These epithelia were then treated or not (untreated) with three consecutive doses of diltiazem (90 μM) alone, baloxavir alone at concentrations of 10 nM or 100 nM, or the combination of both molecules (diltiazem 90 μM and baloxavir 10 nM or 100 nM), delivered at 5, 24, and 48 hours postinfection (hpi), respectively (experimental chromograms are shown in Figure 5a).

[0199] Samples from the apical pole of infected epithelia, treated or untreated, were taken at 24, 48 and 72 hpi to measure virus replication in MDCK cells by infection titration in TCID50 / mL.

[0200] Before each sampling at the apical pole, transepithelial resistance (a physiological marker of epithelial integrity) was also measured using an EVOM2 device and STX2 probe (World Precision Instruments).

[0201] Figure 5b shows that without treatment (untreated), epithelial infection with baloxavir-resistant A / H1N1 I38T virus resulted in a 10% increase in HIV-1 virion count at 72 hours post-infection. 8 This indicates that diltiazem treatment (90 μM) induces the production of infectious particles with titers approaching TCID50 / mL. Diltiazem treatment (90 μM) can reduce A / H1N1 I38T infectious particle production by 2 log10 at 48 hours post-infection and by at least 2 log10 at 72 hours post-infection.

[0202] In contrast, baloxavir treatment (10 nM) is as ineffective as its solvent alone (DMSO), which has no effect on infection.

[0203] The combination of these two molecules, diltiazem and baloxavir, at each concentration was able to reduce the production of A / H1N1 I38T infectious particles, but did not have an additive or synergistic effect compared to treatment with diltiazem alone at 48 and 72 hours post-infection.

[0204] This result differs from that obtained for the A / H1N1 WT virus (which is not resistant to baloxavir, see Figure 3b), where the combination of these two molecules, diltiazem and baloxavir at the same concentrations, has a synergistic antiviral effect compared to monotherapy treatment, particularly by inducing a reduction in virus production of approximately 6 log10 at 48 and 72 hours post-infection.

[0205] Tracking transepithelial resistance (TEER) measurements during infection, shown in Figure 5c, showed a significant decrease in the integrity of untreated epithelia or epithelia treated with the control solvent DMSO at 48 and 72 h postinfection, which correlates with the highly efficient infection shown in Figure 5b.

[0206] In contrast, treatment with diltiazem was able to maintain the integrity of epithelia infected with A / H1N1 I38T, with TEER values ​​remaining stable at 48 and 72 h postinfection, correlating with the antiviral effect demonstrated by infection titration (Fig. 5b).

[0207] Treatment with baloxavir (10 nM) failed to maintain the integrity of epithelia infected with A / H1N1 I38T, consistent with its lack of antiviral efficacy reported in (Fig. 5b).

[0208] Also consistent with its significant antiviral efficacy, the diltiazem plus baloxavir combination is associated with stable TEER measurements at 48 and 72 hours post-infection.

[0209] Figure 5d shows that without treatment (untreated), epithelial infection with baloxavir-resistant A / H1N1 I38T virus resulted in a 10 8 This indicates that production of infectious particles with a titer close to TCID50 / mL is induced.

[0210] Diltiazem treatment (90 μM) can reduce the production of A / H1N1 I38T infectious particles by more than 2 log 10 at 48 hours post-infection.

[0211] When used at a high concentration (100 nM), baloxavir treatment reduced the production of A / H1N1 I38T infectious particles by approximately 1 log at 48 hours postinfection, whereas treatment at a concentration of 10 nM had no effect on the resistant virus I38T (see Figure 5b).

[0212] The combination of these two molecules, diltiazem and baloxavir, at the same concentrations further reduced the production of A / H1N1 I38T infectious particles at 48 hours and especially at 72 hours post-infection compared to monotherapy treatment (up to a 3 log reduction in A / H1N1 I38T infectious particles). Thus, the benefits of combining diltiazem with baloxavir (high concentration = 100 nM) against resistant A / H1N1 I38T viruses in a human respiratory epithelium model are significant.

[0213] This combination is particularly suitable for baloxavir-resistant virus strains, which require significantly increased baloxavir doses.

[0214] Tracking transepithelial resistance (TEER) measurements during infection, shown in Figure 5e, showed a significant decrease in the integrity of the intact epithelium at 48 and 72 h postinfection, which correlates with the highly efficient infection shown in Figure 5d.

[0215] In contrast, treatment with diltiazem or a high dose (100 nM) of baloxavir maintained the integrity of the epithelium infected with A / H1N1 I38T, with TEER values ​​remaining stable at 48 and 72 h postinfection, correlating with the antiviral effect demonstrated by infection titration (Fig. 5d).

[0216] Also consistent with its significant antiviral efficacy (see Figure 5d), the diltiazem plus baloxavir combination is associated with stable TEER measurements at 48 and 72 hours post-infection.

[0217] In conclusion, the results obtained and presented in Examples 4 and 5 indicate that: (i) These results confirm that the reference treatment with 10 nM baloxavir was ineffective against the recombinant A / H1N1 I38T virus (I38T mutation in the PA subunit of the polymerase) with a baloxavir-resistant phenotype (Fig. 4d). (ii) Figure 4d also demonstrates that a significantly increased baloxavir treatment dose (100 nM) was required to obtain, however still limited, antiviral activity against this resistant A / H1N1 I38T virus. (iii) Importantly, these results also demonstrate that diltiazem has significantly more effective antiviral activity against the baloxavir-resistant I38T recombinant A / H1N1 virus in a reconstituted human respiratory epithelium model than against the wild-type (WT) recombinant A / H1N1 virus in the same epithelium model (compare Figures 4b and 4d). (iv) The results shown in Figure 5b also confirm that combined treatment with diltiazem (90 μM) and baloxavir (10 nM) is capable of significantly reducing viral replication in the respiratory epithelium of the upper respiratory tract infected with the wild-type strain of A / H1N1. (v) In contrast, this combination of diltiazem (90 μM) + baloxavir (10 nM) does not provide any additional benefit compared to treatment with diltiazem in monotherapy against resistant A / H1N1 I38T virus (Figure 5b). (vi) Diltiazem (90 μM) when combined with 100 nM baloxavir provides a significant benefit in terms of antiviral activity against resistant A / H1N1 I38T virus compared to monotherapy treatment with the same concentrations of each of the two molecules in the same human respiratory epithelium model.

[0218] Example 6 The use of diltiazem in combination with baloxavir makes it possible to prevent the emergence of baloxavir-resistant viruses in standard tests of serial cell passage of viruses under antiviral selective pressure. MDCK cells in 24-well plates were infected with the prototype A / H1N1 WT virus (against which the half maximal inhibitory concentrations (IC50) of baloxavir and diltiazem are 0.2 nM and 5 μM, respectively) at an MOI of 0.001, thereby constituting the first cell passage (P0) of the virus.

[0219] Four different arms of serial cell passage of this virus were performed under conditions with or without selective pressure, as defined below: (i) no treatment (NT); (ii) treatment with diltiazem (Dil) at a constant concentration of 25 μM; (iii) treatment with increasing concentrations of baloxavir from 1 nM to 128 nM; and (iv) Treatment with diltiazem (Dil) at a constant concentration of 25 μM in combination with the same increasing concentrations of baloxavir from 1 nM to 128 nM as described above in (iii) above.

[0220] From passage P0, cells were treated or not (NT) according to the different conditions mentioned above at 1 hour post-infection, and the infected supernatants were sampled at 48 hours post-infection (D + 2). These infected samples were serially diluted (10 -1 ~10 -6 ), each dilution was plated onto MDCK cells in a 24-well plate, thereby constituting cell passage (P1) subsequent to cell passage P0.

[0221] One hour after this infection, the cells were either treated or not (NT) according to the different conditions described above. At the end of this cell passage P1 (48 hours post-infection, D+4), for each of the treatment arms (I, ii, iii and iv), the supernatants of the infected wells were taken at the so-called "limiting" dilution, i.e., the 10% dilution obtained from P0, at which a significant cytopathic effect (CPE) was observed.-1 ~10 -6 The last dilution of the dilution was observed.

[0222] These infected supernatants were serially diluted (10-1 to 10-6), and each dilution was plated onto MDCK cells in a 24-well plate, thereby constituting cell passage P2 following cell passage P1.

[0223] This procedure was followed for each successive cell passage. The diltiazem concentration remained constant throughout the experiment. The baloxavir concentration was fixed at 1 nM for the first two passages (P0 and P1), then doubled for the next two passages (P3 and P4), and then doubled every passage thereafter to 128 nM at passage 10 (see experimental chronogram, Figure 6a).

[0224] At the end of the experiment, infected supernatants from the last cell passage of each treatment arm showing significant CPE were titrated at TCID50 / mL in MDCK cells. To characterize the emergence of a baloxavir-resistant phenotype compared to the initial wild-type virus used, the half-maximal inhibitory concentrations of baloxavir against these supernatant viruses were also calculated by limiting dilution titration of TCID50 / mL in MDCK cells.

[0225] For the untreated and diltiazem-treated experimental arms, infectious supernatants from cell passage 10 were used. For the baloxavir-treated arm, infectious supernatants from cell passage 8 were used. For the diltiazem + baloxavir combination-treated arm, infectious supernatants from cell passage 6 were used (because the virus had disappeared in these experimental arms by cell passages 7 and 9, respectively; see Figures 6a and 6b).

[0226] MDCK cells were infected with the infection supernatants in this manner and then treated separately with various increasing concentrations of baloxavir (0.04 nM to 160 nM) 1 hour postinfection. Concurrently, MDCK cells were also infected with the A / H1N1 WT virus initially used for cell passage P0 at an MOI of 0.001 (see Figures 6a and 6b), and then treated separately with various increasing concentrations of baloxavir (0.04 nM to 160 nM) or not 1 hour postinfection.

[0227] Infected supernatants were sampled at 48 hpi, and viral loads were then titrated in MDCK cells in TCID50 by limiting dilution. Results were expressed relative to untreated control cells infected with A / H1N1 virus (Figures 6c-6g). From these infectious virus titers, the half-maximal inhibitory concentrations of baloxavir were calculated in this manner and are reported in Table 1 below.

[0228] Detailed explanation of the results The graph in Figure 6b shows the concentrations of baloxavir and diltiazem used in the different experimental arms (untreated, treated with baloxavir, treated with diltiazem, and treated with a combination of diltiazem and baloxavir) for each cell passage, as previously described in Figure 6a. The table shows the limiting dilutions of infection supernatants from each cell passage (where significant cytopathic effect was observed) used to infect MDCK cells from each subsequent cell passage in each of the four experimental arms (untreated, treated with baloxavir, treated with diltiazem, and treated with a combination of diltiazem and baloxavir).

[0229] The untreated and diltiazem-treated experimental arms were maintained for more than 10 cell passages. In the untreated arm, the limiting dilution used for each cell passage was always 10. -6 In the diltiazem-treated arm, the limiting dilution used was 10 as shown in the graph and table. -5 From 10 -6 It fluctuated up to.

[0230] For the baloxavir-treated arm, the limiting dilution used for each cell passage was 10 up to the 6th cell passage, where the concentration of baloxavir used was 8 nM. -5 From this cell passage, the limiting dilution used for subsequent cell passages was gradually decreased (from 8 nM to 32 nM) using increasing concentrations of baloxavir until replicating virus disappeared at cell passage 9, where the concentration of baloxavir used was 64 nM (10 -4 From 10 -1 to).

[0231] In the diltiazem + baloxavir combination treatment arm, the limiting dilution used (where significant cytopathic effects were observed) was the first cell passage (10 -3 ) and up to the 6th cell passage, 10 -3 ~10 -2 The disappearance of replicating virus was observed during the seventh cell passage when the concentrations of baloxavir and diltiazem used were 16 nM and 25 μM, respectively.

[0232] These results indicate that in the baloxavir-treated arm, A / H1N1 virus with an initial half-maximal inhibitory concentration (IC50) of 0.2 nM replicated up to the 8th cell passage at baloxavir concentrations up to 32 nM. These results suggest that the selective pressure exerted by baloxavir under these experimental conditions induces a resistant virus phenotype. In contrast, this virus resistant phenotype was not observed in the diltiazem-treated arm. This is because, just as in the untreated arm, the limiting dilutions used for each cell passage were 10, without the virus disappearing until the 10th cell passage. -5 ~10 -6 This is because it remained stable at

[0233] These results indicate that the addition of diltiazem to baloxavir in the combination treatment arm (diltiazem 25 μM + increasing concentrations of baloxavir from 1 nM to 8 nM) resulted in synergistic viral inhibition compared with treatment with the same concentrations of diltiazem or baloxavir alone. This is because (i) the limiting dilution used at each cell passage was always low in this experimental arm, starting from the first cell passage (10 -3 ), and (ii) because replicating virus disappeared more rapidly (from the seventh cell passage).

[0234] Figure 6c reports the determination of the half-maximal inhibitory concentration of baloxavir against the A / H1N1 virus obtained from the first cell passage P0. The initially determined IC50 for the A / H1N1 virus used in the experiments is confirmed to be 0.2 nM.

[0235] Figure 6d reports the determination of the half-maximal inhibitory concentration of baloxavir against the virus obtained from cell passage P10 of the untreated experimental arm. The calculated IC50 of baloxavir against the A / H1N1 virus derived from cell passage 10 without treatment (untreated) is 0.9 nM. This slight increase can be attributed to viral replication of the A / H1N1 virus adapted to continuous cell growth in MDCK cells in this experimental arm.

[0236] Figure 6e reports the determination of the half-maximal inhibitory concentration of baloxavir against virus obtained from cell passage P10 of the diltiazem-treated experimental arm. The calculated IC50 of baloxavir against A / H1N1 virus derived from diltiazem-treated cell passage 10 is 0.3 nM. This IC50 is essentially similar to the IC50 originally determined for the A / H1N1 virus used in this experiment. This result confirms that treatment with a constant concentration of 25 μM diltiazem over 10 serial cell passages does not alter the susceptibility of A / H1N1 virus to baloxavir (and does not induce the emergence of a baloxavir-resistant phenotype).

[0237] FIG. 6f reports the determination of the half-maximal inhibitory concentration of baloxavir against viruses obtained from cell passage P8 of the experimental arm treated with baloxavir.

[0238] The calculated IC50 of baloxavir against A / H1N1 virus from cell passage 8 treated with baloxavir (at increasing concentrations) is 7 nM, which is much higher than the IC50 initially determined for the A / H1N1 virus used in this experiment (IC50 = 0.2 nM).

[0239] This increase, equivalent to 35-fold over the initial IC50, confirms the emergence of a virus with a baloxavir-resistant phenotype, consistent with the observations described in Figure 6b (effective virus replication down to the lower dilution limit used in this experimental arm from the first cell passage, 32 nM baloxavir concentration at cell passage 8).

[0240] FIG. 6g reports the determination of the half-maximal inhibitory concentration of baloxavir against viruses obtained from cell passage P6 of the experimental arm treated with the diltiazem+baloxavir combination.

[0241] The calculated IC50 of baloxavir against A / H1N1 virus derived from cell passage 6 treated with diltiazem + baloxavir combination (at increasing concentrations) is 0.8 nM. This IC50 is very similar to the IC50 calculated for baloxavir in A / H1N1 virus derived from cell passage 10 in the untreated experimental arm (0.9 nM, see Figure 6d), and slightly higher than the IC50 initially calculated for A / H1N1 virus obtained from cell passage P0 required for the experiment (IC50 = 0.2 nM, see Figure 6c). Apart from the fact that this slight increase can be attributed to viral replication of A / H1N1 virus adapted to continuous cell growth in MDCK cells in this experimental arm, it is clearly confirmed that this IC50 is significantly lower than the IC50 calculated for A / H1N1 virus from cell passage P8 in the baloxavir-treated experimental arm (IC50 = 7 nM, see Figure 6f), indicating that viruses with a baloxavir-resistant phenotype did not emerge in this experimental arm. These results indicate that the use of diltiazem in combination with baloxavir can prevent the emergence of baloxavir-resistant viruses in a standard test of continuous cell passage of viruses under antiviral selection pressure (increasing concentrations of baloxavir).

[0242] The half inhibitory concentrations of baloxavir for A / H1N1 viruses obtained from the last cell passage in different experimental arms are shown in Table 1 below.

[0243] [Table 1]

[0244] In conclusion, the results of this experiment of serial passage of A / H1N1 viruses in MDCK cells under selective pressure conditions show that, as expected, the use of increasing concentrations of baloxavir induced the emergence of A / H1N1 viruses with a baloxavir-resistant phenotype, but the addition of a constant concentration of 25 μM diltiazem in combination under the same conditions of increasing concentrations of baloxavir made it possible to prevent the emergence of A / H1N1 viruses with a baloxavir-resistant phenotype.

[0245] Similarly, as expected, untreated experimental conditions did not induce the emergence of A / H1N1 viruses with a baloxavir-resistant phenotype.

[0246] Similarly, experimental conditions of diltiazem treatment alone also did not induce the emergence of A / H1N1 viruses with a baloxavir-resistant phenotype.

[0247] References in the order in which they are cited in the specification (References) TIFF0007727641000003.tif216170TIFF0007727641000004.tif182170

Claims

1. A combination of diltiazem and baloxavir marboxil for use in the prevention and / or treatment of influenza virus infections.

2. 2. The combination of claim 1, wherein the influenza virus is resistant to the inhibitory action of at least one antiviral compound.

3. The combination of claim 2, wherein the influenza virus is resistant to the inhibitory action of the anti-influenza compound.

4. The combination described in claim 2, characterized in that the influenza virus is resistant to the inhibitory action of a viral polymerase inhibitor.

5. 5. A combination according to any one of claims 1 to 4, characterized in that the combination comprises at least one other active agent.

6. The combination of claim 5, further comprising an antiviral agent.

7. The combination according to claim 5 or 6, characterized in that the combination further comprises an antibiotic.

8. A pharmaceutical composition for use in the prevention and / or treatment of influenza virus infections, comprising a combination of diltiazem and baloxavir marboxil in a pharmaceutical vehicle.

9. 9. The pharmaceutical composition according to claim 8, characterized in that the composition is in a form suitable for administration by inhalation.

10. A combination product comprising diltiazem and baloxavir marboxil for simultaneous, separate or sequential use in the prevention and / or treatment of viral infections caused by influenza virus.

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