COMBINATION OF DILTIAZEM AND BALOXAVIR MARBOXIL FOR THE PREVENTION AND / OR TREATMENT OF VIRAL INFECTION Caused by the Influenza Virus

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a novel combination of diltiazem and baloxavir marboxil, for use in the prevention and / or treatment of a viral infection by the influenza virus. The present invention also relates to a combination product comprising diltiazem and baloxavir marboxil, for simultaneous, separate or sequential use in the prevention and / or treatment of a viral infection by an influenza virus. STATE OF THE ART

[0002] Human acute respiratory infections (ARI) represent one of the main causes of consultations, hospitalizations and deaths worldwide, being in particular the leading cause of mortality in young children with nearly 2 million deaths per year.

[0003] Viruses are the most common etiological agents responsible for acute respiratory infections. They are found in the majority of cases of childhood pneumonia and are a predisposing factor for bacterial pneumonia in adults.

[0004] Among the most representative viruses in terms of frequency (seasonal epidemics) and morbidity, influenza A and B viruses are prevalent and also constitute a recurring pandemic risk factor. Influenza viruses are also etiological agents contributing to increased co-morbidity and co-mortality in the case of respiratory co-infections and also contribute to the emergence of antibiotic-resistant strains of bacteria, which fundamentally call into question the effectiveness of antibiotic treatments in both humans and animals.

[0005] In terms of prophylactic or therapeutic treatments against viruses, particularly influenza viruses, the current arsenal is rather limited.

[0006] To date, vaccination is the primary strategy for influenza prevention. However, the use of vaccines has drawbacks. For example, when a new seasonal viral strain emerges, a delay of 6 to 9 months is required for the development and delivery of a new vaccine. In addition, vaccine inadequacies are observed when circulating strains mutate. Finally, it is difficult or impossible to produce vaccines against certain pandemic strains of avian origin, such as H5N1 or H7N9, using the conventional vaccine production process based on embryonated chicken eggs.

[0007] The review (Pizzorno et al.,2019b) presents the main therapeutic compounds used in the therapeutic treatment of influenza, as well as their modes of action.

[0008] When therapeutic action is required, the main antiviral compounds currently available are: Oseltamivir / Tamiflu ®< (ROCHE); Zanamivir / Relenza ®< (GSK); and Baloxavir Marboxil / Xofluza ®< (ROCHE) which is available in a few countries including the United States and Japan.

[0009] Zanamivir and Oseltamivir are neuraminidase inhibitors.

[0010] Baloxavir marboxil is an inhibitor of the polymerase complex of influenza viruses; it has also recently been characterized as an inhibitor of polymerases of other negative-sense RNA genomic viruses (Wang et al., 2020).

[0011] Although relatively effective, these antiviral treatments also have significant drawbacks: firstly, the increasing emergence of viruses resistant to said antiviral compounds, and secondly, the lack of broad-spectrum antiviral activity of these compounds.

[0012] Indeed, these antiviral compounds target viral determinants and not the host cell. Due to the high frequency of mutations in influenza viruses (inherent in their polymerase, which is not very faithful in terms of replication, their segmented genome and a source of genetic reassortment between different viruses), conventional antivirals induce recurrent viral resistance.

[0013] In the case of Baloxavir marboxil, several recent studies indicate that the risk of developing resistance is very high. In a phase 3 clinical study, the incidence of resistant strains was nearly 10%, mainly in A(H3N2) viruses. More worryingly, the resistance rate was nearly 20% in a pediatric study (Omoto et al., 2018). In addition, a recent complementary study on Baloxavir marboxil resistance mutants showed that one of the main resistance mutations (PA I38T) leads to a delay in the onset of symptom reduction and a prolongation of viral shedding (Uehara et al., 2019).

[0014] Finally, some infected patients not treated with Baloxavir marboxil excreted a resistant strain, which demonstrates good transmissibility between individuals of viruses resistant to Baloxavir and raises fears of possible large-scale dissemination of these mutant viruses (Takashita et al., 2019). However, the clinical impact of resistance is particularly problematic in hospitalized subjects and in immunocompromised individuals, among others.

[0015] New antiviral compounds are therefore being actively sought, with the aim of limiting the emergence of this resistance. In particular, combination therapies based on the association of at least two antiviral compounds appear particularly promising.

[0016] In particular, when two combined antiviral compounds exhibit synergy of action, this makes it possible to reduce the doses of active compounds used and thus limit the risks of the appearance of resistance while ensuring a significant antiviral therapeutic effect.

[0017] Among the new antiviral compounds identified in recent years, we can mention Diltiazem, which is an active compound that acts on the host cell rather than the virus, thus making it possible to treat various viral infections.

[0018] International application WO 87 / 07508 describes the use of Diltiazem for the treatment of viral infections linked to cytomegalovirus or herpes.

[0019] International 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.

[0020] International application WO 2016 / 146836 and the article (Pizzorno et al.,2019a) describe the use of diltiazem to treat influenza virus infections, possibly in combination with other antiviral agents. A combination of diltiazem and oseltamivir is proposed to treat influenza viral infections.

[0021] International application WO 2019 / 224489 details some of the biological effects of Diltiazem, which induces the expression of genes encoding type III interferons in respiratory epithelial cells. Diltiazem can therefore be used in various therapeutic applications, including the treatment of viral and bacterial respiratory infections in the respiratory and intestinal epithelia.

[0022] Thus, Diltiazem represents a very promising new therapeutic option for the treatment of viral infections.

[0023] However, the many benefits of using this compound therapeutically have yet to be identified. In particular, its use in combination with other antiviral agents may offer surprising, unforeseen benefits. STATEMENT OF THE INVENTION

[0024] The present invention relates to a combination of Diltiazem and Baloxavir marboxil for use in the prevention and / or treatment of a viral infection by an influenza virus.

[0025] Finally, the invention relates to a combination product comprising Diltiazem and Baloxavir marboxil for its simultaneous, separate or sequential use in the prevention and / or treatment of a viral infection by an influenza virus. DESCRIPTION OF FIGURES

[0026] In the figures and their legends, for the purpose of simplification, the following names and abbreviations are used: Dil or DIL refers to Diltiazem; FAV refers to favipiravir; BLX, Bal or Baloxavir refers to the active form of Baloxavir (Baloxavir acid). There Figure 1 represents the antiviral effects of the combination Diltiazem + Baloxavir on cultured cells infected with the H1N1nLuc virus. Figure 1a : timeline of the experiment. Figure 1b : Dose-effect curves of the molecules Diltiazem and Baloxavir in monotherapy on the H1N1nLuc virus (determination of EC50). Figure 1c : Graphical representation of the synergistic effects, according to the concentrations used, of Diltiazem (DIL) and Baloxavir (BLX), in combination against a recombinant H1N1nLuc virus. Figure 2 represents the antiviral effects of the combination Diltiazem + Favipiravir on cultured cells infected with the H1N1nLuc virus. Figure 2a : timeline of the experiment Figure 2b: Dose-effect curves of Diltiazem and Favipiravir in monotherapy on the H1N1nLuc virus (determination of EC50). Figure 2c : Graphical representation of the synergistic or antagonistic effects, depending on the concentrations used, of Diltiazem (DIL) and Favipiravir (FAV) in combination against a recombinant H1N1nLuc virus. Figure 3 represents the effects of treatments with Diltiazem and Baloxavir, as well as the combination of the two compounds, on the replication of the A / H1N1 virus and then the A / H3N2 virus in a human respiratory epithelium model. Figure 3a : timeline of the experiment. Figure 3b : Viral infection by A / H1N1. Comparison of viral titration over time post-infection (pi) on untreated epithelia / treated with DMSO solvent / treated with Diltiazem (90µM) / treated with Baloxavir (10 nM) / treated with the combination Diltiazem (90µM) and Baloxavir (10 nM) Figure 3c: Viral infection by A / H1N1. Monitoring of transepithelial resistance (TEER) measurements during the post-infection time (pi), on the epithelia as described above in the legend of Fig. 3b . Figure 3d : Viral infection by A / H3N2. Comparison of viral titration over time post-infection (pi) on untreated epithelia / treated with DMSO solvent / treated with Diltiazem (90 µM) / treated with Baloxavir (10 nM) / treated with the combination of Diltiazem (90 µM) and Baloxavir (10 nM) Figure 3e : Viral infection by A / H3N2. Monitoring of transepithelial resistance (TEER) measurements during the post-infection time (pi), on the epithelia as described above in the legend of Fig. 3d . There Figure 4 represents the effects of Diltiazem and Baloxavir monotherapy treatments on the replication of Baloxavir-resistant A / H1N1 I38T virus ( Figures 4d, 4e ) compared to their respective effects on the A / H1N1 WT virus ( Figures 4b, 4c ) in a human respiratory epithelium model. Figure 4a : Timeline of the experiment. Figure 4b : Viral infection by A / H1N1. Comparison over the post-infection (pi) time course of viral titration on untreated epithelia / treated with DMSO solvent / treated with Diltiazem (90µM) / treated with Baloxavir (10 nM). Figure 4c : Viral infection by A / H1N1. Monitoring of transepithelial resistance (TEER) measurements during the post-infection time, on the epithelia as described above in the legend of Fig. 4b . Figure 4d : Viral infection with A / H1N1 I38T (Baloxavir-resistant strain). Comparison of viral titration over time post-infection (pi) on untreated epithelia / treated with DMSO solvent / treated with Diltiazem (90µM) / treated with Baloxavir (10 nM) / treated with Baloxavir (100 nM). Figure 4e: Viral infection with A / H1N1 I38T (Baloxavir-resistant strain). Monitoring of transepithelial resistance (TEER) measurements during the post-infection time, on the epithelia as described above in the legend of Fig. 4d . There Figure 5 represents the effects of Diltiazem and Baloxavir treatments, as well as the combination of the two compounds, on the replication of the Baloxavir-resistant viral strain A / H1N1 I38T, in a human respiratory epithelium model. Figure 5a : Timeline of the experiment. Figure 5b : Viral infection with / H1N1 I38T (Baloxavir-resistant strain). Comparison over the post-infection time (pi) of viral titration on untreated epithelia / treated with DMSO solvent / treated with Diltiazem (90µM) / treated with Baloxavir (10 nM) / treated with the combination of Diltiazem (90µM) and Baloxavir (10 nM). Figure 5c: Viral infection by / H1N1 I38T. Monitoring of transepithelial resistance (TEER) measurements during the post-infection time, on the epithelia as described above in the legend of Fig. 5b . Figure 5d : Viral infection with A / H1N1 I38T (Baloxavir-resistant strain). Comparison of viral titration over time post-infection (pi) on untreated epithelia / treated with Diltiazem (90 µM) / treated with Baloxavir (100 nM) / treated with the combination of Diltiazem (90 µM) and Baloxavir (100 nM). Figure 5e : Viral infection by A / H1N1 I38T. Monitoring of transepithelial resistance (TEER) measurements during the post-infection time, on the epithelia as described above in the legend of Fig. 5d . There Figure 6 illustrates the experiments carried out to observe the appearance of viral strains resistant to Baloxavir in a classic test of successive cell passages of viruses under antiviral selection pressure. Figure 6a: Timeline of the experiment. Figure 6b : The graph represents the concentrations used of Baloxavir and Diltiazem in the different experimental arms (untreated, Baloxavir treatment, Diltiazem treatment and Diltiazem + Baloxavir combination treatment) for each cell passage, according to the experimental protocol described previously in Fig 6a .

[0027] The table shows the limiting dilutions of infectious supernatants from each cell passage (for which significant cytopathic effects were observed) and which were used to infect MDCK cells from each subsequent cell passage in each of the four experimental arms (untreated, Baloxavir treatment, Diltiazem treatment and Diltiazem + Baloxavir combination treatment) described previously in Fig 6a .

[0028] The following five figures illustrate the determination of the median inhibitory concentrations (IC50) of Baloxavir on A / H1N1 viruses from the last cell passage of each of the experimental arms (untreated, Baloxavir treatment, Diltiazem treatment and Diltiazem + Baloxavir combination treatment) described previously in Fig 6a .

[0029] Figure 6c : Determination of the median inhibitory concentration of Baloxavir on the A / H1N1 virus from the initial cell passage P0, in MDCK cells treated with different increasing concentrations of Baloxavir. The IC50 initially determined on the A / H1N1 virus used for the experiment is confirmed at 0.2 nM.

[0030] Figure 6d: Determination of the median inhibitory concentration of Baloxavir on the virus from cell passage P10 of the untreated experimental arm, in MDCK cells treated with different increasing concentrations of Baloxavir. The calculated IC50 of Baloxavir on the A / H1N1 virus from the 10th cell passage without treatment (untreated), is 0.9 nM. This slight increase can be attributed to viral replication of the A / H1N1 virus that has adapted to successive cellular amplification on MDCK cells in this experimental arm.

[0031] Figure 6e: Determination of the median inhibitory concentration of Baloxavir on the virus from cell passage P10 of the experimental arm of Diltiazem treatment, in MDCK cells treated with different increasing concentrations of Baloxavir. The calculated IC50 of Baloxavir on the A / H1N1 virus from the 10th cell passage with Diltiazem treatment is 0.3 nM. This IC50 is substantially similar to that initially determined on the A / H1N1 virus used for the experiment.

[0032] Figure 6f: Determination of the median inhibitory concentration of Baloxavir on the virus from cell passage P8 of the experimental arm of Baloxavir treatment, in MDCK cells treated with different increasing concentrations of Baloxavir. The calculated IC50 of Baloxavir on the A / H1N1 virus from the 8th cell passage with Baloxavir treatment (in increasing concentration), is 7 nM. This IC50 is much higher than that initially determined on the A / H1N1 virus used for the experiment (IC50 = 0.2 nM). This increase, corresponding to 35 times the initial IC50, confirms the emergence of a virus with a Baloxavir resistance phenotype.

[0033] Figure 6g: Determination of the median inhibitory concentration of Baloxavir on the virus from cell passage P6 of the experimental arm of treatment by combination Diltiazem + Baloxavir, in MDCK cells treated with different increasing concentrations of Baloxavir. The calculated IC50 of Baloxavir on the A / H1N1 virus from the 6th cell passage with treatment by combination Diltiazem + Baloxavir (in increasing concentration), is 0.8 nM. This IC50 is very similar to that calculated for Baloxavir on the A / H1N1 virus from the 10th cell passage in the experimental arm without treatment (0.9 nM, cf. Fig 6d ) and slightly higher than that initially calculated on the A / H1N1 virus used for the experiment and at the end of the P0 cell passage (IC50=0.2 nM, cf. Fig 6c ). DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention relates to a combination of Diltiazem and Baloxavir marboxil. The scope of the invention is defined by the claims.

[0035] For the purposes of the invention, the term "combination" means a composition comprising at least two distinct active compounds, both compounds having an antiviral action.

[0036] This combination includes either the same amount by weight of each antiviral compound, i.e., a combination of 50% Diltiazem and 50% Baloxavir marboxil by weight, or unequal doses of each compound, such as 90% Diltiazem and 10% Baloxavir marboxil, 80% Diltiazem and 20% Baloxavir marboxil, 70% Diltiazem and 30% Baloxavir marboxil, 60% Diltiazem and 40% Baloxavir marboxil, 40% Diltiazem and 60% Baloxavir marboxil, 30% Diltiazem and 70% Baloxavir marboxil, 20% Diltiazem and 80% Baloxavir marboxil, or 10% of Diltiazem and 90% Baloxavir marboxil, the percentages being expressed as the weight of the compound relative to the total weight of the combination.

[0037] According to the invention, the combination comprises Diltiazem and Baloxavir marboxil. More particularly, the combination consists of Diltiazem and Baloxavir marboxil. In a variant of the invention, the combination comprises Diltiazem and Baloxavir, in its active form (Baloxavir acid).

[0038] Also described herein is a combination comprising Diltiazem and Pimodivir.

[0039] Also described herein is a combination comprising Diltiazem and RO-7.

[0040] Also described herein is a combination comprising Diltiazem and CC-42344.

[0041] According to other implementations, the combination includes: Diltiazem, Baloxavir marboxil and CC-42344; Diltiazem, Baloxavir marboxil and Pimodivir; Diltiazem, Baloxavir marboxil and RO-7.

[0042] Thus, the combination according to the invention comprises or consists of at least two active compounds with antiviral activity, which are described in detail below.

[0043] This combination has synergistic antiviral effects, one of its advantages is that the doses used for each antiviral compound can be reduced compared to the doses traditionally used in monotherapy. This makes it possible, on the one hand, to limit the occurrence of negative side effects on the treated organisms, and on the other hand to limit the occurrence of resistance in viruses to said antiviral compounds.

[0044] This patent application also describes a combination of Diltiazem and at least one viral polymerase(s) inhibitor compound as described below. Diltiazem

[0045] Diltiazem is a molecule belonging to the benzothiazepine family, referenced under CAS number 42399-41-7. This molecule can be in the form of two enantiomers L-cis and D-cis, or a racemic mixture.

[0046] The structural chemical formula of Diltiazem hydrochloride is shown below, as formula (I):

[0047] 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. Its most common trade names are Cardizem ®< , Cartia ®< , Taztia ®< and Dilacor ®<.

[0048] Many formulations are available, especially extended-release formulations. Diltiazem is available in various dosage forms, such as cream for topical application, tablets or capsules for oral administration, powder for preparation of injectable solution, or pharmaceutical preparations for inhalation (WO 02 / 094238, US 4,605,552).

[0049] The typical dosage in humans is 180 to 360 mg / day, administered in capsules or tablets, for its therapeutic use as a calcium channel blocker.

[0050] The first physiological property identified in this compound is the inhibition of calcium channels, and therefore the inhibition of intracellular calcium fluxes. Diltiazem notably slows the entry of transmembrane calcium into the myocardial muscle fiber and the smooth muscle fiber of the vessels. This reduces the intracellular calcium concentration reaching the contractile proteins.

[0051] In humans, Diltiazem is indicated for its vasodilatory action, with the aim of reducing cardiac workload. It is thus used in the management of cardiac and circulatory disorders such as angina pectoris, high blood pressure, myocardial ischemia and tachycardia.

[0052] Diltiazem also works by reversing the effects of angiotensin II, both renally and peripherally.

[0053] Topically applied, Diltiazem may be indicated in cases of chronic anal fissures.

[0054] Patent EP 1 117 408 describes the use of Diltiazem, as a calcium channel inhibitor compound, to treat pathologies linked to the degeneration of retinal photoreceptors.

[0055] Regarding the use of Diltiazem for the treatment of viral infections, as previously mentioned, this has already been described in several patent applications. In addition, a clinical trial is currently underway (FLUNEXT PHRC #15-0442 ClinicalTrials.gov Identifier: NCT03212716), with the aim of obtaining marketing authorization for this new antiviral therapeutic indication. Viral polymerase complex inhibitors

[0056] The polymerase complex of influenza viruses is composed of three subunits: PB1 (Basic Protein 1), PB2 (Basic Protein 2), and PA (Acid Protein). This complex enables transcription and replication of the viral genome.

[0057] Antiviral treatments currently under development and / or recently approved for marketing target the different subunits of this polymerase complex and act according to different mechanisms of action: Baloxavir marboxil is a PA subunit-mediated endonuclease inhibitor, Pimodivir is a PB2 subunit inhibitor, and Favipiravir is a nucleoside analogue, an inhibitor of RNA elongation by the polymerase complex.

[0058] The first compound inhibiting this polymerase complex to be approved by regulatory authorities, and thus marketed, was Baloxavir marboxil. Today, other PB2 (Pimodivir) and PB1 (Favipiravir) inhibitors are currently being tested in phase 3 clinical trials or have limited approval, respectively. Baloxavir marboxil

[0059] Baloxavir marboxil (S-033188), also referred to as Baloxavir in the present application (in particular in the figures), is a molecule that inhibits the cap-dependent endonuclease activity of the PA subunit of the polymerase complex of influenza viruses. This molecule is a prodrug that, after administration within a living organism, is metabolized to its active form “Baloxavir acid” which inhibits the initiation of viral mRNA synthesis by forming stable bonds with two manganese ions in the active site of the PA subunit of the viral polymerase complex. Metabolization of the prodrug to the active metabolite is rapid but the hepatic elimination of Baloxavir acid is long (half-life of 50 to 90 hours) and allows for single-dose oral administration (Hayden & Shindo, 2019).In this application, the term “Baloxavir” means the active form “baloxavir acid” with CAS number 1985605-59-1, obtained after metabolization of the pro-drug Baloxavir marboxil with CAS number 1985606-14-1.

[0060] This active form is used for all experiments in vitro. The first studies in vitroshowed very good efficacy in inhibiting influenza A (mean EC50 of 1.4 to 3.1 nM) and B (mean EC50 of 4.5 to 8.9 nM) viruses. Baloxavir was also effective against viruses resistant to adamantanes and neuraminidase inhibitors (H274Y) and against different subtypes of influenza A viruses from the avian reservoir. The combination of Baloxavir and a neuraminidase inhibitor (Oseltamivir) showed a synergistic effect to protect mice from lethal infection by influenza A(H1N1) virus. This combination also allows a significant decrease in viral titer in the lungs, 24 hours after the start of treatment (Fukao et al., 2019).

[0061] Baloxavir marboxil has the potential to revolutionize the treatment of influenza infections given its longer half-life allowing a single-dose treatment regimen (as opposed to 5 days of treatment for Oseltamivir) and increased antiviral activity compared to Neuraminidase inhibitors.

[0062] In a recent phase 3 clinical study, the median time to cessation of viral shedding in non-hospitalized adults with influenza infection was 48 h for the baloxavir marboxil group, compared to 72 h for the oseltamivir group, and 96 h for the placebo group, although the difference in symptom duration was not significant between the 2 treatment types (Hayden et al., 2018).

[0063] Baloxavir marboxil is very well tolerated with mainly digestive side effects (nausea, diarrhea, etc.) in 4 to 5% of patients to whom this compound was administered.

[0064] On the other hand, one of the major drawbacks of this molecule is the rapid appearance of resistance mutations within the PA subunit of the polymerase complex of influenza viruses. From the first tests in vitro variant viruses have been isolated with a major substitution on amino acid I38 (I38T / M / or phenylalanine F) of PA resulting in a 10- to 100-fold decrease in virus sensitivity to Baloxavir (Noshi et al., 2018). In the clinic, although very rare to date, these substitutions can be found in the absence of antiviral treatment (Takashita et al., 2019). In clinical trials, I38T / M substitutions in PA were found in 2–24% of patients treated with Baloxavir marboxil. In addition, the duration of clinical signs was less reduced in patients treated with Baloxavir marboxil and with mutated virus.

[0065] Recently, antiviral effects of Baloxavir marboxil have been observed against other types of viruses with segmented negative-sense RNA genomes, such as Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) and Heartland Virus (HRTV), pathogens originating from ticks. (Wang et al., 2020) Pimodivir

[0066] Pimodivir (JNJ63623872 or JNJ-872 or VX-787) is a compound that inhibits the PB2 subunit of the polymerase complex of influenza A viruses by binding to the cap-binding site, preventing binding of the natural ligand 7-methyl GTP and viral mRNA synthesis (Clark et al., 2014).

[0067] Pimodivir is well tolerated with mainly digestive side effects (nausea, diarrhea, etc.) depending on the dose.

[0068] Two phase III clinical trials were conducted during the 2017-2018 season.

[0069] As with any anti-infective treatment, resistance mutations in PB2 have been identified in vitro and in vivo. The M431I substitution in PB2 is thought to be responsible for a decrease in sensitivity of approximately 60 times and was found in 10% of patients treated with Pimodivir monotherapy (Trevejo et al., 2018). RO-7

[0070] RO-7 is an inhibitor of the endonuclease activity of the PA acidic protein of the polymerase complex of influenza viruses, discovered in 2016. This compound has good in vitro activity (EC50 of 1.1 to 21.6 nM) on a wide selection of influenza viruses circulating in humans or present in the avian reservoir. The use of RO-7 protected mice from lethal infection by influenza viruses while significantly reducing the viral load in their lungs. As RO-7 is structurally very similar to Baloxavir marboxil, substitutions at amino acid I38 of PA also lead to a decrease in the susceptibility of influenza viruses to this molecule. CC-42344

[0071] CC-42344 is one of the most recently identified compounds. It works by inhibiting the activity of the PB2 subunit of the polymerase complex of influenza viruses. Preclinical data have not yet been published, but this molecule is believed to exhibit good antiviral activity and could be administered orally, intravenously, or by inhalation. Ribavirin

[0072] Ribavirin (Virazole) is a broad-spectrum antiviral molecule that is active against many DNA and RNA viruses. It is a guanosine analog that, in its monophosphate form, inhibits IMPDH (inosine 5'-monophosphate dehydrogenase) and decreases available GTP levels. In its triphosphate form, it is incorporated into transcribed viral RNA, thus blocking RNA elongation and having a mutagenic effect. It is therefore considered an inhibitor of viral genome replication.

[0073] In vitro studies have shown ribavirin to be effective against all types of influenza viruses. However, clinical studies have concluded that oral ribavirin is not effective against influenza viruses. Furthermore, the many side effects observed have limited its use. However, several clinical trials based on a combination of ribavirin and other antiviral compounds are underway in humans. Favipiravir

[0074] Favipiravir or T-705 is a compound used as an antiviral against RNA viruses, including orthomyxoviruses, including various influenza viruses, West Nile virus, yellow fever virus, foot-and-mouth disease virus, as well as other flaviviruses, arenaviruses, bunyaviruses, and alphaviruses.

[0075] This molecule would act by selective inhibition of the polymerase of these viruses (Yousuke et al., 2009).

[0076] Regarding its effect on influenza viruses, Favipiravir has shown efficacy in MDCK cell culture against influenza A, B, and C viruses, including viruses resistant to neuraminidase inhibitors, as well as efficacy in a mouse model infected with the A / PuertoRico / 8 / 34 strain. Favipiravir is believed to act as a nucleoside analog with low cytotoxicity and capable of specifically inhibiting the polymerase of influenza viruses.

[0077] In vivo, Favipiravir was effective in protecting mice infected with highly pathogenic A / H5N1 virus, susceptible or resistant to Oseltamivir.

[0078] The combination of Oseltamivir and Favipiravir was shown to be synergistic at certain concentrations in mice infected with influenza A viruses.

[0079] Favipiravir resistance mutations have been identified in the polymerase of influenza viruses (Goldhill et al., 2018).

[0080] Therapeutic use of this compound in humans has been authorized by a marketing authorization in Japan since 2014. Therapeutic use of said combination

[0081] The present description describes a combination of Diltiazem and at least one viral polymerase(s) inhibitor compound selected from Baloxavir marboxil, Pimodivir, RO-7 and CC-42344, for use as a medicament.

[0082] The present description discloses in particular a combination of Diltiazem and at least one viral polymerase inhibitor compound chosen from Baloxavir marboxil, Pimodivir, RO-7 and CC-42344, for its therapeutic use in the prevention and / or treatment of a viral infection, in particular of the respiratory and / or intestinal tracts of a human or animal organism.

[0083] The term "prevention" means preventing, or at least reducing the likelihood of, the occurrence of a viral infection in a human or animal organism.

[0084] The term "treatment" refers to the act of fighting a viral infection in a human or animal body. This means administering treatment with the aim of reducing the viral load within the body. The term "treatment" also refers to alleviating the symptoms associated with the viral infection (fever, fatigue).

[0085] The said treatment is applicable to both humans and animals, and in particular to farm animals such as pigs, horses and poultry.

[0086] The present description discloses a combination of Diltiazem and at least one viral polymerase inhibitor compound selected from Baloxavir marboxil, Pimodivir, RO-7 and CC-42344, for its therapeutic use in the prevention of a viral infection.

[0087] The present description also discloses a combination of Diltiazem and at least one viral polymerase inhibitor compound selected from Baloxavir marboxil, Pimodivir, RO-7 and CC-42344, for its therapeutic use in the treatment of a viral infection.

[0088] The present description also discloses a combination of Diltiazem and at least one viral polymerase inhibitor compound selected from Baloxavir marboxil, Pimodivir, RO-7 and CC-42344, for its therapeutic use in the prevention and treatment of a viral infection.

[0089] The said combination comprises in particular Diltiazem and Baloxavir marboxil.

[0090] The present description also discloses a method for treating a patient suffering from a viral infection, in particular of the respiratory and / or intestinal tracts, comprising administering to said patient a combination of Diltiazem and at least one viral polymerase(s) inhibitor compound selected from the group consisting of: Baloxavir marboxil, Pimodivir, RO-7 and CC-42344.

[0091] The present description also discloses a method for preventing the occurrence of a viral infection, in particular of the respiratory and / or intestinal tracts, in an individual susceptible to being infected by a virus, comprising the administration to said individual of a combination of Diltiazem and at least one viral polymerase(s) inhibitor compound chosen from the group consisting of: Baloxavir marboxil, Pimodivir, RO-7 and CC-42344.

[0092] The present description also discloses a veterinary method for preventing and / or treating a viral infection, in particular of the respiratory and / or intestinal tracts, in an infected or susceptible animal, comprising administering to said animal a combination of Diltiazem and at least one viral polymerase(s) inhibitor compound selected from the group consisting of: Baloxavir marboxil, Pimodivir, RO-7 and CC-42344. Viral infections and associated viruses

[0093] For the purposes of the invention, the term "viral infection" means a disease caused by a virus that has entered the body, infecting certain cells of said body, designated by the terms "target cells" or "host cells". A viral infection is generally diagnosed by a healthcare professional, based on the observation of the symptoms of the infected patient or animal. Additional biological tests may be necessary to confirm the diagnosis (blood tests and / or sputum tests and / or bronchoalveolar fluid tests and / or biological samples from the intestinal tract).

[0094] The combination described here is intended to prevent and / or treat a viral infection, in particular of the respiratory and / or intestinal tracts of a human or animal organism, in other words an infection affecting the cells of the respiratory epithelia.

[0095] Respiratory tract infections are viral infections that affect the lungs and airways, the airways through which people breathe. These include the common cold, the flu, and bronchiolitis.

[0096] Viruses involved in viral respiratory infections include, in particular, respiratory syncytial virus (RSV), influenza viruses (the flu), parainfluenza viruses, adenoviruses, and rhinoviruses.

[0097] In children, the main causes of respiratory viral infections are rhinoviruses, influenza viruses, parainfluenza viruses, respiratory syncytial virus (RSV), enteroviruses, coronaviruses, and certain strains of adenovirus.

[0098] In the context of this description, this will particularly concern an acute respiratory infection (ARI).

[0099] By "intestinal tract infection" we mean viral infections affecting the digestive system, which includes, from top to bottom: the mouth, the pharynx (at the intersection of the digestive and respiratory tracts), the esophagus, the stomach, and the intestines.

[0100] The most common causes of gastroenteritis are related to viral infections such as norovirus or rotavirus.

[0101] The therapeutic combination according to the invention is intended for the treatment and / or prevention of an infection by an influenza virus.

[0102] Influenza viruses, which cause the flu, are divided into four types: A, B, C, and D. On the surface of the virus are two glycoproteins that play an important role in infecting the cells of the infected organism: hemagglutinin (HA) and neuraminidase (NA). There are different subtypes of influenza A viruses depending on the nature of the HA and NA glycoproteins on their surface: 16 types of HA and 9 types of NA have been identified in viruses circulating in the animal world, particularly among migratory seabirds. Influenza viruses can thus be defined according to the type of glycoproteins they have on their surface.

[0103] In humans, the subtype A viruses circulating for several decades are the H1N1, H2N2 and H3N2 subtypes, with occasional interspecies transmissions, notably from animals to humans, of avian viruses H5N1, H7N7, H7N9, H5N2 and H9N2. As highlighted by the recent emergence of a new pandemic H1N1 influenza virus of swine, avian and human origin (virus with swine, avian and human reassortment), influenza A viruses represent a serious public health threat. Influenza pandemics are the result in particular of antigenic breaks that correspond to the appearance of viruses with new surface glycoproteins (HA and NA) in the human population. These breaks allow the direct transmission of animal viruses, particularly avian viruses, to humans: this is the case of the highly pathogenic avian H5N1 epidemics since 2003 in Asia, or the H7N7 flu epidemics in the Netherlands in 2003 and H7N9 in South-East Asia in 2013.Furthermore, seasonal influenza epidemics, which are notably the result of genetic drift (appearance of mutations in surface glycoproteins), are a major cause of increased morbidity and mortality in the human population, especially in the very young, the elderly, immunocompromised individuals and those with cardiopulmonary diseases.

[0104] For the purposes of the invention, the term "influenza virus" means the etiological agents of influenza type A, B, C or D, and in particular influenza viruses type A and type B, having humans or animals as hosts. The terms "influenza virus" and "influenza virus" are used interchangeably in the application and designate the same viruses.

[0105] The invention relates to a combination of Diltiazem and Baloxavir marboxil for its therapeutic use in the prevention and / or treatment of an infection by an influenza virus. In a variant of the invention, it relates to a combination of Diltiazem and Baloxavir marboxil for its therapeutic use in the prevention and / or treatment of an infection by an influenza virus.

[0106] According to one aspect of the invention, the combinations of the present invention are used for the prevention and / or treatment of infections by at least one type A influenza virus.

[0107] Advantageously, the combinations of the present invention have a broad spectrum of action against the different subtypes of influenza A viruses.

[0108] In a particular aspect, the influenza virus is a type A virus selected from the subtypes H1N1, H2N2, H3N2, H5N1, H7N7, H7N9, H5N2 and H9N2.

[0109] According to another aspect of the invention, the combinations of the present invention are used for the prevention and / or treatment of infections by at least one type B influenza virus. Virus resistance

[0110] The invention relates in particular to a combination of Diltiazem and Baloxavir marboxil, for its therapeutic use in the prevention and / or treatment of an infection by an influenza virus, said influenza virus being resistant to the inhibitory action of at least one antiviral compound, in particular an anti-influenza compound, and more particularly of an inhibitor of viral polymerase(s).

[0111] Indeed, the combination according to the invention is particularly interesting in the case where the virus causing the viral infection is not or only slightly sensitive to the action of a compound inhibiting viral polymerase(s) conventionally used, and in particular chosen from Baloxavir marboxil, Pimodivir, RO-7 and CC-42344.

[0112] As presented above, the major drawback of the therapeutic use of this family of compounds that target viral polymerases is the development of viral strains resistant to their action, becoming insensitive to the inhibitory action of these compounds.

[0113] The benefits of using Diltiazem and Baloxavir marboxil together are: 1) the emergence of viral strains resistant to at least one administered viral polymerase inhibitor compound is significantly limited, or even completely inhibited, when administered in combination with Diltiazem (see example 6); 2) Diltiazem alone has effective antiviral activity against viral strains resistant to a viral polymerase inhibitor (see example 4, based on a viral strain resistant to Baloxavir); 3) the combination Diltiazem + Baloxavir marboxil has effective antiviral activity against viral strains resistant to Baloxavir, due to the presence of Diltiazem and a high concentration of Baloxavir (see example 5).

[0114] A combination according to the invention comprises Diltiazem and Baloxavir marboxil, and is used for the treatment and / or prevention of infection by an influenza virus resistant to the inhibitory action of a viral polymerase(s) inhibitor, for example a virus having a resistance mutation in the PB2 subunit of the polymerase.

[0115] According to another implementation of the invention, the combination comprises Diltiazem and Baloxavir marboxil, and is used for the treatment and / or prevention of an infection by an influenza virus resistant to the inhibitory action of Baloxavir marboxil. In particular, it may be an influenza A / H1N1 viral strain carrying a mutation in the PA subunit of the polymerase. More precisely, it may be an influenza A / H1N1 viral strain carrying an I38T point mutation in the PA subunit of the polymerase.

[0116] According to another implementation of the invention, the influenza virus is resistant to the inhibitory action of at least one anti-influenza compound, in particular a neuraminidase inhibitor compound such as Oseltamivir (Tamiflu ®).

[0117] Also described herein is Diltiazem for its therapeutic use in the prevention and / or treatment of an infection by an influenza virus, said influenza virus being resistant to the inhibitory action of at least one antiviral compound, in particular an anti-influenza compound, and more particularly a viral polymerase(s) inhibitor. In particular, this influenza virus will be resistant to one of the following compounds: Baloxavir marboxil, Pimodivir, RO-7, CC-42344 and a neuramidase inhibitor, in particular Oseltamivir. Other active agent(s) present in the combination

[0118] The present invention also relates to a combination of Diltiazem and Baloxavir marboxil, further comprising another active agent, in particular an antiviral agent and / or an antibiotic, for its therapeutic use in the prevention and / or treatment of a viral infection by an influenza virus.

[0119] According to a preferred aspect, the antiviral agent is chosen from antiviral agents well known to those skilled in the art, conventionally used to prevent or treat influenza. Such antiviral agents active on at least one influenza virus are commercially available, and described in reference works such as Le Dictionnaire Vidal. Oseltamivir may be mentioned in particular. Thus, an object of the present invention is a combination of Diltiazem, Baloxavir marboxil and Oseltamivir, for its therapeutic use in the prevention and / or treatment of a viral infection by an influenza virus.

[0120] The antibiotic is chosen from antibiotics well known to those skilled in the art, in particular those used during viral infections to prevent bacterial superinfection, and in particular those from the macrolide family, and in particular roxythromycin. Pharmaceutical or veterinary composition

[0121] The present invention also relates to a pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, a combination of Diltiazem and Baloxavir marboxil, for its therapeutic use in the prevention and / or treatment of a viral infection by an influenza virus.

[0122] Also part of the invention is a veterinary composition comprising, in a suitable pharmaceutical vehicle, a combination of Diltiazem and Baloxavir marboxil, for its veterinary use in the prevention and / or treatment of a viral infection by an influenza virus.

[0123] This pharmaceutical or veterinary composition comprises an effective amount of Diltiazem and an effective amount of Baloxavir marboxil.

[0124] For the purposes of the invention, the term "effective amount" means an amount of antiviral compound sufficient to inhibit the proliferation and / or replication of the virus, and / or the development of the viral infection within the organism. This inhibition can be quantified, for example by measuring viral production as presented in the examples of the present application.

[0125] For example, in vitro, the so-called “effective” quantities are as follows: for Diltiazem, a concentration of 5 to 200 µM is preferred, and for Baloxavir (active form), a concentration of 5 to 200 nM is preferred.

[0126] According to the invention, the term "pharmaceutical vehicle" designates one or more pharmaceutical vehicles or excipients acceptable according to the invention, that is to say vehicles or excipients whose administration to an individual or an animal is not accompanied by significant deleterious effects, and which are well known to those skilled in the art.

[0127] The pharmaceutical or veterinary compositions according to the present invention are suitable for oral, sublingual, inhalation, subcutaneous, intramuscular, intravenous, transdermal, ocular or rectal administration.

[0128] According to a preferred aspect of the invention, the pharmaceutical composition is characterized in that it is in a galenic form suitable for administration by inhalation.

[0129] Inhalation refers to absorption through the respiratory tract. It is specifically a method of absorbing compounds for therapeutic purposes, including certain substances in the form of gases, micro-droplets, or suspended powders.

[0130] The administration of pharmaceutical or veterinary compositions by inhalation, i.e. via the nasal and / or oral routes, is well known to those skilled in the art.

[0131] There are two types of administration by inhalation: administration by insufflation when the compositions are in the form of powders, and administration by nebulization when the compositions are in the form of aerosols (suspensions) or in the form of solutions, for example aqueous solutions, put under pressure. The use of a nebulizer or a sprayer will then be recommended to administer the pharmaceutical or veterinary composition.

[0132] The galenic form considered here is therefore chosen from: a powder, an aqueous suspension of droplets or a solution under pressure.

[0133] Also described herein is a pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, a combination of Diltiazem and at least one viral polymerase(s) inhibitor compound selected from Baloxavir marboxil, Pimodivir, RO-7 and CC-42344, for use as a medicament.

[0134] Also described herein is a pharmaceutical composition comprising, in a suitable pharmaceutical vehicle, a combination of Diltiazem and at least one viral polymerase(s) inhibitor compound chosen from Baloxavir marboxil, Pimodivir, RO-7 and CC-42344, for its therapeutic use in the prevention and / or treatment of a viral infection, in particular of the respiratory and / or intestinal tracts of a human being or an animal, more particularly of an infection by an influenza virus. Combination product

[0135] The present invention also relates to a combination product comprising Diltiazem and Baloxavir marboxil, for its simultaneous, separate or sequential use in the prevention and / or treatment of a viral infection by an influenza virus.

[0136] This combination product can be used in humans or animals.

[0137] It may include other active compounds, including Oseltamivir. EXAMPLES

[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 the form administered to a patient which is a prodrug. Example 1 - Diltiazem + Baloxavir combination has synergistic antiviral effect against recombinant H1N1nLuc virus in cellular system

[0139] A549 cells infected with A / H1N1nLuc (Multiplicity of infection (MOI) 0.01) were treated one hour post-infection with increasing concentrations of Diltiazem (6 nM to 100 µM), Baloxavir acid (0.0625 nM to 1 nM, marketed by Med Chem Tronica, ref. HY-109025A) alone and in combination.

[0140] The luminescence measured in the infection cell supernatants (collected at 48 hours post-infection) reflects the level of viral replication (experimental chronogram represented in Fig 1a). The EC50 of each of the two molecules was determined (EC50 Diltiazem = 50102 nM; EC50 Baloxavir = 0.144 nM, cf. Fig 1b ).

[0141] The effects of the combination of molecules were characterized by analyzing the measured luminescence data using Combenefit software (representation in the form of a table with a chromatic scale, cf. Fig 1c ).

[0142] The synergistic or antagonistic effect of the combination is represented by the "synergy coefficient" (Synergy Score, analysis according to the Loewe model), with a chromatic scale to facilitate the interpretation of the results. A coefficient ≥ 5 (grey to dark grey) indicates a synergistic effect of the combination of molecules, while a coefficient ≤ -5 (white) indicates an antagonistic effect of the combination of molecules. Coefficients between -5 and 5 are indicative of a "non-interference" of the molecules in the combination, or even an additive effect between the different treatments. Number of experiments = 3.

[0143] As presented in Figure 1c , the combination of Diltiazem and Baloxavir acid shows synergistic effects.

[0144] Two particular conditions are particularly synergistic: (i) conditions combining a high concentration of Diltiazem (1562 nM) with concentrations of Baloxavir acid close to its IC50 (0.1 nM); (ii) conditions combining lower concentrations of Diltiazem (less than 25 nM) with very low doses of Baloxavir acid (less than 0.1 nM).

[0145] Under all tested conditions of combined concentrations, no antagonistic effects were observed.

[0146] The results show that the combination of Diltiazem and the active form of Baloxavir allows a very significant and synergistic reduction of viral replication (particularly under conditions of use of low doses of Baloxavir, which is an advantage in terms of reducing the risk of occurrence of Baloxavir resistance mutations) in A549 cells in comparison with monotherapy treatment with each of the two molecules alone, at the same concentrations. Example 2 (outside the invention) - The combination Diltiazem + Favipiravir is synergistic or antagonistic, depending on the concentrations of each of the two molecules, against a recombinant H1N1nLuc virus in a cellular system

[0147] A549 cells infected with A / H1N1nLuc (MOI 0.01) were treated one hour post-infection with increasing concentrations of Diltiazem (6nM to 100µM) and Favipiravir (6nM to 100µM) alone and in combination.

[0148] The luminescence measured in the infection cell supernatants (collected at 48 hours post-infection) reflects the level of viral replication (experimental chronogram represented in Fig 2a ).

[0149] The EC50 of each molecule was determined (EC50 Diltiazem = 53791 nM; EC50 Favipiravir = 2494 nM, see Fig 2b ).

[0150] The effects of the combination of molecules were characterized by analyzing the measured luminescence data using Combenefit software (table representation with a color scale, see Fig 2c ).

[0151] The synergistic or antagonistic effect of the combination is represented by the "synergy score", with a color scale to facilitate the interpretation of the results. A coefficient ≥ 5 (gray to dark gray) indicates a synergistic effect of the combination of molecules, while a coefficient ≤ -5 (white) indicates an antagonistic effect of the combination of molecules. Coefficients between -5 and 5 are indicative of "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, in general, would not allow a significant reduction in treatment doses.

[0153] Depending on the concentration of each of the molecules Diltiazem and Favipiravir, their combinations are associated with very heterogeneous effects. To obtain a synergistic effect, it is necessary to use high doses of Favipiravir (greater than 1580 nM, which is a disadvantage since it favors an increased risk of developing resistance to Favipiravir) with different doses of Diltiazem (from 6 nM to 100 µM).

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

[0155] The combination Diltiazem + Baloxavir allows to obtain homogeneous results even when changing the concentrations used, unlike the combination Diltiazem + Favipiravir which is therefore not preferred. Example 3. Synergy of the combination Diltiazem + Baloxavir in a human respiratory epithelium model

[0156] The reduction in the replication of A / H1N1 and A / H3N2 viruses is significantly greater with the combination treatment Diltiazem + Baloxavir (active form) compared to monotherapy treatments with Diltiazem or Baloxavir alone.

[0157] Reconstituted human respiratory epithelia (of nasal origin) (MucilAir ®< HAE, Epithelix) cultured at the air-liquid interface (according to the instructions of the supplier Epithelix), were infected with prototype (non-recombinant) influenza viruses of the A / H1N1 pdm09 (A / Lyon / 969 / 2009 H1N1) (MOI 0.1) or A / H3N2 (A / Texas / 50 / 2012 H3N2) (MOI 0.01) type, then treated or not (untreated) with 3 successive doses of 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 (chronogram of experiment represented in Fig 3a ).

[0158] Samples from the apical pole of infected epithelia, treated or not, were taken at 24, 48 and 72 hpi to measure viral replication by infectious titration in TCID50 / mL on MDCK cells.

[0159] Before each sample at the apical pole, a measurement of transepithelial resistance (physiological marker of epithelial integrity) was also carried out using the EVOM2 device and the STX2 probe (World Precision Instruments) ( Fig 3c And 3e ).

[0160] As illustrated in Figure 3b , without treatment (untreated), viral infection induces production of infectious A / H1N1 particles with titers greater than 10 8< TCID50 / mL from 48 hours post-infection.

[0161] Treatment with Diltiazem (90 µM) reduces the production of infectious A / H1N1 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 exhibits significant in vitro antiviral activity with up to a 4 log10 reduction in the production of infectious A / H1N1 particles at 48 hours post-infection, while its solvent alone (DMSO) has no impact on infection.

[0163] The combination of the two molecules Diltiazem and Baloxavir significantly improves the antiviral effect compared to monotherapy treatments, with a reduction in A / H1N1 viral production of almost 6log10 at 48 and 72 hours post-infection.

[0164] There Figure 3c presents the monitoring of trans-epithelial resistance (TEER) measurements during infection of epithelia by A / H1N1, as described in Fig 3a It shows a significant drop in the integrity of untreated epithelia or those treated with the control solvent DMSO at 48H and 72H post-infection, in correlation with a very efficient infection, as presented in Fig 3b .

[0165] In contrast, Diltiazem treatment maintains the integrity of infected epithelia with TEER values remaining stable, in correlation with an antiviral effect demonstrated by infectious titration ( Fig 3b ).

[0166] Baloxavir treatment also maintains the integrity of infected epithelia with TEER values similar to those obtained with Diltiazem treatment.

[0167] Similarly, the integrity of infected epithelia is also maintained with the combination treatment of both molecules, in correlation with its antiviral efficacy ( Fig 3b ).

[0168] These TEER measurements also confirm an absence of cytotoxic effect of treatment with Diltiazem and Baloxavir in combination.

[0169] As illustrated in 3D figure, without treatment (untreated), A / H3N2 viral infection of the epithelia induces production of infectious particles with titers greater than 10 9< TCID50 / mL from 48 hours post-infection.

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

[0171] Baloxavir treatment exhibits significant in vitro antiviral activity with up to a 3log10 reduction in the production of infectious A / H3N2 particles at 48 hours post-infection, while its solvent alone (DMSO) has no impact on infection.

[0172] The combination of the two molecules Diltiazem and Baloxavir significantly improves the antiviral effect compared to monotherapy treatments, with a reduction in A / H3N2 viral production of nearly 5 log10 at 48 and 4 log 10 at 72 hours post-infection.

[0173] Monitoring of transepithelial resistance (TEER) measurements during A / H3N2 infection of epithelia, as described in Fig 3a , presented in Fig 3e , shows a significant drop in the integrity of untreated epithelia or those treated with the control solvent DMSO at 48H and 72H post-infection, in correlation with a very efficient infection, as measured and presented in Fig 3d .

[0174] In contrast, Diltiazem treatment maintains the integrity of epithelia infected with A / H3N2 with TEER values remaining stable up to 48 hours post-infection, in correlation with an antiviral effect demonstrated by infectious titration ( Fig 3d ).

[0175] Baloxavir treatment also maintains the integrity of epithelia infected with A / H3N2 with TEER values similar to those obtained with Diltiazem treatment at 48 hours post-infection.

[0176] Similarly, the integrity of the epithelia is also maintained with the combination treatment of the two molecules, in correlation with its antiviral efficacy ( Fig 3d ).

[0177] These TEER measurements also confirm an absence of cytotoxic effect on epithelia of treatment with Diltiazem and Baloxavir in combination.

[0178] In conclusion, the Figures 3b And 3ddemonstrate a significant antiviral effect of treatments with Diltiazem or Baloxavir compared to the infection condition without treatment. The results also indicate the beneficial / synergistic effect of a Diltiazem + Baloxavir combination treatment compared to monotherapy treatments with each of the two molecules alone at the same concentrations: the combination in fact allows a significantly greater reduction in viral replication compared to monotherapy treatments in the human respiratory epithelium model. Example 4 (outside the invention) - Diltiazem has effective antiviral activity against a recombinant A / H1N1 virus I38T resistant to Baloxavir, in a reconstituted human respiratory epithelium model, comparable to its antiviral activity against the recombinant wild-type (WT) A / H1N1 virus in the same epithelium model.

[0179] The resistant A / H1N1 virus was generated by reverse genetics. It contains the I38T resistance mutation in the PA subunit of its polymerase.

[0180] Reconstituted human respiratory epithelia (MucilAir ®< HAE, Epithelix) of nasal origin, maintained in culture at an air-liquid interface according to the instructions of the supplier Epithelix, were infected with recombinant influenza viruses of type A H1N1 pdm09 wild (WT, MOI 0.1) or resistant to Baloxavir (mutation I38T on the PA subunit of the viral polymerase) (138T, MOI 0.1).

[0181] These epithelia were then treated or not (untreated) with 3 successive doses of Diltiazem (90 µM) alone, Baloxavir alone at a concentration of 10 nM or 100 nM, or with a combination of the two molecules (Diltiazem at 90 µM and Baloxavir at 10 nM or 100 nM), delivered at 5, 24 and 48 hours post-infection (hpi), respectively (experiment chronogram represented in Fig 4a ).

[0182] Samples from the apical pole of infected epithelia, treated or not, were taken at 24, 48 and 72 hpi to measure viral replication by infectious titration in TCID50 / mL on MDCK cells.

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

[0184] Each experimental condition was performed in duplicate (n=2). The graphs present mean and standard deviation values that were generated via GraphPad software.

[0185] There Figure 4b illustrates the fact that without treatment (untreated), viral infection induces production of infectious A / H1N1 WT particles with titers greater than 10 8< TCID50 / mL from 48 hours post-infection.

[0186] Treatment with Diltiazem (90 µM) reduces the production of infectious A / H1N1 particles by up to 2 log10 at 48 hours post-infection and 0.5 log10 at 72 hours post-infection.

[0187] Baloxavir treatment exhibits significant in vitro antiviral activity with up to a 3 log10 reduction in the production of infectious A / H1N1 particles at 48 hours post-infection, whereas its solvent alone (DMSO) has no significant impact on infection.

[0188] Monitoring of transepithelial resistance (TEER) measurements during infection, presented in Fig. 4c , shows a significant drop in the integrity of untreated epithelia or those treated with the control solvent DMSO at 48H and 72H post-infection, in correlation with a very efficient infection, as presented in Fig 4b .

[0189] In contrast, Diltiazem treatment maintains the integrity of epithelia infected by A / H1N1 with TEER values remaining stable up to 48 hours post-infection, in correlation with an antiviral effect demonstrated by infectious titration ( Fig 4b ).

[0190] Baloxavir treatment also maintains the integrity of epithelia infected with A / H1N1 with stable TEER values measured at 48 and 72 hours post-infection.

[0191] There figure 4d illustrates the fact that without treatment (untreated), infection of epithelia with the Baloxavir-resistant A / H1N1 I38T virus induces production of infectious particles with titers close to 10 8< TCID50 / mL at 48 hours post-infection (see Fig 4d ).

[0192] Baloxavir treatment at 10 nM has no antiviral effect on this resistant A / H1N1 I38T virus in the infected epithelium model. It is necessary to significantly increase the Baloxavir treatment dose (100 nM) to obtain antiviral activity, which remains limited against this resistant A / H1N1 I38T virus (1 log10 reduction in infectious particle production at 48 hours post-infection and 1.5 log10 at 72 hours post-infection).

[0193] In contrast, treatment with Diltiazem alone (90 µM) significantly reduced the production of infectious A / H1N1 I38T particles by nearly 2 log10 at 48 hours post-infection and by nearly 2 log10 at 72 hours post-infection.

[0194] Monitoring of transepithelial resistance (TEER) measurements during A / H1N1 I38T infection, illustrated in Fig 4e, shows a significant drop in the integrity of untreated epithelia at 48H and 72H post-infection, in correlation with a very efficient infection, as presented in Fig 4d .

[0195] On the contrary, treatment with Diltiazem allows maintaining the integrity of epithelia infected by the A / H1N1 I38T virus with TEER values that remain stable at 48H and 72H post-infection, in correlation with an antiviral effect demonstrated by infectious titration ( Fig 4d ).

[0196] On the other hand, treatment with 10 nM Baloxavir does not maintain the integrity of epithelia infected with A / H1N1 I38T at 48H and 72H post-infection, consistent with the absence of antiviral activity as reported in the Fig 4d . Only high-dose treatment with Baloxavir (100 nM), for which significant antiviral activity against resistant A / H1N1 I38T virus has been reported in the Fig 4d, allows the maintenance of epithelial integrity, as reported by measurements of stable TEER values at 48H and 72H post-infection. Example 5 - The combination of Diltiazem and Baloxavir induces a significantly greater reduction in the replication of Baloxavir-resistant A / H1N1 I38T virus compared to monotherapy treatments, but only when Baloxavir is used at a high dose.

[0197] Reconstituted human respiratory epithelia (MucilAir ®< HAE, Epithelix) of nasal origin, maintained in culture at an air-liquid interface according to the instructions of the supplier Epithelix, were infected with a recombinant influenza virus type A H1N1 pdm09 resistant to Baloxavir (mutation I38T on the PA subunit of the viral polymerase) (138T, MOI 0.1).

[0198] These epithelia were then treated or not (untreated) with 3 successive doses of Diltiazem (90 µM) alone, Baloxavir alone at a concentration of 10 nM or 100 nM, or with a combination of the two molecules (Diltiazem at 90 µM and Baloxavir at 10 nM or 100 nM), delivered at 5, 24 and 48 hours post-infection (hpi), respectively (experiment chronogram represented in Fig 5a ).

[0199] Samples from the apical pole of infected epithelia, treated or not, were taken at 24, 48 and 72 hpi to measure viral replication by infectious titration in TCID50 / mL on MDCK cells.

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

[0201] There Figure 5b shows that, without treatment (untreated), infection of epithelia with the Baloxavir-resistant A / H1N1 I38T virus induces production of infectious particles with titers close to 10 8< TCID50 / mL at 72H post-infection. Treatment with Diltiazem (90µM) reduces the production of infectious A / H1N1 I38T particles by up to 2log10 at 48H post-infection and by at least 2log10 at 72H post-infection.

[0202] On the other hand, treatment with Baloxavir (10 nM) is ineffective, as is its solvent alone (DMSO), which has no impact on the infection.

[0203] The combination of the two molecules Diltiazem and Baloxavir at their respective concentrations reduces the production of infectious A / H1N1 I38T particles but without any additional or synergistic effect compared to treatment with Diltiazem alone at 48H and 72H post-infection.

[0204] This result is different from that obtained on the A / H1N1 WT virus (not resistant to Baloxavir, cf. Fig 3b ) on which the combination of the two molecules Diltiazem and Baloxavir at the same concentrations has a synergistic antiviral effect compared to monotherapy treatments, in particular by inducing a reduction in viral production of almost 6log10 at 48 and 72 hours post-infection.

[0205] Monitoring of transepithelial resistance (TEER) measurements during infection, presented in Fig 5c, shows a significant drop in the integrity of untreated epithelia or those treated with the control solvent DMSO at 48H and 72H post-infection, in correlation with a very efficient infection, as measured and presented in Fig 5b .

[0206] In contrast, Diltiazem treatment maintains the integrity of epithelia infected with A / H1N1 I38T with TEER values remaining stable at 48H and 72H post-infection, in correlation with an antiviral effect demonstrated by infectious titration ( Fig 5b ).

[0207] Baloxavir treatment (10 nM) fails to maintain the integrity of A / H1N1 I38T-infected epithelia, consistent with its antiviral inefficacy, as reported in Fig 5b .

[0208] Also consistent with its significant antiviral efficacy, the Diltiazem + Baloxavir combination is associated with stable TEER value measurements at 48H and 72H post-infection.

[0209] There Figure 5d illustrates that, without treatment (untreated), infection of the epithelium by the Baloxavir-resistant A / H1N1 I38T virus induces production of infectious particles with titers close to 10 8< TCID50 / mL at 48 hours post-infection.

[0210] Diltiazem treatment (90 µM) reduces the production of infectious A / H1N1 I38T particles to more than 2log10 at 48 hours post-infection.

[0211] Used at a high concentration (100 nM), Baloxavir treatment reduced the production of infectious A / H1N1 I38T particles by approximately 1log10 at 48 hours post-infection, whereas treatment at a concentration of 10 nM had no effect on the resistant I38T virus (cf. Fig 5b ).

[0212] The combination of the two molecules Diltiazem and Baloxavir at the same respective concentrations further reduces the production of infectious A / H1N1 I38T particles at 48H and especially at 72H post-infection (with a reduction of up to 3log10 of infectious A / H1N1 I38T particles) compared to monotherapy treatments. The gain of the combination of Diltiazem with Baloxavir (at high concentration = 100 nM) is therefore significant against the resistant A / H1N1 I38T virus in the human respiratory epithelium model.

[0213] This combination is particularly relevant against viral strains resistant to Baloxavir, against which it is necessary to significantly increase the doses of Baloxavir.

[0214] Monitoring of transepithelial resistance (TEER) measurements during infection, presented in Fig 5e, shows a significant drop in the integrity of untreated epithelia at 48H and 72H post-infection, in correlation with a very efficient infection, as measured and presented in Fig 5d .

[0215] In contrast, treatments with high-dose Diltiazem or Baloxavir (100 nM) maintain the integrity of epithelia infected with A / H1N1 I38T with TEER values remaining stable at 48H and 72H post-infection, in correlation with antiviral effects demonstrated by infectious titration ( Fig 5d ).

[0216] Also consistent with its significant antiviral efficacy (cf. Fig 5d ), the combination Diltiazem + Baloxavir is associated with stable TEER value measurements at 48H and 72H post-infection.

[0217] In conclusion, the results obtained and presented in examples 4 and 5: (i) confirm the Baloxavir resistance phenotype of the recombinant A / H1N1 I38T virus (I38T mutation in its PA subunit of the polymerase), against which the reference treatment with 10 nM Baloxavir has no efficacy ( Fig 4d ). (ii) The Fig 4d also demonstrates that it is necessary to strongly increase the treatment dose of Baloxavir (100 nM) to obtain antiviral activity, which remains however limited against this resistant A / H1N1 I38T virus. (iii) Importantly, the results also indicate that Diltiazem has significantly effective antiviral activity against a Baloxavir-resistant recombinant A / H1N1 I38T virus in a reconstituted human respiratory epithelium model, in comparison with its antiviral activity against the recombinant wild-type (WT) A / H1N1 virus in the same epithelium model (compare Fig 4b And 4d ). (iv) The results presented in Fig 5bconfirm again that the combined treatment of Diltiazem (90 µM) with Baloxavir (10 nM) allows a significant reduction of viral replication in upper airway respiratory epithelia infected with a wild strain of A / H1N1. (v) In contrast, this combination Diltiazem (90 µM) + Baloxavir (10 nM) does not provide any additional gain compared to treatment with Diltiazem monotherapy against the resistant A / H1N1 I38T virus ( Fig 5b ). (vi) It is in combination with Baloxavir at 100 nM that Diltiazem (90 µM) provides a significant gain in terms of antiviral activity against the resistant A / H1N1 I38T virus compared to monotherapy treatments of each of the two molecules at the same concentrations in the same model of human respiratory epithelium ( Fig 5d ). Example 6 - The use of Diltiazem in combination with Baloxavir prevents the emergence of Baloxavir-resistant viruses in a classic test of successive cell passages of viruses under antiviral selection pressure (increasing concentration of Baloxavir)

[0218] MDCK cells in 24-well plates were infected with the prototype WT A / H1N1 virus (for which the median inhibitory concentrations IC50 of Baloxavir and Diltiazem are 0.2 nM and 5 µM, respectively) at an MOI of 0.001, thus constituting the first cellular passage of the virus (P0).

[0219] Four different arms of successive cell passages of this virus were conducted under conditions of selection pressure or without selection pressure, as defined below: (i) no treatment (NT), (ii) treatment with Diltiazem (Dil) at a fixed concentration of 25 µM, (iii) treatment with Baloxavir at increasing concentrations from 1 nM up to 128 nM and (iv) treatment with Diltiazem (Dil) at a fixed concentration of 25 µM in combination with Baloxavir at the same increasing concentrations from 1 nM up to 128 nM as previously in (iii).

[0220] From the P0 passage, the cells were treated at 1H post-infection according to the different conditions described previously or not treated (NT) and the infection supernatants were collected at 48H post-infection (D+2). These infectious samples were diluted in cascades (from 10-1 to 10-6) and each dilution was deposited on MDCK cells in 24-well plates, thus constituting the cell passage (P1) following the P0 passage.

[0221] One hour after this infection, the cells were treated according to the different conditions described previously or not treated (NT). At the end of this cell passage P1 (48H post-infection, D+4), and for each of the treatment arms (i, ii, iii and iv), the supernatant of the infected well was collected with the so-called "limiting" dilution, i.e. the last of the dilutions from 10-1 to 10-6 from P0 where significant cytopathic effects (CPE) were observed.

[0222] These infection supernatants were diluted in cascades (from 10-1 to 10-6) and each dilution was deposited on MDCK cells in 24-well plates, thus constituting the cell passage (P2) following passage P1.

[0223] The procedure was thus followed for each successive cell passage. The concentration of Diltiazem remained fixed throughout the experiment. That of Baloxavir was fixed at 1nM during the first two passages (P0 and P1), then was doubled during the following two passages (P3 and P4), before being doubled at each passage up to 128 nM at the tenth passage (see Experimental timeline Fig 6a ).

[0224] At the end of the experiment, infectious supernatants from the last cell passage of each treatment arm showing significant CPE were titrated in TCID50 / mL on MDCK cells. The median inhibitory concentration of Baloxavir on the viruses in these supernatants was also calculated by a limiting dilution titration method in TCID50 / mL on MDCK cells, in order to characterize the appearance of a Baloxavir resistance phenotype in comparison to the initial wild-type virus used.

[0225] For the untreated and Diltiazem treatment arms, infectious supernatants from the 10th cell passage were used. For the Baloxavir treatment arm, infectious supernatant from the 8th cell passage was used. For the Diltiazem + Baloxavir combination treatment arm, infectious supernatant from the 6th cell passage was used (because viruses were lost at the 7th and 9th cell passages for these experimental arms, respectively, see Fig 6a And 6b ).

[0226] MDCK cells were thus infected with the infectious supernatants in question, then treated separately one hour post infection with different increasing concentrations of Baloxavir (0.04 nM to 160 nM). In parallel, MDCK cells were also infected with the A / H1N1 WT virus initially used for the P0 cell passage (see Fig 6a And 6b) at an MOI 0.001, then treated or not separately one hour post infection with different increasing concentrations of Baloxavir (0.04 nM to 160 nM).

[0227] Infection supernatants were collected 48 hpi and then the viral load was titrated by the limiting dilution method in TCID50 / mL on MDCK cells. The results were expressed as relative values compared to control cells infected with the A / H1N1 virus and not treated ( Fig 6c to 6g ). From these infectious viral titration results, the median inhibitory concentrations of Baloxavir were calculated and are reported in Table 1 below. Detailed presentation of the results

[0228] The graph of the Figure 6brepresents the concentrations of Baloxavir and Diltiazem used in the different experimental arms (untreated, Baloxavir treatment, Diltiazem treatment and Diltiazem + Baloxavir combination treatment) for each cell passage, according to the experimental protocol described previously in Fig 6a The table shows the limiting dilutions of infectious supernatants from each cell passage (for which significant cytopathic effects were observed) and which were used to infect MDCK cells from each subsequent cell passage in each of the four experimental arms (untreated, Baloxavir treatment, Diltiazem treatment and Diltiazem + Baloxavir combination treatment).

[0229] The experimental arms without treatment (untreated) and with diltiazem treatment were maintained over the 10 cell passages. For the untreated arm, the limiting dilution used for each cell passage was always that at 10 -6< . For the diltiazem-treated arm, the limiting dilution used varied between 10 -5< and 10 -6< , as indicated in the graph and table.

[0230] For the Baloxavir treatment arm, the limiting dilution used for each cell passage remained stable at 10 -5< until the 6th< cell passage, for which the Baloxavir concentration used was 8 nM. From this cell passage, the limiting dilution used for subsequent cell passages gradually decreased (from 10 -4< to 10 -1< ) as increasing concentrations of Baloxavir were used (from 8nM up to 32nM) until the loss of replicative virus at the 9th< cell passage, for which the Baloxavir concentration used was 64nM.

[0231] In the Diltiazem + Baloxavir combination treatment arm, the limiting dilutions used (for which significant cytopathic effects were observed) were lower from the first cell passage (10 -3< ) and remained stable between 10 -3< and 10 -2< until the 6th< cell passage. Loss of replicative virus was observed during the 7th< cell passage for which the concentrations used of Baloxavir and Diltiazem were 16 nM and 25 µM, respectively.

[0232] These results indicate that in the Baloxavir treatment arm, the A / H1N1 virus, for which the initial median inhibitory concentration (IC50) of Baloxavir was 0.2 nM, replicated up to the 8th cell passage up to conditions of Baloxavir concentration of 32 nM. These results suggest that the selection pressure exerted by Baloxavir under these experimental conditions induced a viral resistance phenotype. In contrast, this viral resistance phenotype was not observed in the Diltiazem treatment arm, since, as in the untreated arm, the limiting dilution used for each cell passage remained stable between 10 -5< and 10 -6< , with no virus loss up to the 10th cell passage.

[0233] These results also indicate that the addition of Diltiazem to Baloxavir in the combination treatment arm (Diltiazem 25µM + Baloxavir at increasing concentrations from 1 nM to 8 nM) provided a synergistic viral inhibition effect compared to treatments with Diltiazem or Baloxavir alone at the same concentrations, since (i) the limiting dilutions used at each cell passage were always lower in this experimental arm, starting from the first cell passage (10 -3< ), and (ii) the replicative virus was lost more rapidly (from the 7th< cell passage).

[0234] There Figure 6c reports the determination of the median inhibitory concentration of Baloxavir on the A / H1N1 virus from the initial cell passage P0. The IC50 initially determined on the A / H1N1 virus used for the experiment is confirmed at 0.2 nM.

[0235] There Figure 6dreports the determination of the median inhibitory concentration of Baloxavir on the virus from cell passage P10 of the untreated experimental arm. The calculated IC50 of Baloxavir on the A / H1N1 virus from the 10th cell passage without treatment (untreated), is 0.9 nM. This slight increase can be attributed to viral replication of the A / H1N1 virus that has adapted to successive cellular amplification on MDCK cells in this experimental arm.

[0236] There Figure 6ereports the determination of the median inhibitory concentration of Baloxavir on the virus from cell passage P10 of the experimental arm of Diltiazem treatment. The calculated IC50 of Baloxavir on the A / H1N1 virus from the 10th cell passage with Diltiazem treatment is 0.3 nM. This IC50 is substantially similar to that initially determined on the A / H1N1 virus used for the experiment. This result confirms that treatment with Diltiazem at a constant concentration of 25µM over 10 successive cell passages does not modify the sensitivity of the A / H1N1 virus to Baloxavir (and does not induce the emergence of viruses with a Baloxavir resistance phenotype).

[0237] There Figure 6f reports the determination of the median inhibitory concentration of Baloxavir on the virus from the P8 cell passage of the experimental Baloxavir treatment arm.

[0238] The calculated IC50 of Baloxavir on the A / H1N1 virus from the 8th cell passage with Baloxavir treatment (in increasing concentration) is 7 nM. This IC50 is much higher than that initially determined on the A / H1N1 virus used for the experiment (IC50 = 0.2 nM).

[0239] This increase, corresponding to 35 times the initial IC50, confirms the emergence of a virus with a Baloxavir resistance phenotype, consistent with the observations described in Fig 6b (lower limiting dilutions used from the first cell passage in this experimental arm, effective viral replication up to Baloxavir concentration conditions of 32 nM at the 8th cell passage).

[0240] There figure 6g reports the determination of the median inhibitory concentration of Baloxavir on the virus from the P6 cell passage of the experimental arm of treatment with the combination Diltiazem + Baloxavir.

[0241] The calculated IC50 of Baloxavir on the A / H1N1 virus from the 6th cell passage with treatment by combination with Diltiazem + Baloxavir (in increasing concentration), is 0.8 nM. This IC50 is very similar to that calculated for Baloxavir on the A / H1N1 virus from the 10th cell passage in the experimental arm without treatment (0.9 nM, cf. Fig 6d ) and slightly higher than that initially calculated on the A / H1N1 virus used for the experiment and at the end of the P0 cell passage (IC50=0.2 nM, cf. Fig 6c ). In addition to the fact that this slight increase can be attributed to viral replication of the A / H1N1 virus which has adapted to successive cellular amplification on MDCK cells in this experimental arm, it is clearly established that this IC50 is very clearly lower than that calculated for the A / H1N1 virus from the P8 cell passage of the experimental Baloxavir treatment arm (IC50=7 nM, cf. Fig 6f), indicating the absence of emergence of viruses with a Baloxavir resistance phenotype in this experimental arm. These results indicate that the use of Diltiazem in combination with Baloxavir prevents the emergence of Baloxavir-resistant viruses in a classic test of successive cell passages of viruses under antiviral selection pressure (increasing concentration of Baloxavir).

[0242] The median inhibitory concentrations of Baloxavir for A / H1N1 viruses from the last cell passages of the different experimental arms are presented in Table 1 below. Table 1 Experimental arm IC50 Baloxavir (active form) H1N1 WT Initial PO 0.2 nM H1N1 NT P10 0.9 nM H1N1 Dil P10 0.3 nM H1N1 Baloxavir marboxil P8 7 nM H1N1 Combination P6 0.8 nM

[0243] In conclusion, the results of this experiment of successive cell passages in MDCK cells of A / H1 N1 virus under conditions of selection pressure, indicate that, as expected, the use of increasing concentrations of Baloxavir induced the emergence of an A / H1N1 virus with a phenotype of resistance to Baloxavir, whereas under the same conditions of increasing concentration of Baloxavir, the addition in combination of Diltiazem at a constant concentration of 25 µM makes it possible to prevent the emergence of an A / H1N1 virus with a phenotype of resistance to Baloxavir.

[0244] Similarly and as expected, the experimental condition without treatment also did not induce the emergence of an A / H1N1 virus with a Baloxavir resistance phenotype.

[0245] Similarly, the experimental condition with Diltiazem treatment alone did not induce the emergence of an A / H1N1 virus with a Baloxavir resistance phenotype. BIBLIOGRAPHICAL REFERENCES in the order of citation in the description BREVETS

[0246] WO 87 / 07508 WO 2011 / 066657 WO 2016 / 146836 WO 2019 / 224489 WO 02 / 094238 US 4,605,552 ARTICLES

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Claims

1. Combination of Diltiazem and Baloxavir marboxil, for use in the prevention and / or treatment of viral infection by an influenza virus.

2. Combination for use according to claim 1, characterized in that said influenza virus is resistant to the inhibitory action of at least one antiviral compound, in particular an anti-influenza compound, and more particularly a viral polymerase inhibitor compound.

3. A combination for use according one of claims 1 or 2, characterised in that said combination comprises at least one other active agent, in particular an antiviral agent and / or an antibiotic.

4. Pharmaceutical composition comprising, in a pharmaceutical vehicle, a combination of Diltiazem and Baloxavir marboxil, for its therapeutic use in the prevention and / or treatment of a viral infection by an influenza virus.

5. Pharmaceutical composition for use according to claim 4, characterized in that it further comprises another active agent, in particular an antiviral agent and / or an antibiotic.

6. Pharmaceutical composition for use according to one of claims 4 or 5, characterised in that said composition is in a galenic form suitable for administration by inhalation.

7. Combination product comprising Diltiazem and Baloxavir marboxil, for simultaneous, separate or sequential use in the prevention and / or treatment of viral infection by an influenza virus.