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    • 专利摘要:
    The present invention relates to a pharmaceutical or veterinary composition for its use in the prevention and / or treatment of an influenza virus infection, characterized in that it comprises, in a suitable pharmaceutical vehicle, at least one compound selected from the group consisting of Diltiazem and Etileurine.
    公开号:FR3033701A1
    申请号:FR1552284
    申请日:2015-03-19
    公开日:2016-09-23
    发明作者:Manuel Rosa-Calatrava；Olivier Terrier；Julien Textoris；Guy Boivin；Mario Pizzorno
    申请人:De Laval, University of；Universite Laval；Universite Claude Bernard Lyon 1 UCBL；Institut National de la Sante et de la Recherche Medicale INSERM；Hospices Civils de Lyon HCL；
    IPC主号:
    专利说明:

    [0001] The invention relates to compositions for their use in the treatment of viral infections related to influenza viruses, in humans and in animals. The influenza viruses are influenza viruses that are divided into three types: A, B and C. On the surface of the virus are two glycoproteins that play an important role in the infection of cells of the infected organism: hemagglutinin (HA) and neuraminidase (NA). There are different subtypes of influenza A virus 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 circulating viruses in the animal world and in particular among marine migratory birds . It is thus possible to define the influenza viruses according to the type of glycoproteins they possess on their surface. In humans, viruses circulating for several decades are subtypes H1N1, H2N2 and H3N2, with occasional interspecies transmissions, notably from animals to humans, avian viruses H5N1, H7N7, H7N9, H5N2 and H9N2.
    [0002] As the recent emergence of a new H1N1 pandemic influenza virus of porcine, avian and human origin (swine, avian and human reassortant viruses) indicates, influenza A viruses represent a serious threat to public health. Influenza pandemics are the result 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 to humans of animal and especially avian viruses: this is the case of highly pathogenic avian H5N1 outbreaks since 2003 in Asia, or H7N7 influenza outbreaks in the Netherlands in 2003 and H7N9 in the Netherlands. South-East Asia in 2013. Antigenic breaks are the result of genetic rearrangements between avian, porcine and human viruses, with pigs acting as intermediary hosts, for example. These genetic rearrangements were the cause of the H1N1 pandemic in 2009. Moreover, seasonal influenza epidemics, which are notably the result of genetic drift (appearance of mutations in surface glycoproteins) are a major cause morbidity and increased mortality, especially in the human population, especially in very young individuals, the elderly, immunocompromised individuals and carriers of cardiopulmonary diseases.
    [0003] 3033701 2 Vaccination remains the cornerstone of influenza prevention. However, when a new virus appears, 6 to 9 months is needed for the development and delivery of a new vaccine, and the use of antiviral drugs should be considered for treatment. and / or prevention. Common antivirals are M2 channel inhibitors such as amantadine and neuraminidase inhibitors such as zanamivir and Oseltamivir. The use of these drugs is limited by the recurrent appearance of resistance, particularly observed for the pandemic H1N1 virus. Moreover, it is not excluded that new emerging viruses are already resistant to these molecules. Finally, most of these molecules are not administrable, which is a problem in case of serious infections. It therefore appears necessary to develop or identify new antiviral compounds that are more effective and have a broad spectrum of action. One solution for the development of novel broad-spectrum therapeutic molecules is to identify molecules having an action on the pathways and cellular factors that viruses target to carry out their replicative cycle. This strategy, presented in the article by Josset et al. (Plos One, 2010), has allowed the identification of a first series of molecules with unexpected antiviral activity, such as the molecules presented in the patent application FR 2 953 410. In the context of the present invention, several molecules were selected and evaluated in a viral infection cell assay as well as in vivo murine model. Some molecules have thus presented an antiviral effect not only on an influenza A strain of subtype H1N1 but also on a strain of H3N2 subtype. The compounds selected had previously been described as active principles in the treatment of pathologies far removed from viral infections. Unexpectedly, it has now been shown that some of these compounds have antiviral activity and in particular against different influenza virus subtypes A. SUMMARY OF THE INVENTION The invention relates to pharmaceutical or veterinary compositions for their use in the prevention and / or treatment of influenza virus infections, comprising at least one compound selected from Diltiazem and Etiléfrine.
    [0004] These pharmaceutical or veterinary compositions may advantageously also comprise at least one other antiviral agent and / or an antibacterial agent. These compositions may be in the form of combination products for simultaneous, separate or sequential use in human or veterinary therapy, particularly in the prevention and / or treatment of influenza virus infections. The invention also relates to pharmaceutical or veterinary compositions comprising at least one antiviral agent in combination with at least one compound selected from Diltiazem and Etiléfrine, or a combination of both. The invention also relates to pharmaceutical or veterinary compositions, comprising in a suitable pharmaceutical vehicle, a combination of Diltiazem and Etiléfrine. FIGURES Figure 1. (A) Experimental Protocol; (B) In vitro evaluation of the antiviral effect of the compounds Diltiazem, Etiléfrine and Lanatoside C, and compounds known for their antiviral activity (Ribavirin, Monensin) on A549 cells infected with the H1N1 virus pdm09. The solid curve represents the evolution of normalized viral production (percentage) as a function of the concentration of the compounds (1..tM). The dashed line represents the normalized cell viability (percent) as a function of the concentration of the compounds (1..tM). Figure 2. In vitro evaluation of the antiviral effect of Etilofine on A549 cells infected with H3N2 virus. The continuous line curve (round dots) represents the evolution of normalized viral production (percentage) as a function of the concentration of Etilefrine (1..tM). The dotted curve (square dots) represents the normalized cell viability (percent) versus the concentration of Etilefrine (1..tM). Figure 3. In vitro evaluation of the antiviral effect of Etilofrin (A) and Diltiazem (B), in combination with Oseltamivir, on A549 cells infected with the H1N1 virus pdm09. The graph represents normalized viral production (percent) for each of the conditions. Figure 4. In vivo evaluation of the toxicity of the following molecules: Oseltamivir, Monensin, Diltiazem, Etilefrine and Isoxicam. The "Saline" control indicates the weight of the treated mice with PBS buffer. The curve represents the change in weight (percentage gain / loss) of different groups of mice over time. Figure 5. In vivo evaluation of the antiviral effect of the compounds Oseltamivir, Monensin, Diltiazem, Etiléfrine and Isoxicam, administered before the H1N1 infection of the mice: (A) experimental protocol; (B) survival rate over time, up to 14 days post-infection; (C) weight loss observed over time; (D) Virus titre measured in the lungs of infected mice at 5 days post-infection. Figure 6. In vivo evaluation of the antiviral effect of Oseltamivir and Diltiazem compounds, administered 24 hours after H1N1 infection of mice. Experimental Protocol (A); Survival rate over time, after in vivo treatment with PB S alone Saline (B), Oseltamivir (C) and Diltiazem (D) administered 24 h after H1N1 infection in mice. DETAILED DESCRIPTION OF THE INVENTION The subject of the present invention is a pharmaceutical or veterinary composition for its use in the prevention and / or treatment of an influenza virus infection, characterized in that it comprises, in a pharmaceutical vehicle suitable, at least one compound selected from Diltiazem, Etilofine and their derivatives. These compounds are known for use in other therapeutic applications unrelated to antiviral activity against influenza viruses in humans or animals. It has now been shown that these compounds unexpectedly have antiviral activity against different influenza A virus subtypes. Diltiazem is commonly used in the management of angina, arterial hypertension, myocardial ischemia, and tachycardia. Diltiazem 25 is a potent vasodilator. Several patent applications describe processes for producing Diltiazem (EP0728751, EP0561861), as well as derivatives of Diltiazem (EP0450705, EP0355395). This molecule, a member of the benzothiazepine family, has the following chemical structural formula: ## STR2 ## Formula (1) Diltiazem works by curbing the entry of transmembrane calcium into myocardial muscle fiber and muscle fiber smooth vessels, and thus decreases the intracellular calcium concentration reaching the contractile proteins. Diltiazem reduces cardiac work and slows down the ventricular rate. The doses conventionally used in humans are preferably between 200 and 300 mg / day. For the purposes of the present invention, the term "Diltiazem" means Diltiazem 10 in all its forms, especially in the form of salts, as well as all the molecules derived from the formula (1) presented above, having the same biological activity. Etileurine has been described as a cardiac stimulant, and is used as an anti-hypotensive agent, particularly in the treatment of orthostatic neurological, cardiovascular, endocrine or metabolic hypotension. Its use has in particular been described in the patent application EP0162320. Etilofine increases cardiac output, stroke volume, venous return, and elevates central venous pressure and blood pressure, via a direct sympathomimetic effect on alpha-1 and beta-adrenergic receptors, by its positive inotropic action. In addition, it increases venous tone and causes an increase in circulating blood volume. It is a sympathomimetic amine of the 3-hydroxyphenylethanolamine series of chemical formula developed: 3033701 6 HO Formula (2) The doses conventionally used in humans are preferably about 30 mg / day, generally administered in three doses .
    [0005] Within the meaning of the present invention, the term "Etilofine" means Etilofine in all its forms, especially in the form of salts, as well as all the molecules derived from the formula (2) presented above, having the same biological activity. . According to the invention, the influenza viruses are type A viruses having for host the human or the animal. The terms "influenza virus" and "influenza virus" are used interchangeably in the application and refer to the same viruses. According to the invention, the term "suitable pharmaceutical vehicle" designates pharmaceutically acceptable vehicles or excipients according to the invention, ie vehicles or excipients whose administration to an individual or an animal is not accompanied by significant deleterious effects, which are well known to those skilled in the art. The compositions of the present invention are used for the prevention and / or treatment of influenza A virus infections. Advantageously, the compositions of the present invention have a broad spectrum of action against the different subtypes of influenza viruses. type A.
    [0006] In one embodiment, the compositions of the present invention are used for the prevention and treatment of circulating type A virus infections primarily in humans and animals. There are different subtypes of influenza A virus depending on the nature of the HA and NA glycoproteins on their surface. 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. The invention thus relates to the prevention and / or treatment of infections with influenza viruses in humans. The invention also relates to the prevention and / or treatment of infections with influenza viruses in animals, in particular, in farm animals such as pigs, horses and poultry. More particularly, the subject of the invention is the prevention and / or treatment of influenza virus infections in poultry, and more particularly in hens, ducks, geese, turkeys and turkeys. The invention also relates to the prevention and treatment of influenza virus infections in other animals such as horses, cats, dogs and felines. According to a first aspect of the invention, the pharmaceutical or veterinary composition comprises an effective amount of Diltiazem or a derivative thereof for use in the prevention and / or treatment of influenza virus infection. According to a second aspect of the invention, the pharmaceutical or veterinary composition comprises an effective amount of Etilofine or a derivative thereof for use in the prevention and / or treatment of influenza virus infection. According to a third aspect of the invention, the pharmaceutical or veterinary composition comprises a combination of Diltiazem and Etilofine or derivatives thereof for use in the prevention and / or treatment of an infection with one or more influenza. This combination comprises either the same dose of each compound (50/50 by weight composition) or unequal doses of each compound, such as 90% Diltiazem and 10% Etilofine, 80% Diltiazem and 20% Etilofine, 70% of Diltiazem and 30% of Etilofine, 60% of Diltiazem and 40% of Etilofine, 40% of Diltiazem 20 and 60% of Etilofine, 30% of Diltiazem and 70% of Etilofine, 20% of Diltiazem and 80% of Etilofine, or else 10% of Diltiazem and 90% of Etilofine. According to a particular aspect of the invention, the pharmaceutical or veterinary composition for its use as described above is characterized in that it further comprises another antiviral agent.
    [0007] Indeed, Diltiazem and Etilofine or mixtures thereof can be used in therapy alone, or in combination with at least one other active agent. They may be compounds which make it possible to improve the activity of the compounds, or else other active agents known for their use as antiviral agents in the treatment of influenza virus infections.
    [0008] In particular, the antiviral agent has an inhibitory activity on influenza viruses.
    [0009] Such active agents are well known to those skilled in the art, available commercially or described in reference books such as The Vidal Dictionary. According to a first aspect of the invention, the pharmaceutical or veterinary composition comprises Diltiazem and an antiviral agent for use in the prevention and / or treatment of influenza virus infection. According to a second aspect of the invention, the pharmaceutical or veterinary composition comprises Etilofine and an antiviral agent for its use in the prevention and / or treatment of influenza virus infection.
    [0010] According to a third aspect of the invention, the pharmaceutical or veterinary composition comprises a combination of Diltiazem and Etilofine, and an antiviral agent, for use in the prevention and / or treatment of influenza virus infection. In particular, this antiviral agent is selected from Oseltamivir (also called Tamiflu), zanamivir, peramivir, amantadine, rimantadine, ribavirin and arbidol. This antiviral agent may also be selected from midodrine, desglymidodrine, rilmenidine, harmol, harmol dimers and brinzolamide. This antiviral agent may also be chosen from: viral polymerase inhibitors (ex: T705); - nucleoprotein inhibitors; - hemagglutinin inhibitors; - neuraminidase inhibitors; - NS1 protein inhibitors.
    [0011] This antiviral agent may also be chosen from: recombinant sialidases (ex DAS181); - NFKB inhibitors; - HSP90 inhibitors; and inhibitors of Raf, Mek, Erk or PKC cellular protein kinases.
    [0012] It is understood that this antiviral agent will be used at the doses necessary to have an antiviral action, this dose being designated as "effective", this dosage being easily determinable by those skilled in the art.
    [0013] According to another preferred aspect of the invention, the pharmaceutical or veterinary composition comprises Diltiazem, in combination with Oseltamivir, for use in the prevention and / or treatment of influenza virus infection. According to a second preferred aspect of the invention, the pharmaceutical or veterinary composition comprises Etilofine, in combination with Oseltamivir, for use in the prevention and / or treatment of influenza virus infection. According to a third preferred aspect of the invention, the pharmaceutical or veterinary composition comprises a combination of Diltiazem and Etilofine, in combination with Oseltamivir, for use in the prevention and / or treatment of influenza viruses. The pharmaceutical or veterinary composition for its use according to the invention may also comprise an antibacterial agent. Such an agent will be in particular an antibiotic, intended to prevent bacterial superinfections conventionally observed in complications of influenza virus infections. According to a first aspect of the invention, the pharmaceutical or veterinary composition comprises Diltiazem, in combination with an antibacterial agent, for its use in the prevention and / or treatment of influenza virus infection. According to a second aspect of the invention, the pharmaceutical or veterinary composition comprises Etilofine, in combination with an antibacterial agent, for use in the prevention and / or treatment of influenza virus infection. According to a third aspect of the invention, the pharmaceutical or veterinary composition comprises a combination of Diltiazem and Etilofine, in combination with an antibacterial agent, for its use in the prevention and / or treatment of virus infection. influenza. According to a fourth aspect of the invention, the pharmaceutical or veterinary composition comprises a combination of Diltiazem and Etiléfrine, in combination with an antiviral agent, in particular Oseltamivir, and an antibacterial agent, for its use in the prevention and / or or treating an infection with influenza viruses.
    [0014] The present invention also relates to a combination product comprising at least one compound selected from Diltiazem and Etilofine, and at least one antiviral agent and / or an antibacterial agent, for simultaneous, separate or simultaneous use. sequential method for preventing and / or treating influenza virus infection in humans or animals. According to one embodiment, said combination product comprises Diltiazem and at least one antiviral agent such as Oseltamivir. In another embodiment, said combination product comprises Etilearin and at least one antiviral agent such as Oseltamivir. According to another embodiment, said combination product comprises a combination of Diltiazem and Etiléfrine and at least one antiviral agent such as 0 seltamivir. In another embodiment, said combination product comprises Diltiazem and at least one antibacterial agent. According to another embodiment, said combination product comprises Etilofine and at least one antibacterial agent. In another embodiment, said combination product comprises a combination of Diltiazem and Etilofine and at least one antibacterial agent.
    [0015] According to another embodiment, said combination product comprises Diltiazem, at least one antiviral agent and at least one antibacterial agent. According to another embodiment, said combination product comprises Etilofine, at least one antiviral agent and at least one antibacterial agent. In another embodiment, said combination product comprises a combination of Diltiazem and Etilofine, at least one antiviral agent and at least one antibacterial agent. Antiviral agents, especially having an inhibitory action against influenza viruses, are well known to those skilled in the art, and will be chosen in particular from the following agents: oseltamivir, zanamivir, peramivir, amantadine, rimantadine, ribavirin and arbidol; Midodrine, desglymidodrine, rilmenidine, harmol, harmol dimers and brinzolamide; viral polymerase inhibitors (eg T705); nucleoprotein inhibitors; Hemagglutinin inhibitors; neuraminidase inhibitors; inhibitors of NS1 protein. recombinant sialidases (ex DAS181); inhibitors of NFKB; Inhibitors of HSP90; and inhibitors of Raf, Mek, Erk or PKC cellular protein kinases. The invention also relates to a combination product comprising at least one compound selected from Diltiazem and Etilofine, as well as at least one other antiviral agent and / or an antibacterial agent, for simultaneous, separate or sequential use. to prevent and / or treat infections with influenza viruses. The invention also relates to a therapeutic method for the prevention and / or treatment of influenza virus (influenza virus) infection in humans in which a patient is administered an effective amount of a compound Selected from Etilefrine and Diltiazem, or a mixture of both, optionally in combination with another antiviral compound and / or an antibacterial compound. The invention also relates to a therapeutic method for the prevention and / or treatment of an influenza virus infection in animals in which an animal is administered an effective amount of a compound selected from among Etilefrine and Diltiazem, optionally in combination with another antiviral compound and / or an antibacterial compound. Advantageously, the animal is a farm animal such as, for example, pork, horse or even poultry and domestic animals (dogs, cats, etc.).
    [0016] Pharmaceutical or Veterinary Compositions The present invention also relates to a pharmaceutical or veterinary composition, comprising in a suitable pharmaceutical carrier, at least one antiviral agent in combination with at least one compound selected from Diltiazem and Etilofine, or a combination of two. According to a preferred aspect, the antiviral agent is chosen from antiviral agents well known to those skilled in the art, and conventionally used to prevent or treat influenza, in particular having an inhibitory action against influenza viruses. The antiviral agent is in particular chosen from: Oseltamivir, zanamivir, peramivir, amantadine, rimantadine, ribavirin and arbidol. This antiviral agent may also be selected from midodrine, desglymidodrine, rilmenidine, harmol, harmol dimers and brinzolamide.
    [0017] This antiviral agent may also be chosen from: viral polymerase inhibitors (ex: T705); - nucleoprotein inhibitors; - hemagglutinin inhibitors; neuraminidase inhibitor 15 - NS1 protein inhibitors. This antiviral agent may also be chosen from: recombinant sialidases (ex DAS181); - NFKB inhibitors; - HSP90 inhibitors; and 20 - Raf, Mek, Erk or PKC cellular protein kinase inhibitors. In a preferred aspect, the antiviral agent is Oseltamivir. The invention therefore relates specifically to: - a pharmaceutical or veterinary composition, comprising in a suitable pharmaceutical vehicle, at least one antiviral agent with Diltiazem; and a pharmaceutical or veterinary composition, comprising in a suitable pharmaceutical vehicle, at least one antiviral agent with Etilofine; and a pharmaceutical or veterinary composition, comprising in a suitable pharmaceutical vehicle, at least one antiviral agent with Etilofine and Diltiazem. This antiviral agent will preferably be selected from those listed herein, and will include Oseltamivir.
    [0018] In yet another aspect, the pharmaceutical or veterinary composition according to the invention comprises, in a suitable pharmaceutical vehicle, a combination of Diltiazem and Etiléfrine. As previously indicated, any combination of Diltiazem and Etilofine comprises either the same dose of each compound (50/50 by weight composition) or unequal doses of each compound, such as 90% Diltiazem and 10% Etilofine, 80% of Diltiazem and 20% of Etilofine, 70% of Diltiazem and 30% of Etilofine, 60% of Diltiazem and 40% of Etilofine, 40% of Diltiazem and 60% of Etilofine, 30% of Diltiazem and 70% of Etilofine, 20% of Diltiazem and 80% of Etilofine, or else 10% of Diltiazem and 90% of Etilofine. The pharmaceutical compositions of the present invention are suitable for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, ocular, local or rectal administration, wherein the active compound can be administered in unit dosage forms in admixture with carriers. pharmaceuticals, animals or humans. Suitable unit dosage forms include oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, sublingual and oral forms of administration, subcutaneous forms of administration intramuscular, intravenous, intranasal or intraocular and forms of rectal administration. When a solid composition in tablet form is prepared, the main active compound is mixed with a suitable pharmaceutical carrier such as gelatin, starch, lactose, magnesium stearate, talcum, gum arabic or the like. . The tablets may be coated with sucrose or other suitable materials or may be treated so that they have prolonged or delayed activity and continuously release a predetermined amount of active ingredient. A preparation in capsules is obtained by mixing the active compound with a diluent and pouring the resulting mixture into soft or hard gelatin capsules. A syrup or elixir preparation may contain the active compound together with a sweetener, an antiseptic, as well as a flavoring agent and a suitable colorant. The water-dispersible powders or granules may contain the active compound in admixture with dispersants or wetting agents, or suspending agents, as well as with taste correctors or sweeteners.
    [0019] EXAMPLES Introduction Studies of the cellular transcriptomic response during influenza infection have made it possible to highlight the signaling pathways solicited and / or diverted during infection, in vitro in cells in culture. This allowed to characterize in vitro "transcriptomic signatures" specific for infection with different influenza viruses, both human and avian (Josset et al., PLoS One, 2010, Terrier et al., Virol J, 2011, Terrier et al., J Gen Virol, 2013). This same strategy was applied to cell samples from nasal washings of 9 patients to characterize the in vivo signatures of pandemic H1N1 infection (pdm09). These transcriptomic signatures were obtained on the Affymetrix 450 fluid platform, using different bioinformatic analysis tools. At the end of an "in silico" screening in public databases such as Connectivity Map (Broad Institute, MIT), 34 molecules were selected, 15 including Etiléfrine, Diltiazem, Monensin, Lanatoside C, Isoxicam. These molecules were evaluated for their antiviral activity in cellular infection model in vitro, on the A549 line of human lung epithelium, and in vivo in an infectious murine model.
    [0020] EXAMPLE 1 In vitro evaluation of the antiviral effect of various selected compounds on H1N1 infected A549 cells. Detailed Culture and Infection Protocol: A. Culture of A549 Human Cells A549 cells (lung carcinoma) are maintained in culture in DMEM medium (modified Dulbecco medium, BioWhittaker) supplemented with 10% (v / v) serum fetal calf, 2 mM L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin, in an incubator at 37 ° C, at a saturated humidity atmosphere containing 5% CO 2. At confluence, the cells are detached from the support by treatment with trypsin-EDTA and reseeded in a culture flask containing 15 ml of fresh medium. Three days before the infection stage, 0.75 × 10 6 cells are distributed by 25 cm 2 flasks (T25), in medium to 10% fetal calf serum so as to obtain 70-80% confluence during infection. .
    [0021] B. Infection of cells with representative influenza viruses The cells are infected with the different influenza viruses at a multiplicity of infection (moi) of 0.1 in DMEM, 2 mM of L-glutamine, 100 U / mL of penicillin and 100 μg / mL streptomycin, and 0.5 μg / mL trypsin.
    [0022] The viruses used are: strains of human influenza A virus (H1N1 A / Pdm / 09) and H3N2 (113N2 A / Moscow / 10/99) C. Pre-treatment and treatment The molecules were tested on A549 cells at 70-80% confluence.
    [0023] The cells are incubated for 6 h with different concentrations of molecules, then infected with the different viruses for 1 h, and returned to the presence of the molecules at the same concentrations. D. Measurements A cytotoxicity test and a virus quantification test are performed after 48 hours of incubation at 37 ° C. under 5% CO 2. The cytotoxicity of the different molecules is determined at each test in a non-infected cell plate by a viability test (MTS test, Promega). This test is based on measuring the metabolic activity of the cells, which transforms a substrate (MTS tetrazolium) into a product (Formazan), soluble in the medium and whose absorbance measured at 490 nm proportionally reflects the number of cells. alive. The ratio of the absorbance in each well to the average absorbance of the control cell wells (not treated by the molecules) is calculated and indicated on the schemes as an index of cell viability (relative cell viability). The effect on viral production is estimated by determination of the infectious titres (DITC50 / ml, infectious dose 50%) carried out in MDCK cells, the titres being calculated according to the technique of Reed and Muench. The ratio of infectious titer in each condition was expressed as a function of the infectious titer measured in control condition E. Results The pre-treatment and cell treatment protocol is shown schematically in FIG. 1A. Several concentrations were tested. The cells are pre-treated for 6 hours with the various molecules tested, then are infected with the virus H1N1 pdm09 for one hour. The treatment with the different molecules tested is then resumed for 48 hours. The measurements made at the end of the treatment as described above make it possible to determine the median inhibitory concentration (IC 50), which is dependent on the viral production as a function of the different concentrations tested. Figure 1B shows the results obtained for the following molecules: Ribavirin, Monensin, Lanatoside C, Diltiazem and Etiléfrine. As expected, the compounds known for their antiviral activity: Ribavirin and Monensin, induce a significant decrease in normalized viral production. More surprisingly, Diltiazem and Etiléfrine also show a significant decrease in normalized virus production. Among the compounds selected as described in the introduction, Lanatoside C was tested to evaluate its antiviral activity - the results obtained did not confirm the antiviral activity of this compound.
    [0024] EXAMPLE 2 In Vitro Evaluation of the Antiviral Effect of Etilefrine on A549 Cells Infected with H3N2 Viral Subtype The pre-treatment and treatment protocol is shown schematically in FIG. 1A, and the experimental conditions are the same as those described in example 1. The H3N2 virus used for the infection is: strain A / Moscow / 10/99 ( H3N2).
    [0025] FIG. 2 shows that the antiviral effects of Etilofen are significant from the lowest concentration tested and make it possible to totally inhibit the viral production at 0.018 μM of Etiléfrine. Example 3 In vitro evaluation of the antiviral effect of (A) Etilefrine and (B) Diltiazem, in combination with Oseltamivir, on A549 cells infected with H1N1 virus pdm09. The pre-treatment and treatment protocol is shown diagrammatically in FIG. 1A, and the experimental conditions are the same as those described in example 1. As expected, Oseltamivir makes it possible to significantly reduce viral production to around 40% ( Figures 3A and 3B). The combinations Etiléfrine and Oseltamivir are clearly more efficient than the compounds alone, and at the two concentrations of Etiléfrine tested.
    [0026] The combination of Oseltamivir and 0.9 nM Diltiazem is more effective than the compounds alone. The best results are obtained with Diltiazem alone at a concentration of 9 nM.
    [0027] Example 4 In vivo evaluation in mice of the toxicity of the following molecules: Oseltamivir, Monensin, Diltiazem, Etiléfrine and Isoxicam. The "Saline" control indicates untreated mice, having been treated with PB S buffer. The following daily amounts were administered by gavage to the 10 mice: - Oseltamivir: 10 mg / kg / day - Etilofine: 3 mg / kg / day - Diltiazem: 90 mg / kg / day The compound with the highest cellular toxicity is Oseltamivir, however, this toxicity is acceptable, the mice maintaining positive weight gain during treatment. Diltiazem (diamond) and Etilofine (star) are clearly non-toxic at the doses administered. Example 5 In Vivo Evaluation of the Anti-Viral Effect of the Oseltamivir, Monensin, Diltiazem, Etilearin and Isoxicam Compounds on Infected B57BL / 6 Mice: (A) Administration Protocol; (B) survival rate over time, up to 14 days post-infection; (C) weight loss observed over time; (D) Virus titre in the lungs. The B57BL / 6 mice at 7-8 weeks of age are treated by gavage before 25 (day -1), the same day and the three days following infection with the H1N1 pdm09 virus, according to the protocol shown schematically in FIG. 5A. The following parameters are evaluated over time: Fig 5B: survival rate over time, up to 14 days post-infection; Fig 5C: loss and weight gain observed over time; Fig 5D: Virus titre in the lungs of infected and treated mice. Etilofine (represented by stars) and Diltiazem (represented by lozenges) allow maximum survival of the infected mice, namely 70% and 30% survival at 14 days, which is a better result than that obtained. for Oseltamivir (represented by circles, 60% survival). Example 6. In Vivo Evaluation of the Anti-Viral Effect of Oseltamivir and Diltiazem Compounds Administered 24 Hours After Infection of Mice with H1N1 Virus pdm09: Survival Rate Over Time, Up to 14 Days Post-Infection The B57BL / 6 mice at 7-8 weeks of age are treated by gavage after infection with the H1N1 pdm09 virus, according to the protocol shown schematically in FIG. 6A. The survival rate over time is measured for infected, untreated mice (Fig.
    [0028] 6B); For mice treated with Oseltamivir (Fig.
    [0029] 6C); and for Diltiazem treated mice (Fig.
    [0030] 6D). The survival rate of mice treated with Oseltamivir, and those treated with Diltiazem, is 100%. REFERENCES 15 - FR 2 953 410 - EP01628751 - EP0450595 - Josset L, Textoris J, Loriod B, Ferraris 0, Mussels V, Lina B, N'guyen C, Diaz JJ, Rosa-Calatrava M. Gene expression signature-based screening identifies new broadly effective influenza antiviral. »PLoS One.
    [0031] 2010 Oct 4; 5 (10). Terrier 0, Josset L, Textoris J, Marcel V, Cartet G, Ferraris 0, N'guyen C, 25 Lina B, Diaz JJ, Bourdon JC, Rosa-Calatrava M. "Cellular transcriptional profiling in human lung epithelial cells infected by Different subtypes of influenza A viruses reveals an overall down-regulation of the host p53 pathway. »Virol J.
    [0032] 2011 Jun 8; 8: 285. - Terrier 0, Textoris J, Carron C, Marcel V, JC Bourdon, Rosa-Calatrava M. "Host microRNA molecular signatures associated with human HINI and H3N2 influenza 30 A viruses reveal anantanticipated antiviral activity for miR-146a." J Gen Virol .
    [0033] 2013 May; 94 (Pt 5): 985-95.
    权利要求:
    Claims (12)
    [0001]
    REVENDICATIONS1. A pharmaceutical or veterinary composition for use in the prevention and / or treatment of influenza virus infection, characterized in that it comprises, in a suitable pharmaceutical carrier, at least one compound selected from Diltiazem and etilefrine.
    [0002]
    2. Pharmaceutical or veterinary composition for its use according to claim 1, characterized in that the influenza viruses are selected from type A viruses, such as subtypes H1N1, H2N2, H3N2, H5N1, H7N7 and H7N9. 10
    [0003]
    3. Pharmaceutical or veterinary composition for use according to one of claims 1 or 2, characterized in that it comprises a combination of Diltiazem and Etiléfrine.
    [0004]
    4. Pharmaceutical or veterinary composition for use according to one of claims 1 to 3, characterized in that it further comprises another antiviral agent. 15
    [0005]
    5. Pharmaceutical or veterinary composition for its use according to claim 4, characterized in that the antiviral agent has an inhibitory activity on influenza viruses.
    [0006]
    6. Pharmaceutical or veterinary composition for its use according to claim 5, characterized in that the antiviral agent is selected from Oseltamivir, Zanamivir, Peramivir, Amantadine, Rimantacline, Ribavirin and Arbidol.
    [0007]
    7. Pharmaceutical or veterinary composition for use according to one of claims 1 to 6, characterized in that it further comprises an antibacterial agent.
    [0008]
    8. A combination product comprising at least one compound selected from Diltiazem and Etiléfine, and at least one antiviral agent and / or an antibacterial agent, for use in preventing and / or treating influenza virus infection.
    [0009]
    9. A pharmaceutical or veterinary composition, comprising in a suitable pharmaceutical vehicle, at least one antiviral agent in combination with at least one compound selected from Diltiazem and Etiléfrine, or a combination of both.
    [0010]
    10. The pharmaceutical or veterinary composition according to claim 9, wherein the antiviral agent has an influenza virus inhibitory activity.
    [0011]
    11. The pharmaceutical or veterinary composition according to claim 10, wherein the antiviral agent is Oseltamivir.
    [0012]
    12. A pharmaceutical or veterinary composition comprising in a suitable pharmaceutical vehicle a combination of Diltiazem and Etilofine.
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    同族专利:
    公开号 | 公开日
    CN107743402A|2018-02-27|
    HK1249430A1|2018-11-02|
    IL254548D0|2017-11-30|
    CN113876781A|2022-01-04|
    JP2018508587A|2018-03-29|
    BR112017019845A2|2018-06-05|
    WO2016146836A2|2016-09-22|
    KR20180006370A|2018-01-17|
    CA2979784A1|2016-09-22|
    EP3270968A2|2018-01-24|
    US20180042937A1|2018-02-15|
    WO2016146836A3|2016-11-03|
    FR3033701B1|2021-01-15|
    JP6884112B2|2021-06-09|
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    FR3108033A1|2020-03-10|2021-09-17|Universite Claude Bernard Lyon 1|ANTIVIRAL COMPOUNDS AND THEIR COMBINATIONS FOR TREATING VIRAL INFECTION WITH SARS-CoV-2|
    法律状态:
    2016-03-31| PLFP| Fee payment|Year of fee payment: 2 |
    2016-09-23| PLSC| Publication of the preliminary search report|Effective date: 20160923 |
    2017-02-27| PLFP| Fee payment|Year of fee payment: 3 |
    2018-03-12| PLFP| Fee payment|Year of fee payment: 4 |
    2020-03-10| PLFP| Fee payment|Year of fee payment: 6 |
    2021-03-23| PLFP| Fee payment|Year of fee payment: 7 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1552284A|FR3033701B1|2015-03-19|2015-03-19|NEW ANTIVIRAL COMPOSITIONS FOR THE TREATMENT OF INFLUENZA|FR1552284A| FR3033701B1|2015-03-19|2015-03-19|NEW ANTIVIRAL COMPOSITIONS FOR THE TREATMENT OF INFLUENZA|
    JP2017567547A| JP6884112B2|2015-03-19|2016-03-18|A novel antiviral composition for treating influenza|
    EP16714789.1A| EP3270968A2|2015-03-19|2016-03-18|Novel antiviral compositions for treating the flu|
    PCT/EP2016/056036| WO2016146836A2|2015-03-19|2016-03-18|Novel antiviral compositions for treating the flu|
    CN202111122486.2A| CN113876781A|2015-03-19|2016-03-18|Novel antiviral compositions for the treatment of influenza|
    CA2979784A| CA2979784A1|2015-03-19|2016-03-18|Novel antiviral compositions for treating the flu|
    CN201680016574.5A| CN107743402A|2015-03-19|2016-03-18|For treating the novel antiviral composition of influenza|
    US15/557,170| US20180042937A1|2015-03-19|2016-03-18|Novel antiviral compositions for treating the flu|
    BR112017019845-2A| BR112017019845A2|2015-03-19|2016-03-18|new antiviral compositions for treating flu|
    KR1020177029873A| KR20180006370A|2015-03-19|2016-03-18|Novel antiviral compositions for treating the flu|
    IL254548A| IL254548D0|2015-03-19|2017-09-18|Novel antiviral compositions for treating the flu|
    HK18109070.4A| HK1249430A1|2015-03-19|2018-07-12|Novel antiviral compositions for treating the flu|
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