EP3796916A1 - Diltiazem pour son utilisation dans le traitement des infections microbiennes - Google Patents

Diltiazem pour son utilisation dans le traitement des infections microbiennes

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Publication number
EP3796916A1
EP3796916A1 EP19737813.6A EP19737813A EP3796916A1 EP 3796916 A1 EP3796916 A1 EP 3796916A1 EP 19737813 A EP19737813 A EP 19737813A EP 3796916 A1 EP3796916 A1 EP 3796916A1
Authority
EP
European Patent Office
Prior art keywords
diltiazem
virus
infection
epithelia
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19737813.6A
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German (de)
English (en)
French (fr)
Inventor
Manuel Rosa-Calatrava
Olivier Terrier
Claire NICOLAS DE LAMBALLERIE
Guy Boivin
Mario Andres PIZZORNO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite Laval
Centre National de la Recherche Scientifique CNRS
Universite Claude Bernard Lyon 1 UCBL
Institut National de la Sante et de la Recherche Medicale INSERM
Ecole Normale Superieure de Lyon
Original Assignee
Universite Laval
Centre National de la Recherche Scientifique CNRS
Universite Claude Bernard Lyon 1 UCBL
Institut National de la Sante et de la Recherche Medicale INSERM
Ecole Normale Superieure de Lyon
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Application filed by Universite Laval, Centre National de la Recherche Scientifique CNRS, Universite Claude Bernard Lyon 1 UCBL, Institut National de la Sante et de la Recherche Medicale INSERM, Ecole Normale Superieure de Lyon filed Critical Universite Laval
Publication of EP3796916A1 publication Critical patent/EP3796916A1/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/554—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one sulfur as ring hetero atoms, e.g. clothiapine, diltiazem
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to a compound for use in preventing and / or treating infections with pathogenic microorganisms.
  • the present invention relates to a compound for use in the prevention and / or treatment of infections by pathogenic microorganisms of the epithelia of the respiratory and / or intestinal tracts.
  • Acute respiratory infections are one of the leading causes of consultations, hospitalizations and deaths worldwide, including the leading cause of death among young children with nearly 2 million deaths per year. Each year, in an attempt to treat these various respiratory infections, the cost to companies is estimated at between 1, 5 and 2 billion euros.
  • viruses occupy a prominent place. They are found in most cases of childhood pneumonia and are a predisposing factor for bacterial pneumonia in adults. Among the most representative viruses in terms of frequency and morbidity, influenza viruses type A and B, which are also a recurrent pandemic risk factor, as well as respiratory syncytial virus (hRSV or hVRS), viruses parainfluenza (hPIV) and the human metapneumovirus (hMPV).
  • hRSV or hVRS respiratory syncytial virus
  • hPIV viruses parainfluenza
  • hMPV human metapneumovirus
  • influenza viruses With the exception of influenza viruses, there is currently no vaccine or antiviral molecule effective to prevent or treat infections with these different pathogenic respiratory viruses. In addition, in the case of influenza viruses, delays and the variable effectiveness of vaccination, as well as the increasing emergence of antiviral-resistant viruses, are of great concern today.
  • hVRS respiratory syncytial virus
  • hMPV human metapneumovirus
  • hPIV human parainfluenza virus
  • Pseudomonas aeruginosa is a gram-negative bacillus found in the environment and in more than 50% of the respiratory tract in hospitalized patients.
  • P. aeruginosa is a ubiquitous microorganism that has the ability to survive under multiple environmental conditions. This microorganism causes not only diseases in plants and animals, but also in humans, causing serious infections in immunocompromised patients with cancer and patients with severe burns or cystic fibrosis.
  • Standard antibiotic therapy for Pseudomonas aeruginosa infections combines a beta-lactam (ceftazidime, imipenem or meropenem, piperacillin / tazobactam) with aminoside (tobramycin, amikacin) and / or fluoroquinolone.
  • beta-lactam ceftazidime, imipenem or meropenem, piperacillin / tazobactam
  • aminoside tobramycin, amikacin
  • fluoroquinolone fluoroquinolone
  • Intestinal infections are also an important cause of hospitalization and in some cases of death.
  • the most common causes of acute gastroenteritis in immunocompetent adults are noroviruses and rotaviruses, as well as the following species and genera of bacteria: Campylobacter spp., Salmonella spp., Escherichia coli, Staphylococcus aureus , Bacillus cereus, and Clostridium difficile.
  • Type III interferons also known as lambda interferons (1), constitute the first line of antimicrobial defense in the epithelium of the respiratory and intestinal tracts, contributing to the initial inhibition of the spread of pathogens, without, however, triggering inflammatory response (Andreakos et al., 2017). These type III interferons are actually produced very early in response to pathogen activation of host cell sensors, Toll-like receptors or cytoplasmic effectors.
  • ISGs Interferon - stimulated genes
  • IFITM gene the cell entry of viruses
  • OAS gene OASL, IFIT1, IFIT2, IFIT13, ISG15
  • type III interferon type This early anti-pathogenic response, called “type III interferon type", is particularly prevalent in the initial innate response of the respiratory epithelium to influenza virus infection (Galani et al., 2017).
  • Type III interferons have been previously evaluated in clinical trials to treat hepatitis B and C infections, and have demonstrated lower side effects compared to type I interferon therapy (Chan et al., 2016).
  • the use of type III interferons as a non-inflammatory antiviral treatment against influenza virus infections has recently been proposed (Davidson et al., 2016).
  • Diltiazem is a molecule member of the benzothiazepine family, registered under the number CAS 42399-41-7. This molecule may be in the form of two cis- and cis-cis enantiomers, or a racemic mixture.
  • Diltiazem has been known for over 30 years and is approved in Europe and the United States by regulatory authorities for the drug. It can be administered as diltiazem hydrochloride. Cardizem®, Cartia®, Taztia® and Dilacor® are its most common brand names.
  • Diltiazem is available in various dosage forms, such as in the form of cream for topical application, in the form of tablets or capsules for oral administration, in powder form for preparation of solution for injection or in the form of pharmaceutical preparations for inhalation (WO 02 / 094238, US 4,605,552). Its known physiological action is the inhibition of calcium channels, and thus the inhibition of intracellular calcium fluxes. In particular, diltiazem inhibits the entry of transmembrane calcium into the myocardial muscle fiber and the smooth muscle fiber of the vessels. This makes it possible to reduce the intracellular calcium concentration reaching the contractile proteins.
  • diltiazem In humans, administration of diltiazem is indicated for its vasodilator action, in order to reduce cardiac work. It is thus used in the management of cardiac and circulatory disorders such as angina pectoris, arterial hypertension, myocardial ischemia and tachycardia.
  • WO 201 1/066657 describes the use of a calcium channel blocker, such as verapamil or diltiazem, for the treatment or prevention of viral and / or bacterial infections, or autoimmune diseases .
  • the viruses concerned include those of oral herpes, genital herpes and shingles.
  • diltiazem inhibits the interaction between the virus and the calcium channels, and thus blocks the entry of the virus into the cells.
  • EP 1 1 17 408 describes the use of diltiazem, as a calcium channel blocker compound, to treat pathologies related to the degeneration of photo receptors in the retina.
  • diltiazem as a calcium channel blocker compound
  • the technical problem underlying the present invention relates to the identification of molecules for stimulating the expression of genes encoding interferon III type proteins, for the purpose of preventing and / or treating infections by pathogenic microorganisms of the epithelia respiratory and / or intestinal tracts.
  • Therapeutic compounds having the ability to stimulate the expression of genes encoding proteins such as interleukin 29, interleukin 28A and / or interleukin 28B are therefore actively sought.
  • diltiazem a calcium channel blocker
  • diltiazem also stimulates the expression of these genes encoding interferon III type proteins, both in vitro and in vitro. vivo.
  • ISG genes Interferon-stimulated genes
  • the proteins encoded by the ISG genes participate in the antimicrobial action and / or participate in "interferon” signaling.
  • diltiazem can be used for various therapeutic applications, and in particular for preventing and / or treating infections with at least one pathogenic microorganism of the epithelia of the respiratory and / or intestinal tracts.
  • Diltiazem as an activating agent for the expression of genes encoding one or more interferon (s) type III, is particularly suitable for the treatment of viral or bacterial infections, but also co-infections related to the presence of at least one virus and at least one bacterium, or at least two viruses, or at least two bacteria, and in particular is suitable for the treatment of bacterial superinfections concomitant with viral primo-infection.
  • interferon (s) type III is particularly suitable for the treatment of viral or bacterial infections, but also co-infections related to the presence of at least one virus and at least one bacterium, or at least two viruses, or at least two bacteria, and in particular is suitable for the treatment of bacterial superinfections concomitant with viral primo-infection.
  • the present invention relates to diltiazem for its use as an activating agent for the expression of at least one gene encoding a type III interferon.
  • the present invention relates to diltiazem for its use as an activating agent for the expression of at least one gene encoding a type III interferon, in the prevention and / or treatment of infections by at least one pathogenic microorganism of the epithelia of the respiratory and / or intestinal tracts.
  • the present invention also relates to diltiazem for its use as an activating agent for the expression of at least one gene called "ISG", in the prevention and / or treatment of infections with at least one pathogenic microorganism of the epithelia of respiratory and / or intestinal tracts.
  • ISG diltiazem
  • the present invention also relates to a pharmaceutical or veterinary composition
  • a pharmaceutical or veterinary composition comprising diltiazem as an activating agent for the expression of at least one gene encoding a type III interferon, for its use in the prevention and / or treatment of infections by pathogenic microorganisms of the epithelia of the respiratory and / or intestinal tracts.
  • the pharmaceutical or veterinary composition is characterized in that it is in a dosage form suitable for administration by inhalation.
  • Figure 1 Characterization of the induction of expression of type III interferon genes by basolateral treatment with diltiazem of the 3D model of reconstituted human respiratory epithelium of nasal origin (MucilAir® HAE, Epithelix).
  • Reconstituted human respiratory epithelia (MucilAir® HAE, Epithelix) were treated or not (mock) with a single dose of 90 ⁇ M diltiazem administered by the apical pole on day 0.
  • the cells were lysed and the total RNA was extracted.
  • RT-qPCR TaqMan, Thermo Fisher Scientific. Data were normalized via the household GAPDH gene. The expression ratios were made by calculations based on the 2AACt method (Livak and Schmittgen, 2001).
  • FIG. 4 Diltiazem treatment reduces respiratory syncytial virus (RSV) replication in infected reconstituted human respiratory epithelia (MucilAir® HAE, Epithelix) and BALB / c mouse model after infectious challenge (in vivo).
  • RSV respiratory syncytial virus
  • 4A Schematic representation of the chronology of treatments via the basolateral pole of human respiratory epithelia: after infection with RSV Court 0), the reconstituted epithelia are treated or not (untreated) at 5h post-infection (5hpi) and daily the next three days (1, 2 and 3 days pi) diltiazem. Viral quantification is performed at 6 days pi.
  • 5A- Schematic representation of the chronology of treatments via the basolateral pole of human respiratory epithelia: after infection with the hPIV-3 virus at a MOI of 0, 1 Court 0), the reconstituted epithelia are treated or not (untreated) at 5h post-infection (5hpi), then daily the following four days (24, 48, 72 and 96 hours pi) diltiazem.
  • the viral quantification is carried out at 120 hours pi by infectious titration (TCID50 / mL).
  • TEER transepithelial electrical resistance
  • the viral titres (DITC50 / mL) of samples taken at the apical pole of the epithelia were determined in LLC-MK2 cells from washings taken from 48 h post-infection and up to 120 hpi.
  • viral titers measured at the apical surface of reconstituted human respiratory epithelia peaked at 72 h post-infection at a value of approximately 10 8 TCID50 / mL and at 96 h post-infection at a value of 10 7 TCID50 / mL, unlike diltiazem-treated reconstituted human respiratory epithelia, which show significantly lower values at 72 hours post-infection (approximately 10 6 TCID50 / mL) and 96 hours post-infection (10 5 TCID50 / mL ).
  • FIG. 6A Schematic representation of the chronology of treatment of human respiratory epithelia: reconstituted human respiratory epithelia were treated 24 hours before infection with 90 ⁇ M diltiazem via their basolateral medium or alternatively with 10 ⁇ L of diltiazem (90 ⁇ M) at their apical surface, then infected with Pseudomonas aeruginosa (PAK strain) at an MOI of 1 and again treated with diltiazem 2 hours after infection under the same conditions, respectively.
  • PAK strain Pseudomonas aeruginosa
  • FIG. 7A Schematic representation of the chronology of treatments for human respiratory epithelia: after infection with a recombinant hMPV-GFP virus (strain C-85473) at an MOI of 0.1, the epithelia were treated (via their basolateral medium) or not (3 epithelia per condition), with 3 successive doses of diltiazem at 90 ⁇ M at day (5 hpi), D1 and D3 post-infection. Viral quantification is performed at 3 days pi (supernatant) and at 5 days pi (total epithelium).
  • RNAs corresponding to the viral N gene of hMPV were quantified by RT-qPCR (Biosystems TM PowerUp TM SYBR TM Green, Thermo Fisher Scientific), using a range made from a plasmid containing the N gene. of HMPV. The results are expressed as copies of the N / pg gene of total RNA extracted.
  • mice are treated (20 mg / kg) or not (PBS control group "mock-treated") at day 0 (OJ), then treated or not, either daily on the following two days (OJ, D1 and D2) or every 48 hours until day 4 (OJ, D2 and D4).
  • OJ intranasal instillation
  • IFNk2 The expression of the IFNk2 gene (IFNL2) was measured by RT-qPCR and is represented as the ratio of expression relative to the basal level (equal to 1) of the expression of the gene measured in untreated mice (Mock -treaty).
  • the present invention relates to a new therapeutic use of a known drug, diltiazem, a molecule member of the benzothiazepine family referenced under the CAS number 42399-41-7, of formula (1):
  • the term "diltiazem” means diltiazem in all its forms, especially in the form of salts and in particular in the form of diltiazem hydrochloride. This term includes the racemic mixture as well as each of the enantiomers when these are isolated. This term also includes diltiazem derivatives, ie molecules derived from formula (1), having the same biological activity for stimulating the expression of type III interferon proteins.
  • the present invention relates to diltiazem for its use as an activating agent for the expression of at least one gene encoding a type III interferon.
  • diltiazem has been used for its calcium channel inhibitory action; by this inhibition of intracellular calcium transport, the administration of diltiazem generates many physiological effects on the organisms of humans and animals.
  • the inventors have demonstrated a new technical effect of diltiazem, namely the activation of the expression of one or more genes coding for type III interferons.
  • the gene or genes whose expression is activated are endogenous genes, that is to say the native genes of the organism considered, which have not undergone any genetic modification.
  • the present invention relates to diltiazem for use as an activating agent for the expression of genes encoding interleukin 29, interleukin 28A and interleukin 28B.
  • a therapeutic compound for activating the "interferon III pathway” that is to say an activating agent for the expression of one or more genes coding for type interferons. III. This is the first compound with this activity to be identified.
  • Type III interferons also known as lambda interferons (1), constitute the first line of defense of a human or animal organism against infection by pathogenic microorganisms.
  • This new family of interferons described for the first time in 2003, includes the following four proteins in humans:
  • Interferon lambda-1 IFN-11
  • interleukin 29 IL-29
  • Interferon lambda-2 IFN-72
  • interleukin 28A IL-28A
  • Interferon lambda-3 IFN-73
  • interleukin 28B IL-28B
  • Interferon lambda-4 (IFN-74).
  • mice In mice, only two proteins belonging to this interferon III family have so far been identified (IFN-A2 / IL-28A and IFN-A3 / IL-28B).
  • IFN-7R1 receptor also known as IL-28RA.
  • a heteromeric complex is formed between this receptor and the IL-10R2 receptor to bind FN-1 monomers (see Donnelly & Kotenko, 2010, for review).
  • gene encoding a type III interferon means one of the following genes, or their homologs:
  • genes mentioned above are the human genes, but that if diltiazem is used in a different animal species, the genes encoding the interferons type
  • Example 1 of the present application demonstrates that diltiazem induces the expression of several so-called “interferon-stimulated” (ISG) genes, and in particular the “immunity” genes, following an apical or basolateral treatment.
  • ISG interferon-stimulated
  • diltiazem induces the expression of ISG well described in the literature such as IFI44L, IFIT2 OAS1, IRF7, MX1 or IFITM1.
  • IFN-RI receptor Infections by pathogenic microorganisms of the epithelia of the respiratory and / or intestinal tracts
  • the IFN-RI receptor is exclusively expressed by epithelial cells, thus limiting the effects of type III interferons to epithelia.
  • the epithelia of the respiratory and intestinal tracts are the first affected organs, due to their direct contact, respectively, with the air drawn in, and the water and food ingested.
  • the present invention particularly relates to diltiazem for its use as an activating agent for the expression of at least one gene encoding a type III interferon, in the prevention and / or treatment of infections with at least one a pathogenic microorganism of the epithelia of the respiratory and / or intestinal tracts.
  • infection by at least one pathogenic microorganism of the epithelia of the respiratory and / or intestinal tracts must be understood as being an infection generated by the presence of at least one pathogenic microorganism, this microorganism having infected the individual or the animal, or likely to infect the individual or animal.
  • the present invention relates both to the prevention and / or treatment of infections affecting humans (also referred to as "individuals”), as infections affecting animals, including livestock.
  • prevention refers to preventing, or at least decreasing the probability of occurrence, an infection in a human or animal body by at least one pathogenic microorganism. Under the action of type III interferons produced, the tissues of the body and especially the epithelia become more resistant and are more likely not to be infected and / or to limit infection by said microorganism.
  • diltiazem is used as an activating agent for the expression of at least one gene coding for a type III interferon, in the prevention of infections by at least one pathogenic microorganism. epithelia from the respiratory and / or intestinal tracts
  • treatment refers to fighting infection with at least one pathogenic microorganism in a human or animal organism. Thanks to the administration of diltiazem, the rate of viral, bacterial, fungal or parasitic infection in the body will gradually decrease or even disappear completely.
  • treatment also means attenuating the symptoms associated with the infection (fever, fatigue, etc.) and / or preventing / reducing the risk of complications, especially superinfection.
  • diltiazem is used as an activating agent for the expression of at least one gene encoding a type III interferon, in the treatment of infections with at least one pathogenic microorganism. epithelia from the respiratory and / or intestinal tracts.
  • the present invention relates to diltiazem for its use as an activating agent for the expression of at least one gene encoding a type III interferon, in the prevention and / or treatment of infections with less a pathogenic microorganism of the epithelia of the respiratory and / or intestinal tracts, characterized in that the said at least one gene is chosen from the gene coding for interleukin 29, the gene coding for interleukin 28A and the gene coding for interleukin 28B.
  • diltiazem is used as an activating agent for the expression of the three genes coding for interleukin 29, for interleukin 28A and for interleukin 28B.
  • pathogenic microorganisms denotes any microorganism capable of creating a disease in other organisms, such as in humans or animals. This term includes viruses, bacteria, fungi, protozoa, worms, and other unicellular pathogenic microorganisms.
  • the "pathogenic microorganism” is a microorganism sensitive to the cellular response of "interferon III” type, that is to say a microorganism whose cell infection will be prevented or inhibited, totally or partially, by the expression and secretion of interferon III-like proteins.
  • the pathogenic microorganism is a microorganism that specifically infects the epithelia of the respiratory tract.
  • the pathogenic microorganism is a microorganism that specifically infects the epithelia of the intestinal tracts.
  • the pathogenic microorganism is a microorganism capable of infecting all types of epitheliums and in particular those of the respiratory and intestinal tracts.
  • the infection is a viral infection, that is to say that the pathogenic microorganism of the epithelia of the respiratory and / or intestinal tracts is a virus.
  • Proteins belonging to the type III interferon family are essential players in the anti-viral response of virus target epithelia.
  • an activating agent for the expression of the genes coding for said type III interferon proteins makes it possible to stimulate and optimize the anti-viral response of the infected epithelia.
  • the invention relates to diltiazem for its use as an activating agent for the expression of at least one gene encoding a type III interferon, in the prevention and / or treatment of infections by at least one a virus infecting the epithelia of the respiratory and / or intestinal tracts.
  • virus includes any type of virus but more particularly the viruses infecting vertebrate eukaryotic organisms. These may be DNA viruses or
  • RNA infecting organisms via the epithelia of the respiratory and / or intestinal tracts.
  • the infection is not an infection with an influenza virus.
  • diltiazem for treating influenza virus infections has already been proposed in WO 2011/126071 and WO 2016/146836.
  • diltiazem is used in the prior art for its action on calcium channels, and not for its activating action of the expression of genes encoding type III interferons.
  • the pathogenic microorganism is a group I virus having a double-stranded DNA genome.
  • This group of viruses includes viruses of the order of Herpes, those of the family of papillomaviruses, as well as polyomaviruses and poxviridae.
  • the pathogenic microorganism is a group II virus having a single-stranded DNA genome, in particular comprising viruses of the parvoviridae family.
  • the pathogenic microorganism is a group III virus having a double-stranded RNA genome, including in particular viruses of the rheoviridae family, such as rotaviruses.
  • the pathogenic microorganism is a Group IV virus having a positive-strand single-stranded RNA genome.
  • This group includes:
  • viruses of the order Nidovirales such as those of the coronaviridae family
  • viruses of the family Caliciviridae including the Norwalk virus
  • viruses of the Flaviviridae family including, but not limited to, yellow fever virus, West Nile virus, hepatitis C virus and dengue virus;
  • viruses of the family Picornaviridae including poliovirus, rhinovirus, and hepatitis A virus;
  • viruses of the family Togaviridae including rubella virus, Ross River virus, Sindbis virus and Chikungunya virus.
  • the pathogenic microorganism is a group V virus having a negative-strand single-stranded RNA genome (ssRNA).
  • This group includes viruses of the order Mononegavirales, such as:
  • viruses of the Filoviridae family including Ebola virus and Marburg virus, viruses of the family Paramyxoviridae, including measles virus, mumps virus and Flenipaviruses,
  • viruses of the family Rhabdoviridae including the rabies virus
  • viruses of the family Arenaviridae including the Lassa fever virus
  • viruses of the family Bunyaviridae including Flantavirus and fever virus
  • the pathogenic microorganism is a virus chosen from human respiratory syncytial virus (hVRS), parainfluenza virus (hPIV), human metapneumovirus (hMPV), Nipah virus, novovirus, Swine virus. fever, adenovirus, coronavirus, Zika virus, yellow fever virus, rheovirus, rotavirus, Dengue virus, and West Nile virus.
  • hVRS human respiratory syncytial virus
  • hPIV parainfluenza virus
  • hMPV human metapneumovirus
  • Nipah virus novovirus
  • Swine virus fever, adenovirus, coronavirus, Zika virus, yellow fever virus, rheovirus, rotavirus, Dengue virus, and West Nile virus.
  • the pathogenic microorganism is a virus selected from human respiratory syncytial virus (hVRS), parainfluenza viruses (hPIV) and human metapneumovirus (hMPV).
  • hVRS human respiratory syncytial virus
  • hPIV parainfluenza viruses
  • hMPV human metapneumovirus
  • the invention relates to diltiazem for its use as an activating agent for the expression of at least one gene coding for a type III interferon, in the prevention and / or treatment of infections. by human respiratory syncytial virus (hRSV).
  • hRSV human respiratory syncytial virus
  • the invention relates to diltiazem for use in the prevention and / or treatment of human respiratory syncytial virus (hRSV) infections.
  • the invention relates to diltiazem for its use as an activating agent for the expression of at least one gene coding for a type III interferon, in the prevention and / or treatment of infections. by human parainfluenza virus (hPIV).
  • hPIV human parainfluenza virus
  • the invention relates to diltiazem for use in preventing and / or treating infections with human parainfluenza virus (hPIV).
  • hPIV human parainfluenza virus
  • the invention relates to diltiazem for its use as an activating agent for the expression of at least one gene coding for a type III interferon, in the prevention and / or treatment of infections. by the human metapneumovirus (hMPV).
  • hMPV human metapneumovirus
  • the invention relates to diltiazem for use in the prevention and / or treatment of human metapneumovirus (hMPV) infections.
  • hMPV human metapneumovirus
  • the infection is a non-viral infection.
  • the pathogenic microorganism may be selected from a bacterium, a fungus and a parasite.
  • the pathogenic microorganism is a bacterium.
  • bacteria of the following species Enterococcus faecalis, Borrelia burgdorferi, Listeria monocytogenes,
  • Mycobacterium tuberculosis Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Salmonella Typhimurium, Streptococcus pneumoniae and Haemophilus influenzae
  • bacteria specific for intestinal infections such as Campylobacter spp., Salmonella spp., Shigella spp., Yersinia enterocolitica, Vibrio cholerae, Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Clostridium difficile.
  • the pathogenic microorganism is not a bacterium of the genus Chlamydiae.
  • the pathogenic microorganism is a bacterium chosen from the following bacterial species: Enterococcus faecalis, Borrelia burgdorferi, Listeria monocytogenes, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Salmonella Typhimurium, Streptococcus pneumoniae, Haemophilus influenzae, Campylobacter spp., Salmonella spp. , Shigella spp., Yersinia enterocolitica, Vibrio cholerae, Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Clostridium difficile.
  • the pathogenic microorganism is a bacterium of the species Pseudomonas aeruginosa.
  • the invention relates to diltiazem for its use as an activating agent for the expression of at least one gene coding for a type III interferon, in the prevention and / or treatment of infections. by Pseudomonas aeruginosa.
  • the invention relates to diltiazem for use in the prevention and / or treatment of Pseudomonas aeruginosa infections.
  • the pathogenic microorganism is a fungus.
  • Aspergillus fumigatus Candida albicans and Pneumocystis jiroveci, Fusarium solari.
  • the invention relates to diltiazem for its use as an activating agent for the expression of at least one gene coding for a type III interferon, in the prevention and / or treatment of infections.
  • a pathogenic microorganism selected from the following group: Pseudomonas aeruginosa, human respiratory syncytial virus (hVRS), parainfluenza viruses (hPIV) and human metapneumovirus (hMPV).
  • the invention relates to diltiazem for its use in the prevention and / or treatment of infections by a pathogenic microorganism selected from the following group: Pseudomonas aeruginosa, human respiratory syncytial virus (hVRS), parainfluenza viruses (hPIV) and the human metapneumovirus (hMPV).
  • a pathogenic microorganism selected from the following group: Pseudomonas aeruginosa, human respiratory syncytial virus (hVRS), parainfluenza viruses (hPIV) and the human metapneumovirus (hMPV).
  • At least one pathogenic microorganism means that one or more pathogenic microorganisms are present in the infected organism, thus generating an immune response of the organism.
  • the body may have been infected with both a virus and a bacterium, which is referred to as a co-infection of the body.
  • the invention relates to diltiazem for its use as described above, characterized in that the infection is a co-infection with at least one virus and at least one bacterium.
  • diltiazem is then used for the concomitant treatment of a viral infection and a bacterial infection, which makes it possible to limit the number of active compounds administered to an individual or an animal suffering from this co-infection.
  • this co-infection may be related to the presence in the infected epithelia of a combination of pathogenic microorganisms chosen from the following:
  • a human respiratory syncytial virus and at least one bacterium of a species selected from Enterococcus faecalis, Borrelia burgdorferi, Listeria monocytogenes, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Salmonella typhimurium, Streptococcus pneumoniae and Haemophilus influenzae.
  • hRSV human respiratory syncytial virus
  • hPIV parainfluenza virus
  • bacterium of a species selected from Enterococcus faecalis, Borrelia burgdorferi, Listeria monocytogenes, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Salmonella typhimurium, Streptococcus pneumoniae and Haemophilus influenzae,
  • human metapneumovirus and at least one bacterium of a species selected from Enterococcus faecalis, Borrelia burgdorferi, Listeria monocytogenes, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Salmonella typhimurium, Streptococcus pneumoniae and Haemophilus influenzae,
  • West Nile virus and at least one bacterium of a species selected from Enterococcus faecalis, Borrelia burgdorferi, Listeria monocytogenes, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Salmonella typhimurium, Streptococcus pneumoniae and Haemophilus influenzae, and Influenza virus and at least one bacterium of a species selected from Enterococcus faecalis, Borrelia burgdorferi, Listeria monocytogenes, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Salmonella typhimurium, Streptococcus pneumoniae and Haemophilus influenzae.
  • Influenza virus and at least one bacterium of a species selected from
  • ii) Prevent co-infection by at least one other pathogenic microorganism of epithelia from the respiratory and / or intestinal tracts, such as bacterial superinfection.
  • diltiazem as an activating agent for the expression of at least one gene coding for a type III interferon will be used for both:
  • diltiazem as an activating agent for the expression of at least one gene coding for a type III interferon will be used for both:
  • the present invention also relates to diltiazem for its therapeutic use as presented above, characterized in that it is used in combination with at least one other active agent, that is to say with one or more active agents.
  • this other active agent is a therapeutic compound having a beneficial action on a human or animal body, for the purpose of preventing or treating a pathology.
  • This other active agent will be chosen in particular from an anti-viral compound, an antibacterial compound, an anti-parasitic compound, an anti-fungal compound, and a preventive or therapeutic vaccine.
  • this other active agent is chosen from an antiviral compound and an anti-bacterial compound.
  • antiviral compound means a compound having either a direct inhibitory action on at least one virus (for example, inhibiting its replication) or an action on a target cell of the virus (for example, in inducing a cellular state unfavorable to a viral infection, thus preventing the viral infection).
  • Anti-viral agents are classified into different categories according to their mode of action. We can mention in particular:
  • nucleotide analogues which interfere with or stop the synthesis of DNA or RNA; as well as inhibitors of the enzymes involved in the synthesis of DNA or RNA (helicase, replicase);
  • antiviral agent (s) is (are) chosen from:
  • viral agents having a direct inhibitory action on viruses, such as for example ribavirin and favipiravir;
  • Inhibitors of the penetration of a virus into the cells such as, for example, arbidol; Monoclonal antibodies, such as, for example, palivizumab, directed against an epitope of the antigenic site A of the respiratory syncytial virus (RSV) fusion protein; and
  • RSV respiratory syncytial virus
  • Antisense oligonucleotides having a virus inhibitory action are provided.
  • anti-bacterial compound means a compound having an anti-bacterial activity, that is to say, inhibiting the replication of bacteria (bacteriostatic compounds) or destroying them (bactericidal compounds) or by inhibiting their biosynthesis and / or secretion of toxic products. These are in particular antibiotics.
  • anti-bacterial agent (s) is (are) chosen from:
  • antibiotics especially those of the macrolide family, and especially roxithromycin
  • the other active agent used in combination with diltiazem is an anti-fungal compound or an anti-parasitic compound, especially chosen from systemic anti-fungal compounds (amphotericin B, azoles, echinocandins). and systemic anti-parasitic compounds (e.g., anti-malarial, antiamibian, toxoplasmosis, leishmaniasis, pneumocytosis, anthelmintic).
  • systemic anti-fungal compounds amphotericin B, azoles, echinocandins
  • systemic anti-parasitic compounds e.g., anti-malarial, antiamibian, toxoplasmosis, leishmaniasis, pneumocytosis, anthelmintic.
  • the at least one other active agent is a vaccine.
  • the term "vaccine” means a compound or a combination of compounds that specifically stimulate the immune system of a human or animal organism.
  • a vaccine will include in particular an antigen, ie a compound inducing a specific immune response in the body, which will keep the memory.
  • Such a vaccine may be preventive vaccine type, that is to say, intended to stimulate a specific immune response before infection of an organism by a pathogenic microorganism.
  • Nonlimiting examples that may be mentioned as examples are different types of vaccines, classified according to the nature of the antigens from which they are prepared.
  • the antigens conventionally used are the following: inactivated infectious agents, agents live attenuated subunits of infectious agents, toxoids, viral vectors expressing antigens from pathogens, vectors carrying nucleic acids (DNA or RNA), and antibodies.
  • Such a vaccine may also be therapeutic vaccine type, that is to say, intended to stimulate a specific immune response concomitantly with the infection of an organism with a pathogenic microorganism.
  • this vaccine may be administered before, during or after treatment with diltiazem.
  • the present invention also relates to a pharmaceutical or veterinary composition
  • a pharmaceutical or veterinary composition comprising diltiazem as an activating agent for the expression of at least one gene encoding a type III interferon.
  • the present invention also relates to a pharmaceutical or veterinary composition
  • a pharmaceutical or veterinary composition comprising diltiazem as an activating agent for the expression of at least one gene encoding a type III interferon, for its use in the prevention and / or treatment of infections by pathogenic microorganisms of the epithelia of the respiratory and / or intestinal tracts.
  • composition according to the invention will comprise diltiazem as well as a suitable pharmaceutical vehicle, and optionally another active agent.
  • suitable pharmaceutical carrier means pharmaceutically acceptable vehicles or excipients, ie vehicles or excipients whose administration to an individual or animal is not accompanied by significant deleterious effects, which are well known to those skilled in the art.
  • composition according to the invention can be adapted for any type of administration and in particular for oral, sublingual, nasal and / or oral administration by inhalation, subcutaneous, intramuscular, intravenous, transdermal, ocular or rectal administration.
  • Suitable dosage forms of administration may for example be tablets, capsules, powders, granules and solutions or suspensions.
  • the pharmaceutical or veterinary composition is characterized in that it is in a dosage form suitable for administration by inhalation, that is to say by the nasal and / or oral routes.
  • Inhalation refers to absorption through the respiratory tract. It is a method of absorbing therapeutic compounds in the form of gas, microdroplets or powder in suspension.
  • compositions are in the form of aerosols (suspensions) or in the form of solutions, for example of aqueous solutions, put under pressure.
  • a nebulizer or a sprayer will then be recommended for administering the pharmaceutical or veterinary composition.
  • the dosage form suitable for administration of diltiazem by inhalation is selected from: a powder, an aqueous suspension of droplets or a solution under pressure.
  • the pharmaceutical or veterinary composition comprises an effective amount of diltiazem, for its use as an activating agent for the expression of at least one gene encoding a type III interferon, in the prevention of and / or treating infections with at least one pathogenic microorganism of the epithelia of the respiratory and / or intestinal tracts.
  • the present invention also relates to a combination product comprising: diltiazem as an activating agent for the expression of at least one gene encoding a type III interferon, and
  • At least one other active agent selected from an anti-viral compound, an anti-bacterial compound, and an agent for preventing infections by pathogenic microorganisms,
  • the present invention also relates to a method for preventing and / or treating an infection in an individual infected or susceptible to infection by at least one pathogenic microorganism of the epithelia of the respiratory and / or intestinal tracts, including the administration of diltiazem to this individual, to activate the expression of at least one gene encoding a type III interferon.
  • the present invention also relates to a method for preventing and / or treating an infection in an individual infected or susceptible to infection by at least one pathogenic microorganism of the epithelia of the respiratory and / or intestinal tracts, including the administration of diltiazem to this individual, diltiazem being used as an activating agent for the expression of at least one gene encoding a type III interferon.
  • the present invention also relates to a method for preventing and / or treating an infection in an animal infected or susceptible to infection by at least one pathogenic microorganism of the epithelia of the respiratory and / or intestinal tracts, comprising the administration of diltiazem to this animal, to activate the expression of at least one gene encoding a type III interferon.
  • This administration of diltiazem will be carried out, preferably, by inhalation.
  • Reconstituted human respiratory epithelia (MucilAir® HAE, Epithelix) and maintained in culture at the air-liquid interface according to the instructions of the supplier Epithelix were treated or not with diltiazem (90 ⁇ M) via their culture medium at their basolateral pole .
  • Diltiazem treatment was repeated for 3 consecutive days (3 administrations in total).
  • CDNA libraries were prepared from 200 ng of total RNA using the Scriptseq TM kit supplemented with Gold kit-Low Input (SCL6EP, Epicenter) according to the supplier's instructions. Each library was amplified, quantified and indexed with primers provided in the ScriptSeq TM PCR Kit Primers kit (RSBC10948, Epicenter) and sequenced. Sequencing was conducted on an Illumina HiSeq 2500 system with a minimum requirement of 40 million sequenced reads per sample.
  • the demultiplexing of the data and the conversion of the BCL files resulting from sequencing into FASTQ files were carried out using the Illumina tool bcl2fastq, version 1.8.4.
  • the FastQC software http://www.bioinformatics.babraham.ac.uk/projects/fastqc) provided the necessary quality controls on the raw data. Trimming was done with the Trimmomatic tool, with a minimum quality threshold equal to Q30.
  • a pseudo-alignment of the reads trimmers on the human genome (Homo Sapiens: GRCh38.p1 1) by the software Kallisto was realized, followed by a statistical analysis using the software R 3.3.1 and the package EdgeR 3.14.0.
  • FIG. 1A shows the expression ratios of the IFN-II, IRN-12 and IFN-10 type III interferon genes observed between diltiazem-treated human respiratory epithelia and untreated human respiratory epithelia.
  • FIG. 1B shows the expression ratios of the type III interferon response genes such as IFI44L, IFIT1, IFIT2, IFIT3, IFITM1, ISG15, MX1, MX2, OAS1, OAS2, OAS3, OASL, RSAD2 and STAT1 observed between diltiazem-treated human respiratory epithelia and untreated control human respiratory epithelia.
  • diltiazem treatment of reconstituted human respiratory epithelia for 3 days significantly alters the level of expression of genes encoding type III IFN-11, IRN-12 and IRN-13 increasing significantly (from 100 to more than 200 fold in comparison with untreated control epithelia).
  • this treatment also very significantly stimulates the expression of type III interferon response genes such as IFI44L, IFIT1, IFIT2, IFIT3, IFITM1, ISG15, MX1, MX2, OAS1, OAS2, OAS3, OASL, RSAD2 and STAT1. .
  • Figure 1C presents the results obtained 24 hours after the apical pole treatment of reconstituted human epithelial models (MucilAir® HAE, Epithelix) with diltiazem, on endogenous IFNL1 (interferon lambda I) gene expression, in comparison endogenous untreated epithelium (control). The measurement was performed by RT-qPCR.
  • results indicate that the treatment of diltiazem-reconstituted human respiratory epithelia via their apical pole induces significant overexpression of the IFNL1 gene, with an expression ratio greater than 6: 1 compared to control epithelia (untreated).
  • Reconstituted human respiratory epithelia (MucilAir® HAE, Epithelix) were maintained in culture at the air-liquid interface according to the instructions of the Epithelix supplier, and were treated or not diltiazem (90 ⁇ M) via their culture medium at their temperature. basolateral pole. Diltiazem treatment was repeated for 3 consecutive days (3 administrations in total).
  • Quantitative PCR primers (GAPDH: Hs02758991_g1, IFNL1: Hs00601677_g1, IFNL2: Hs00820125_g1, IFIT1: Hs01675197_m1, IFIT2: Hs00533665_m1, IFIT3: Hs00382744_m1, IFI27: Hs01086373_g1, IFI44L: Hs00915292_m1, IFITM1: Hs01652522_g1) and probes (TaqMan gene assays term ) were provided by Thermo Fisher Scientific.
  • the expression ratios of the IFIT1, IFIT2, IFIT3, IF127, IFN-II, IFN-72 IFI44L and IFITM1 genes between the diltiazem treated and untreated cells were determined by the 2AACt method (Livak Schmittgen, 2001).
  • Figure 2 presents the results obtained: after treatment with diltiazem, the reconstituted epithelia show a significant increase in the expression of IFIT1, IFIT2, IFIT3, IF127, IFN-II, IFN-72, IFI44L and IFITM1 genes compared to untreated epithelia
  • Example 3 ELISA assay of diltiazem-stimulated secretion of type III lambda interferon (IL-29).
  • Reconstituted human respiratory epithelia (MucilAir® HAE, Epithelix) were maintained in culture at the air-liquid interface according to the instructions of the Epithelix supplier, and were treated or not diltiazem (90 ⁇ M) via their culture medium at their temperature. basolateral pole. Diltiazem treatment was repeated for 3 consecutive days (3 administrations in total).
  • Figure 3 shows the results obtained: the secretion of IFN-II (IL-29) is very clearly increased at the apical pole following treatment with diltiazem via the epithelial culture medium; a significant but less marked increase is also observed at the basolateral pole of treated epithelia.
  • Example 4 Diltiazem treatment significantly reduces replication of respiratory syncytial virus.
  • Reconstituted human respiratory epithelia (MucilAir® HAE, Epithelix) were maintained in culture at the air-liquid interface according to the instructions of the Epithelix supplier, and were infected with respiratory syncytial virus (Long strain, ATCC VR-26) at a multiplicity of infection (MOI) 1, then treated or not with diltiazem (90 ⁇ M) via their culture medium at their basolateral pole. Diltiazem treatment was performed at 5 hours post-infection and repeated for 3 consecutive days (4 administrations in total).
  • Figures 4B and 4C show that when reconstituted human respiratory epithelia were treated post-infection with diltiazem (90 ⁇ M) via their basolateral pole, the amount of viral genome is much lower both in the culture supernatant (FIG. 4B) and in the epithelium ( Figure 4C) with respect to untreated epithelia.
  • the diltiazem therefore has an effect significant antiviral against respiratory syncytial virus in model of reconstituted human respiratory epithelia.
  • Diltiazem (oral) treatment reduces the replication of respiratory syncytial virus (RSV) in the BALB / c mouse model after infectious challenge.
  • RSV respiratory syncytial virus
  • Figure 4D schematically shows the chronology of infection and treatment of mice:
  • mice BALB / c mice were randomized into 3 groups of 5 individuals and then infected intranasally with 50 ⁇ l of a virus suspension containing 5 ⁇ 10 5 PFU of RSV (Day 0). Six hours before infection, the mice were treated with either 40 mg / kg of intraperitoneal Ribavirin (RSV RIB) or 50 mg / kg of oral diltiazem (RSV DIL), or PBS (RSV PBS) as a negative control.
  • RSV RIB intraperitoneal Ribavirin
  • RSV DIL oral diltiazem
  • PBS PBS
  • mice of each group were euthanized and their lungs were recovered aseptically to measure their pulmonary viral titers by RTqPCR (F gene).
  • RSV DIL Diltiazem-treated animals
  • RV PBS mice in the control group
  • RV IBR ribavirin-treated group
  • Diltiazem therefore has a significant antiviral effect against respiratory syncytial virus in murine model in vivo.
  • Example 5 Diltiazem treatment significantly reduces parainfluenza virus type 3 replication
  • the experimental scheme is shown in Figure 5A.
  • Reconstituted human respiratory epithelia were infected with human parainfluenza virus type 3 (strain C243 ATCC VR-93) at an MOI of 0, 1, and treatment of basolateral medium with 90 ⁇ M diltiazem was started 2 hours post-infection and continued daily for 4 consecutive days.
  • the transepithelial electrical resistance (TEER) which reflects the integrity of the epithelium, was measured once daily from 48 hours post-infection up to 120 hours post-infection.
  • Apical infectious viral titers were determined in LLC-MK2 cells from washings taken as early as 48 h post-infection (hpi), and up to 120 hpi, as shown in Figure 5A.
  • FIG. 5B shows the results obtained after carrying out the following experiment: at 96 and 120 hpi, the untreated reconstituted human respiratory epithelia (images on the left) and treated with diltiazem (right images) were observed under a microscope.
  • the cytopathic effects induced by hPIV-3 were larger and easily visible under the microscope in untreated reconstituted human respiratory epithelia, unlike those treated with diltiazem.
  • Figure 5C shows TEER values as a function of post-infection time, measured on reconstituted human respiratory epithelia:
  • Figure 5D shows the measured viral titers (in LLC-MK2 (DITC50 / mL) over time (48 to 120 hpi) at the apical surface of diltiazem-treated, diluted, reconstituted human respiratory epithelia.
  • infectious viral titres measured at the apical surface of reconstituted human respiratory epithelia peaked at 72 hours post-infection at a value of 10 8 TCID50 / mL) and 96 hours post-infection at a value of 10 7 TCID50 / mL, unlike diltiazem-treated reconstituted human respiratory epithelia, which show significantly lower values at 72 hours post-infection (10 6 TCID50 / mL) and 96 hours post-infection (10 5 TCID50 / mL) . Overall, these results indicate that diltiazem treatment reduces the viral replication of hPIV-3 in the model of infected reconstituted human respiratory epithelium.
  • Example 6 Diltiazem treatment significantly reduces the multiplication of the bacterium Pseudomonas aeruginosa
  • Reconstituted human respiratory epithelia were pretreated 24 hours before infection with 90 ⁇ M diltiazem in basolateral medium or alternatively with 10 ⁇ L of 90 ⁇ M diltiazem at the apical surface, then infected with Pseudomonas aeruginosa (PAK strain) at an MOI of 1 and treated again. by diltiazem 2 hours after infection.
  • Pseudomonas aeruginosa PAK strain
  • dilitiazem treatment limits the replication of Pseudomonas aeruginosa in the human respiratory epithelium.
  • Diltiazem treatment significantly reduces the multiplication of human metapneumovirus (hMPV).
  • Viral replication in reconstituted human epithelial (MucilAir® FIAE, Epithelix) models of nasal origin has been studied in diltiazem-treated or non-dilated epithelia.
  • fluorescence optical microscopy photos were used to evaluate the efficacy and progression of the infection and
  • viral genome quantification by RTqPCR was performed, on the one hand at the apical pole of the epithelium (harvest at the surface) to quantify the excretion of the viral offspring, and secondly in the lysates Epithelial cell totals end-point Cour 5 post-infection) to quantify intracellular viral replication within the epithelium.
  • the human respiratory epithelia (Mucilair, Epithelix) were infected with a recombinant hMPV-GFP virus (strain C-85473) at a MOI of 0.1, then treated (via the basolateral epithelium medium) or not with 3 successive doses of diltiazem at 90pM at OJ, D1 and D3 post-infection (3 epithelia per condition).
  • RNA samples were harvested and lysed to extract total RNA.
  • the total RNAs corresponding to the viral N gene of hMPV were quantified by Rt-qPCR (Biosystems TM PowerUp TM SYBR TM Green, Thermo Fisher Scientific), using a range made from a plasmid containing the N gene. of HMPV.
  • the results are expressed as copies of the N / pg gene of extracted RNA ( Figure 7D).
  • Example 8 In vivo diltiazem induction of IRN-12 gene expression (IFNL2) measured by RT-qPCR
  • FIG. 8A schematically represents the chronology of intranasal instillation (in) treatment of BALB / c mice: the mice were treated (20 mg / kg) or not (PBS control group "mock-treated") at day 0 (OJ) , then treated or not, either daily on the following two days (OJ, D1 and D2), or every 48 hours until day 4 (OJ, D2 and D4). At day 5, the nasal cavities were removed after euthanasia of the animals.
  • IRN-12 gene (IFNL2) was measured by RT-qPCR, and is represented with respect to the basal level (equal to 1) measured on untreated (Mock-treated) mice in Figure 8B.
  • Galani IE Triantafyllia V, Eleminiadou EE, Koltsida O, Stavropoulos A, Manioudaki M, Thanos D, Doyle SE, Kotenko SV, Thanopoulou K, Andreakos E. Interferon-l Medias Non-redundant Front-Line Antiviral Protection against Influenza Virus Infection without Compromising Host Fitness. Immunity. 2017 May 16; 46 (5): 875-890.e6.
  • IFNA is a potent anti-influenza therapeutic with the inflammatory side effects of IFNa treatment. EMBO Mol Med. 2016 Sep 1; 8 (9): 1099-1 12.

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JP2017538749A (ja) * 2014-12-18 2017-12-28 ヘルパービー セラピューティクス リミテッドＨｅｌｐｅｒｂｙ Ｔｈｅｒａｐｅｕｔｉｃｓ Ｌｉｍｉｔｅｄ 抗微生物剤の組み合わせおよび微生物感染の治療におけるそれらの使用
FR3033701B1 (fr) * 2015-03-19 2021-01-15 Univ Claude Bernard Lyon Nouvelles compositions antivirales pour le traitement de la grippe
US11197904B2 (en) * 2018-01-23 2021-12-14 Shoei-Yn Lin-Shiau Pharmaceutical compositions for preventions and managements of dementia, infectious diseases, cancers, periodontitis, dental caries, diabetes, obesity, osteoporosis and chronic pain and methods thereof

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WO2019224489A1 (fr) 2019-11-28
IL278895A (en) 2021-01-31
BR112020023675A2 (pt) 2021-02-17
CN112789046A (zh) 2021-05-11
AU2019273741A1 (en) 2020-12-24
KR20210013136A (ko) 2021-02-03
JP7412353B2 (ja) 2024-01-12
CN112789046B (zh) 2024-02-20
FR3081325A1 (fr) 2019-11-29
FR3081325B1 (fr) 2020-10-09
US20210154205A1 (en) 2021-05-27
CA3101006A1 (fr) 2019-11-28
JP2021525250A (ja) 2021-09-24
MX2020012543A (es) 2021-05-13

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