WO2021018786A1 - Use of ffar2 agonists for the treatment of bacterial superinfections post-viral infection - Google Patents

Use of ffar2 agonists for the treatment of bacterial superinfections post-viral infection Download PDF

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WO2021018786A1
WO2021018786A1 PCT/EP2020/071015 EP2020071015W WO2021018786A1 WO 2021018786 A1 WO2021018786 A1 WO 2021018786A1 EP 2020071015 W EP2020071015 W EP 2020071015W WO 2021018786 A1 WO2021018786 A1 WO 2021018786A1
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carboxylic acid
pyrrolidine
chlorophenyl
carbonyl
biphenyl
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PCT/EP2020/071015
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French (fr)
Inventor
François TROTTEIN
Trond Ulven
Valentin SENCIO
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université de Lille
Institut Pasteur De Lille
Centre National De La Recherche Scientifique (Cnrs)
University Of Copenhagen
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Publication of WO2021018786A1 publication Critical patent/WO2021018786A1/en

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    • 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
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to method and compositions for the treatment of bacterial superinfections post-viral infection (e.g. post-influenza).
  • influenza A virus IAV
  • IAV influenza A virus
  • This inability to control bacterial infection is associated with changes in the numbers and/or functions of innate immune cells, including alveolar macrophages, conventional dendritic cells, neutrophils and non-conventional T cells 4-8 .
  • innate immune cells including alveolar macrophages, conventional dendritic cells, neutrophils and non-conventional T cells 4-8 .
  • the gastrointestinal tract hosts a complex, highly diverse microbial ecosystem.
  • the tightly regulated interplay between the microbiota and the host enables the establishment and persistence of immune homeostasis 11-13 .
  • the commensal microbes impact on host immune responses is not limited to the gut compartment (i.e. barrier functions and gut homeostasis) but also extends to systemic compartments and distant mucosal interfaces, such as the lungs 9 10 .
  • the mechanism by which the gut microbiota regulates the size and/or the functions of the steady-state immune cell pool depends on microbial-associated molecular patterns, microbial metabolites, and their interactions with progenitor cells and mature immune cells 14-18 .
  • nucleotide-binding oligomerization domain (NOD)-like receptor agonist(s) modulate the functions of effector immune cells, including alveolar macrophages and neutrophils.
  • Pathological situations such as infections and chronic inflammatory or metabolic disorders
  • Changes in intestinal bacterial communities can influence disease outcomes even in distant organs, as demonstrated by transfer experiments with dysbiotic microbiota.
  • sub-lethal infection with the H3N2 and H1N1 subtypes of influenza is associated with a drop in the production of short chain fatty acids (SCFAs), the end products of dietary fiber fermentation.
  • SCFAs short chain fatty acids
  • Supplementation of acetate (the predominant SCFA) during influenza infection reinforced lung defenses against secondary pneumococcal infection and ameliorated the survival rate of double- infected mice.
  • Acetate can act through multiple free fatty acid receptors (FFARs) as well as through histone deacetylase (HD AC) inhibition.
  • FFAR2 free fatty acid receptor 2
  • the inventors showed that activation of free fatty acid receptor 2 (FFAR2) by a highly specific agonist mimicked the effects of acetate and protected against bacterial superinfection post-influenza. Accordingly, these results might be of value in developing appropriate therapeutic approaches against post-viral infection (e.g. post- influenza) bacterial superinfection.
  • the present invention relates to method and compositions for the treatment of bacterial superinfections post-viral infection (e.g. post-influenza).
  • the present invention is defined by the claims.
  • Acetate can act through the FFAR2, (formerly GPR43), a receptor amenable to pharmacological manipulation in vivo.
  • the inventors have surprisingly shown that pharmacological activation of FFAR2 during influenza reduced bacterial superinfection. These findings might have therapeutic applications in diseases associated with dysbiosis and secondary bacterial infections.
  • the first object of the present invention relates to a method of treating a bacterial superinfection post-viral infection (e.g. post-influenza) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a synthetic free fatty acid receptor 2 (FFAR2) agonist.
  • a bacterial superinfection post-viral infection e.g. post-influenza
  • FFAR2 synthetic free fatty acid receptor 2
  • the term“subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate.
  • the subject according to the invention is a human. More particularly, the subject of the present invention can be an elderly subject (>65 years of age) or a non-elderly subject ( ⁇ 65 years of age) or a subject suffering from co-morbidities.
  • the term“subject” encompasses“patient”.
  • sub-lethal influenza (H3N2 and H1N1) infection leads to transient gut dysbiosis.
  • the subject suffers or has suffered from dysbiosis.
  • the term“dysbiosis” has its general meaning in the art and refers to a microbial imbalance or maladaptation on or inside the body, such as an impaired microbiota.
  • a part of the human microbiota such as the skin flora, gut flora, or vaginal flora, can become deranged, with normally dominating species underrepresented and normally outcompeted or contained species increasing to fill the void.
  • the subject suffers or has suffered from a viral infection.
  • the viral infection is caused by a virus selected from the group consisting of influenza virus, human immunodeficiency viruses (HIV), Zika virus, Cytomegalovirus (CMV), respiratory syncytial virus, adenovirus, metapneumovirus, cytomegalovirus, parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), rhinovirus, coxsackie virus, echo virus, herpes simplex virus, coronavirus (SARS-coronavirus such as SARS-CoVl or SARS- CoV2), and smallpox.
  • HAV human immunodeficiency viruses
  • CMV Cytomegalovirus
  • respiratory syncytial virus e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4
  • rhinovirus e.g., hPIV-1, h
  • the viral infection may be due to a member of the Pneumoviridae, Paramyxoviridae and/or Coronaviridae families are in particular selected from the group consisting of upper and lower respiratory tract infections due to: human respiratory syncytial virus (hRSV), type A and type B, human metapneumovirus (hMPV) type A and type B; parainfluenza virus type 3 (PIV-3), measles virus, endemic human coronaviruses (HCoV- 229E, -NL63, -OC43, and -HKU1), severe acute respiratory syndrome (SARS) and Middle- East respiratory syndrome (MERS) coronaviruses.
  • hRSV human respiratory syncytial virus
  • hMPV human metapneumovirus
  • PIV-3 parainfluenza virus type 3
  • measles virus endemic human coronaviruses
  • HoV- 229E endemic human coronaviruses
  • SARS severe
  • influenza infection has its general meaning in the art and refers to the disease caused by an infection with an influenza virus.
  • influenza infection is associated with Influenza virus A or B.
  • influenza infection is associated with Influenza virus A.
  • influenza infection is cause by influenza virus A that is H1N1, H2N2, H3N2 or H5N1.
  • the term“bacterial superinfection post-influenza” has its general meaning in the art and refers to a bacterial infection (e.g. bacterial pneumonia) which occurs in a subject who suffers or has suffered from an influenza infection. Typically, the bacterial superinfection occurs within 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days after influenza infection.
  • the method of the present invention is particularly suitable for the treatment of a bacterial superinfection post-viral infection (e.g. post-influenza) such as, but not limited to infections of the lower respiratory tract (e.g., pneumonia), middle ear infections (e.g., otitis media) and bacterial sinusitis.
  • the bacterial superinfection may be caused by numerous bacterial pathogens. For example, they may be mediated by at least one organism selected from the group consisting of: Streptococcus pneumoniae ; Staphylococcus aureus ; Haemophilus influenza , Myoplasma species and Moraxella catarrhalis.
  • the subject suffers or is susceptible to suffer from enteric infections.
  • enteric infection refers to a disease of the intestine caused by any infection and characterized by diarrhea, abdominal discomfort, nausea and vomiting, and anorexia.
  • bacteria involved in enteric infections are Escherichia coli, Vibrio cholerae , and several species of Salmonella, Shigella , and anaerobic streptococci.
  • FFAR free fatty acid receptor
  • FFAR2 also known as“GPR43” has its general meaning in the art and refers to a G-protein-coupled receptor that is activated by physiological concentrations of short-chain fatty acids (SCFAs).
  • SFAs short-chain fatty acids
  • MLPD WK S SLILM A YIIIFLT GLP ANLL ALR AF V GRIRQPQP AP VHILLL SLTL ADLLLLL LLPFKIIEA ASNFRWYLPK VVC ALT SFGF YS SI Y C STWLL AGISIERYLGVAFP V Q YKL S RRPL Y GVIAALVAWVMSF GHCTIVIIVQYLNTTEQVRSGNEITCYENFTDNQLD VVLP VRLELCL VLFFIPMAVTIFC YWRF VWIML SQPL V GAQRRRRAV GL AVVTLLNFL V CF GPYNV SHLVGYHQRKSPWWRSIAVVF S SLNASLDPLLF YFS S S VVRRAF GRGLQ VLR NQGS SLLGRRGKDT AEGTNEDRGVGQGEGMP S SDFTTE
  • the term“synthetic” has its general meaning in the art and refers to products made from artificial substances.
  • the term“synthetic FFAR2 agonist” refers to a synthetic agonist of FFAR2.
  • the term“agonist” has its general meaning in the art and refers to a substance that binds to a receptor and activates the receptor to produce a biological response.
  • the synthetic FFAR2 agonist is not a SCFA.
  • SCFA short-chain fatty acid
  • Suitable short-chain fatty acids include: formic acid; acetic acid; propionic acid; butyric (butanoic) acid; isobutyric (2-methylpropanoic) acid; valeric (pentanoic) acid; isovaleric (3-methylbutanoic); and caproic (hexanoic) acid and analogues including halogenated derivatives, such as dichloroacetate (DCA).
  • DCA dichloroacetate
  • the SCFA are highly abundant in the colon but can also be detected in the blood.
  • the SCFA is also a salt or an ester selected from formate, acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, caproate.
  • the synthetic FFAR2 agonist of the present invention is not formic acid, acetic acid, propionic acid, butyric (butanoic) acid, isobutyric (2-methylpropanoic) acid, valeric (pentanoic) acid, isovaleric (3- methylbutanoic) acid or caproic (hexanoic) acid and analogues including halogenated derivatives, such as dichloroacetate (DCA).
  • DCA dichloroacetate
  • the synthetic FFAR2 agonist of the present invention is not a salt or an ester selected from formate, acetate, propionate, isobutyrate, valerate, isovalerate, caproate, preferably acetate, propionate or butyrate.
  • the synthetic FFAR2 agonist is a compound of formula I:
  • Ar 1 is a 5- to 6-membered aryl or heteroaryl group, 3- to 8-membered cycloalkyl group, a 3 - to 8-membered heterocycloalkyl group, or a linear or branched C3-C6 alkyl group, each of which being optionally substituted by one or more group(s) selected from halo, cyano, alkyl, hydroxyalkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, heteroalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, hydroxyl, alkoxy, haloalkoxy, cycloalkyloxy, heterocyclyloxy, aryloxy, amino, alkylamino, aminoalkyl, carboxy, alkoxycarbonyl, cycloalkyloxycarbonyl, heterocyclyl
  • L 1 is a single bond, C1-C3 alkylene, C3-C 6 cycloalkyl ene, C2-C 3 alkenylene, C2-C 3 alkynylene, each of which being optionally substituted by one or more group(s) selected from halo, alkyl, haloalkyl, hydroxyl, alkoxy, haloalkoxy, hydroxyalkyl, alkoxyalkyl;
  • R 1 is H, linear or branched C 1 -C 4 alkyl
  • E is N, C-R 5 where R 5 is H, linear or branched C 1 -C 4 alkyl; linked to E eiher
  • R 6 is H, alkyl, C 2 -C 4 alkenyl, C 2 - C 4 alkynyl, haloalkyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl or alkoxyalkyl, and under the condition that E is C-R 5 ;
  • L 2 is a single bond, C1-C4 alkylene, C 3 -C 6 cycloalkyl ene, C2-C 3 alkenylene, C2-C 3 alkynylene each of which being optionally substituted by one or more group(s) selected from halo, alkyl, haloalkyl, hydroxyl, alkoxy, haloalkoxy, hydroxyalkyl or alkoxyalkyl;
  • Ar 2 is an aryl or heteroaryl, cycloalkyl, heterocyclyl or C 2 -C 6 alkyl group, each of which being optionally substituted by one or more group(s) selected from halo, cyano, nitro, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, heteroalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, benzoxazol- 2-yl, heteroarylalkyl, hydroxyl, hydroxyalkyl, alkoxy, haloalkoxy, alkoxyalkoxy, cycloalkyloxy, heterocyclyloxy, aryloxy, heteroaryl oxy, alkoxyalkyl, haloalkoxyalkyl, cycloalkylalkyloxy, aryloxy, heteroaryl oxy, alkoxyalkyl, halo
  • R 2 is H
  • L 3 is a single bond, C1-C3 alkylene, C3-C 6 cycloalkylene, C2-C 3 alkenylene or C2- C 3 alkynylene each of which being optionally substituted by one or more group(s) selected from halo, alkyl, haloalkyl, hydroxyl, alkoxy, haloalkoxy, hydroxyalkyl, alkoxyalkyl;
  • Z is selected from the group consisting of -COOR
  • R is H or linear or branched alkyl, aryl, acyloxyalkyl, dioxolene, R 7 is H, methyl or ethyl, and R 7 is hydroxyl -S02CH3j -S02cyclopropyl or -S02CF3; the bond represented by the dotted line is either absent or present;
  • R 3 is H, halo, cyano, alkyl, hydroxyalkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, heteroalkyl, 5-membered heterocyclyl, heterocyclylalkyl, aryl, aralkyl, 5- membered heteroaryl, heteroarylalkyl, hydroxyl, alkoxy, alkoxy alkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, aryloxy, amino, alkylamino, aminoalkyl, alkylcarbonyloxy, cycloalkylcarbonyloxy, heterocyclylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, arylalkyloxy, acetyl, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, heterocyclyl
  • R 3 is H or Ci -C 4 alkyl, or R 3 is absent if the dotted line is present;
  • R 4 is H, halo, cyano, alkyl, hydroxyalkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroaryl alkyl, hydroxyl, alkoxy, haloalkoxy, cycloalkyloxy, heterocyclyloxy, aryloxy, amino, alkylamino, aminoalkyl, carboxy, alkoxycarbonyl, cycloalkyloxycarbonyl, heterocyclyloxy carbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylcarbonyloxy, cycloalkylcarbonyloxy, heterocyclylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, alkylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, alkylcarbonyloxy
  • R 4 is H, C 1 -C 4 alkyl, or R 4 is absent if the dotted line is present.
  • the synthetic FFAR2 agonist is selected from the group consisting of: (2S,5R)-5-(2-chlorophenyl)-l-(2'-methoxy-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-methyl-[l, G- biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(3-((4-chlorobenzyl)oxy)-5- methoxybenzoyl)-5-(2-chlorophenyl)pyrro 2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (2'-fluoro-[l,r-biphenyl]
  • (2chlorophenyl)pyrrolidine-2carboxylic acid (2S,5R)-5-(2-chlorophenyl)-l-(2 (cyclopropylmethoxy)-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S.5R)-l-(2'- methoxy-[l,r-biphenyl]-4-carbonyl)-5-phenylpyrro lidine-2-carboxylic acid, (2S,5R)-5-(3- chlorophenyl)-l-(2'-methoxy-[l, -biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid,
  • the synthetic FFAR2 agonist is generally described in the International Publication No. WO2011151436A2 which is incorporated herein by reference in in its entirety.
  • the synthetic FFAR2 agonist is generally described in Hansen et al, Journal of Medicinal Chemistry (2016) 32 and Wang et al, Bioorganic & Medicinal Chemistry Letters 20 (2009) 33 which are incorporated herein by reference in their entirety.
  • the synthetic FFAR2 agonist is TUG-1375.
  • TUG-1375 also called (2R,4R)-2-(2-chlorophenyl)-3-(4- (3,5-dimethylisoxazol-4-yl)benzoyl)thiazolidine-4-carboxylic acid has its general meaning in the art and refers to the compound characterized by the formula of :
  • the synthetic FFAR2 agonist is AMG7703.
  • AMG7703 also called (2S)-2-(4-chlorophenyl)-3-methyl-N- (l,3-thiazol-2-yl)butanamide has its general meaning in the art and refers to the compound characterized by the formula of :
  • the synthetic FFAR2 agonist is AZ1729.
  • AZ1729 also called N-[3-(2-carbamimidamido-4-methyl- l,3-thiazol-5-yl)phenyl]-4-fluorobenzamide has its general meaning in the art and refers to the compound characterized by the formula of :
  • the synthetic FFAR2 agonist is 4-CMTB.
  • 4-CMTB also called 4-chloro-a-(l-methylethyl)-N-2- thiazolylbenzeneacetamide has its general meaning in the art and refers to the compound characterized by the formula of:
  • the synthetic FFAR2 agonist is 4-CDPB.
  • the term“4-CDPB” also called (S)-2-(4-chlorophenyl)-3,3-dimethyl-N- (5-phenylthiazol-2-yl)butanamide in its racemic form and (S)-2-(4-chlorophenyl)-3,3- dimethyl-N-(5-phenylthiazol-2-yl)butanamide in its enantiopure form has its general meaning in the art and refers to the compound characterized by the formula of:
  • the synthetic FFAR2 agonist is Cpd A.
  • Cpd A also called 3-benzyl-4-(cyclopropyl(4-(2,5- dichlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid in its racemic form and (f?)-3-benzyl- 4-(cyclopropyl(4-(2,5-dichlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid in its enantiopure form has its general meaning in the art and refers to the compound characterized by the formula of:
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • a “therapeutically effective amount” refers to an amount effective (e.g., a prophylactic or therapeutic agent), at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of drug may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of drug to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for drug depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • a suitable dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen.
  • Such an effective dose will generally depend upon the factors described above.
  • a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • An exemplary, non-limiting range for a therapeutically effective amount of drug is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg.
  • An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g.
  • the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.
  • treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
  • 0.1-100 mg/kg such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4,
  • the therapy which is sufficient to reduce the severity and/or duration of a disease, ameliorate one or more symptoms thereof, prevent the advancement of a disease or cause regression of a disease, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of a disease or one or more symptoms thereof, or enhance or improve the prophylactic and/or therapeutic effect(s) of another therapy (e.g., another therapeutic agent) useful for treating a disease.
  • another therapy e.g., another therapeutic agent
  • the method of the present invention is particularly suitable for subjects who are identified as at high risk for developing a bacterial superinfection post-viral infection (e.g. post- influenza), including subjects who are at least 50 years old, subjects who reside in chronic care facilities, subjects who have chronic disorders of the pulmonary or cardiovascular system, subjects who required regular medical follow-up or hospitalization during the preceding year because of chronic metabolic diseases (including diabetes mellitus), renal dysfunction, hemoglobinopathies, or immunosuppression (including immunosuppression caused by medications or by human immunodeficiency [HIV] virus); children less than 14 years of age, patients between 6 months and 18 years of age who are receiving long-term aspirin therapy, and women who will be in the second or third trimester of pregnancy during the influenza season.
  • a bacterial superinfection post-viral infection e.g. post- influenza
  • subjects who are at least 50 years old subjects who reside in chronic care facilities, subjects who have chronic disorders of the pulmonary or cardiovascular system, subjects who required regular medical follow-up or hospitalization during the preceding
  • the method of the invention is suitable for the treatment of bacterial superinfection post- viral infection (e.g. post-influenza) in subjects older than 1 year old and less than 14 years old (i.e., children); subjects between the ages of 50 and 65, and adults who are older than 65 years of age.
  • bacterial superinfection post- viral infection e.g. post-influenza
  • the synthetic FFAR2 agonist is administered to the subject in a form of a pharmaceutical composition.
  • the synthetic FFAR2 agonist may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • pharmaceutically acceptable excipients such as a pharmaceutically acceptable polymers
  • pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the synthetic FFAR2 agonist can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the composition comprises any other ingredients or excipients known to be employed in the type of composition in question.
  • ingredients include: proteins, amino acids, carbohydrates, oligosaccharides, lipids, prebiotics or probiotics, nucleotides, nucleosides, other vitamins, minerals and other micronutrients.
  • the composition typically comprises carriers or vehicles.
  • Carriers or“vehicles” mean materials suitable for administration and include any such material known in the art such as, for example, any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is non-toxic and which does not interact with any components of the composition in a deleterious manner.
  • nutritionally acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.
  • FIGURES
  • FIG. 1 Altered fermentative activity (SCFA production) of the gut microbiota during IAV infection. Seven and fourteen days after infection, cecal contents were collected for SCFA quantification.
  • A Cecal concentrations of total SCFAs in mock-treated and IAV- (H3N2) infected mice.
  • D Blood concentrations of total SCFAs (9-10 pooled sera, 4 mice/pool). The repartition of individual SCFAs in the blood is represented (mock). Significant differences were determined using the Kruskal-Wallis ANOVA test (a and b) and the Mann- Whitney U test (c and d) (*p ⁇ 0.05; ***p ⁇ 0.001).
  • FIG. 2 Effect of acetate supplementation in mice doubly infected with IAV and S. pneumoniae.
  • A-C IAV (HlNl)-infected mice were treated with acetate (Ace) (200 mM in drinking water) or vehicle (Vh) at day 2 post-infection, five days before the pneumococcal challenge (lxlO 3 c.f.u).
  • B Body weight evolution (in % initial body weight, means ⁇ SD).
  • C The survival of superinfected animals was monitored.
  • B) and (C), n 14 (two pooled experiments). Survival of mice was compared using Kaplan-Meier analysis and log-rank test (*p ⁇ 0.05, **p ⁇ 0.01).
  • FIG. 3 Effect of FFAR2 agonist treatment on bacterial superinfection postinfluenzal.
  • a and B IAV (HlNl)-infected mice were treated with the selective FFAR2 agonist TUG-1375 (A) or with the selective FFAR3 agonist AR420626 (B) (1 mM in 50 m ⁇ , i.n. route) or vehicle (Vh) 16 h before the pneumococcal challenge.
  • the number of bacteria in the lungs and spleen was determined 30 h after the S. pneumoniae challenge.
  • A), n 13-14 (two pooled experiments) and
  • B), n 6-8 (one representative experiment out of two).
  • Significant differences were determined using the Mann- Whitney U test (*p ⁇ 0.05, **p ⁇ 0.01).
  • mice Specific pathogen-free C57BL/6J mice (6 week-old, male) were purchased from Janvier (Le Genest-St-Isle, France). Mice were maintained in a biosafety level 2 facility in the Animal Resource Center at the Lille Pasteur Institute for at least two weeks prior to usage to allow appropriate acclimatation. Unless specified, mice were fed a standard rodent chow (SAFE A04, SAFE, Augy, France) and water ad libitium. This diet contains -11.8% fiber including -10% water-insoluble fiber (3.6% cellulose) and 1.8% water-soluble fiber. All experiments complied with current national and institutional regulations and ethical guidelines (B59-350009, Institut Pasteur detechnisch protocol number: 2015121722376405 and 13743-2018022211144403).
  • mice were anesthetized by intramuscular injection of 1.25 mg of ketamine plus 0.25 mg of xylazine in 100 m ⁇ of phosphate buffered saline (PBS), and then intranasally (i.n.) infected with 50 m ⁇ of PBS containing (or not, in a mock sample) 30 plaque forming units (p.f.u.) of the H3N2 IAV strain A/Scotland/20/1974, or 100 p.f.u. ofHINl A/Califomia/04/2009 (pdm09) 3 ’ 7 . These doses correspond to sub-lethal doses, which are necessary to investigate secondary bacterial infection.
  • PBS phosphate buffered saline
  • IAV H1N1, pdm2009
  • S. pneumoniae serotype 1 a serotype linked to invasive pneumococcal disease
  • This dose is largely sufficient to allow bacterial outgrowth and dissemination.
  • bacteria in the lungs and spleen were counted 3 Oh after the S. pneumoniae challenge by plating serial 10-fold dilutions of lung or spleen homogenates onto blood agar plates. The plates were incubated at 37°C with 5% C02 overnight and viable bacteria were counted 24h later. Survival and body weight were monitored daily after IAV infection and mice were euthanized when they lost in excess of 20% of their initial body weight.
  • the FFAR2 agonist TUG-1375 and the FFAR3 agonist AR420626 (stock solutions in DMSO at 20mM) was inoculated by the i.n. route (50m1, 1 mM) 16h before S. pneumoniae infection.
  • mice are expressed as the mean ⁇ standard deviation unless otherwise stated. All statistical analysis was performed using GraphPad Prism v6 software. A Mann- Whitney U test was used to compare two groups unless otherwise stated. Comparisons of more than two groups with each other were analyzed with the One-way ANOVA Kruskal- Wallis test (nonparametric), followed by the Dunn’s posttest. Survival of mice was compared using Kaplan-Meier analysis and log-rank test.*, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001.
  • Sub-lethal influenza infection transiently alters the composition and the metabolic output of the gut microbiota
  • influenza infection alters the metabolic (fermentative) output of the gut microbiota at 7 dpi and that it affects local (gut) and systemic (blood) concentration of SCFAs, an emerging group of dietary derived metabolites endowed with immune regulatory functions 42 .
  • Acetate can act through the G-protein coupled receptors FFAR2 (formerly GPR43) and, to a lesser extent, FFAR3 (formerly GPR41).
  • FFAR2 and FFAR3 are amenable to pharmacological manipulation in vivo 43 .
  • mice were treated with TUG-1375 (a selective FFAR2 agonist) or AR420626 (a selective FFAR3 agonist) by intra nasal administration.
  • Pharmacological FFAR2 activation just before pneumococcal challenge, led to a significant reduction of lung bacterial burden and dissemination to blood (Figure 3A).
  • acetate supplementation reduced the bacterial burden after the episode influenza - despite the immunosuppressive environment imposed by IAV. Of importance, this translated into improved survival rate of double-infected mice.
  • the inventors showed that local activation of FFAR2 resulted in reduced bacterial burden in superinfected mice. This later result suggests that the use of synthetic FFAR2 agonists as therapeutics might be envisaged to lower bacterial superinfection post-influenza.
  • FFAR2 agonists are viewed as a promising treatment of metabolic syndromes such as type 2 diabetes and obesity 43 ’ 45 47 .
  • Our study shows for the first time the positive effect of a synthetic FFAR2 agonist in respiratory infection and highlights a new opportunity for further development against bacterial pneumonia.

Abstract

Severe influenza is associated with defects in pulmonary innate immunity, a phenomenon leading to secondary bacterial infections. The gut microbiota can control immune/inflammatory responses locally and at distant sites. The inventors hypothesized that perturbation of the gut microbiota during severe influenza might participate in bacterial superinfection. Their data demonstrated that influenza infection profoundly altered the functionality of the gut microbiota as assessed by the altered production of short chain fatty acids (SCFAs). Remarkably, treatment of IAV-infected mice with acetate, the main SCFA found systematically, reinforced host defenses against S. pneumoniae. Lastly, the inventors showed that pharmacological manipulation of the FFAR2 agonist TUG-1375 provides the same benefit as acetate in the treatment of bacterial superinfection post-influenza. The present invention thus relates to the use of synthetic free fatty acid receptor 2 (FFAR2) agonist for the treatment of bacterial superinfections post-viral infection (e.g. post-influenza).

Description

USE OF FFAR2 AGONISTS FOR THE TREATMENT OF BACTERIAL
SUPERINFECTIONS POST- VIRAL INFECTION
FIELD OF THE INVENTION:
The present invention relates to method and compositions for the treatment of bacterial superinfections post-viral infection (e.g. post-influenza).
BACKGROUND OF THE INVENTION:
Despite the widespread application of vaccination programs and antiviral drug treatments, influenza A virus (IAV) infections are responsible for significant morbidity and mortality. Influenza infections can also result in sporadic and often devastating pandemics; the 1918 pandemic led to the death of 50 million people. Severe bacterial infections (e.g. pneumococcal infections) can occur in the aftermath of viral infection, and contribute significantly to the excess morbidity and mortality of influenza1. Infection with IAV disrupts pulmonary barrier integrity and dampens innate antibacterial immunity, thus favoring local bacterial outgrowth and dissemination from the lungs1-3. This inability to control bacterial infection is associated with changes in the numbers and/or functions of innate immune cells, including alveolar macrophages, conventional dendritic cells, neutrophils and non-conventional T cells4-8. Regarding the importance of the gut-lung axis in diseases9 10, the inventors hypothesized that perturbation of the gut microbiota functionality during IAV infection might favor bacterial superinfection.
The gastrointestinal tract hosts a complex, highly diverse microbial ecosystem. The tightly regulated interplay between the microbiota and the host enables the establishment and persistence of immune homeostasis11-13. The commensal microbes impact on host immune responses is not limited to the gut compartment (i.e. barrier functions and gut homeostasis) but also extends to systemic compartments and distant mucosal interfaces, such as the lungs9 10. The mechanism by which the gut microbiota regulates the size and/or the functions of the steady-state immune cell pool depends on microbial-associated molecular patterns, microbial metabolites, and their interactions with progenitor cells and mature immune cells14-18. It has recently been shown that a healthy microbiota has a critical role in the host’s defense against respiratory tract infections, including IAV19-21 and Streptococcus pneumoniae22-24. In the latter context, nucleotide-binding oligomerization domain (NOD)-like receptor agonist(s) modulate the functions of effector immune cells, including alveolar macrophages and neutrophils. Pathological situations (such as infections and chronic inflammatory or metabolic disorders) can modify the diversity and composition of the gut microbiota, leading to dysbiosis25. Changes in intestinal bacterial communities can influence disease outcomes even in distant organs, as demonstrated by transfer experiments with dysbiotic microbiota. Only a few studies have investigated the impact of an acute respiratory infection on the gut microbiota, and most of these involved animal (murine) models of influenza. In this system, severe infections with H1N1 and H5N1 IAV were associated with alteration of the gut microbiota26- 30 - a finding that seems also to apply to infections in humans31. This perturbation of the microbiota is associated with enhanced susceptibility to secondary enteric infections27,29. Influenza-associated dysbiosis has yet to be fully characterized (e.g. metabolic output) and functionally explored. In particular, it remains to be seen whether changes in the composition of the gut microbiota during influenza infection affect remote (respiratory tract) bacterial infections. In the present study, the inventors found that sub-lethal infection (mouse system) with the H3N2 and H1N1 subtypes of influenza is associated with a drop in the production of short chain fatty acids (SCFAs), the end products of dietary fiber fermentation. Supplementation of acetate (the predominant SCFA) during influenza infection reinforced lung defenses against secondary pneumococcal infection and ameliorated the survival rate of double- infected mice. Acetate can act through multiple free fatty acid receptors (FFARs) as well as through histone deacetylase (HD AC) inhibition. The inventors showed that activation of free fatty acid receptor 2 (FFAR2) by a highly specific agonist mimicked the effects of acetate and protected against bacterial superinfection post-influenza. Accordingly, these results might be of value in developing appropriate therapeutic approaches against post-viral infection (e.g. post- influenza) bacterial superinfection.
SUMMARY OF THE INVENTION:
The present invention relates to method and compositions for the treatment of bacterial superinfections post-viral infection (e.g. post-influenza). In particular, the present invention is defined by the claims.
DETAILED DESCRIPTION OF THE INVENTION:
Secondary bacterial (pneumococcal) infections often complicate viral respiratory infections. Regarding the importance of the gut-lung axis in diseases, the inventors hypothesized that perturbation of the gut microbiota functionality during influenza A virus (IAV) infection might favor respiratory bacterial superinfection. Sub-lethal infection with influenza transiently altered the fermentative activity of the gut microbiota in mice. In particular, the production and the systemic (blood) concentration of the predominant short- chain fatty acid (SCFA) acetate were strongly reduced at day 7 post-infection. In the context of influenza infection, acetate supplementation reduced local and systemic bacterial loads and improved survival of double-infected mice. Acetate can act through the FFAR2, (formerly GPR43), a receptor amenable to pharmacological manipulation in vivo. The inventors have surprisingly shown that pharmacological activation of FFAR2 during influenza reduced bacterial superinfection. These findings might have therapeutic applications in diseases associated with dysbiosis and secondary bacterial infections.
Accordingly, the first object of the present invention relates to a method of treating a bacterial superinfection post-viral infection (e.g. post-influenza) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a synthetic free fatty acid receptor 2 (FFAR2) agonist.
As used herein, the term“subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human. More particularly, the subject of the present invention can be an elderly subject (>65 years of age) or a non-elderly subject (<65 years of age) or a subject suffering from co-morbidities. As used herein, the term“subject” encompasses“patient”.
Accordingly, sub-lethal influenza (H3N2 and H1N1) infection leads to transient gut dysbiosis. According to the invention the subject suffers or has suffered from dysbiosis.
As used herein, the term“dysbiosis” has its general meaning in the art and refers to a microbial imbalance or maladaptation on or inside the body, such as an impaired microbiota. For example, a part of the human microbiota, such as the skin flora, gut flora, or vaginal flora, can become deranged, with normally dominating species underrepresented and normally outcompeted or contained species increasing to fill the void.
According to the invention the subject suffers or has suffered from a viral infection. In some embodiments, the viral infection is caused by a virus selected from the group consisting of influenza virus, human immunodeficiency viruses (HIV), Zika virus, Cytomegalovirus (CMV), respiratory syncytial virus, adenovirus, metapneumovirus, cytomegalovirus, parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), rhinovirus, coxsackie virus, echo virus, herpes simplex virus, coronavirus (SARS-coronavirus such as SARS-CoVl or SARS- CoV2), and smallpox. In some embodiments, the viral infection may be due to a member of the Pneumoviridae, Paramyxoviridae and/or Coronaviridae families are in particular selected from the group consisting of upper and lower respiratory tract infections due to: human respiratory syncytial virus (hRSV), type A and type B, human metapneumovirus (hMPV) type A and type B; parainfluenza virus type 3 (PIV-3), measles virus, endemic human coronaviruses (HCoV- 229E, -NL63, -OC43, and -HKU1), severe acute respiratory syndrome (SARS) and Middle- East respiratory syndrome (MERS) coronaviruses.
According to the invention the subject suffers or has suffered from an influenza infection. As used herein, the term "influenza infection" has its general meaning in the art and refers to the disease caused by an infection with an influenza virus. In some embodiments of the invention, influenza infection is associated with Influenza virus A or B. In some embodiments of the invention, influenza infection is associated with Influenza virus A. In some specific embodiments of the invention, influenza infection is cause by influenza virus A that is H1N1, H2N2, H3N2 or H5N1.
As used herein, the term“bacterial superinfection post-influenza” has its general meaning in the art and refers to a bacterial infection (e.g. bacterial pneumonia) which occurs in a subject who suffers or has suffered from an influenza infection. Typically, the bacterial superinfection occurs within 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days after influenza infection. The method of the present invention is particularly suitable for the treatment of a bacterial superinfection post-viral infection (e.g. post-influenza) such as, but not limited to infections of the lower respiratory tract (e.g., pneumonia), middle ear infections (e.g., otitis media) and bacterial sinusitis. The bacterial superinfection may be caused by numerous bacterial pathogens. For example, they may be mediated by at least one organism selected from the group consisting of: Streptococcus pneumoniae ; Staphylococcus aureus ; Haemophilus influenza , Myoplasma species and Moraxella catarrhalis.
In one embodiment, the subject suffers or is susceptible to suffer from enteric infections. As used herein, the term“enteric infection” refers to a disease of the intestine caused by any infection and characterized by diarrhea, abdominal discomfort, nausea and vomiting, and anorexia. In particular, the bacteria involved in enteric infections are Escherichia coli, Vibrio cholerae , and several species of Salmonella, Shigella , and anaerobic streptococci.
As used herein, the term“free fatty acid receptor” or“FFAR” has its general meaning in the art and refers to a G-protein coupled receptors which binds free fatty acids. FFAR includes 4 receptors: FFARl, FFAR2, FFAR3 and FFRA4. In some embodiment, the FFAR is selected from free fatty acid receptor 2 (FFAR2).
As used herein, the term“FFAR2” also known as“GPR43” has its general meaning in the art and refers to a G-protein-coupled receptor that is activated by physiological concentrations of short-chain fatty acids (SCFAs). An exemplary amino acid sequence of FFAR2 is represented by SEQ ID NO: 1 :
SEQ ID N0: l>sp|015552.1 |FFAR2_HUMAN RecName: Full=Free fatty acid receptor 2; AltName: Full=G-protein coupled receptor 43
MLPD WK S SLILM A YIIIFLT GLP ANLL ALR AF V GRIRQPQP AP VHILLL SLTL ADLLLLL LLPFKIIEA ASNFRWYLPK VVC ALT SFGF YS SI Y C STWLL AGISIERYLGVAFP V Q YKL S RRPL Y GVIAALVAWVMSF GHCTIVIIVQYLNTTEQVRSGNEITCYENFTDNQLD VVLP VRLELCL VLFFIPMAVTIFC YWRF VWIML SQPL V GAQRRRRAV GL AVVTLLNFL V CF GPYNV SHLVGYHQRKSPWWRSIAVVF S SLNASLDPLLF YFS S S VVRRAF GRGLQ VLR NQGS SLLGRRGKDT AEGTNEDRGVGQGEGMP S SDFTTE
As used herein, the term“synthetic” has its general meaning in the art and refers to products made from artificial substances.
As used herein, the term“synthetic FFAR2 agonist” refers to a synthetic agonist of FFAR2. As used herein, the term“agonist” has its general meaning in the art and refers to a substance that binds to a receptor and activates the receptor to produce a biological response.
In some embodiments, the synthetic FFAR2 agonist is not a SCFA. As used herein, the term“short-chain fatty acid” or“SCFA” has its general meaning in the art and refers to aliphatic carboxylic acids composed of 1 to 6 carbon atoms, which may be linear or branched. Suitable short-chain fatty acids include: formic acid; acetic acid; propionic acid; butyric (butanoic) acid; isobutyric (2-methylpropanoic) acid; valeric (pentanoic) acid; isovaleric (3-methylbutanoic); and caproic (hexanoic) acid and analogues including halogenated derivatives, such as dichloroacetate (DCA). SCFA are highly abundant in the colon but can also be detected in the blood. The SCFA is also a salt or an ester selected from formate, acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, caproate. In another word, in this embodiment, the synthetic FFAR2 agonist of the present invention is not formic acid, acetic acid, propionic acid, butyric (butanoic) acid, isobutyric (2-methylpropanoic) acid, valeric (pentanoic) acid, isovaleric (3- methylbutanoic) acid or caproic (hexanoic) acid and analogues including halogenated derivatives, such as dichloroacetate (DCA). In this embodiment, the synthetic FFAR2 agonist of the present invention is not a salt or an ester selected from formate, acetate, propionate, isobutyrate, valerate, isovalerate, caproate, preferably acetate, propionate or butyrate.
In a particular embodiment, the synthetic FFAR2 agonist is a compound of formula I:
Figure imgf000007_0001
and pharmaceutically acceptable salts, solvates and prodrugs thereof, wherein
Ar1 is a 5- to 6-membered aryl or heteroaryl group, 3- to 8-membered cycloalkyl group, a 3 - to 8-membered heterocycloalkyl group, or a linear or branched C3-C6 alkyl group, each of which being optionally substituted by one or more group(s) selected from halo, cyano, alkyl, hydroxyalkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, heteroalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, hydroxyl, alkoxy, haloalkoxy, cycloalkyloxy, heterocyclyloxy, aryloxy, amino, alkylamino, aminoalkyl, carboxy, alkoxycarbonyl, cycloalkyloxycarbonyl, heterocyclyloxy carbonyl, aryloxy carbonyl, heteroaryloxycarbonyl, alkylcarbonyloxy, cycloalkyl carbonyloxy, heterocyclylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, arylalkyloxy, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, heterocyclylcarbonylamino arylcarbonylamino, heteroarylcarbonylamino, alkylcarbonylaminoalkyl, carbamoyl, hydroxycarbamoyl, alkyl carbamoyl, arylcarbamoyl, heteroarylcarbamoyl, carbamoylalkyl, carbamoylamino, alkylcarbamoylamino, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocyclylsulfonyl, arylsulfonyl, heteroaryl sulfonyl sulfamoyl, alkyl sulfamoyl, arylsulfamoyl, heteroaryl sulfamoyl, alkylsulfonylamino, cycloalkylsulfonylamino, heterocyclylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, haloalkylsulfonylamino, or two substituents form an alkylenedioxy group or a haloalkylenedioxy group, or two substituents form a cycloalkyl or heterocycloalkyl moiety together with the cycloalkyl or heterocycloalkyl group they are attached to, or fused to the aryl, heteroaryl, cycloalkyl or heterocycloalkyl group may be one or more cycloalkyl, aryl, heterocyclyl or heteroaryl moiety, each of said substituents being optionally substituted by one or more further substituents selected from halo, cyano, alkyl, hydroxyalkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, hydroxyl, alkoxy, haloalkoxy, cycloalkyloxy, alkylamino, carboxy, alkoxycarbonyl, alkylcarbonyloxy, cycloalkylcarbonyloxy, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, alkylcarbonylaminoalkyl, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, carbamoylalkyl, carbamoylamino, alkylcarbamoylamino, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, sulfamoyl, alkyl sulfamoyl, alkylsulfonylamino, cycloalkylsulfonylamino, haloalkylsulfonylamino or oxo;
L1 is a single bond, C1-C3 alkylene, C3-C6 cycloalkyl ene, C2-C3 alkenylene, C2-C3 alkynylene, each of which being optionally substituted by one or more group(s) selected from halo, alkyl, haloalkyl, hydroxyl, alkoxy, haloalkoxy, hydroxyalkyl, alkoxyalkyl;
R1 is H, linear or branched C1-C4 alkyl;
E is N, C-R5 where R5 is H, linear or branched C1-C4 alkyl;
Figure imgf000008_0001
linked to E eiher
on the nitrogen or the carbonyl and R6 is H, alkyl, C2-C4 alkenyl, C2- C4 alkynyl, haloalkyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl or alkoxyalkyl, and under the condition that E is C-R5;
L2 is a single bond, C1-C4 alkylene, C3-C6 cycloalkyl ene, C2-C3 alkenylene, C2-C3 alkynylene each of which being optionally substituted by one or more group(s) selected from halo, alkyl, haloalkyl, hydroxyl, alkoxy, haloalkoxy, hydroxyalkyl or alkoxyalkyl;
Ar2 is an aryl or heteroaryl, cycloalkyl, heterocyclyl or C2-C6 alkyl group, each of which being optionally substituted by one or more group(s) selected from halo, cyano, nitro, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, heteroalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, benzoxazol- 2-yl, heteroarylalkyl, hydroxyl, hydroxyalkyl, alkoxy, haloalkoxy, alkoxyalkoxy, cycloalkyloxy, heterocyclyloxy, aryloxy, heteroaryl oxy, alkoxyalkyl, haloalkoxyalkyl, cycloalkylalkyloxy, arylalkyloxy, heteroarylalkyloxy, aryloxyalkyl, heteroaryloxyalkyl, amino, alkylamino, aminoalkyl, arylcarbonyl, carboxy, alkoxycarbonyl, cycloalkyloxycarbonyl, heterocyclyloxy carbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylcarbonyloxy, cycloalkylcarbonyloxy, heterocyclylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, heterocyclylcarbonylamino arylcarbonylamino, heteroarylcarbonylamino, alkylcarbonylaminoalkyl, carbamoyl, hydroxycarbamoyl, alkyl carbamoyl, arylcarbamoyl, heteroarylcarbamoyl, carbamoylalkyl, carbamoylamino, alkylcarbamoylamino, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, sulfamoyl, alkylsulfamoyl, aryl sulfamoyl, heteroaryl sulfamoyl, alkylsulfonylamino, cycloalkylsulfonylamino, heterocyclylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, haloalkylsulfonylamino, oxo, or two substituents form an alkylenedioxy group or a haloalkylenedioxy group, or two substituents form a cycloalkyl or heterocycloalkyl moiety together with the cycloalkyl or heterocyclyl group they are attached to, or fused to the aryl, heteroaryl, cycloalkyl or heterocyclyl group may be one or more cycloalkyl, aryl, heterocyclyl or heteroaryl moiety, each of said substituents being optionally substituted by one or more further substituents selected from halo, cyano, nitro, alkyl, hydroxyalkyl, haloalkyl, cyanomethyl, cycloalkyl, heterocyclyl, aryl optionally substituted by a chloro or methyl group, heteroaryl, cycloalkylalkyl, heteroalkyl, hydroxyl, alkoxy, haloalkoxy, cycloalkyloxy, cycloalkylalkyloxy, aryloxy, aralkyloxy optionally substituted by a fluoro group, alkylamino, carboxy, alkoxycarbonyl, alkylcarbonyloxy, cycloalkylcarbonyloxy, amino, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, alkylcarbonylaminoalkyl, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, carbamoylalkyl, carbamoylalkyloxy, carbamoylamino, alkylcarbamoylamino, carbamimidoyl, hydroxycarbamimidoyl, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocyclylsulfonyl, arylsulfonyl, sulfamoyl, alkylsulfamoyl, alkylsulfonylamino, cycloalkylsulfonylamino, haloalkylsulfonylamino, oxo, aralkyl, heteroarylalkyl, alkoxyalkoxy, alkoxyalkyl, and haloalkoxy alkyl.
R2 is H;
L3 is a single bond, C1-C3 alkylene, C3-C6 cycloalkylene, C2-C3 alkenylene or C2- C3 alkynylene each of which being optionally substituted by one or more group(s) selected from halo, alkyl, haloalkyl, hydroxyl, alkoxy, haloalkoxy, hydroxyalkyl, alkoxyalkyl;
Z is selected from the group consisting of -COOR,
wherein R is H or linear or branched alkyl, aryl, acyloxyalkyl, dioxolene, R7 is H, methyl or ethyl, and R7 is hydroxyl -S02CH3j -S02cyclopropyl or -S02CF3; the bond represented by the dotted line is either absent or present;
R3 is H, halo, cyano, alkyl, hydroxyalkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, heteroalkyl, 5-membered heterocyclyl, heterocyclylalkyl, aryl, aralkyl, 5- membered heteroaryl, heteroarylalkyl, hydroxyl, alkoxy, alkoxy alkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, aryloxy, amino, alkylamino, aminoalkyl, alkylcarbonyloxy, cycloalkylcarbonyloxy, heterocyclylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, arylalkyloxy, acetyl, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, heterocyclyl carb ony 1 amino aryl carb ony 1 amino, heteroaryl carb ony 1 amino, alkylcarbonylaminoalkyl, carbamoylalkyl, carbamoylamino, alkylcarbamoylamino, alkylsulfonyl, haloalkylsulfonyl, cycloalkyl sulfonyl, heterocyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, sulfamoyl, alkyl sulfamoyl, arylsulfamoyl, heteroarylsulfamoyl, alkylsulfonylamino, cycloalkylsulfonylamino, heterocyclylsulfonylamino, arylsulfonylamino, heteroaryl sulfonylamino, haloalkylsulfonylamino, or fused to the aryl, heteroaryl, cycloalkyl or heterocycloalkyl group may be one or more cycloalkyl, aryl, heterocyclyl or heteroaryl moiety, each of said substituents being optionally substituted by one or more further substituents selected from halo, cyano, alkyl, hydroxyalkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heteroalkyl, hydroxyl, alkoxy, haloalkoxy, cycloalkyloxy, alkylamino, carboxy, alkoxycarbonyl, alkylcarbonyloxy, cycloalkylcarbonyloxy, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, alkylcarbonylaminoalkyl, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, carbamoylalkyl, carbamoylamino, alkylcarbamoylamino, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl, sulfamoyl, alkylsulfamoyl, alkylsulfonylamino, cycloalkylsulfonylamino, haloalkylsulfonylamino or oxo,
R3 is H or Ci -C4 alkyl, or R3 is absent if the dotted line is present;
R4 is H, halo, cyano, alkyl, hydroxyalkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroaryl alkyl, hydroxyl, alkoxy, haloalkoxy, cycloalkyloxy, heterocyclyloxy, aryloxy, amino, alkylamino, aminoalkyl, carboxy, alkoxycarbonyl, cycloalkyloxycarbonyl, heterocyclyloxy carbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylcarbonyloxy, cycloalkylcarbonyloxy, heterocyclylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, arylalkyloxy, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, heterocyclylcarbonylamino arylcarbonylamino, heteroarylcarbonylamino, alkylcarbonylaminoalkyl, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, arylcarbamoyl, heteroarylcarbamoyl, carbamoylalkyl, carbamoylamino, alkylcarbamoylamino, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocyclylsulfonyl, arylsulfonyl, heteroaryl sulfonyl sulfamoyl, alkylsulfamoyl, aryl sulfamoyl, heteroaryl sulfamoyl, alkylsulfonylamino, cycloalkylsulfonylamino, heterocyclylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, haloalkylsulfonylamino, or fused to the aryl, heteroaryl, cycloalkyl or heterocycloalkyl group may be one or more cycloalkyl, aryl, heterocyclyl or heteroaryl moiety, each of said substituents being optionally substituted by one or more further substituents selected from halo, cyano, alkyl, hydroxyalkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heteroalkyl, hydroxyl, alkoxy, haloalkoxy, cycloalkyloxy, alkylamino, carboxy, alkoxycarbonyl, alkylcarbonyloxy, cycloalkylcarbonyloxy, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, alkylcarbonylaminoalkyl, carbamoyl, hydroxycarbamoyl, alkylcarbamoyl, carbamoylalkyl, carbamoylamino, alkylcarbamoylamino, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, sulfamoyl, alkylsulfamoyl, alkylsulfonylamino, cycloalkylsulfonylamino, haloalkylsulfonylamino or oxo, or R4 forms together with R3 a cyclopropane ring optionally substituted by one or more group selected from halo, alkyl, haloalkyl, hydroxyl, alkoxy, or haloalkoxy, under the condition that the dotted line is absent;
R4 is H, C1-C4 alkyl, or R4 is absent if the dotted line is present. In a particular embodiment, the synthetic FFAR2 agonist is selected from the group consisting of: (2S,5R)-5-(2-chlorophenyl)-l-(2'-methoxy-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-methyl-[l, G- biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(3-((4-chlorobenzyl)oxy)-5- methoxybenzoyl)-5-(2-chlorophenyl)pyrro 2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (2'-fluoro-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4'-methyl-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)- 5-(2-chlorophenyl)-l-(3-methoxy-5-phenethoxybenzoyl)pyrrolidine-2-carboxylic acid,
(2S,5R)-l-([l,F-biphenyl]-4-carbonyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3-(3,3-diphenylpropoxy)-5-methoxybenzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3'-fluoro-[l,r-biphenyl]-4-carbonyl)pyrro lidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3’-methyl-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3-methoxy-5-((4- (methylsulfonyl)benzyl)oxy)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(3’-methoxy-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3,5-dimethoxybenzoyl)pyrrolidine-2-carboxylic acid,
(2S,5R)5-(2-chlorophenyl)-l-(4-(phenoxymethyl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-((2-fluorobenzyl)oxy)benzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-l-(3-chloro-5-methoxybenzoyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4'-fluoro-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-phenethoxybenzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(chroman-3- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3,5-diethoxybenzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3-phenethoxybenzoyl)pyrrolidine-2-carboxylic acid (2S)-1- ([l,F-biphenyl]-4-carbonyl)-4-benzyl-5-phenylpyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(l,2,3,4-tetrahydronaphthalene-2-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-isobutylbenzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(2,2-difluorobenzo [d][l,3]dioxole-6-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-([l,F-biphenyl]-4-carbonyl)-5-phenylpyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(3-fluoro-5- methoxybenzoyl)pyrro lidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(6-phenylnicotinoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chl orophenyl)-l-(3-methoxy-5-(2-methoxy ethoxy )benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)- 1 -(3’-methoxy-[l,r-biphenyl]-3-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3-methoxy-5- (trifluoromethyl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)- 1 -( 1 -(4- methoxyphenyl)-5 -phenyl- lH-pyrazo le-3-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5- (2-chlorophenyl)-l-(4-isopropoxybenzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(3-((3,5- dimethylisoxazo l-4-yl)methoxy)-5-methoxybenzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2,3-dihydro-lH-indene-2-carbonyl)pyrrolidine- 2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3-methyl-5-
(trifluoromethoxy)benzoyl)pyrrolidine-2-carboxylic acid (2S,5R)-l-(3-(benzyloxy)benzoyl)-5- (2- chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3- methoxybenzoyl)pyrro lidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2- phenylpyrimidine-5-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (4-(trifluoromethoxy)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4- (5-cyclopropyl- l,2,4-oxadiazol-3-yl)benzoyl)pyrrolidine-2- carboxylic acid 4-((2S,5R)-2- carboxy-5-(2-chlorophenyl)pynOlidine-l-carbonyl)-2,6-dimethoxypyrimidin-l-ium formate, (2S,5R)-5-(2-chlorophenyl)-l-(4-phenylbutanoyl)pyrro lidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(3-methyl-5-(trifluoromethyl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)- l-([l,r-biphenyl]-4-carbonyl)-5-(3-chloropyridin-2-yl)pyrrolidine-2-carboxylic acid, (2S,5R)- 5-(2-chlorophenyl)-l-(3-hydroxy-5-(trifluoromethyl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5S)-5-(2-chlorophenyl)-l-(3-methoxybenzoyl)pyrro lidine-2-carboxylic acid, (2S,5R)-1- (3,5-dimethoxybenzoyl)-5-phenylpyrrolidine-2-carboxylic acid (S)-5-([l,r-biphenyl]-3-yl)-l- (3-methoxybenzoyl)pyrro lidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3- phenylpropanoyl)pyrrolidine-2-carboxylic acid, (2S,5S)-5-(2-chlorophenyl)-l-(2-methoxy-[l,r- biphenyl]-4-carbonyl)pyrro lidine-2-carboxylic acid, (2S,5R)-l-([l,r-biphenyl]-4-carbonyl)-5- (pyridin-2-yl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(5- phenylpicolinoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-fiuorophenyl)-l-(3- methoxybenzoyl)pyrro lidine-2-carboxylic acid, (2S,5R)-l-(2-([l,r-biphenyl]-4-yl)acetyl)-5-(2- chlorophenyl)pyrrolidine-2-carboxylic acid, (2R,5S)-l-([l,r-biphenyl]-4-carbonyl)-5- phenylpyrrolidine-2-carboxylic acid, (2S,5R)-5-phenyl-l-(2-phenylacetyl)pyrrolidine-2- carboxylic acid, (2R,5 S)-5-phenyl-l-(2-phenylacetyl)pyrrolidine-2-carboxylic acid, (2S,5R)- l-(3-methoxybenzoyl)-5-(2-methoxyphenyl)pyrrolidine-2-carboxylic acid, (2R,5S)-5-(2- chlorophenyl)-l-(3-methoxybenzoyl)pyrrolidine-2-carboxylic acid, (2R,5R)-5-(2- chlorophenyl)-l-(3- methoxybenzoyl)pyrrolidine-2-carboxylic acid, (2S)-5-(4-chlorophenyl)-l- (3-methoxybenzoyl)pyrrolidine-2-carboxylic acid, (2S)-5-([l,l'-biphenyl]-4-yl)-l-(3- methoxybenzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-methyl5-(2-chlorophenyl)-l-(3- methoxybenzoyl)pyrrolidine-2-carboxylate, (2S)-5-(2-chlorobenzyl)-l-(3- methoxybenzoyl)pyrrolidine-2-carboxylic acid, (2S)-5-cyclohexyl-l-(3- methoxybenzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2-(3- methoxyphenyl)acetyl)pyrrolidine-2-carboxylic acid, (2S,5S)-5-(2-chlorophenyl)-l-(3,5- dimethoxybenzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-([l,r-biphenyl]-2-yl)-l-(3- methoxybenzoyl)pyrrolidine-2-carboxylic acid, 2-((2S,5R)-5-(2-chlorophenyl)-l-(3- methoxybenzoyl)pyrrolidin-2-yl)acetic acid, (2S,5R)-5-(2-chlorophenyl)-l-(6- phenylpyrimidine-4-carbonyl)pyrrc>lidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (6-(2-fluorophenyl)nicotinoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(6- (2-chlorophenyl)nicotinoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(6- (2-methoxyphenyl)nicotinoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(6- (3- fluorophenyl)nicotinoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(6-(3- methoxyphenyl)nicotinoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(6- (4- methoxyphenyl)nicotinoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (6-(4-fluorophenyl)nicotinoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2- (2-chlorophenyl)pyrimidine-5-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(2-methyl-6-phenylnicotinoyl)pynOlidine-2-carboxylic acid, (2S,5R)-l-(4- chloro-2-(pyridin-3-yl)pyrimidine-5-carbonyl)-5-(2-chlorophenyl)pynOlidine-2-carboxylic acid, (2S,5R)-l-(4-chloro-2-(pyridin-2-yl)pyrimidine-5-carbonyl)-5-(2-chlorophenyl)pyrro lidine-2-carboxylic acid, (2S,5R)-l-(4-chloro-2-(pyridin-4-yl)pyrimidine-5-carbonyl)-5-(2- chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(pyridin-2- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)4-(4-((4-chlorophenoxy)methyl)benzoyl)- 5- (2-chlorophenyl)pyrrolidine-2-carboxylic acid (2S,5R)-5-(2-chlorophenyl)-l-(4-((4- fluorophenoxy)methyl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (4-((4-methoxyphenoxy)methyl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4-((2- chlorophenoxy)methyl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5- (2-chlorophenyl)- 1 -(4-((2-methoxyphenoxy)methyl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-((3-methoxyphenoxy)methyl)benzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-l-(4-((3-chlorophenoxy)methyl)benzoyl)-5-(2- chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-((p- tolyloxy)methyl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-((3- methoxybenzyl)oxy)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4-((3- chlorobenzyl)oxy)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4-((3,5-dimethylisoxazol-4-yl)methoxy)benzoyl)pyrrolidine-2-carboxylic acid, (2 S, 5R)-5-(2-chlorophenyl)-l-(4-((3 , 5 -dimethyl- 1 H-pyrazo 1 - 1 - yl)methoxy)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(pyridin- 2-ylmethoxy)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4- (pyridin-4-ylmethoxy)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (4-(pyridin-3-ylmethoxy)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-
1-(4-(5-methyl-lH-pyrazol-l-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4-(isoxazol-5-yl)benzoyl)pyrrolidine-2-carboxylic, (2S,5R)-l-(4-(4H-l,2,4- triazol-4-yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)- 1 -(4-(5-(p-tolyl)- 1H- 1 ,2,3 -triazol- 1 -yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(5-oxo-3-phenyl-4,5-dihydro-IH-pyrazol- lyl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(5-methyl-3- (trifluorom ethyl)- lH-pyrazo l-l-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4-(lH- pyrazol-l-yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4-(oxazol-5-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4-(3,5-dimethyl-l H-pyrazo l-l-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2,5’-dichloro-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(pyrimidin-5-yl)benzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(furan-3-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(6-methoxypyridin-3-yl)benzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(3-fluoropyridin-4-yl)benzoyl)pyrrolidine-
2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(pyridin-3-yl)benzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(6-(dimethylamino)pyridin-3- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(pyridin-4- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(6- methylpyridin-
3-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2- methoxypyridin-3-yl)benzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (4'-methoxy-[l, r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4'-cyano-[l, r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)- 5-(2-chlorophenyl)-l-(4-(4-methoxypyridin-3-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4'-chloro-[l,l'-biphenyl]-4-carbonyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(3’-chloro-[l,r-biphenyl]-4-carbonyl)-5-(2-chlorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-l-(2'-chloro-[l, l'-biphenyl]-4-carbonyl)-5-(2- chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4'-
(methylsulfonamido)-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(3,-(methylsulfonamido)-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-(methylsulfonamido)-[l, -biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(naphthalen-2- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3',5'-difluoro-[l, - biphenyl] -4-carbonyl)pyrro lidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'- hydroxy-[l, -biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)- 1 -(2'-(trifluoromethoxy)-[ 1 , 1 '-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(2'-(benzyloxy)-[l,r-biphenyl]-4-carbonyl)-5-(2-chlorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-phenoxy-[l, -biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-isopropoxy-[l, G- biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'- isobutoxy-[l, -biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)- 1 -(2'-(cy clopropylmethoxy)-[ 1 , 1 '-biphenyl]-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-((4- fluorobenzyl)oxy)-[l,l'-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(6-chloropyridin-3- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(6-fluoropyridin-3- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2-chloropyridin-4- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4-(2-chloro-3-fluoropyridin-4- yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2- carboxylic , (2S,5R)-5-(2-chlorophenyl)-l-(4-(2- chloropyridin-3-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4-(6-
(benzyloxy)pyridin-3-yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S ,5R)-1 -(4-(lH-pyrazol-4-yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4-(thiophen-3-yl)benzoyl)pynOlidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4-cyclohexylbenzoyl)pynOlidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4'-(methylsulfonyl)-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(9-oxo-9H-fluorene-2-carbonyl)pyrrolidine-2-carboxylic acid, (2 S, 5R)-5 -(2-chlorophenyl)- 1 -(2'-(methyl sulfonyl)- [ 1 , 1 '-biphenyl] -4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(tetrahydro-2H- pyran-4-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(9-methyl- 9H- carbazole-2-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4- phenoxybenzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4-benzylbenzoyl)-5-(2- chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4-benzoylbenzoyl)-5-(2- chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)- 1 -(4-(pyrimidin-2- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(4,6- dimethoxypyrimidin-2-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)- l-(4-(2,4-dimethoxypyrimidin-5-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4-(2-methoxypyrimidin-5-yl)benzoyl)pyrrolidine-2-carboxylic acid,
(2S,5R)-5-(2-chlorophenyl)-l-(4-(2-(dimethylamino)pyrimidin-5-yl)benzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2-morpholinopyrimidin-5- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2-(piperidin-l- yl)pyrimidin-5-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (cyclohexanecarbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4- methylpentanoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(4- methylpiperidin-l-yl)-3-nitrobenzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4-(2-oxopiperidin-l-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)- 1 -(3-methyl-4- morpholinobenzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5- (2-chlorophenyl)-l-(4-(piperidin-l-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4-morpholinobenzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(l-(2-cyanophenyl)piperidine-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(4-chlorophenyl)cyclohexanecarbonyl)pynOlidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-phenylcyclohexanecarbonyl)pynOlidine-2- carboxylic acid, ((2R,5S)-2-(2-chlorophenyl)-5-(lH-tetrazol-5-yl)pyrrolidin-l-yl)(2'-methoxy- [l,r-biphenyl]-4-yl)methanone, (2R,5 S)-5-(2-chlorophenyl)- 1 -(2'-methoxy-[l, - biphenyl]- 4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(6-(2- fluorophenyl)nicotinoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(5- methoxy-6- phenylnicotinoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4- (2- methoxyphenoxy)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4- (3- methoxypyridin-4-yl)benzoyl)pyrrolidine-2- carboxylic acid, (2S)-5-(2-chlorophenyl)-l- (2'-methoxy-[l, r-biphenyl]-4-carbonyl)-4,4-dimethylpyrrolidine-2-carboxylic acid, (2S)-5- (2-chlorophenyl)-l-(2'-methoxy-[l,r-biphenyl]-4-carbonyl)-4-methylpyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2-methoxy-[l, -biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-cyano-[l, - biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2',6’- dimethoxy-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(2',4'-dichloro-[l, -biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-(trifluoromethyl)-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2,2'-dimethoxy-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4'-chloro-2-methoxy-[l,r-biphenyl]-4- carbonyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic, (2S,5R)-5-(2-chlorophenyl)-l-(4-(4- methoxypyrimidin-5-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (2',4-dimethoxy-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-([l,r- biphenyl]-4-carbonyl)-5-(pyridin-3-yl)pyrrolidine-2-carboxylic acid, (2R,5R)-5-(2- chlorophenyl)-l-(2’-methoxy-[l, -biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(l-phenyl-lH-benzo[d]imidazole-5-carbonyl)pynOlidine-2- carboxylic acid, (2S,5R)-methyl5-(2-chlorophenyl)-l-(2'-methoxy-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylate, (2S,4S,5R)-5-(2-chlorophenyl)-4-(hydroxymethyl)-l -(2 - methoxy-[l,l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,4S,5S)-5-(2- chlorophenyl)- 1 -(2'-methoxy-[l, -biphenyl]-4-carbonyl)-4-(phenylsulfonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-4-cyano-l-(2'-methoxy-[l, l'-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,3R,5R)-5-(2-chlorophenyl)-3-cyano-l-(2'- methoxy-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(2-chloro-[l,r- biphenyl]-4-carbonyl)-5-(2-chlorophenyl)pynOlidine-2-carboxylic acid, (2S,5R)-l-(2'-chloro- 2-methoxy-[l,r-biplienyl]-4-carbonyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid,
(2S,5R)-5-(2-chlorophenyl)-l-(2'-(2-methoxyethoxy)-[l,r-biphenyl]-4-carbonyl)pyrrolidine- 2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2-methylthiophen-3- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2,6’-dichloro-[l,r- biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(2-chloro-4'-methoxy-[l,r- biphenyl]-4-carbonyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(3-methoxy-4-(pyrimidin-5-yl)benzoyl)pyrrolidine-2-carboxylic acid,
(2S.5R)-l-(2'-carbamimidoyl-[l,r-biphenyl]-4-carbonyl)-5-(2-chlorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-fluorophenyl)-l-(2'-methoxy-[l, -biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)4-(2’-methoxy-[l,r-biphenyl]-4-carbonyl)-5- (o-tolyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(2'-methoxy-[l,r-biphenyl]-4-carbonyl)-5-(2- methoxyphenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-
(methoxymethyl)-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4-(2,6-dimethoxypyridin-3-yl)benzoyl)pyrrolidine-2-carboxylic acid,
(2S,5R)-5-(2-chlorophenyl)-l-(3-methoxy-4-(2- methoxypyrimidin-5-yl)benzoyl)pyrrolidine- 2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(5-methoxypyrazin-2- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2-(2- methoxyethoxy)pyridin-3-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(4-(3-methoxypyrazin-2-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-1- (4-(2-chloro-4-(dimethylamino)pyrimidin-5-yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2,6-dimethoxypyrimidin-4- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-(dimethylamino)- [1, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2- methoxypyrimidin-4-yl)benzoyl)pyrrolidine-2-carboxylic acid , (2S,5R)-5-(2-chlorophenyl)-l- (3-methoxy-4-(2-methoxypyrimidin-4-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5- (2-fluorophenyl)-l-(4-(2-methoxypyridin-3-yl)benzoyl)pyrrolidine-2-carboxylic acid,
(2S,5R)-l-(4-(2,4-dimethoxypyrimidin-5-yl)benzoyl)-5-(2-fluorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2-methyl-[l,l'-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)- 1 -(3-methoxy-[l, - biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2’-(2- oxopyrrolidin-l-yl)-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(5-phenylpyrazine-2-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(5-methoxy-6-(2-methoxyphenyl)nicotinoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(5- methoxypyrimidin-4-yl)benzoyl)pyrrolidine-2- carboxylic acid , (2S,5R)-5-(2-chlorophenyl)-l-(4-(pyridazin-4- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4-(lH-l,2,3-triazol-l-yl)benzoyl)-5-(2- chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(4-(p-tolyl)-lH-l,2,3-triazol-l-yl)benzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(l-(2-methoxyphenyl)piperidine-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2- methoxyphenyl)piperazine-l-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(l-(4-methoxypyrimidin-5-yl)piperidine-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(4-methoxypyrimidin-5-yl)piperazine-l- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3-methoxy-4-(4- methylpiperidin-l-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3- methoxy-4-( l-methylpiperidin-4-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(2-cyano-[l, r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)- 5-(2-chlorophenyl)-l-(2-isobutoxy-[l, -biphenyl] -4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2,4-dichloropyrimidin-5-yl)benzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2,4-dimethoxypyrimidin-5-yl)-3- methoxybenzoyl)pyrro idine-2-carboxylic acid, (2S,5R)-l-(4-(2-chloro-4-methoxypyrimidin-5- yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2- carboxylic acid, (2S,3S,5S)-5-(2-chlorophenyl)- l-(2'-methoxy-[l,r-biphenyl]-4-carbonyl)-3-methylpyrrolidine-2-carboxylic acid, (2S,5R)-l-(4- (2,6-dimethoxypyridin-3-yl)benzoyl)-5-(2-fluorophenyl)pyrrolidine-2-carboxylic acid,
(2S,5R)-l-(2'-(2-amino-2-oxoethoxy)-[l, -biphenyl]-4-carbonyl)-5-
(2chlorophenyl)pyrrolidine-2carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2 (cyclopropylmethoxy)-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S.5R)-l-(2'- methoxy-[l,r-biphenyl]-4-carbonyl)-5-phenylpyrro lidine-2-carboxylic acid, (2S,5R)-5-(3- chlorophenyl)-l-(2'-methoxy-[l, -biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid,
(2S,5R)-5-(4-chlorophenyl)-l-(2'-methoxy-[l, -biphenyl]-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(3-fluorophenyl)-l-(2'-methoxy-[l, -biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(4-fluorophenyl)-l-(2'-methoxy-[l, 1- biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-4-acetyl-5-(2-chlorophenyl)-l- (2'- methoxy-[l,l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,4S,5R)-5-(2- chlorophenyl)-l-(2-methoxy-[l,r-biphenyl]-4-carbonyl)-4-(methoxymethyl)pynOlidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2-methoxypyrimidin-4- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-cyclohexyl-l-(2'-methoxy-[l, - biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4-(2-chloro-4- methoxypyrimidin-5-yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5- (2-chlorophenyl)-l-(4-(3-methoxypyridin-2-yl)benzoyl)pyrrolidine-2-carboxylic acid,
(2R,5R)-5-(2-fluorophenyl)-l-(2'-methoxy-[l, -biphenyl]-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5S)-5-(2-fluorophenyl)-l-(2'-methoxy-[l, -biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2R,5 S)-5-(2-fluorophenyl)-l-(2'-methoxy-[l, - biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2- (trifluoromethyl)-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(2',4'-difluoro-[l,biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid,
(2S,5R)-5-(2-chlorophenyl)-l-(2-methyl-[l,r- biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2,6-difluorophenyl)-l-(2'-methoxy-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2,4-difluorophenyl)-l-(2'-methoxy-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2,4-dichlorophenyl)-l-(2'-methoxy-[l, G- biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-isobutyl-l-(2'-methoxy-[l, G- biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-isopropyl-l-(2’-methoxy-[l,l '-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(3-chloro-4-(pyrimidin-4- yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (2-fluoro-[l, r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(2'-fluoro-4'- methoxy-[ 1 , 1 '-biphenyl]-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4'-fluoro-2'-methoxy-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(6-ethoxypyridin-3- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(6- isopropoxypyridin-3-yl)benzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (4-(6-methoxy-2-methylpyridin-3-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(3- chloro-4-(2-methoxypyrimidin-4-yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(3-chloro-4-(pyrimidin-5-yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-4-cyano-l-(2'-methoxy-[l, l'-biphenyl]-4- carbonyl)-3-methylpyrrolidine-2-carboxylic acid, (2S,4S,5R)-5-(2-chlorophenyl)-4-cyano-l- (2'-methoxy-[l, l'-biphenyl]-4-carbonyl)-4-methylpyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(2,3-dimethoxy-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3,,4-dimethoxy-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2,3’,4-trimethoxy-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2,3’,6-trimethoxy- [1, r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (3’,5’dimethoxy-[ 1 , 1 '-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(2',5,-dimethoxy-[l,r-biphenyl]-4-carbonyl)pynOlidine-2carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-isopropyl-[l, -biphenyl]-4-carbonyl)pyrrolidine-2- carboxylic acid, 2S,5R)-l-(2,2'-dimethoxy-[l,r-biphenyl]-4-carbonyl)-5-(2- fluorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)- l-(2-fluoro-2'-methoxy-[l,r-biphenyl]-
4-carbonyl)-5-(2-fluorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-cyclopentyl- 1 -(2 - methoxy-[l, -biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(2'-ethyl-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-
5-(2-chlorophenyl)-l-(4-(2,6-dimethylpyridin-3-yl)benzoyl)pyrrolidine-2-carboxylic acid,
(2S,5R)-l-(4-(2,4-bis(benzyloxy)pyrimidin-5-yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-l-([l,4', -terphenyl]-4-carbonyl)-5-(2- chlorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4'-propyl-[l, biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid 324 (2S.5R)- l-(4'-(tert-butyl)-[l, -biphenyl]-4- carbonyl)-5-(2-chlorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-l-(3-chloro-4-(2,4- dimethoxypyrimidin-5- yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)- 5-(2-chlorophenyl)-l-(5-(2-methoxyphenyl)pyrazine-2-carbonyl)pynOlidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3-methoxy-4-(4-methoxypyridin-3- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3-methoxy-4-(6- methoxypyridin-3-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(3-chloro-4-(2- methoxypyrimidin-5- yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2- carboxylic acid, (2S ,5R)- 1 -(3 -chi oro-4-(6-methoxypyri din-3- yl)benzoyl)-5-(2-chlorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(l-(4-(4-chlorophenyl)thiazol-2-yl)piperidine-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-fluorophenyl)-l-(5-methoxy-6-(2- methoxyphenyl)nicotinoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(l-(benzo[d]oxazol-2- yl)piperidine-4- carbonyl)-5 -(2-chlorophenyl)pyrro lidine-2- carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(3-methoxy-4-(pyrrolidin-l-yl)benzoyl)pyrrolidine-2-carboxylic acid,
(2S,5R)-5-(2-chlorophenyl)-l-(5-methoxy-6-(2-methoxyphenyl)nicotinoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(l-(2-methoxyphenyl)piperidine-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2,4- dimethoxypyrimidin-5 yl)-3-methoxybenzoyl)pyrro lidine-2-carboxylic acid, 2S,5R)-5-(2- bromophenyl)-l-(2'-methoxy-[l, -biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3’-cyano-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3,-cyano-2'-methoxy-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3’-cyano-2',4'- bis(2,2,2-trifluoroethoxy)-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-1- (3’-amino-2'-methyl-[l,r-biphenyl]-4- carbonyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-methyl-3,-(methylsulfonamido)-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(3’-acetamido-2'-methyl-[l,r-biphenyl]-4- carbonyl)-5-(2-chlorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(5’- cyano-2'-methoxy-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(5'-cyano-2’-methyl-[l,r-biphenyl]-4-carbonyl)pynOlidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(4,6-dimethoxypyridin-3-yl)benzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(3,6-dimethoxypyridazin-4- yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5S)-5-isopentyl-l-(2'-methoxy-[l,r-biphenyl]- 4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-methoxy-4'- (methylsulfonamido)-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4'- acetamido-2'-methoxy-[l,r- biphenyl]-4-carbonyl)-5-(2- chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(3’-carbamimidoyl-[l,r-biphenyl]-4-carbonyl)-5-(2-chlorophenyl)pynOlidine- 2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3,-((E)-N’- hydroxycarbamimidoyl)-[l,r- biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-fluorophenyl)-l-(2'- methoxy-4'-(methylsulfonamido)-[l,r-biphenyl]-4-carbonyl)pynOlidine-2-carboxylic acid 355 (2S,5R)-5-(2,4-difluorophenyl)-l-(4-(2,6-dimethoxypyridin-3-yl)benzoyl)pynOlidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3-methoxy-4-(5-methoxypyridin-3- yl)benzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-l-(4-amino-2-methoxy-[l,r-biphenyl]-4- carbonyl)-5 -(2-chlorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (23,6’-trimethoxy- [2,3'-bipyridine]-5-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(3’- carbamoyl-[l,r-biphenyl]-4-carbonyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(5’-cyano-2,3’-dimethoxy-[l, -biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-cyano-4,5- dimethoxy-[l,l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(3,4,5,-trimethoxy- [1,1 '-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid (2S,5R)-5-(2-chlorophenyl)-l-(2’-(cyanomethyl)-4',5'-dimethoxy-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3’,4'-dicyano-[l,r- biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(5’-cyano- 2'-fluoro-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, 2S,5R)-5-(2- chlorophenyl)-l-(2-fluoro-3,4-dimethoxy-[l,r-biphenyl]-4-carbonyl)pyrrolidine- 2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(2,6-dimethoxypyridin-3-yl)-3- fluorobenzoyl)pyrrolidine-2-carboxylic acid, 2S,5R)-5-(2-chlorophenyl)-l-(3-fluoro-4-(6- methoxypyridin-3-yl)benzoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)- 1 - ( 1 -(2-cyano-4- (trifluoromethyl)phenyl)piperidine-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(l-(2-chloro-4-(trifluoromethyl)phenyl)piperidine-4-carbonyl)-5(2- chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(5’-cyano-2-methoxy-[l,r-biphenyl]-4- carbonyl)-5-(2-fluorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4-(2,6- dimethoxypyridin-3-yl)-3- fluorobenzoyl)-5-(2-fluorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(3-fluoro-4-(6-methoxypyridin-3-yl)benzoyl)-5-(2-fluorophenyl)pynOlidine-2- carboxylic acid, (2S,5R)-l-(4-(3,6-dimethoxypyridazin-4-yl)benzoyl)-5-(2- fluorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-l-(3’-carbamoyl-4'-cyano-[l,r-biphenyl]- 4-carbonyl)-5-(2-chlorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l- (l-(2-nitro-4-(trifluoromethyl)phenyl)piperidine-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(l-(4-(morpholinosulfonyl)-2-nitrophenyl)piperidine-4- arbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(l-(2-nitro-4-(piperidin- l-ylsulfonyl)phenyl)piperidine-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(l-(4-(N,N-diethylsulfamoyl)-2-nitrophenyl)piperidine-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(l-(4-methyl-2- nitrophenyl)piperidine-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-
1-(l-(2-cyano-4-nitrophenyl)piperidine-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5- (2-chlorophenyl)-l-(l-(4-nitrophenyl)piperidine-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(l-(2-fluoro-4-nitrophenyl)piperidine-4-carbonyl)pynOlidine-
2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(l-(3-methoxy-4-nitrophenyl)piperidine-4- carbonyl)pyrrolidine-2-carboxylic acid,(2S,5R)-l-(l-(5-chloro-2-nitrophenyl)piperidine-4- carbonyl)-5-(2-chlorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-cyanophenyl)-l-(2'- methoxy-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(2'-cyano-4'-methoxy-[l, r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2-fluoro-3,-(methylsulfonamido)-[l,'-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2’-cyano-2-fluoro- [1, 1 '-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)- 1 -( 1 -(2- cyano-4-(methylsulfonamido)phenyl)piperidine-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(l-(2-cyano-4-methoxyphenyl)piperidine-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(l-(2-
(methylsulfonamido)-4-(trifluoromethyl)phenyl)piperidine-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(l-(2-nitrophenyl)piperidine-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(l-(4- cyanophenyl)piperidine-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(3,5- difluorophenyl)-l-(2'-methoxy-[l, r-biphenyl]-4-carbonyl)pynOlidine-2-carboxylic acid, (2S,5R)-5-(3,4-difluorophenyl)-l-(2'-methoxy-[l, r-biphenyl]-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2,3-difluorophenyl)-l-(2'-methoxy-[l, r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2,5-difluorophenyl)-l-(2'-methoxy-[l, G- biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid 400 (2S,5R)-5-([l, l-biphenyl]-2-yl)-l-(2'- methoxy- [l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S.5R)- 1 -(2’-cyano-4’- methoxy-[l, -biphenyl]-4-carbonyl)-5-(2-fluorophenyl)pyrrolidine-2-carboxylic acid,
(2S,5R)-5-(4-cyanophenyl)-l-(2'-methoxy-[l, -biphenyl]-4-carbonyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4-(5-methyl-4- (phenylsulfonyl)-lH-l,2,3- triazol- 1 - yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(3’-cyano- 4’-fluoro-[l,r-biphenyl]-4-carbonyl)pynOlidine-2-carboxylic acid, (2S,5R)-l-(2'-chloro-5’- cyano-[l,r-biphenyl]-4- carbonyl)-5 -(2-chlorophenyl)pyrro lidine-2-carboxylic acid, (2S,5R)- 5-(2-chlorophenyl)-l-(2-cyano-4'- (trifluorom ethyl)- [ 1 , 1 '-biphenyl]-4- carbonyl)pyrrolidine- 2-carboxylic acid, 2S,5R)-5-(2-chlorophenyl)-l-(l-(2-methoxy-4-
(trifluoromethyl)phenyl)piperidine-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- chlorophenyl)-l-(2'-methyl-3 ,-(N-methylmethylsulfonamido)-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(2'-methoxy-4-(N- methylmethylsulfonamido)-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid,(2S,5R)- 5-(2-chlorophenyl)- 1 -(6-(5-cyano-2- methoxyphenyl)-5-methoxynicotinoyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(6-(2,4-dimethoxyphenyl)-5- methoxynicotinoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(6-(2,4- dimethoxyphenyl)nicotinoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(2'-cyano-4- (trifluoromethyl)-[l, -biphenyl]-4-carbonyl)-5-(2-fluorophenyl)pyrrolidine-2-carboxylic acid, (2 S, 5R)- 1 -(3’ -cyano-4'-fluoro- [ 1 , 1 '-biphenyl] -4-carbonyl)-5 -(2- fluorophenyl)pyrrolidine-2- carboxylic acid, (2S.5R)-l-(2'-chloro-5’-cyano-[l,r-biphenyl]-4- carbonyl)-5-(2-fluorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-chlorophenyl)-l-(4- (3,6-dimethoxypyridazin-4-yl)-3-fluorobenzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2- fluorophenyl)-l-(2'-methyl-3,-(N-methylmethylsulfonamido)-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-fluorophenyl)-l-(2'-methoxy-4'-(N methylmethylsulfonamido)-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)- 5-(2-chlorophenyl)-l-(4-(4,6-dimethoxypyrimidin-5-yl)benzoyl)pyrrolidine-2- arboxylic acid, (2S,5R)-5-(2,3-difluorophenyl)-l-(4-(2,4-dimethoxypyrimidin-5-yl)benzoyl)pyrrolidine-2- carboxylic acid, (2S.5R)-l-(5'-cyano-2'-methyl-[l,l’-biphenyl]-4-carbonyl)-5-(2,3- difluorophenyl)pyrro lidine-2-carboxylic acid, (2S,5R)-5-(2,3-difluorophenyl)-l-(2-methoxy- 4'- (methylsulfonamido)-[l,r-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5- (2,3-difluorophenyl)-l-(2-methyl-3,-(methylsulfonamido)-[l,r-biphenyl]-4- carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2-fluorophenyl)-l-(2-methyl-3 (methylsulfonamido)-[l, l'-biphenyl]-4-carbonyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5- (2,3-difluorophenyl)-l-(4-(2-methoxypyridin-3-yl)benzoyl)pynOlidine-2-carboxylic acid, (2S,5R)-5-(2,3-difluorophenyl)-l-(3-methoxy-4-(2-methoxypyrimidin-5-yl)benzoyl)pyrro lidine-2- carboxylic acid, (2S,5R)-5-(2-fluorophenyl)-l-(3-methoxy-4-(2- methoxypyrimidin-5 -yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-5-(2,3-difluorophenyl)-l-(4-(3,6- dimethoxypyridazin-4-yl)benzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(5’-cyano-2- methoxy-[l,r-biphenyl]-4-carbonyl)-5-(2,3-difluorophenyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(5'-cyano-2'-methyl-[l,l-biphenyl]-4-carbonyl)-5-(2-fluorophenyl)pyrrolidine-2- carboxylic acid, (2S,5R)-5-(2,3-difluorophenyl)-l-(4-(3,6-dimethoxypyridazin-4-yl)-3 fluorobenzoyl)pyrrolidine-2-carboxylic acid, (2S,5R)-l-(4-(3,6-dimethoxypyridazin-4-yl)-3- fluorobenzoyl)-5-(2-fluorophenyl)pyrrolidine-2-carboxylic acid (see the International Publication No. WO2011073376, the complete contents of which is hereby incorporated by reference)
In some embodiment, the synthetic FFAR2 agonist is generally described in the International Publication No. WO2011151436A2 which is incorporated herein by reference in in its entirety.
In some embodiment, the synthetic FFAR2 agonist is generally described in Hansen et al, Journal of Medicinal Chemistry (2018)32 and Wang et al, Bioorganic & Medicinal Chemistry Letters 20 (2009)33 which are incorporated herein by reference in their entirety. In a particular embodiment, the synthetic FFAR2 agonist is TUG-1375.
As used herein, the term“TUG-1375” also called (2R,4R)-2-(2-chlorophenyl)-3-(4- (3,5-dimethylisoxazol-4-yl)benzoyl)thiazolidine-4-carboxylic acid has its general meaning in the art and refers to the compound characterized by the formula of :
Figure imgf000026_0001
In a particular embodiment, the synthetic FFAR2 agonist is AMG7703.
As used herein, the term“AMG7703” also called (2S)-2-(4-chlorophenyl)-3-methyl-N- (l,3-thiazol-2-yl)butanamide has its general meaning in the art and refers to the compound characterized by the formula of :
Figure imgf000026_0002
In a particular embodiment, the synthetic FFAR2 agonist is AZ1729.
As used herein, the term“AZ1729” also called N-[3-(2-carbamimidamido-4-methyl- l,3-thiazol-5-yl)phenyl]-4-fluorobenzamide has its general meaning in the art and refers to the compound characterized by the formula of :
Figure imgf000026_0003
In a particular embodiment, the synthetic FFAR2 agonist is 4-CMTB. As used herein, the term “4-CMTB” also called 4-chloro-a-(l-methylethyl)-N-2- thiazolylbenzeneacetamide has its general meaning in the art and refers to the compound characterized by the formula of:
Figure imgf000027_0001
In a particular embodiment, the synthetic FFAR2 agonist is 4-CDPB.
As used herein, the term“4-CDPB” also called (S)-2-(4-chlorophenyl)-3,3-dimethyl-N- (5-phenylthiazol-2-yl)butanamide in its racemic form and (S)-2-(4-chlorophenyl)-3,3- dimethyl-N-(5-phenylthiazol-2-yl)butanamide in its enantiopure form has its general meaning in the art and refers to the compound characterized by the formula of:
Figure imgf000027_0002
In a particular embodiment, the synthetic FFAR2 agonist is Cpd A.
As used herein, the term “Cpd A” also called 3-benzyl-4-(cyclopropyl(4-(2,5- dichlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid in its racemic form and (f?)-3-benzyl- 4-(cyclopropyl(4-(2,5-dichlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid in its enantiopure form has its general meaning in the art and refers to the compound characterized by the formula of:
Figure imgf000027_0003
As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
A "therapeutically effective amount" refers to an amount effective (e.g., a prophylactic or therapeutic agent), at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of drug may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of drug to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for drug depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of drug employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of drug is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered overtime or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
The therapy, which is sufficient to reduce the severity and/or duration of a disease, ameliorate one or more symptoms thereof, prevent the advancement of a disease or cause regression of a disease, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of a disease or one or more symptoms thereof, or enhance or improve the prophylactic and/or therapeutic effect(s) of another therapy (e.g., another therapeutic agent) useful for treating a disease.
The method of the present invention is particularly suitable for subjects who are identified as at high risk for developing a bacterial superinfection post-viral infection (e.g. post- influenza), including subjects who are at least 50 years old, subjects who reside in chronic care facilities, subjects who have chronic disorders of the pulmonary or cardiovascular system, subjects who required regular medical follow-up or hospitalization during the preceding year because of chronic metabolic diseases (including diabetes mellitus), renal dysfunction, hemoglobinopathies, or immunosuppression (including immunosuppression caused by medications or by human immunodeficiency [HIV] virus); children less than 14 years of age, patients between 6 months and 18 years of age who are receiving long-term aspirin therapy, and women who will be in the second or third trimester of pregnancy during the influenza season. More specifically, it is contemplated that the method of the invention is suitable for the treatment of bacterial superinfection post- viral infection (e.g. post-influenza) in subjects older than 1 year old and less than 14 years old (i.e., children); subjects between the ages of 50 and 65, and adults who are older than 65 years of age.
In some embodiments, the synthetic FFAR2 agonist is administered to the subject in a form of a pharmaceutical composition. For instance, the synthetic FFAR2 agonist may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The synthetic FFAR2 agonist can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
In some embodiments, the composition comprises any other ingredients or excipients known to be employed in the type of composition in question. Non limiting examples of such ingredients include: proteins, amino acids, carbohydrates, oligosaccharides, lipids, prebiotics or probiotics, nucleotides, nucleosides, other vitamins, minerals and other micronutrients.
In some embodiments, the composition typically comprises carriers or vehicles. “Carriers” or“vehicles” mean materials suitable for administration and include any such material known in the art such as, for example, any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is non-toxic and which does not interact with any components of the composition in a deleterious manner. Examples of nutritionally acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:
Figure 1: Altered fermentative activity (SCFA production) of the gut microbiota during IAV infection. Seven and fourteen days after infection, cecal contents were collected for SCFA quantification. (A) Cecal concentrations of total SCFAs in mock-treated and IAV- (H3N2) infected mice. (B) Cecal concentrations of individual SCFAs. A and B (n = 21-33, five pooled experiments). (C) Cecal concentrations of total SCFAs in mock-treated and IAV (HlNl)-infected mice (7 dpi) (n = 6-8, one experiment performed). (D) Blood concentrations of total SCFAs (9-10 pooled sera, 4 mice/pool). The repartition of individual SCFAs in the blood is represented (mock). Significant differences were determined using the Kruskal-Wallis ANOVA test (a and b) and the Mann- Whitney U test (c and d) (*p < 0.05; ***p < 0.001).
Figure 2: Effect of acetate supplementation in mice doubly infected with IAV and S. pneumoniae. A-C, IAV (HlNl)-infected mice were treated with acetate (Ace) (200 mM in drinking water) or vehicle (Vh) at day 2 post-infection, five days before the pneumococcal challenge (lxlO3 c.f.u). (A), The number of bacteria was determined 30 h after the S. pneumoniae challenge. The solid lines correspond to the median values (n = 8-9, a representative experiment out of two is shown). Significant differences were determined using the Mann- Whitney U test. (B), Body weight evolution (in % initial body weight, means ± SD). (C), The survival of superinfected animals was monitored. (B) and (C), n=14 (two pooled experiments). Survival of mice was compared using Kaplan-Meier analysis and log-rank test (*p < 0.05, **p < 0.01).
Figure 3: Effect of FFAR2 agonist treatment on bacterial superinfection postinfluenzal. A and B, IAV (HlNl)-infected mice were treated with the selective FFAR2 agonist TUG-1375 (A) or with the selective FFAR3 agonist AR420626 (B) (1 mM in 50 mΐ, i.n. route) or vehicle (Vh) 16 h before the pneumococcal challenge. The number of bacteria in the lungs and spleen was determined 30 h after the S. pneumoniae challenge. (A), n = 13-14 (two pooled experiments) and (B), n = 6-8 (one representative experiment out of two). Significant differences were determined using the Mann- Whitney U test (*p < 0.05, **p < 0.01).
EXAMPLE:
Material & Methods
Mice and ethics statement
Specific pathogen-free C57BL/6J mice (6 week-old, male) were purchased from Janvier (Le Genest-St-Isle, France). Mice were maintained in a biosafety level 2 facility in the Animal Resource Center at the Lille Pasteur Institute for at least two weeks prior to usage to allow appropriate acclimatation. Unless specified, mice were fed a standard rodent chow (SAFE A04, SAFE, Augy, France) and water ad libitium. This diet contains -11.8% fiber including -10% water-insoluble fiber (3.6% cellulose) and 1.8% water-soluble fiber. All experiments complied with current national and institutional regulations and ethical guidelines (B59-350009, Institut Pasteur de Lille protocol number: 2015121722376405 and 13743-2018022211144403).
Reagents and antibodies
Acetate as from Sigma-Aldrich (St quentin valladier, france). The FFAR2 agonist TUG- 1375 (2R,4R)-2-(2-chlorophenyl)-3-(4-(3,5-dimethylisoxazol-4-yl)benzoyl)thiazolidine-4- carboxylic acid) and the FFAR3 agonist AR420626 (N-(2,5-dichlorophenyl)-4-(furan-2-yl)-2- methyl-5-oxo-l,4,5,6,7,8-hexahydroquinoline-3-carboxamide were produced as described34’35.
Infections and assessment of bacterial loads
For infection with IAV alone, mice were anesthetized by intramuscular injection of 1.25 mg of ketamine plus 0.25 mg of xylazine in 100 mΐ of phosphate buffered saline (PBS), and then intranasally (i.n.) infected with 50 mΐ of PBS containing (or not, in a mock sample) 30 plaque forming units (p.f.u.) of the H3N2 IAV strain A/Scotland/20/1974, or 100 p.f.u. ofHINl A/Califomia/04/2009 (pdm09)3’7. These doses correspond to sub-lethal doses, which are necessary to investigate secondary bacterial infection. For secondary pneumococcal infection, IAV (H1N1, pdm2009)-infected mice were challenged at 7 dpi with a low dose (lxlO3 c.f.u.) of S. pneumoniae serotype 1, a serotype linked to invasive pneumococcal disease (clinical isolate El 586). This dose is largely sufficient to allow bacterial outgrowth and dissemination. In double infected mice, bacteria in the lungs and spleen were counted 3 Oh after the S. pneumoniae challenge by plating serial 10-fold dilutions of lung or spleen homogenates onto blood agar plates. The plates were incubated at 37°C with 5% C02 overnight and viable bacteria were counted 24h later. Survival and body weight were monitored daily after IAV infection and mice were euthanized when they lost in excess of 20% of their initial body weight.
Measurement of SCFA concentrations and treatment with acetate or FFAR2/FFAR3 agonists
Concentrations of SCFAs in the cecal content were determined after extraction with diethyl ether using GC-2014 gas chromatography with AOC-20i auto injector (Shimadzu, Hertogenbosch, the Netherlands) as described36. Concentrations of SCFAs in plasma were determined after extraction with acetonitrile. Results are expressed as pmol/g of cecal content or as mM (blood). To assess the effects of SCFAs on lung defense against bacterial infection, mice infected with IAV were treated with acetate (200 mM, drinking water) five days before the S. pneumoniae challenge. The FFAR2 agonist TUG-1375 and the FFAR3 agonist AR420626 (stock solutions in DMSO at 20mM) was inoculated by the i.n. route (50m1, 1 mM) 16h before S. pneumoniae infection.
Statistical analysis
Results are expressed as the mean ± standard deviation unless otherwise stated. All statistical analysis was performed using GraphPad Prism v6 software. A Mann- Whitney U test was used to compare two groups unless otherwise stated. Comparisons of more than two groups with each other were analyzed with the One-way ANOVA Kruskal- Wallis test (nonparametric), followed by the Dunn’s posttest. Survival of mice was compared using Kaplan-Meier analysis and log-rank test.*, p < 0.05; **, p < 0.01; ***, p < 0.001.
Results
Sub-lethal influenza infection transiently alters the composition and the metabolic output of the gut microbiota
We have recently shown that sub-lethal influenza (H3N2 and H1N1) infection leads to transient gut dysbiosis (Sencio et al. submitted). Since changes in the composition of the gut microbiota can alter its functionality (e.g. metabolic activity), we quantified the production of SCFAs, major metabolites of the gut microbiota, during the course of influenza infection. SCFAs are generated by bacterial fermentation of colonic dietary fibers, reaching high concentrations in the gut lumen under physiological conditions18,37. As shown in Figure 1A, the total SCFA concentration in the cecum 7 days after H3N2 infection was lower relative to non-infected mice. The concentrations of acetate (the predominant SCFA), propionate and butyrate were all lower (Figure IB). At 14 dpi, the cecal SCFA concentrations returned to basal levels. A significantly reduced concentration of SCFAs was also observed at 7 dpi in mice infected with H1N1 IAV (Figure 1C). It is known that SCFAs produced in the gut can pass into the systemic circulation and then exert remote biological effects (particularly acetate and, to a lesser extent, propionate and butyrate)38^40. As seen in Figure ID, influenza infection resulted in a lowered concentration of SCFAs in the blood at 7 dpi. In agreement with other studies38-41, acetate was the predominant SCFA found in the blood. These data show that influenza infection alters the metabolic (fermentative) output of the gut microbiota at 7 dpi and that it affects local (gut) and systemic (blood) concentration of SCFAs, an emerging group of dietary derived metabolites endowed with immune regulatory functions42.
Supplementation of acetate during influenza protects against bacterial superinfection We then investigated the potential contribution of altered SCFA production during IAV infection on secondary bacterial infection. To this end, IAV-infected mice were treated with acetate and secondarily infected with S. pneumoniae at day 7, the peak of susceptibility. Remarkably, acetate supplementation lowered the bacterial load in the lungs and resulted in reduced systemic spread of bacteria from the lungs in double-infected mice (Figure 2A). We then determined whether the positive effect of acetate on bacterial loads extended to ameliorated morbidity and mortality outcomes. Whilst acetate treatment had no effect on weight loss due to IAV infection, it favored weight regain after secondary pneumococcal infection (Figure 2B). Most notably, supplementation of acetate during the course of IAV infection effectively and significantly improved survival of double-infected mice (-50% survival rate, Figure 2C). Taken as a whole, low acetate production during influenza infection influences susceptibility to secondary bacterial infection and supplementation of acetate is sufficient to improve disease outcomes.
Exogenous administration of a synthetic FFAR2 agonist protects against postinfluenza secondary bacterial infection
Acetate can act through the G-protein coupled receptors FFAR2 (formerly GPR43) and, to a lesser extent, FFAR3 (formerly GPR41). Of interest, FFAR2 and FFAR3 are amenable to pharmacological manipulation in vivo43. To investigate the potential consequences of local FFAR2 or FFAR3 activation on post-influenza bacterial superinfection, mice were treated with TUG-1375 (a selective FFAR2 agonist) or AR420626 (a selective FFAR3 agonist) by intra nasal administration. Pharmacological FFAR2 activation, just before pneumococcal challenge, led to a significant reduction of lung bacterial burden and dissemination to blood (Figure 3A). In contrast, the selective FFAR3 agonist AR420626 failed to confer any protection (Figure 3B). Hence, the FFAR2 agonist TUG-1375 provided the same benefit as acetate in the treatment of post-influenza bacterial superinfection. This latter finding opens up important new possibilities for pharmacological management of post-influenza bacterial superinfection.
Discussion
A large body of research indicates that alterations in the gut microbiota have a role in the pathogenesis of various chronic diseases. The present study sought to analyze the impact of an acute respiratory infection on the gut microbiota functionality (SCFA production), and study the consequences of any functional perturbations on disease outcomes. The results showed that influenza infection alters the production of gut SCFAs and that these changes account for enhanced susceptibility to secondary pulmonary bacterial infections. This study also showed for the first time the positive effect of a synthetic FFAR2 agonist in respiratory infection.
Recent data indicate that dysbiosis in the upper respiratory compartment contributes to post-influenza secondary bacterial infections44. The present study is the first to have addressed the question of whether alterations in the gut microbiota functionality might predispose to secondary bacterial infections of the lung. A continuous input from complex microbiota is necessary to maintain the (pulmonary) innate immune system. The inventors hypothesized that a loss of input from gut microbiota-derived signals during IAV infection may have negative consequences on pulmonary defenses against bacterial infection. The inventors showed that at 7 days post-IAV infection, there is a drop of production of SCFAs, major metabolites of the gut microbiota. Surprisingly, acetate supplementation reduced the bacterial burden after the episode influenza - despite the immunosuppressive environment imposed by IAV. Of importance, this translated into improved survival rate of double-infected mice. Lastly, the inventors showed that local activation of FFAR2 resulted in reduced bacterial burden in superinfected mice. This later result suggests that the use of synthetic FFAR2 agonists as therapeutics might be envisaged to lower bacterial superinfection post-influenza. FFAR2 agonists are viewed as a promising treatment of metabolic syndromes such as type 2 diabetes and obesity43’45 47. Our study shows for the first time the positive effect of a synthetic FFAR2 agonist in respiratory infection and highlights a new opportunity for further development against bacterial pneumonia.
REFERENCES:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
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Claims

CLAIMS:
1. A method of treating a bacterial superinfection post-viral infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a synthetic free fatty acid receptor 2 (FFAR2) agonist.
2. The method of claim 1 wherein the synthetic FFAR2 agonist is selected from TUG- 1375, AMG7703, AZ1729, 4-CMTB, 4-CDPB, Cpd A.
3. The method of claim 2 wherein the synthetic FFAR2 agonist is TUG-1375.
4. The method of claim 1 to 3 wherein the viral infection is influenza infection.
5. The method of claim 1 to 3 wherein the influenza infection is associated with Influenza virus A or B.
6. The method of claim 5 wherein the influenza infection is caused by an influenza virus A that is H1N1, H2N2, H3N2 or H5N1.
7. The method of claim 1 to 3 wherein viral infection is caused by a virus selected from the group consisting of influenza virus, human immunodeficiency viruses (HIV), Zika virus, Cytomegalovirus (CMV), respiratory syncytial virus, adenovirus, metapneumovirus, cytomegalovirus, parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV- 3, hPIV-4), rhinovirus, coxsackie virus, echo virus, herpes simplex virus, coronavirus (SARS-coronavirus such as SARS-CoV-1 or SARS-CoV-2), and smallpox.
8. The method of claim 1 to 3 wherein viral infection may be due to a member of the Pneumoviridae, Paramyxoviridae and/or Coronaviridae families.
9. The method of claim 1 to 8 wherein the subject suffers or is susceptible to suffer from enteric infections.
10. The method of claim 1 wherein the bacterial superinfection is selected from the group consisting of lower respiratory tract infections, middle ear infections and bacterial sinusitis.
11. The method of claim 1 wherein the bacterial superinfection may be mediated by at least one organism selected from the group consisting of Streptococcus pneumoniae ; Staphylococcus aureus ; Haemophilus influenza , Myoplasma species and Moraxella catarrhalis.
12. The method of claim 1 wherein the subject is selected from the group consisting of subjects who are at least 50 years old, subjects who reside in chronic care facilities, subjects who have chronic disorders of the pulmonary or cardiovascular system, subjects who required regular medical follow-up or hospitalization during the preceding year because of chronic metabolic diseases, renal dysfunction, hemoglobinopathies, or immunosuppression, children less than 14 years of age, patients between 6 months and 18 years of age who are receiving long-term aspirin therapy, and women who will be in the second or third trimester of pregnancy during the influenza season
13. The method of claim 1 which is suitable for the prevention of bacterial superinfection post-viral infection in subjects older than 1 year old and less than 14 years old; subjects between the ages of 50 and 65, and adults who are older than 65 years of age, in subject suffering from co-morbidities.
14. The method of claim 1 wherein the synthetic FFAR2 agonist is administered to the subject in a form of a pharmaceutical composition.
PCT/EP2020/071015 2019-07-26 2020-07-24 Use of ffar2 agonists for the treatment of bacterial superinfections post-viral infection WO2021018786A1 (en)

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Cited By (2)

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CN116421606A (en) * 2023-04-24 2023-07-14 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Application of small molecular medicine AMG7703 in resisting influenza virus infection
CN116421606B (en) * 2023-04-24 2023-09-19 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Application of small molecular medicine AMG7703 in resisting influenza virus infection

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