WO2012064654A1 - Méthodes de prévention et de traitement de la grippe - Google Patents

Méthodes de prévention et de traitement de la grippe Download PDF

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Publication number
WO2012064654A1
WO2012064654A1 PCT/US2011/059590 US2011059590W WO2012064654A1 WO 2012064654 A1 WO2012064654 A1 WO 2012064654A1 US 2011059590 W US2011059590 W US 2011059590W WO 2012064654 A1 WO2012064654 A1 WO 2012064654A1
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alkyl
noscapine
analog
aralkyl
hydrogen
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PCT/US2011/059590
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English (en)
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Milton H. Werner
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Inhibikase Therapeutics, Inc.
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Publication of WO2012064654A1 publication Critical patent/WO2012064654A1/fr

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    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • A61K9/2018—Sugars, or sugar alcohols, e.g. lactose, mannitol; Derivatives thereof, e.g. polysorbates

Definitions

  • Influenza commonly referred to as the flu
  • Symptoms of influenza in human may include: fever and extreme coldness (e.g., chills, shivering, and shaking); cough; nasal congestion; body aches, especially in the joints and throat; fatigue; headache; irritated, watering eyes; and reddened eyes, skin (especially face), mouth, throat and nose.
  • fever and extreme coldness e.g., chills, shivering, and shaking
  • cough nasal congestion
  • body aches especially in the joints and throat
  • fatigue fatigue
  • headache irritated, watering eyes
  • reddened eyes skin (especially face), mouth, throat and nose.
  • gastrointestinal symptoms such as diarrhea and abdominal pain may also occur.
  • Current treatments for influenza include prevention by vaccination with inactivated or live attenuated virus, or administration of antiviral drugs prophylactically or therapeutically.
  • Standard flu vaccine grown in chicken eggs can provide some protective benefit for the most at-risk sectors of the human population (children ⁇ 2 years old and adults > 62 years old (Root et al. (2000) Entry of influenza viruses into cells is inhibited by a highly specific protein kinase C inhibitor. J Gen Virol 81:2697- 2705), but vaccine effectiveness is waning as the prevalence of vaccination is leading to ever more resistant strains of the virus.
  • influenza advisory committees at the Center for Disease Control and Prevention and the World Health Organization (“WHO”) are tasked to predict which flu strains are likely to infect the human population one full year in advance of treatment in order to meet production timelines for the flu vaccine.
  • SARS severe acute respiratory syndrome
  • the virus is a novel coronavirus and has been named SARS-associated coronavirus (SARS-CoV).
  • SARS-CoV SARS-associated coronavirus
  • the present invention relates to methods of preventing or treating influenza or a coronavirus-associated disease (e.g., SARS).
  • a method of preventing or treating influenza or a coronavirus-associated disease can comprise administering to a mammal in need thereof an effective amount of a comprising noscapine or an analog thereof, or a pharmaceutically acceptable prodrug thereof, or a pharmaceutically acceptable salt of noscapine, the analog, or the prodrug.
  • the analog of noscapine is a compound having a structure of Formula (I):
  • R 1 and R 2 are each independently selected from: H, a substituted or unsubstituted alkyl, alkenyl, or alkynyl group or a substituted or unsubstituted carbocyclyl, heterocyclyl, aryl, or heteroaryl group, provided that if R 1 is a methyl group, then R 2 is not H.
  • the analog is selected from:
  • the analog of noscapine is a compound having a structure of Formula (II):
  • Ar is aryl or heteroaryl.
  • the analog of noscapine is a compound having a structure of Formula (III):
  • R 3 and R 4 are each independently selected from hydrogen, alkyl, or aryl, or R 3 and R 4 together with the nitrogen to which they are bound form a heterocycle.
  • the analog of noscapine is a compound having a structure of Formula (IV):
  • R 3 and R 4 are each independently selected from hydrogen, alkyl, or aryl, or R 3 and R 4 together with the nitrogen to which they are bound form a heterocycle.
  • the analog of noscapine is a compound of having a structure of Formula (V):
  • R is selected from: H, SH, OH, OTf, SBn, NHBn, NH 2 , NHMe, and Ar, wherein Ar is selected from:
  • the analog of noscapine is a compound of Formula (V), wherein R is OH or NH and the analog has a structure of V-1 or V-2:
  • the analog of noscapine is a compound having a structure of Formula (VI):
  • the analog of noscapine is a compound having a structure of Formula (VII):
  • the analog of noscapine is a compound having a structure of Formula (VIII):
  • R is selected from:
  • the analog of noscapine is a compound having a structure of Formula (IX):
  • B is-OH, halogen, or a single bond that forms a six-membered heterocyclyl ring with A;
  • C is hydrogen, Q_ 6 alkyl, or halogen
  • D is (i) -CH 2 -halogen, -CH(O), -COOH, -C(0)-0-Ci_ 6 alkyl, -C(0)-0-Ci_ 6 aralkyl, -CH 2 OH, or -(CH 2 )n-CH 3 , wherein n is 1,2, or 3, or
  • E forms a five-or six-membered cycloalkyl or heterocyclyl ring
  • E is-OH or Ci_ 6 alkyl
  • D forms a five-or six-membered cycloalkyl or heterocyclyl ring, wherein this heterocyclyl ring contains- C(O) 0-, -C(0)NH-, -C(S)0-, or-C(S)NH-;
  • F is hydrogen, -0-Ci_ 6 alkyl,-0-Ci_ 6 aralkyl, -0-Ci_ 6 alkylheteroaryl, halogen, aryl, Ci_ 6 alkyl, -SH, thio-Ci_ 6 alkyl, -S-aryl, -0-S0 2 -Ci_ 6 alkyl, -0-S0 2 -Ci_ 6 aralkyl, cyano, or NR R 2 , where R 1 and R 2 are independently hydrogen, Ci_ 6 alkyl, Ci_ 6 aralkyl, cyano, aryl, heteroaryl, -S0 2 -Ci_ 6 alkyl, or-S0 2 -N(Ci_ 6 alkyl)(Ci_ 6 alkyl);
  • Gl to G4 independently represent hydrogen, aryl, halogen, Ci_ 6 alkyl, hydroxyl, -S-Ci-6 alkyl, nitro, -0-Ci_ 6 alkyl, -0-Ci_ 6 aralkyl, or -(CH 2 ) X NR 1 R 2 , where x is 0, 1, or 2 and where R 1 and R 2 are independently hydrogen, Ci_ 6 alkyl, Ci_ 6 aralkyl, cyano, aryl, heteroaryl, or acyl, or
  • two adjacent G2 to G4 groups together comprise an alkylene -(CH 2 )m-, where m is 3 or 4, to form a cycloalkyl ring, or together comprise an alkylene dioxy -O- (CH 2 ) n -0-, where n is 1 , 2, or 3, to form a heterocyclyl ring; and
  • K is Ci_6 alkyl, halogen, cyano, aryl, hydrogen, hydroxyl, thio-Ci_ 6 alkyl, sulfonyl, sulfoxyl, nitro, -0-Ci_ 6 alkyl, -0-Ci_ 6 aralkyl, or NR ⁇ 2 , where R 1 and R 2 are independently hydrogen, Ci_ 6 alkyl, Ci_ 6 aralkyl, cyano, aryl, heteroaryl, or acyl ;
  • alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, and aralkyl groups are optionally substituted with one or more suitable substituents.
  • the methods of the present invention can be used to prevent or treat influenza caused by any strain of the influenza virus, e.g., A/CA/04/2009, A/Bris/59/2007, A/Bris/10/2007, and B/Bris/60/2008.
  • the methods of the present invention can be used to prevent or treat influenza caused by any species of the influenza virus, e.g., Seasonal A influenza virus, and Seasonal B influenza virus.
  • any species of the influenza virus e.g., Seasonal A influenza virus, and Seasonal B influenza virus.
  • the method of the present invention can also be used to prevent or treat influenza caused by HINl-like and H3N2-like influenza viruses.
  • compositions of the present invention can be used to prevent or treat a disease associated with or caused by any strain of coronavirus, e.g., SARS caused by any strain of SARS-CoV.
  • the compositions of the present invention can further comprise a
  • the carrier can comprise cyclodextrins, e.g., ⁇ -cyclodextrins.
  • the present invention provides methods of preventing or treating influenza or a coronavirus-associated disease (e.g., SARS) in a human.
  • the composition can be administered orally.
  • the composition administered orally can be in a solid formulation.
  • the compounds of the present invention can be administered at a dose from about 300 mg/day to about 3 g/day.
  • the compositions of the present invention can be administered for a period from one day, up to 8 weeks or longer.
  • the methods of the prevent invention can be used to prevent or treat influenza in mammals that exhibit the symptom of coughing.
  • the methods of the present invention can also be used to prevent or treat influenza in mammals that do not exhibit the symptom of coughing.
  • FIG. 1A is a graph showing the efficacy of noscapine (dissolved in 100% dimethyl sulfoxide (“DMSO”)) in reducing cytopathic effect (“CPE”) on Madin
  • MDCK Darby canine kidney cells induced by infection of influenza virus strain A/CA/04/2009 (also known as novel H1N1 or the "swine flu” virus).
  • FIG. IB is a graph comparing the reduction of CPE by noscapine (as shown in FIG. 1 A) to its cytotoxicity across a range of noscapine concentrations.
  • FIGS. 2 A and 2B are tables comparing the antiviral activities
  • noscapine pharmacological parameters of noscapine and other anti-influenza drugs.
  • Human pharmacology parameters for noscapine are published (see, e.g., Karlsson et al. (1990) Pharmacokinetics of oral noscapine. Eur J Clin
  • FIG. 3 is a table showing the efficacy of noscapine against various respiratory pathogens.
  • FIG. 4 is a graph showing the effects of noscapine on survival of BALB/c mice infected with influenza virus strain A/C A/04/2009.
  • noscapine in beta-cyclodextrin was delivered by intraperitoneal (i.p.) administration twice a day for five days (b.i.d. x 5). 20 and 30% of the mice survived the virus infection when treated with noscapine at 62.3 or 19.9 mg/kg/d.
  • mice receiving that dose were likely to die almost 10 times less rapidly than mice receiving the excipient as opposed to mice receiving ribavirin at 75 mg/kg/d, which protected all mice from death; they were likely to die 47 times less rapidly than mice receiving the placebo PSS.
  • the mean day of death for those mice treated with this dose of noscapine that did succumb to infection was prolonged by almost four days compared to the mean day of death for mice treated with excipient.
  • FIGs. 5A, 5B and 5C are graphs showing the efficacy of noscapine, compound V-l and compound V-2 delivered by subcutaneous delivery on survival of BALB/c Mice infected with influenza virus strain A/C A/04/09: FIG.
  • FIG. 5A is a graph showing the efficacy of noscapine
  • FIG. 5B is a graph showing the efficacy of compound V-l
  • FIG. 5C is a graph showing the efficacy of compound V-2.
  • groups of seven mice were administered noscapine, V-1 or V-2 by subcutaneous infusion using Alzet® pumps (flow rate of 0.5 ⁇ /h) for 10 days beginning 24 h before virus exposure.
  • noscapine 200 or 20 mg/kg was administered by infusion
  • compound V-1 40 or 10 mg/kg was administered by infusion
  • for compound V-2 1.0 or 0.5 mg/kg was administered by infusion.
  • Ribavirin 75 mg/kg/d was intranasally (i.p.) administered b.i.d.
  • Seven mice per group were treated with 50% DMSO in unbuffered water by infusion as described above, these mice constituting the vehicle control. Animal deaths were recorded for up to 21 days post virus exposure. Mice treated with either dose of V-2 and the top dose of V- 1 survived 1-2 days longer than mice in the placebo group.
  • the day calculated as the day when most mice died in these three treatment groups was greatly prolonged compared to the mean day of death of mice treated with placebo; day 12 and 14 for the mice receiving either dose of V-2 and day 13 for mice treated with the top dose of V-1 compared to day 9 for mice treated with placebo.
  • the present invention is based on the discovery that the compound noscapine has anti-influenza and anti-coronavirus (e.g., against SARS-CoV) activity, and provides methods of preventing or treating influenza or a coronavirus-associated disease (such as SARS), comprising administering to a mammal in need thereof an effective amount of a composition comprising noscapine or an analog thereof, or a pharmaceutically acceptable prodrug of noscapine or the analog thereof, or a pharmaceutically acceptable salt of noscapine, the analog thereof, or the prodrug.
  • SARS-CoV coronavirus-associated disease
  • Noscapine also known as Narcotine, Nectodon, Nospen, and Anarcotine
  • Narcotine also known as Narcotine, Nectodon, Nospen, and Anarcotine
  • Anarcotine is a benzylisoquinoline alkaloid from plants of the Papaveraceae family. This compound is a tubulin inhibitor and has been primarily used for its anti-tussive (cough-suppressing) effects in over-the-counter formulations of cough syrup, with a 40-year safety history in humans.
  • the chemical structure of noscapine is shown below:
  • Analogs of noscapine include, but are not limited to, the compounds disclosed in International Patent Application Nos. PCT/RU2007/000138 and
  • the analog of noscapine is a compound having a structure of Formula (I):
  • R 1 and R 2 are each independently selected from: H, a substituted or unsubstituted alkyl, alkenyl, or alkynyl group or a substituted or unsubstituted carbocyclyl, heterocyclyl, aryl, or heteroaryl group, provided that if R 1 is a methyl group, then R 2 is not H.
  • the analog is selected from:
  • Ar is aryl or heteroaryl.
  • the analog is selected from: U 2011/059590
  • the analog of noscapine is a compound having a structure of Formula (III):
  • R 3 and R 4 are each independently selected from hydrogen, alkyl, or aryl, or R 3 and R 4 together with the nitrogen to which they are bound form a heterocycle.
  • the analog is selected from:
  • the analog of noscapine is a compound having a structure of Formula (IV):
  • R 3 and R 4 are each independently selected from hydrogen, alkyl, or aryl, or R 3 and R 4 together with the nitrogen to which they are bound form a heterocycle.
  • the analog is selected from:
  • the analog of noscapine is a compound of having a structure of Formula (V):
  • R is selected from: H, SH, OH, OTf, SBn, NHBn, NH 2 , NHMe, and Ar, wherein Ar is selected from:
  • the analog of noscapine is a compound of Formula (V), wherein R is OH or NH 2 , and the analog has a structure of V-1 or V-2:
  • the analog of noscapine is a compound having a structure of Formula (VI):
  • R is NH 2 , and R' is Br.
  • the analog of noscapine is a compound having a structure of Formula (VII):
  • the analog of noscapine is a compound having a structure of Formula (VIII):
  • R is selected from:
  • the analog of noscapine is a compound having a structure of Formula (IX):
  • B is-OH, halogen, or a single bond that forms a six-membered heterocyclyl ring with A;
  • C is hydrogen, Q_ 6 alkyl, or halogen
  • E is-OH or Ci_ 6 alkyl, or together with D forms a five-or six-membered cycloalkyl or heterocyclyl ring, wherein this heterocyclyl ring contains- C(O) 0-, -C(0)NH-, -C(S)0-, or-C(S)NH-;
  • F is hydrogen, -0-Ci_ 6 alkyl,-0-Ci_ 6 aralkyl, -0-Ci_ 6 alkylheteroaryl, halogen, aryl, Q_ 6 alkyl, -SH, thio-Ci_ 6 alkyl, -S-aryl, -0-S0 2 -Ci_ 6 alkyl, -0-S0 2 -Ci_ 6 aralkyl, cyano, or NR R 2 , where R 1 and R 2 are independently hydrogen, Ci_ 6 alkyl, Ci_ 6 aralkyl, cyano, aryl, heteroaryl, -S0 2 -Ci_ 6 alkyl, or-S0 2 -N(Ci_ 6 alkyl)(Ci_ 6 alkyl);
  • Gl to G4 independently represent hydrogen, aryl, halogen, Q_ 6 alkyl, hydroxyl, -S-Ci_6 alkyl, nitro, -0-Ci_ 6 alkyl, -0-Ci_ 6 aralkyl, or -(CH 2 ) X NR 1 R 2 , where x is 0, 1 , or 2 and where R 1 and R 2 are independently hydrogen, Ci_ 6 alkyl, Ci_ 6 aralkyl, cyano, aryl, heteroaryl, or acyl, or
  • two adjacent G2 to G4 groups together comprise an alkylene -(CH 2 ) m -, where m is 3 or 4, to form a cycloalkyl ring, or together comprise an alkylene dioxy -O- (CH 2 ) n -0-, where n is 1 , 2, or 3, to form a heterocyclyl ring; and
  • K is Ci_6 alkyl, halogen, cyano, aryl, hydrogen, hydroxyl, thio-Ci_ 6 alkyl, sulfonyl, sulfoxyl, nitro, -0-Ci_ 6 alkyl, -0-Ci_ 6 aralkyl, or NR ⁇ 2 , where R 1 and R 2 are independently hydrogen, Ci_ 6 alkyl, Ci_ 6 aralkyl, cyano, aryl, heteroaryl, or acyl ; wherein one or more of said alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, and aralkyl groups are optionally substituted with one or more suitable substituents.
  • A is-(CH 2 ) 2 -N(Y)- and forms a nitrogen-containing heterocyclyl ring with B.
  • Y is hydrogen, Ci_ 6 alkyl, or Ci_6 aralkyl.
  • D together with E forms a substituted or
  • D together with E forms a five-membered heterocyclyl ring that contains -C(0)0-.
  • A is-(CH 2 ) 2 -N(Y)- and forms a nitrogen-containing heterocyclyl ring with B, and D together with E forms a substituted or unsubstituted five-or six-membered heterocyclyl ring that contains-C(0)0-, - C(0)NH-, -C(S)0-, or -C(S) NH-.
  • A is-(CH 2 ) 2 -N(Y)- and forms a nitrogen-containing heterocyclyl ring with B, and D together with E forms a five-membered heterocyclyl ring that contains-C(0)0-.
  • Y is hydrogen, Ci_ 6 alkyl, or Ci_ 6 aralkyl.
  • K is hydrogen.
  • Gi to G 4 each independently represents hydrogen or -0-Ci_ 6 alkyl.
  • the analog of noscapine is a compound having a structure of Formula (IX) :
  • B is-OH, halogen, or a single bond that forms a six-membered heterocyclyl ring with A;
  • C is hydrogen, Q_ 6 alkyl, or halogen
  • D is (i) -CH 2 -halogen, -CH(O), -COOH, -C(0)-0-Ci_ 6 alkyl, -C(0)-0-Ci_ 6 aralkyl, -CH 2 OH, or -(CH 2 ) n -CH 3 , wherein n is 1, 2, or 3, or
  • E is-OH or Ci_ 6 alkyl, or together with D forms a five-or six-membered cycloalkyl or heterocyclyl ring, wherein this heterocyclyl ring contains- C(O) O- , -C(0)NH-, -C(S)0-, or-C(S)NH-;
  • F is hydrogen, -0-Ci_ 6 alkyl,-0-Ci_ 6 aralkyl, -0-Ci_ 6 alkylheteroaryl, halogen, aryl, Ci_ 6 alkyl, -SH, thio-Ci_ 6 alkyl, -S-aryl, -0-S0 2 -Ci_ 6 alkyl, -0-S0 2 -Ci_ 6 aralkyl, cyano, or NR R 2 , where R 1 and R 2 are independently hydrogen, Ci_ 6 alkyl, Ci_ 6 aralkyl, cyano, aryl, heteroaryl, -S0 2 -Ci_ 6 alkyl, or-S0 2 -N(Ci_ 6 alkyl)(Ci_ 6 alkyl);
  • Gl to G4 independently represent hydrogen, aryl, halogen, Q_ 6 alkyl, hydroxyl, - S-Ci_6 alkyl, nitro, -0-Ci_ 6 alkyl, -0-Ci_ 6 aralkyl, or -(CH 2 ) X NR 1 R 2 , where x is 0, 1, or 2 and where R 1 and R 2 are independently hydrogen, Ci_ 6 alkyl, Ci_ 6 aralkyl, cyano, aryl, heteroaryl, or acyl, or two adjacent G2 to G4 groups together comprise an alkylene -(CH 2 )m-, where m is 3 or 4, to form a cycloalkyl ring, or together comprise an alkylene dioxy -O- (CH 2 ) n -0-, where n is 1, 2, or 3, to form a heterocyclyl ring; and
  • K is Ci_6 alkyl, halogen, cyano, aryl, hydrogen, hydroxyl, thio-Ci_ 6 alkyl, sulfonyl, sulfoxyl, nitro, -0-Ci_ 6 alkyl, -0-Ci_ 6 aralkyl, or NR R 2 , where R 1 and R 2 are independently hydrogen, Ci_ 6 alkyl, Ci_ 6 aralkyl, cyano, aryl, heteroaryl, or acyl ;
  • alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, and aralkyl groups are optionally substituted with one or more suitable substituents, provided that when A is-(CH 2 ) 2 -N(Y)- and forms a nitrogen-containing heterocyclyl ring with B, and D together with E forms an unsubstituted five- membered heterocyclyl ring that contains-C(O) 0-, then:
  • F is not unsubstituted-0-Ci_6 alkyl when Gl and G4 are the same unsubstituted-0-Ci_6 alkyl and Y is unsubstituted Ci_ 6 alkyl, carbamoyl- substituted Ci_ 6 alkyl, thiocarbamoyl-substituted Ci_ 6 alkyl, hydroxy-substituted Ci_6 alkyl, or heteroaryl, and
  • F is not unsubstituted-0-Ci_ 6 alkyl when Gl is unsubstituted-0-Ci_ 6 alkyl, G4 is hydroxyl, and Y is unsubstituted Ci_ 6 alkyl
  • A is-(CH 2 ) 2 -N(Y)- and forms a nitrogen-containing heterocyclyl ring with B.
  • Y is hydrogen, Ci_ 6 alkyl, or Ci_6 aralkyl.
  • D together with E forms a substituted or
  • D together with E forms a five-membered heterocyclyl ring that contains -C(0)0-.
  • A is-(CH 2 ) 2 -N(Y)- and forms a nitrogen-containing heterocyclyl ring with B, and D together with E forms a substituted or unsubstituted five-or six-membered heterocyclyl ring that contains-C(0)0-, - C(0)NH-, -C(S)0-, or -C(S) NH-.
  • A is-(CH 2 ) 2 -N(Y)- and forms a nitrogen-containing heterocyclyl ring with B, and D together with E forms a five-membered heterocyclyl ring that contains-C(0)0-.
  • Y is hydrogen, Ci_ 6 alkyl, or Ci_ 6 aralkyl.
  • K is hydrogen.
  • Gi to G 4 each independently represents hydrogen or -0-Ci_ 6 alkyl.
  • alkyl refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3- C30 for branched chains), and more preferably 20 or fewer.
  • preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
  • alkyl (or “lower alkyl) as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • Such substituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety.
  • a halogen
  • the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
  • the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF 3 , -CN and the like.
  • substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl- substituted alkyls, -CF 3 , -CN, and the like.
  • substituted refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by
  • substituted is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic mo
  • alkenyl refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyls" and “substituted alkenyls", the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents may occur on one or more carbons that are included or not included in one or more double bonds. Moreover, such substituents include all those contemplated for alkyl groups, as discussed below, except where stability is prohibitive. For example, substitution of alkenyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.
  • alkynyl refers to an aliphatic group containing at least one triple bond and is intended to include both "unsubstituted alkynyls" and “substituted alkynyls", the latter of which refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the alkynyl group. Such substituents may occur on one or more carbons that are included or not included in one or more triple bonds. Moreover, such substituents include all those contemplated for alkyl groups, as discussed above, except where stability is prohibitive. For example, substitution of alkynyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.
  • C x _ y when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain.
  • C x _ y alkyl refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-tirfluoroethyl, etc.
  • Co alkyl indicates a hydrogen where the group is in a terminal position, a bond if internal.
  • C2- y alkenyl and C2- y alkynyl refer to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
  • carbocyclyl refers to a non-aromatic saturated or unsaturated ring in which each atom of the ring is carbon.
  • a carbocycle ring contains from 3 to 10 atoms, more preferably from 5 to 7 atoms.
  • heterocyclyl refers to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7- membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms.
  • heterocyclyl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.
  • aryl as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon.
  • the ring is a 5- to 7-membered ring, more preferably a 6-membered ring.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls,
  • Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.
  • heteroaryl includes substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6- membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms.
  • heteroaryl and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
  • heterocycle and “heterocyclyl” refer to substituted or
  • heterocyclyl and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.
  • aralkyl refers to an alkyl group substituted with an aryl group.
  • acyl is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.
  • prevention of influenza includes, for example, reducing the number of or severity of occurrences of one or more symptoms of influenza in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the onset of one or more symptoms of influenza in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • an "effective amount" of an agent as used herein is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an "effective amount” depends upon the context in which it is being applied.
  • an effective amount of an agent is, for example, an amount sufficient to achieve such a relief of influenza symptoms as compared to the response obtained without administration of the agent.
  • compositions, excipients, adjuvants, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • prodrugs of the compounds of the invention include within its scope, prodrugs of the compounds of the invention.
  • prodrugs will be functional derivatives of a compound of the invention which are readily convertible in vivo into the compound from which it is notionally derived.
  • Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs” ed. H. Bundgaard, Elsevier, 1985, the contents of which are hereby incorporated by reference herein in their entirety.
  • pharmaceutically acceptable salt means an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.
  • Influenza viruses are RNA viruses that make up three of the five genera of the family Orthomyxoviridae: Influenzavirus A, Influenzavirus B, and
  • Influenzavirus C (Kawaoka (editor) (2006) Influenza Virology: Current Topics. Caister Academic Press).
  • the genus Influenzavirus A has one species, influenza A virus.
  • the type A viruses are the most virulent human pathogens among the three influenza types and cause the most severe disease.
  • the influenza A virus can be subdivided into different serotypes based on the antibody response to these viruses (Hay et al. (2001) The evolution of human influenza viruses. Philos Trans R Soc Lond B Biol Sci 356: 1861-70).
  • the serotypes that have been confirmed in humans include: H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, and H10N7.
  • the genus Influenzavirus B has one species, influenza B virus. Influenza B almost exclusively infects humans (Hay et al. (2001)) and is less common than influenza A.
  • the genus Influenzavirus C has one species, influenza C virus.
  • Each species of influenza virus may have multiple strains, e.g.,
  • the present invention provides methods of preventing or treating influenza caused by any species, serotype, or strain of the influenza viruses.
  • Coronaviruses are viruses belonging to the subfamily Coronavirinae in the Coronaviridae family. Coronaviruses are single stranded (+) RNA viruses, and include genus Cononavirus, genus Torovirus, infectious bronchitis virus (IBV), feline infectious peritonitis virus (FIP), canine coronavirus (CCV), swine transmissible gastroenteritis virus (TGEV), equine torovirus (EqTV) and the like. The name was given because the viruses have, on the surface of the envelope thereof, projections which show an appearance resembling the corona of the sun.
  • the SARS epidemic in Southeast Asia in 2003 was cased by a new coronavirus species - SARS-associated coronavirus (SARS-CoV).
  • SARS-CoV coronavirus species - SARS-associated coronavirus
  • the present invention provides methods of preventing or treating a coronavirus-associated disease caused by any species, serotypes, or strain of coronavirus. In one embodiment, the present invention provides a method of preventing or treating SARS.
  • the compounds or salts of the present invention may be administered to a mammal alone or in combination with pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.
  • Each carrier must be “acceptable” in the sense of being compatible with other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar;
  • buffering agents such as magnesium hydroxide and aluminum hydroxide
  • alginic acid (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non- toxic compatible substances employed in pharmaceutical formulations.
  • a preferred carrier of the compounds of the present invention is cyclodextrins ("CDs").
  • CDs, cyclic oligosaccharides have potentials to alter physical, chemical and biological properties of guest molecules through the formation of inclusion complexes.
  • the ⁇ -, ⁇ -, and ⁇ -CDs are the most common natural CDs consisting of six, seven and eight D-glucopyranose residues, respectively, linked by a- 1,4 glycosidic linkage into a macrocycle (Szejtli and Szente (2005) Elimination of bitter, disgusting taste of drugs and foods by cyclodextrins. Eur J Pharm Biopharm 61:115-125).
  • Preparation of an inclusion complex of noscapine and ⁇ -CD is described in Madan (2009) Inclusion complexes of noscapine in ⁇ -cyclodextrin offer better solubility and improved
  • a mammal refers to any mammal that is susceptible to influenza viral infection or coronavirus infection. Examples of such mammals include, but are not limited to, humans, pigs, dogs, seals and ferrets. A preferred example of mammals to which compositions of the present invention can be administered is humans.
  • compositions as described herein can be administered in various forms, depending on the severity of influenza or coronavirus-associated disease (e.g., asthma, diabetes, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, neurological disorders, a virus-associated disease (e.g., a virus, e.g., a virus-associated disease
  • compositions for example, they may be formulated as tablets, capsules, granules, powders, or syrups; or for parenteral administration, they may be formulated as injections (intravenous, intramuscular, or subcutaneous), drop infusion preparations, or suppositories.
  • injections intravenous, intramuscular, or subcutaneous
  • drop infusion preparations or suppositories.
  • ophthalmic mucous membrane route they may be formulated as eye drops or eye ointments.
  • compositions of the present invention can be prepared by conventional means, and if desired, the active ingredient may be mixed with any conventional additive or excipient, such as a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent, a coating agent, a cyclodextrin, and/or a buffer.
  • a preferred route of administration is oral administration.
  • a preferred formulation for oral administration is a solid formulation, e.g., as tablets, capsules, granules, or powders.
  • the compositions of the present invention are formulated in tablets or pills for oral administration. More preferably, the tablets or pills have a multi-year shelf-life.
  • the dosage will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the influenza or coronavirus-associated disease (e.g., SARS) to be prevented or treated, the route of administration, and the form of the drug.
  • a daily dosage of from 300 to 3000 mg or more of the compound for an adult human patient may be administered, e.g., in a single dose or in divided doses.
  • the compounds of the present invention can be
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • the precise time of administration and/or amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a particular compound, physiological condition of the patient (including age, sex, type and severity of the influenza or coronavirus-associated disease to be treated, general physical condition, responsiveness to a given dosage, and type of medication), route of administration, etc.
  • physiological condition of the patient including age, sex, type and severity of the influenza or coronavirus-associated disease to be treated, general physical condition, responsiveness to a given dosage, and type of medication
  • route of administration etc.
  • the above guidelines can be used as the basis for fine-tuning the treatment, e.g., determining the optimum time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing.
  • compositions of the present invention can be administered at the above-mentioned dosages for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, or longer.
  • the compositions of the present invention can be administered at the above-mentioned dosages for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks, or longer, e.g., throughout a high-risk period for seasonal flu.
  • the methods of the present invention can be used to prevent or treat influenza in a mammal, preferably a human, irrespective of the presence or the absence of certain symptoms that can be exhibited by influenza infection.
  • the methods of the present invention can be used to prevent or treat influenza in a mammal which does or does not exhibit the symptom of fever.
  • the methods of the present invention are used to prevent or treat influenza in a mammal which does or does not exhibit the symptom of coughing.
  • the methods of the present invention are used to prevent or treat influenza in a mammal which does or does not exhibit the symptom of nasal congestion.
  • influenza may not be noticed during the first few days of the disease when other symptoms are more severe. For example, as the initial fever goes away, symptoms of coughing, runny nose and dry or sore throat may become more evident.
  • the methods of the present invention can be used to treat influenza at any stage of the disease progression, e.g., commencing during the first few days of infection when high fever is most evident and other symptoms (e.g., coughing) are not evident (e.g., within the first one day, two days, three days, four days, five days, six days, or seven days of the onset of a fever), whether before the onset of the symptom of coughing or runny nose or dry or sore throat, or after the onset of the symptom of coughing or runny nose or dry or sore throat.
  • Example 1 In vitro efficacy of compounds against influenza virus
  • influenza antiviral evaluation assay examines the effects of compounds at designated dose-response concentrations. MDCK cells were used in the assay to test the efficacy of the compounds in preventing the CPE induced by influenza infection.
  • the plate layout is shown in Table 1. Oseltamivir carboxylate (the active metabolite of Tamiflu) was included in each run as a positive control compound.
  • Subconfluent cultures of MDCK cells were plated into 96-well plates for the analysis of cell viability (cytotoxicity) and antiviral activity (CPE). Drugs were preincubated with cells up to 48 hours later, the optimal time of which was determined as described below. At a designated time, the CPE wells also received 100 tissue culture infectious doses (100 TCID50s) of titered influenza virus. 24, 48, and 72 hours later the cell viability was determined. The effective compound concentrations which reduced viral-induced CPE by 25% (IC25), 50% (IC50), and 90%> (IC90) were calculated by regression analysis with semilog curve fitting. Cell viability was assessed using CellTiter-Glo (Promega). The toxic concentration of drug that reduced cell numbers by 50%> and 90%> (TC50 and TC90, respectively) were calculated as well. The selectivity
  • Measurement of influenza-induced CPE was based on quantitation of ATP, an indicator of metabolically active cells.
  • the CPE assay employed a
  • cytotoxicity and cell proliferation in culture involved adding the single reagent (CellTiter-Glo® Reagent) directly to previously cultured, subconfluent cells in media. This induced cell lysis and the production of a bio luminescent signal (half-life greater than 5 hours, depending on the cell type) that was proportional to the amount of ATP present (which is a biomarker for viability).
  • the compounds were screened for intrinsic cytotoxicity at 5 ⁇
  • the neutral red uptake assay provided a quantitative estimation of the number of viable cells in a culture.
  • the assay was based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes.
  • Vera 76 cells were used in this assay to test the efficacy of the compounds in preventing the CPE induced by influenza infection. Cells were seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates were then incubated for 2 hours with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer.
  • a virus yield reduction assay was used, for example, to confirm the results of the CPE inhibition and/or neutral red uptake assays.
  • Vero 76 cells were used in this assay to test the efficacy of the compounds in preventing the CPE induced by influenza infection. Infectious virus yields from each well from a CPE inhibition assay were determined according to published procedures (see, e.g., Barnard et al. Antiviral Chemistry & Chemotherapy 12: 241-250). After CPE was scored, each plate was frozen at -80 °C and thawed. Sample wells were pooled and titred in Vero cells for infectious virus by CPE assay according to published procedures (see, e.g., Barnard et al. Antiviral Chemistry &
  • MDCK cells were obtained from ATCC (Cat. No. CCL-34).
  • the human influenza viruses used in the experiment included strain A/C A/04/2009 (novel HlNl; "swine fiu"), A/Bris/59/2007 (Seasonal A, current vaccine strain, H1N1- like), and B/Bris/60/2008 (Seasonal B).
  • CellTiter GLO kit was obtained from Promega (Substrate Cat. No. G755B; Buffer Cat. No. G756B). The control drug,
  • Oseltamivir carboxylate was provided by Southern Research Institute. Test compounds, including noscapine, were provided by Inhibikase Therapeutics.
  • MDCK cells were grown to 90% confluency, trypsinized, recovered, centrifuged, and washed twice in phosphate-buffered saline to remove residual serum. The cells were then diluted in bovine serum albumin
  • BSA Dulbeco's Modified Eagle's Medium
  • the test compounds were diluted to the appropriate test concentrations in BSA- containing media and added to each plate efficacy and toxicity test well in the format indicated in Table 1.
  • the test compounds were pre-incubated with cells for the specified period of time determined by the Pre-inbucation Test outlined above.
  • Raw data and dose response curves for each treatment and virus strain were prepared. Inhibitory concentrations were calculated for: IC25, IC50, and IC90. Raw data and toxicity profile for each treatment and virus strain were prepared. Toxic concentrations were calculated for TC50 and TC90. Therapeutic indices (SI50 and SI90) for each test compound and virus strain were finally determined.
  • FIG. 1A is a graph showing the efficacy of noscapine (dissolved in 100% DMSO) in reducing CPE on MDCK cells induced by infection of influenza virus strain A/CA/04/2009.
  • the dose-response curve shown in FIG. 1 A was re- graphed in FIG. IB (squares; error bars removed) to compare with the
  • Noscapine when dissolved in 100% DMSO, was found to have in vitro anti-flu activity in MDCK cells with an IC50 of about 5 ⁇ . Noscapine showed no cytotoxicity at any concentration tested.
  • noscapine When dissolved in water, noscapine was found to be 100-fold more effective than dissolved in 100% DMSO, with an IC50 of 0.05 ⁇ against strain
  • noscapine had an IC50 of 0.05 ⁇ across all three strains tested.
  • the small deviation of IC50s of noscapine against different influenza strains is in contrast to the viral NA inhibitors, which are 100 fold more potent against seasonal A than seasonal B (FIG. 2A).
  • noscapine was found to be effective against a number of respiratory pathogens.
  • noscapine was effective in reducing CPE on MDCK cells induced by infection of a number of influenza strains including
  • Noscapine was also found effective in increasing cell viabilities in the cells infected with virus strain SARS CoV Urbani. Furthermore, noscapine was found to reduce virus yield in cells infected with various SARS virus strains, including SARS CoV CuHK v2157, SARS CoV Frank V 1940 and SARS CoV Hot v2225. These results are shown in FIG 3.
  • Example 2 In vivo efficacy of compounds in the standard ferret model for flu Compounds of the present invention are tested in the standard ferret model for flu (Noah, Twu, and Krug (2003) Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS1A protein via its binding to a cellular protein required for the 3 end processing of cellular pre-mRNAS. Virology 307:386-395). This is a non-GLP study, performed at Biosafety Level 2 for the Wisconsin strain. The study is performed with the Fitch strain of ferret using 69 males, 4-8 months old (800- 1600g).
  • Table 2 Groupings and treatments for ferret animal screen
  • Influenza viral strain H3N2 (A/Wisconsin/67/2005) at 10 EID50 on Day 0 or Day 3 is used. Verification of the virus innoculum is performed by EID50 using the virus formulation as administered (i.e. undiluted) and a 10-fold dilution of the formulation to confirm the dosage given for each day of dosing (Day 0 and Day 3). Data is reported and a range of +/- 1 log is considered acceptable and used in conjunction with animal control data to assess the overall study. Antiviral evaluation
  • the dose administered is calculated based on Day 0 body weights (so that dose is in mg Drug/kg of animal body weight as specified in the table above).
  • Compounds of the present invention can be used in combination with any solvent/adjuvant/carrier which effect on the efficacy of the compounds may be tested in parallel with the compounds in the absence of the solvent/adjuvant/carrier in the same experiment.
  • Animals infected with H3N2, but not receiving any drug treatment (only receiving solvent/control vehicle) are used for positive controls. Animals receiving the compounds and not challenged with virus are used as test article controls at Low and High Dose mg/kg for 6 days. Tamiflu and Ribavirin are used as positive controls.
  • Group 1 is treated with Low Dose mg/kg/animal DRUG 1 daily, D0- D5, and infected on Day 3 with A/Wisconsin at ⁇ 10 7 EID50/ML 3-4 hours after the D3 treatment.
  • Group 2 is treated with High Dose mg/kg/animal DRUG 1 daily, D0-D5, and infected on Day 3 with A/Wisconsin at ⁇ 10 7 EID50/mL 3-4 hours after the D3 treatment.
  • Group 3 is infected with A/Wisconsin at ⁇ 10 7 EID50/ML and treated with Low Dose mg/kg/animal DRUG 1 3-4 hours post- infection with subsequent daily treatments for 6 days.
  • Group 4 is infected with A/Wisconsin at ⁇ 10 7 EID50/ML and treated with 30 mg/kg/animal DRUG 1 3-4 hours post-infection with subsequent daily treatments for 6 days.
  • Group 5 is treated with Low Dose mg/kg/animal DRUG 2 daily, D0-D5, and infected on Day 3 with A/Wisconsin at ⁇ 10 7 EID50/ML 3-4 hours after the D3 treatment.
  • Group 6 is treated with High Dose mg/kg/animal DRUG 2 daily, D0-D5, and infected on Day 3 with A/Wisconsin at ⁇ 10 7 EID50/mL 3-4 hours after the D3 treatment.
  • Group 7 is infected with A/Wisconsin at ⁇ 10 7 EID50/ML and treated with Low Dose mg/kg/animal DRUG 2 3-4 hours post-infection with subsequent daily treatments for 6 days.
  • Group 8 is infected with A/Wisconsin at ⁇ 10 7 EID50/ML and treated with 30 mg/kg/animal DRUG 2 3-4 hours postinfection with subsequent daily treatments for 6 days.
  • Group 9 is infected with A/Wisconsin at ⁇ 10 7 EID50/ML on DO 3-4 hrs prior to Vehicle administration and serve as the virus positive control group.
  • Group 10 is infected with
  • Group 11 is treated with Low Dose mg/kg/animal DRUG 1 Days 0 -5 and serve as the product control group.
  • Group 12 is treated with High Dose mg/kg/animal DRUG 1 Days 0 -5 and serve as the product control group.
  • Group 13 is treated with Low Dose mg/kg/animal DRUG 2 Days 0 -5 and serve as the product control group.
  • Group 14 is treated with High Dose mg/kg/animal DRUG 2 Days 0 -5 and serve as the product control group.
  • Virus challenge is performed intranasally. Animals are anesthetized and inoculated with 1 mL (500 ⁇ ⁇ per nare) at Day 0 or Day 3. Clinical
  • Nasal Wash/Viral Load is tested by nasal washes collected on Days -3, 0, 3, 5, and 7; animals are gently anesthetized. Nasal washes are snap frozen and stored at -80°C for future analysis for viral load by EID50.
  • Turbinates and the right lung are snap frozen on liquid nitrogen and stored at - 80°C for future viral load analysis.
  • the left lung and spleen are stored in 10% neutral buffered formalin at ⁇ -20°C for future histopathology.
  • Animals found dead or euthanized in moribund condition have the same procedures performed as those euthanized on Day 14 or 17.
  • Animals found dead or moribund are necropsied and lungs, spleens, and nasal turbinates removed and stored for future processing.
  • Turbinates and the right lung are snap frozen on liquid nitrogen and stored at -80°C for future viral load analysis.
  • the left lung and spleen are stored in 10% neutral buffered formalin at ⁇ -20°C for future histopathology.
  • Noscapine, compound V-l, and compound V-2 are inhibitors of normal cellular function associated with response to virus infections of cells. Because of the potentially novel mechanism of inhibiting virus infections, these compounds were evaluated in a pandemic HlNl influenza A BALB/c mouse model of infection. In addition, to enhance the bioavailability of the drugs, subcutaneous infusion was used to try to continuously deliver compound throughout the infection.
  • mice Female 18-20 g BALB/c mice were obtained from Charles River Laboratories (Wilmington, MA) for this study. They were maintained on Wayne Lab Blox and tap water ad libitum. They were quarantined for 24 h prior to use.
  • Virus The novel HlNl strain was originally obtained from Jennifer Govorkova (St. JudeChildren's Research Hospital, Memphis, TN). It has been designated as influenza A/C A/04/09 pandemic HlNl virus (SJ#175190) and was adapted to mice by Natalia A. Ilyushina (St. Jude Children's Research Hospital, Memphis, TN).
  • influenza A/California/04/09 strain used in this experiment was first passaged in MDCK cells and then grown in embryonated chicken eggs. It was then adapted to mice by 9 sequential passages through mouse lungs. The virus from the last lung passage was then plaque purified in MDCK cells and amplified in embryonated chicken eggs. The virus was then amplified in MDCK cells to prepare stocks for mouse studies. Mice were exposed to 3 LD 50 (10 339 PFU) of virus by the intranasal (i.n.) route.
  • mice Groups of seven mice were administered noscapine, V-l, or V-2, by subcutaneous infusion using Alzet® pumps (flow rate of 0.5 ⁇ /h) for 10 days beginning 24 h before virus exposure.
  • For compound noscapine 200 or 20 mg/kg was administered by subcutaneous osmotic pump, for compound V-l 40 or 10 mg/kg was administered by subcutaneous osmotic pump, and for compound V-2 1.0 or 0.5 mg/kg was administered by subcutaneous osmotic pump.
  • Ribavirin 75 mg/kg/d
  • Seven mice per group were treated with 50% DMSO in unbuffered water by infusion as described above, these mice constituting the vehicle control. Animal deaths were recorded for up to 21 days post virus exposure.
  • Toxicity was evaluated in terms of weight change and adverse events. Mice were weighed every day from 24 h prior to virus infection to day 9 post virus exposure and then on days 14 and 21 post virus exposure. Adverse events for which observations were made included ruffling of fur, lethargy, paralysis, incontinence, repetitive circular motion, and aggression.

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  • Medical Informatics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Communicable Diseases (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des méthodes de prévention ou de traitement de la grippe ou d'une maladie associée à un coronavirus chez les mammifères, comprenant l'administration à un mammifère en ayant besoin d'une quantité efficace d'une composition comprenant de la noscapine ou l'un de ses analogues, ou un précurseur pharmaceutiquement acceptable de la noscapine ou de son analogue, ou un sel pharmaceutiquement acceptable de la noscapine, de son analogue ou du précurseur. L'invention concerne également les formulations des compositions et les posologies.
PCT/US2011/059590 2010-11-08 2011-11-07 Méthodes de prévention et de traitement de la grippe WO2012064654A1 (fr)

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US61/411,393 2010-11-08

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023196632A1 (fr) * 2022-04-07 2023-10-12 Veru Inc. Méthodes de traitement d'infections virales de la grippe et du poxvirus
EP4146208A4 (fr) * 2020-05-04 2024-05-22 Prilenia Neurotherapeutics Ltd. Traitement d'une infection virale, d'une maladie ou d'un trouble à l'aide d'un agoniste de s1r sélectif

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000064446A1 (fr) * 1999-04-26 2000-11-02 Emory University Derives de noscapine utilises en tant que compositions adjuvantes et procedes d'utilisation correspondants
WO2010030582A2 (fr) * 2008-09-11 2010-03-18 Emory University Noscapine et analogues de la noscapine et leur utilisation dans le traitement de maladies infectieuses par inhibition par liaison à la tubuline

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000064446A1 (fr) * 1999-04-26 2000-11-02 Emory University Derives de noscapine utilises en tant que compositions adjuvantes et procedes d'utilisation correspondants
WO2010030582A2 (fr) * 2008-09-11 2010-03-18 Emory University Noscapine et analogues de la noscapine et leur utilisation dans le traitement de maladies infectieuses par inhibition par liaison à la tubuline

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHALLA, R. ET AL.: "Cyclodextrins in Drug Delivery: An Updated Review", AAPS PHARMASCITECH, vol. 6, no. 2, 2005, pages E329 - E357 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4146208A4 (fr) * 2020-05-04 2024-05-22 Prilenia Neurotherapeutics Ltd. Traitement d'une infection virale, d'une maladie ou d'un trouble à l'aide d'un agoniste de s1r sélectif
WO2023196632A1 (fr) * 2022-04-07 2023-10-12 Veru Inc. Méthodes de traitement d'infections virales de la grippe et du poxvirus

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