WO2013120040A1 - Inhibition d'une voie ciblée pour améliorer la structure, le fonctionnement et l'activité musculaires dans la dystrophie musculaire - Google Patents

Inhibition d'une voie ciblée pour améliorer la structure, le fonctionnement et l'activité musculaires dans la dystrophie musculaire Download PDF

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WO2013120040A1
WO2013120040A1 PCT/US2013/025464 US2013025464W WO2013120040A1 WO 2013120040 A1 WO2013120040 A1 WO 2013120040A1 US 2013025464 W US2013025464 W US 2013025464W WO 2013120040 A1 WO2013120040 A1 WO 2013120040A1
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substituted
unsubstituted
compound
alkynyl
alkenyl
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Louis M. Kunkel
Genri KAWAHARA
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Children's Medical Center Corporation
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
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    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
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    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
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    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/566—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol having an oxo group in position 17, e.g. estrone
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • A61K31/5685—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone having an oxo group in position 17, e.g. androsterone
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin

Definitions

  • Muscular dystrophy is a muscle degenerative disease in which the muscle at first forms normally, but starts to degenerate faster than it can be repaired.
  • the most common form of muscular dystrophy is Duchenne Muscular Dystrophy (DMD) representing over 90% of the diagnosed cases.
  • DMD Duchenne Muscular Dystrophy
  • mutations in the dystrophin gene were found to be the cause of both Duchenne and Becker Muscular Dystrophy. Shortly thereafter antibodies were developed against dystrophin and used to improve diagnosis of the disease.
  • the predominant muscle dystrophin isoform is translated from the largest gene in the human genome. The gene encodes a large protein of 427 KDa that positions just inside of the sarcolemmal membrane and links the internal cytoskeleton with the muscle cell membrane. This linkage is vital to maintaining muscle membrane integrity during repeated cycles of cell contraction.
  • Almost all known human dystrophin mutations that cause DMD typically result in the loss or degradation of the dystrophin protein at the sarcolemmal membrane
  • muscular dystrophies are a heterogeneous group of genetic disorders for which there are now emerging rational therapies. Despite these advances, there are few small molecules that have been developed which might target disease progression in the most common form of muscular dystrophy - DMD. There are also only a few targets for therapy, which do not involve direct modulation of the dystrophin gene.
  • DMD Duchenne Muscular Dystrophy
  • a method of treating Duchenne Muscular Dystrophy comprises administering to a patient in need thereof a pharmaceutical composition comprising a compound that selectively targets the cAMP-PKA pathway, wherein the compound is present in an amount effective to upregulate heme oxygenase 1.
  • a method of treating Duchenne Muscular Dystrophy comprises administering to a patient in need thereof a pharmaceutical composition comprising a compound that selectively targets the cAMP-PKA pathway to upregulate heme oxygenase 1, wherein the compound is present in an amount effective to restore muscle function or phenotype.
  • the compound activates heme oxygenase 1 directly in an amount effective to inhibit NFKB and increase vasodilation, the compound activates heme oxygenase 1 by directly activating PKA in an amount effective to inhibit nuclear factor kappa-light-chain-enhancer of activated B cell (NFKB) and increase vasodilation, or a combination thereof.
  • the compound is selected from the group consisting of aminophylline, sildenafil, ibudilast, rolipram, equilin, androsterone acetate, crassin acetate acetate, cerulenin, 9a,l lb-prostaglandin F2, and salts and derivatives thereof.
  • the compound has formula I:
  • the compound has formula II:
  • Ri-Rn are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • the compound has formula III:
  • the compound has formula IV:
  • R' and R" are individually hydrogen or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, arylalkyl, heteroalkyls, alkylaryl, alkylheteroaryl, and r is an integer from 1 to 6.
  • the compound is equilin or salts or derivatives thereof.
  • the compound has formula VI:
  • the compound has formula VII:
  • the compound has formula VIII:
  • the compound has formula IX:
  • R4-R5 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • the compound has formula X:
  • the compound has formula XI:
  • R' and R" are individually hydrogen or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, arylalkyl, heteroalkyls, alkylaryl, alkylheteroaryl, and r is an integer from 1 to 6.
  • the compound is androsterone or salts or derivatives thereof.
  • the compound has formula XII:
  • R4-R43 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • the compound has formula XIII:
  • the compound has formula XIV:
  • a method of treating Duchenne Muscular Dystrophy comprises administering to a patient in need thereof a pharmaceutical composition comprising a phosphodiesterase (PDE) inhibitor in an amount effective to treat the DMD.
  • PDE phosphodiesterase
  • the PDE inhibitor is selected from the group consisting of aminophylline, sildenafil, tadalifil, vardenafil, ibudilast, rolipram, and salts and derivatives thereof.
  • a method of treating Duchenne Muscular Dystrophy comprises administering to a patient in need thereof a pharmaceutical composition comprising a compound that induces vasodilation in an amount effective to treat the DMD.
  • the compound is selected from the group consisting of aminophylline, equilin, androsterone acetate, crassin acetate acetate, cerulenin, 9a,l lb-prostaglandin F2, and salts and derivatives thereof.
  • Muscular Dystrophy (DMD) is provided.
  • the method comprises administering to a patient in need thereof a pharmaceutical composition comprising a compound selected from the group consisting of aminophylline, sildenafil, tadalifil, vardenafil, ibudilast, rolipram, epirizole, homochlorcyclizine, conessine, equilin, pentetic acid, proscillaridin A, nitromide, propantheline bromide, androsterone acetate, crassin acetate acetate, pomiferin, cerulenin, 9a, 11b- prostaglandin F2, and salts and derivatives thereof, in an amount effective to treat the DMD.
  • a pharmaceutical composition comprising a compound selected from the group consisting of aminophylline, sildenafil, tadalifil, vardenafil, ibudilast, rolipram, epirizole, homochlorcyclizin
  • a pharmaceutical composition for use in treating Duchenne Muscular Dystrophy comprising a compound that selectively targets the cAMP-PKA pathway, wherein the compound is present in an amount effective to upregulate heme oxygenase 1.
  • a pharmaceutical composition for use in treating Duchenne Muscular Dystrophy comprising a compound that selectively targets the cAMP-PKA pathway to upregulate heme oxygenase 1, wherein the compound is present in an amount effective to restore muscle function or phenotype.
  • the pharmaceutical composition for use in treating DMD of either of the above two preceeding aspects of the disclosure further comprises that the compound activates heme oxygenase 1 directly in an amount effective to inhibit NFKB and increase vasodilation, the compound activates heme oxygenase 1 by directly activating PKA in an amount effective to inhibit NFKB and increase vasodilation, or a combination thereof.
  • the compound is selected from the group consisting of aminophylline, sildenafil, ibudilast, rolipram, equilin, androsterone acetate, crassin acetate acetate, cerulenin, 9a,l lb-prostaglandin F2, and salts and derivatives thereof.
  • the compound has formula I:
  • the compound has formula II:
  • R Rn are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • the compound has formula III:
  • the compound has formula IV:
  • R' and R" are individually hydrogen or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, arylalkyl, heteroalkyls, alkylaryl, alkylheteroaryl, and r is an integer from 1 to 6.
  • the compound is equilin or salts or derivatives thereof.
  • the compound has formula VI:
  • the compound has formula VII:
  • the compound has formula VIII:
  • the compound has formula IX:
  • R4-R5 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • the compound has formula X:
  • the compound has formula XI:
  • R' and R" are individually hydrogen or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, arylalkyl, heteroalkyls, alkylaryl, alkylheteroaryl, and r is an integer from 1 to 6.
  • the compound is androsterone or salts or derivatives thereof.
  • the compound has formula XII:
  • R4-R43 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • the compound has formula XIII:
  • the compound has formula XIV:
  • a pharmaceutical composition for use in treating Duchenne Muscular Dystrophy comprises a phosphodiesterase (PDE) inhibitor in an amount effective to treat the DMD.
  • PDE phosphodiesterase
  • the PDE inhibitor is selected from the group consisting of aminophylline, sildenafil, tadalifil, vardenafil, ibudilast, rolipram, and salts and derivatives thereof.
  • a pharmaceutical composition for use in treating Duchenne Muscular Dystrophy comprises a compound that induces vasodilation, wherein the compound is in an amount effective to treat the DMD.
  • the compound is selected from the group consisting of aminophylline, equilin, androsterone acetate, crassin acetate acetate, cerulenin, 9a,l lb-prostaglandin F2, and salts and derivatives thereof.
  • a pharmaceutical composition for use in treating Duchenne Muscular Dystrophy comprises a compound selected from the group consisting of aminophylline, sildenafil, tadalifil, vardenafil, ibudilast, rolipram, epirizole, homochlorcyclizine, conessine, equilin, pentetic acid, proscillaridin A, nitromide, propantheline bromide, androsterone acetate, crassin acetate acetate, pomiferin, cerulenin, 9a,l lb-prostaglandin F2, and salts and derivatives thereof.
  • a pharmaceutical composition for the treatment of Duchenne Muscular Dystrophy comprises a compound that selectively targets the cAMP-PKA pathway, wherein the compound is present in an amount effective to upregulate heme oxygenase 1.
  • a pharmaceutical composition for the treatment of Duchenne Muscular Dystrophy comprises a compound that selectively targets the cAMP-PKA pathway to upregulate heme oxygenase 1, wherein the compound is present in an amount effective to restore muscle function or phenotype.
  • the composition of either of the two preceding apsects of the disclosure further comprises that the compound activates heme oxygenase 1 directly in an amount effective to inhibit NFKB and increase vasodilation, that the compound activates heme oxygenase 1 by directly activating PKA in an amount effective to inhibit NFKB and increase vasodilation, or a combination thereof.
  • the compound is selected from the group consisting of aminophylline, sildenafil, ibudilast, rolipram, equilin, androsterone acetate, crassin acetate acetate, cerulenin, 9a, 11b- prostaglandin F2, and salts and derivatives thereof.
  • the compound has formula I:
  • Ri-R 6 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • the compound has formula II:
  • the compound has formula III:
  • the compound has formula IV:
  • R' and R" are individually hydrogen or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, arylalkyl, heteroalkyls, alkylaryl, alkylheteroaryl, and r is an integer from 1 to 6.
  • the compound is equilin or salts or derivatives thereof.
  • the compound has formula VI:
  • the compound has formula VII:
  • the compound has formula VIII:
  • the compound has formula IX:
  • R4-R5 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • the compound has formula X:
  • the compound has formula XI:
  • R' and R" are individually hydrogen or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, arylalkyl, heteroalkyls, alkylaryl, alkylheteroaryl, and r is an integer from 1 to 6.
  • the compound is androsterone or salts or derivatives thereof.
  • the compound has formula XII:
  • R4-R43 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • the compound has formula XIII:
  • the compound has formula XIV:
  • a pharmaceutical composition for the treatment of Duchenne Muscular Dystrophy comprises a phosphodiesterase (PDE) inhibitor in an amount effective to treat the DMD.
  • PDE phosphodiesterase
  • the PDE inhibitor is selected from the group consisting of aminophylline, sildenafil, tadalifil, vardenafil, ibudilast, rolipram, and salts and derivatives thereof.
  • a pharmaceutical composition for the treatment of Duchenne Muscular Dystrophy comprises a compound that induces vasodilation in an amount effective to treat the DMD.
  • the compound is selected from the group consisting of aminophylline, equilin, androsterone acetate, crassin acetate acetate, cerulenin, 9a, 11b- prostaglandin F2, and salts and derivatives thereof.
  • a pharmaceutical composition for the treatment of Duchenne Muscular Dystrophy comprises a compound selected from the group consisting of aminophylline, sildenafil, tadalifil, vardenafil, ibudilast, rolipram, epirizole, homochlorcyclizine, cones sine, equilin, pentetic acid, proscillaridin A, nitromide, propantheline bromide, androsterone acetate, crassin acetate acetate, pomiferin, cerulenin, 9a,l lb-prostaglandin F2, and salts and derivatives thereof, in an amount effective to treat the DMD.
  • compositions for the manufacture of a medicament for treating Duchenne Muscular Dystrophy comprises a compound that selectively targets the cAMP-PKA pathway, wherein the compound is present in an amount effective to upregulate heme oxygenase 1.
  • compositions for the manufacture of a medicament for treating Duchenne Muscular Dystrophy comprises a compound that selectively targets the cAMP-PKA pathway to upregulate heme oxygenase 1, wherein the compound is present in an amount effective to restore muscle function or phenotype.
  • the compound activates heme oxygenase 1 directly in an amount effective to inhibit NFKB and increase vasodilation, that the compound activates heme oxygenase 1 by directly activating PKA in an amount effective to inhibit NFKB and increase vasodilation, or a combination thereof.
  • the compound is selected from the group consisting of aminophylline, sildenafil, ibudilast, rolipram, equilin, androsterone acetate, crassin acetate acetate, cerulenin, 9a, 11b- prostaglandin F2, and salts and derivatives thereof.
  • the compound has a formula selected from the group consisting of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, and XV.
  • formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, and XV The foregoing formulas are described herein.
  • the use of a composition for the manufacture of a medicament for treating Duchenne Muscular Dystrophy comprises a phosphodiesterase (PDE) inhibitor in an amount effective to treat the DMD.
  • PDE phosphodiesterase
  • the PDE inhibitor is selected from the group consisting of aminophylline, sildenafil, tadalifil, vardenafil, ibudilast, rolipram, and salts and derivatives thereof.
  • the use of a composition for the manufacture of a medicament for treating Duchenne Muscular Dystrophy comprises a compound that induces vasodilation, wherein the compound is in an amount effective to treat the DMD.
  • the compound is selected from the group consisting of aminophylline, equilin, androsterone acetate, crassin acetate acetate, cerulenin, 9a,l lb-prostaglandin F2, and salts and derivatives thereof.
  • compositions for the manufacture of a medicament for treating Duchenne Muscular Dystrophy comprises a compound selected from the group consisting of aminophylline, sildenafil, tadalifil, vardenafil, ibudilast, rolipram, epirizole, homochlorcyclizine, cones sine, equilin, pentetic acid, proscillaridin A, nitromide, propantheline bromide, androsterone acetate, crassin acetate acetate, pomiferin, cerulenin, 9a, 1 lb-prostaglandin F2, and salts and derivatives thereof.
  • FIG. 1 is a diagram of the Heme oxygenase pathway, showing where compounds identified using the zebrafish assay are active.
  • cAMP-PKA pathway via heme oxygenase 1 (HMOX1) indirectly induced vasodilation and inhibition of NFkB (e.g., NFK-B).
  • HMOX1 heme oxygenase 1
  • Six drugs of 14 drugs from chemical screening of three drug libraries (2640 chemicals) using DMD model fish e.g., aminophylline (#4), equilin (#5), androsterone acetate (#10), crassin acetate (#11), cerulenin (#13) and prostaglandin (#14)
  • DMD model fish e.g., aminophylline (#4), equilin (#5), androsterone acetate (#10), crassin acetate (#11), cerulenin (#13) and prostaglandin (#14)
  • HMOXl and phosphorylated PKA were upregulated in chemical treated fish with aminophyline (#4), sildenafil citrate (SC), crassin acetate (#11) and cerulenin (#13).
  • Figure 2 is a diagram of the heme degradation pathway.
  • Figure 3 is a graph of the total number of surviving fish over a period of twenty days, comparing wild type; affected no treatment; and compounds 5, 10, 11, 13 and 14.
  • Affected sapje fish were separated at 4 dpf by birefringence assay.
  • Ten affected fish were treated with aminophyline (2.5 ⁇ g/ml), sildenafil (e.g., Viagra) (2.5 ⁇ g/ml) or a combination of aminophyline and sildenafil (each 2.5 ⁇ g/ml) for 20 days in triplicate.
  • treated fish showed some improvement in survival for both drugs but nearly normal survival when the two were given together (aminophyline, green: 4.67, sildenafil, yellow: 4,33, combination of both, red: 5.33, wildtype, black: 5.67).
  • Figure 4 is a graph of percentage of affected dystrophin-null fish with a series of different PDE inhibitors at different dosages. Twenty embryos from matings of heterozygous sapje fish were treated with a series of PDE inhibitors from 1 to 4 dpf (in triplicate at varying concentrations). At 4 dpf, the percentage of fish exhibiting abnormal muscle structure as examined by birefringence is shown.
  • Enoximone PDE3 inhibitor; ⁇
  • milrinone PDE3 inhibitor; O
  • ibudilast PDE4 inhibitor; A
  • rolipram PDE4 inhibitor; ⁇
  • sildenafil citrate salt PDE5 inhibitor; ⁇
  • dipyridamole PDE5 inhibitor; o
  • aminophylline nonselective PDE inhibitor; ⁇
  • DMSO DMSO
  • Figures 5A-D Birefringence assay results in HMOXl mRNA injected sapje fish and uninjected sapje fish. Abnormal birefringence (red, "Abnormal”), normal (blue, “Normal”) and dead (yellow, "Dead”); note the decreased abnormal birefringence in the construct injected fish.
  • Figure 5B HMOXl cDNA in pCS2+.
  • Figure 5C Western blot analysis of HMOXl mRNA injected fish with anti-myc.
  • Figure 5D Immunostaining of wild type, non- treated fish and HMOXl mRNA injected fish with anti-myosin heavy chain (green) and anti- dystrophin antibody (red). Non-treated dystrophin-null fish have broken and disturbed structure of skeletal muscle fibers. The treated dystrophin-null fish with overexpression of HMOXl have normal skeletal muscle structure. DETAILED DESCRIPTION OF THE INVENTION
  • a pathway that can be targeted for the treatment of or alleviation of symptoms of DMD has been identified.
  • Compounds affecting this pathway have been identified and tested in a zebrafish model of DMD and found to be efficacious based on an increase in the survival rate of dystrophin deficient fish from all but 10% surviving to more than 75% of affected fish surviving.
  • Several of the compounds inhibit components that regulate the cAMP- PKA pathway, such as phosphodiesterases (PDEs), e.g., PDE inhibitors.
  • PDEs phosphodiesterases
  • NFK- ⁇ nuclear factor kappa B
  • HMOX1 heme oxygenase- 1
  • specific (or selective) phosphodiesterase inhibitors that are effective in the zebrafish assay are screened for inhibition of NFK- ⁇ and increase of heme oxygenase.
  • Compounds showing utility in the zebrafish screen, and which are active in the cAMP-PKA pathway via heme oxygenase 1 are effective for treatment of or alleviation of symptoms of DMD.
  • the cAMP-PKA pathway via heme oxygenase 1 is
  • NFK& heme oxygenase In some embodiments, compounds selectively activate cAMP, protein kinase A (“PKA”) or heme oxygenase 1, thereby inhibiting nuclear factor kappa B (“NFK- ⁇ ”) and activating heme oxygenase.
  • PKA protein kinase A
  • NFK- ⁇ nuclear factor kappa B
  • compounds e.g. inhibitors of PDE
  • PDEs are known to degrade the phosphodiester bonds of cAMP and cGMP, thus it is contemplated that the inhibition of PDEs would result in an increase in the amount or availability of cAMP and/or cGMP.
  • an increase in cAMP and/or cGMP results in increased activation of e.g., PKA, thereby resulting in e.g., increased inhibition of NFK- ⁇ and activation of heme oxygenase, and/or increased vasodilation.
  • zebrafish models of human forms of dystrophy including two alleles of dystrophin deficiency are used to screen compounds.
  • Many therapies in development are focused towards increasing dystrophin (exon skipping) expression or replacing dystrophin with utrophin.
  • the zebrafish model can be used to identify completely novel mechanisms that completely bypass the absence of dystrophin to ameliorate the disease phenotype.
  • Chemical libraries were screened using muscle birefringence as the method for scoring muscle effects.
  • the drug libraries include many FDA approved drugs. Many of these drugs are already approved for human use. While these drugs have little direct effect on a large structural protein like dystrophin, there are many other ways muscle can be stabilized, including stabilization of other structural components of the membrane such as the integrins.
  • vasodilation plays an important role in the disease process and increasing it may influence membrane stability even in the absence of dystrophin. Even when dystrophin is absent the membrane of the myo fiber remains intact for the majority of contractions and is only more prone to damage. Any compound that increases the life of a dystrophin deficient myofiber could have profound effects of disease progression.
  • Two known zebrafish dystrophin mutants represent excellent small-animal models of human muscular dystrophy.
  • the Prestwick, the NINDS2 and the ICCB known bioactive chemical libraries were screened for small molecules that modulate the muscle phenotype in these fish. Dosages are typically 2.4 micrograms/ml or 12.5 microM. With a quick and easy birefringence assay, fourteen small molecules that influence muscle pathology in dystrophin-null zebrafish without restoration of dystrophin expression were identified.
  • the six compounds identified worked at different levels in this pathway, indicating that zebrafish with absent dystrophin can be used to identify a key pathway in disease pathology and provide targets for drug therapy, the key components being the elevation of hemeoxgenase-1, the suppression of NFK- ⁇ , and/or the increase of vasodilation.
  • DMD a disease described herein, e.g., DMD
  • a disease described herein means to reduce or eliminate a sign or symptom of the disease, to stabilize the disease, and/or to reduce or slow further progression of the disease.
  • treatment of DMD may result in e.g., a slowing of muscle degeneration, decreased fatigue, increased muscle strength, reduced blood levels of creatine kinase (CK), decreased difficulty with motor skills, decreased muscle fiber deformities, reverse, reduce, or prevent cardiac dysfunction (resulting from, e.g., cardiomayopathy) manifested by e.g., congestive heart failure and arrhythmias, etc.
  • CK creatine kinase
  • cardiac dysfunction resulting from, e.g., cardiomayopathy
  • an “effective amount,” or an “amount effective,” as used herein, refers to an amount of a compound and/or an additional therapeutic agent, or a composition thereof that is effective in producing the desired molecular, therapeutic, ameliorative, inhibitory or preventative effect, and/or results in a desired clinical effect.
  • an effective amount of a composition described herein when administered to a patient results in e.g., increased muscle strength, increased motility, restoration of muscle function or phenotype, decreased fatigue, decreased difficulty with motor skills, etc.
  • the desired therapeutic or clinical effect resulting from administration of an effective amount of a composition described herein may be measured or monitored by methods know to those of ordinary skill in the art e.g., by monitoring the creatine kinase (CK) levels in a patient's blood, by electromyography, and/or by histological examination of a muscle biopsy.
  • CK creatine kinase
  • an effective amount can refer to each individual agent or to the combination as a whole, wherein the amounts of all agents administered are together effective, but wherein the component agent of the combination may not be present individually in an effective amount.
  • compositions described herein comprise a compound (e.g., a small molecule, a protein, a peptide, an antibody, an antibody fragment, a ligand, a receptor, etc.) that selectively targets the cAMP-PKA pathway.
  • a compound e.g., a small molecule, a protein, a peptide, an antibody, an antibody fragment, a ligand, a receptor, etc.
  • selectively targets it is meant that the compound binds a target (e.g., a biomolecule or component in the cAMP-PKA pathway), with greater affinity than it binds to a non-target (e.g., a component that is not part of the cAMP- PKA pathway).
  • the compound binds its target with a dissociation constant (K D ) of less than 10 "6 M, of less than 10 "7 M, of less than 10 ⁇ 8 M, of less than 10 "9 M, or of less than 10 ⁇ 10 M.
  • K D dissociation constant
  • a compound may be a small molecule, a chemical, a protein, a peptide, an antibody, an antibody fragment, a ligand, or a receptor, that binds to a target molecule with a K D as specified above.
  • "selectively targets” refers to binding of a compound to a target with high affinity, e.g.
  • the compound binds to the target molecule with high selectivity or specificity, e.g., in that it does not bind to molecules other than the target molecule with a K D of less than 10 "6 M, of less than 10 "7 M, or of less than 10 ⁇ 8 M.
  • the "cAMP-PKA pathway” refers to the collection of
  • biomolecules or components e.g., proteins, peptides, receptors, ligands, enzymes, nucleotides, cyclic nucleotides, etc.
  • PKA which is activated by cAMP, mediates downstream signaling events which results in e.g., inhibition of NFKB, and/or upregulation of heme oxygenase 1 (HMOX1), and/or vasodilation.
  • HMOX1 heme oxygenase 1
  • the present disclosure contemplates methods and compositions for the treatment of DMD, by affecting components of the cAMP-PKA pathway in such a way as to promote inhibition of NFKB, and/or promote upregulation of HMOXl, and/or promote vasodilation.
  • compositions described herein comprise compounds wherein the compound is present in an amount effective to upregulate a component of the cAMP-PKA pathway, e.g. heme oxygenase 1 (HMOXl).
  • upregulate it is meant, in some aspects, that the component (e.g., HMOXl) is e.g., increased in amount or activity, thereby resulting in a desired therapeutic effect or benefit.
  • upregulation e.g., an increase in activity or amount of a component or molecule (e.g., HMOXl)
  • upregulation means a 5%, 10%, 15%, 25%, 35%, 50%, 75%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1250%, 1500%, 1750%, 2000%, 2500%, 3000%, 3500%, 4000%, 4500 %, 5000% increase, or more.
  • upregulation e.g., an increase in activity or amount of a component or molecule (e.g.
  • HMOXl means a statistically significant increase.
  • upregulation may be monitored by e.g., the monitoring of certain clinical parameters (e.g., increased muscle strength, motility, restoration of muscle function or phenotype, CK blood levels, electromyography, muscle biopsy, etc.).
  • certain clinical parameters e.g., increased muscle strength, motility, restoration of muscle function or phenotype, CK blood levels, electromyography, muscle biopsy, etc.
  • the upregulation of HMOXl could be assessed by immunohistochemical analysis of a muscle biopsy using antibodies against HMOXl (e.g. Anti-HMOXl, Sigma-Aldrich, HPA000635).
  • HMOXl is known to cleave heme to form biliverdin, which is subsequently converted to bilirubin by biliverdin reductase, releasing carbon monoxide (CO).
  • biliverdin reductase releasing carbon monoxide
  • CO carbon monoxide
  • less invasive procedures may be used to monitor upregulation of HMOXl by determining and monitoring the blood levels of heme catabolic byproducts such as biliverdin and CO. Methods of determining and monitoring heme catabolic byproducts such as biliverdin and CO are known to those of ordinary skill in the art.
  • compositions described herein comprise compounds that activate HMOXl directly in an amount effective to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B) and increase vasodilation.
  • NF-kappa B nuclear factor kappa-light-chain-enhancer of activated B cells
  • NF-kappa B nuclear factor kappa-light-chain-enhancer of activated B cells
  • NF-kappa B is used interchangeably with “NFK- ⁇ ,” “NFKB,” and “NF- ⁇ .”
  • the term “inhibit” as used herein may refer to the detectable reduction and/or elimination of a biological activity. In some embodiments, the reduction of biological activity is statistically significant, e.g., as compared to a standard or a control. Methods of detecting inhibition of NFKB are known in the art, and include, but are not limited to, immunohistochemical analysis.
  • antibodies against NFKB are used to analyze the amount and/or location of NFKB (e.g., if nuclear localized then active; if cytoplasmic, then inactive) in tissue from a patient's muscle biopsy.
  • NFKB e.g., if nuclear localized then active; if cytoplasmic, then inactive
  • increased vasodilation as a result of e.g., HMOX1 activation, promotes increased blood flow to skeletal muscle resulting in a therapeutic effect in the treatment of DMD.
  • Methods of monitoring or measuring blood flow to skeletal muscle are known to those of ordinary skill in the art e.g., ultrasonic flow meters (e.g., doppler).
  • compositions described herein comprise compounds that activate protein kinase A (PKA) directly in an amount effective to inhibit NFKB and increase
  • Methods for detecting and/or monitoring activation of PKA are known in the art, and include, but are not limited to, the methods as described herein in Example 1 (PepTag Assay for Non-Radioactive Detection of cAMP-Dependent Protein Kinase (Promega)).
  • an increase in vasodilation may be measured by monitoring a decrease in blood pressure, and/or increase in blood flow.
  • ultrasonographic imaging of a patient's brachial artery can measure an increase in vasodilation.
  • an increase in vasodilation means a 5%, 10%, 15%, 25%, 35%, 50%, 75%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1250%, 1500%, 1750%, 2000%, 2500%, 3000%, 3500%, 4000%, 4500 %, 5000% increase, or more.
  • an increase in vasodilation means a statistically significant increase.
  • patient refers to an individual organism. In some embodiments,
  • a patient is a mammal, for example, a human, a non-human primate, a mouse, a rat, a cat, a dog, a cattle, a goat, a pig, or a sheep.
  • the patient is a human having or suspected of having DMD.
  • administering means providing a drug to a patient in a manner that is pharmacologically useful.
  • compositions as described herein may be
  • prodrug refers to compounds which, under physiological conditions, are converted into the therapeutically active agents of the present invention.
  • a common method for making a prodrug is to include selected moieties which are hydrolyzed under physiological conditions to reveal the desired molecule.
  • the prodrug is converted by an enzymatic activity of the host animal.
  • Stereoisomer refers to isomeric molecules that have the same molecular formula and sequence of bonded atoms (constitution), but which differ in the three dimensional orientations of their atoms in space. Examples of stereoisomers include
  • an enantiomer refers to one of the two mirror- image forms of an optically active or chiral molecule.
  • Diastereomers or diastereoisomers are stereoisomers that are not enantiomers (non-superimposable mirror images of each other).
  • Chiral molecules contain a chiral center, also referred to as a stereocenter or stereogenic center, which is any point, though not necessarily an atom, in a molecule bearing groups such that an interchanging of any two groups leads to a stereoisomer.
  • the chiral center is typically a carbon, phosphorus or sulfur atom, though it is also possible for other atoms to be stereocenters in organic and inorganic compounds.
  • a molecule can have multiple stereocenters, giving it many stereoisomers.
  • a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%.
  • Enantiomers and/or diasteromers can be resolved or separated using techniques known in the art.
  • 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., C C3o 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 one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a hosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Throughout the application, preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.
  • the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
  • the substituents of a substituted alkyl may include halogen, hydroxy, nitro, thiols, 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),
  • Cycloalkyls can be substituted in the same manner.
  • heteroalkyl refers to straight or branched chain, or cyclic carbon-containing radicals, or a combination thereof, containing at least one heteroatom.
  • Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized.
  • Heteroalkyls can be substituted as defined above for alkyl groups.
  • alkylthio refers to an alkyl group, as defined above, having a sulfur radical attached thereto.
  • the "alkylthio" moiety is represented by one of -S- alkyl, -S-alkenyl, and -S-alkynyl.
  • Representative alkylthio groups include methylthio, ethylthio, and the like.
  • alkylthio also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.
  • Arylthio refers to aryl or heteroaryl groups.
  • alkenyl and alkynyl refer to 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.
  • alkoxyl or alkoxy refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.
  • An “ether” is two hydrocarbons covalently linked by an oxygen.
  • an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, and -O- alkynyl.
  • Aroxy can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined below.
  • the alkoxy and aroxy groups can be substituted as described above for alkyl.
  • amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:
  • R 9 , R 10 , and R' 10 each independently represent a hydrogen, an alkyl, an alkenyl, aryl, heteroaryl, -(CH 2 ) m -R8 or R 9 and R 10 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure;
  • R9 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and
  • m is zero or an integer in the range of 1 to 8.
  • only one of R 9 or R 10 can be a carbonyl, e.g., R 9 , R 10 and the nitrogen together do not form an imide.
  • the term "amine” does not encompass amides, e.g., wherein one of R9 and R ⁇ represents a carbonyl.
  • R9 and R ⁇ (and optionally R' w) each independently represent a hydrogen, an alkyl or cycloakly, an alkenyl or cycloalkenyl, or alkynyl.
  • alkylamine as used herein means an amine group, as defined above, having a substituted (as described above for alkyl) or unsubstituted alkyl attached thereto, i.e., at least one of R 9 and Rio is an alkyl group.
  • Aryl refers to Cs-Cio-membered aromatic, heterocyclic, fused aromatic, fused heterocyclic, biaromatic, or bihetereocyclic ring systems.
  • aryl includes 5-, 6-, 7-, 8-, 9-, and 10-membered single -ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • aryl heterocycles or "heteroaromatics”.
  • the aromatic ring can be substituted at one or more ring positions with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF 3 , -CN; and combinations thereof.
  • substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (i.e., "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic ring or rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocycles.
  • heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydro furan, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, indolenyl, indolinyl, indolizin
  • aralkyl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
  • carrier refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.
  • Heterocycle refers to a cyclic radical attached via a ring carbon or nitrogen of a monocyclic or bicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ring atoms, consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, (C Cio) alkyl, phenyl or benzyl, and optionally containing 1-3 double bonds and optionally substituted with one or more substituents.
  • heterocyclic ring examples include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, indolenyl, indolinyl, in
  • Heterocyclic groups can optionally be substituted with one or more substituents at one or more positions as defined above for alkyl and aryl, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
  • substituents at one or more positions as defined above for alkyl and aryl, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl
  • carbonyl is art-recognized and includes such moieties as can be represented by the general formula:
  • X is a bond or represents an oxygen or a sulfur
  • Rn represents a hydrogen, an alkyl, a cycloalkyl, an alkenyl, an cycloalkenyl, or an alkynyl
  • R'n represents a hydrogen, an alkyl, a cycloalkyl, an alkenyl, an cycloalkenyl, or an alkynyl
  • X is an oxygen and Rn or R'n is not hydrogen
  • the formula represents an "ester”.
  • X is an oxygen and Rn is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when Rn is a hydrogen, the formula represents a "carboxylic acid".
  • R'n is not hydrogen, the formula represents a "thioester.” Where X is a sulfur and R is hydrogen, the formula represents a "thiocarboxylic acid.” Where X is a sulfur and R' n is hydrogen, the formula represents a "thioformate.” On the other hand, where X is a bond, and Rn is not hydrogen, the above formula represents a "ketone” group. Where X is a bond, and Rn is hydrogen, the above formula represents an "aldehyde” group.
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium. Other heteroatoms include silicon and arsenic.
  • nitro means -N0 2 ;
  • halogen designates -F, -CI, -Br or -I;
  • sulfhydryl means -SH;
  • hydroxyl means -OH;
  • sulfonyl means -S0 2 -.
  • Suitable classes of compounds that can be used to effectively treat DMD include, but are not limited to, the following classes.
  • the classes of compounds can be used alone or in combinations. For example, two or more compounds which target the same point in the pathway and/or two or more compounds which target different points in the pathway can be administered.
  • Non-steroidal anti-inflammatories having the structures below or analogs or derivatives thereof:
  • Ri and R 6 are substituted or unsubstituted lower alkyl, such as methyl, and R2-R5 are as defined above. In some embodiments, Ri and R 6 are substituted or unsubstituted lower alkyl, such as methyl, R 3 and R 4 are substituted or unsubstituted lower alkoxy, such as methoxy, and R 2 and R5 are as defined above. In some embodiments, Ri and R 6 are substituted or unsubstituted lower alkyl, such as methyl, R and R 4 are substituted or unsubstituted lower alkoxy, such as methoxy, and R 2 and R5 are hydrogen.
  • Exemplary compounds include, but are not limited to, epirizole.
  • Ri-R 6 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • Ri is substituted or unsubstituted lower alkyl, such as methyl, ethyl, propyl, or isopropyl
  • R 2 -R6 are as defined above.
  • Ri is substituted or unsubstituted lower alkyl, such as methyl, ethyl, propyl, or isopropyl
  • R 3 -R 6 are as defined above.
  • Ri is substituted or unsubstituted lower alkyl, such as methyl, ethyl, propyl, or isopropyl
  • R 3 -R 6 are hydrogen.
  • Exemplary compounds include, but are not limited, ibudilast.
  • antihistamines having the structures below or analogs or derivatives thereof:
  • R Rn are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • R 3 is a halogen, such as chloro or bromo, and R 1; R 2 , and R 4 -Rn are as defined above.
  • R 3 is a halogen, such as chloro or bromo, Rn is substituted or unsubstituted lower alkyl, such as methyl, and R 1; R 2 , and R 4 -R 10 are as defined above.
  • R 3 is a halogen, such as chloro or bromo, Rn is substituted or unsubstituted lower alkyl, such as methyl, and R 1; R 2 , and R 4 -R 10 are hydrogen.
  • Exemplary compounds include, but are not limited to, homochlorcyclizine.
  • the compound is steroid alkaloid found in one or more plant species. Examples include, but are not limited to:
  • R Rg are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • R 1; R 2 , and/or R 7 are substituted or unsubstituted lower alkyl, such as methyl, and the remaining substituents are as defined above. In other embodiments, R 1; R 2 , and/or R 7 are substituted or unsubstituted lower alkyl, such as methyl, Rg is a primary, secondary, or tertiary amine, and the remaining substituents are as defined above.
  • R 1; R 2 , and/or R 7 are substituted or unsubstituted lower alkyl, such as methyl
  • R 8 is a tertiary amine, wherein the groups attached to nitrogen are alkyl, such as lower alkyl (e.g., methyl, ethyl, and/or propyl), and the remaining substituents are as defined above.
  • R 1; R 2 , and R 7 are substituted or unsubstituted lower alkyl, such as methyl
  • Rg is a tertiary amine, wherein the groups attached to nitrogen are alkyl, such as lower alkyl (e.g., methyl, ethyl, and/or propyl), and the remaining substituents are hydrogen.
  • Exemplary compounds include, but are not limited to, conessine.
  • Other examples of such steroid alkaloids include
  • Ri and R5 are substituted or unsubstituted lower alkyl, such as methyl and R2-R4 are as defined above. In other embodiments, Ri and R 5 are substituted or unsubstituted lower alkyl, such as methyl and R 3 and R 4 are hydrogen and R 2 is as defined above. In other embodiments, Ri and R5 are substituted or unsubstituted lower alkyl, such as methyl and R and R 4 are hydrogen and R 2 is hydroxy.
  • Exemplary compounds include, but are not limited to, proscillaridin.
  • bronchodilators having the formula shown below and analogs and derivatives thereof:
  • Ri is hydrogen or substituted or unsubstituted lower alkyl, and R 2 and R 3 are hydrogen or lower alkyl. In some embodiments, Ri is hydrogen, and R 2 and R 3 are hydrogen or substituted or unsubstituted lower alkyl. In some embodiments, Ri is hydrogen, and R 2 and R 3 are substituted or unsubstituted lower alkyl, such as methyl.
  • Exemplary compounds include, but are not limited to, aminophylline.
  • Aminophylline is typically administered with ethylene diamine to improve solubility.
  • Estrogens having the formula below or analogs or derivatives thereof:
  • R -R 4 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • Ri is substituted or unsubstituted lower alkyl, such as methyl and R2-R 4 are as defined above. In some embodiments, Ri is substituted or unsubstituted lower alkyl, such as methyl, R 2 and R 3 are hydrogen. In still other embodiments, Ri is substituted or unsubstituted lower alkyl, such as methyl, R 2 and R 3 are hydrogen, and R 4 is hydroxy.
  • Exemplary compounds include, but are not limited, equilin.
  • R R ⁇ are hydrogen.
  • Exemplary compounds include, but are not limited, pentetic acid.
  • Non-selective or selective (e.g., PDE4 or PDE5) phosphodiesterase inhibitors such as compounds having the formula below or analogs or derivatives thereof:
  • R 3 is substituted or unsubstituted lower alkoxy, such as methoxy, and R 1; R 2 , R 4 , and R 5 are as defined above.
  • R 3 is substituted or unsubstituted lower alkoxy, such as methoxy
  • R 4 is substituted or unsubstituted cycloalkoxy, such as cyclopentoxy and R 1; R 2 , and R5 are as defined above.
  • R is substituted or unsubstituted lower alkoxy, such as methoxy, and R 4 is substituted or
  • unsubstituted cycloalkoxy such as cyclopentoxy and R 1; R 2 , and R 5 are hydrogen.
  • Exemplary compounds include, but are not limited, rolipram.
  • Other PDE inhibitors include sildenafil, vardenafil, tadalifil, eonoximone, milrinone, and analogs or derivatives thereof.
  • vasodilators such as compounds having the formula below or analogs or derivatives thereof:
  • R R 35 are hydrogen.
  • Exemplary compounds include, but are not limited to, dipyridamole.
  • R1-R 5 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • Ri and R 2 are hydrogen and/or substituted or unsubstituted lower alkyl and R 3 -R 5 are as defined above. In other embodiments, Ri and R 2 are hydrogen and R 3 -R 5 are as defined above. In still other embodiments, Ri-R 5 are hydrogen.
  • Exemplary compounds include, but are not limited to, nitromide.
  • R -R9 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • Ri is and R2-R9 are as defined above.
  • Ri is , R10-R12 are substituted or unsubstituted lower alkyl, such as methyl, ethyl, propyl, and isopropyl, and R 2 -R9 are hydrogen.
  • Ri is , Rio is methyl, Rn and R 12 are isopropyl, and R 2 -R9 are hydrogen.
  • Exemplary compounds include, but are not limited, propantheline, such as propantheline bromide.
  • XL metabolites of testosterone such as compounds having the formula shown below or analogs or derivatives thereof:
  • R -R7 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • Ri and R 5 are lower alkyl, such as methyl, and R 2 -R 4 , R6, and R 7 are as defined above.
  • Ri and R5 are substituted or unsubstituted lower alkyl, such as methyl, and R 2 -R 4 , and R 6 are hydrogen, and R 7 is as defined above.
  • Ri and R 5 are substituted or unsubstituted lower alkyl, such as methyl, and R 2 -R 4 , and R 6 are hydrogen, and R 7 is hydroxy.
  • Exemplary compounds include, but are not limited, androsterone.
  • Exemplary compounds include, but are not limited, crassin, such as crassin acetate. compounds having the formula below or analogs or derivatives thereof:
  • R' and R" are individually hydrogen or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, arylalkyl, heteroalkyls, alkylaryl, alkylheteroaryl, and r is an integer from 1 to 6.
  • R5 is substituted or unsubstituted alkene, such as
  • R3 and R 4 are substituted or unsubstituted lower alkyl, such as methyl
  • R 1; R 2 , and R 6 -Rio are as defined above.
  • Exemplary compounds include, but are not limited, pomiferin.
  • XIV. antifungal antiobiotics that inhibit fatty acid and/or steroid biosynthesis having the formula below or analogs or derivatives thereof:
  • R1-R15 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR' ; -NR'R"; -
  • Ri and R 2 are hydrogen or substituted or unsubstituted lower alkyl and R3-R 15 are as defined above. In other embodiments, Ri and R 2 are hydrogen, and R 3 - R 15 are hydrogen.
  • Exemplary compounds include, but are not limited, cerulenin.
  • R1-R25 are each independently selected from hydrogen; substituted or unsubstituted, linear, branched, or cyclic alkyl, alkenyl, or alkynyl (optionally containing one or more heteroatoms); substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; alkylaryl; alkylheteroaryl; arylalkyl; heteroarylalkyl; halogen; hydroxy; -OR'; -NR'R"; -
  • R1-R25 is hydrogen.
  • Exemplary compounds include, but are not limited to, prostaglandin F2-alpha.
  • the compounds described herein can be formulated for parenteral administration.
  • Parenteral administration means administration by any method other than through the digestive tract or non-invasive topical or regional routes.
  • parenteral administration may include administration to a patient intravenously, intradermally,
  • Parenteral formulations can be prepared as aqueous compositions using techniques is known in the art.
  • such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
  • injectable formulations for example, solutions or suspensions
  • solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
  • emulsions such as water-in-oil (w/o) emulsions
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and a combination thereof.
  • polyols e.g., glycerol, propylene glycol, and liquid polyethylene glycol
  • oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.)
  • 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/or by the use of surfactants.
  • isotonic agents for example, sugars or sodium chloride.
  • pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof.
  • Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents.
  • Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.
  • Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium
  • Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine.
  • nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide.
  • amphoteric surfactants include sodium N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.- iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
  • the formulation can contain a preservative to prevent the growth of microorganisms.
  • Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.
  • the formulation may also contain an antioxidant to prevent degradation of the active agent(s).
  • the formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution.
  • Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.
  • Water soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.
  • Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed 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 listed above.
  • the preferred 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 powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.
  • parenteral formulations described herein can be formulated for controlled release including immediate release, delayed release, extended release, pulsatile release, and a combination thereof.
  • the one or more compounds, and optional one or more additional active agents can be incorporated into microparticles, nanoparticles, or combinations thereof that provide controlled release of the compounds and/or one or more additional active agents.
  • the formulations contains two or more drugs
  • the drugs can be formulated for the same type of controlled release (e.g., delayed, extended, immediate, or pulsatile) or the drugs can be independently formulated for different types of release (e.g., immediate and delayed, immediate and extended, delayed and extended, delayed and pulsatile, etc.).
  • the compounds and/or one or more additional active agents can be incorporated into polymeric microparticles which provide controlled release of the drug(s). Release of the drug(s) is controlled by diffusion of the drug(s) out of the microparticles and/or degradation of the polymeric particles by hydrolysis and/or enzymatic degradation.
  • Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives.
  • Polymers which are slowly soluble and form a gel in an aqueous environment may also be suitable as materials for drug containing microparticles.
  • Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and a combination thereof.
  • the drug(s) can be incorporated into microparticles prepared from materials which are insoluble in aqueous solution or slowly soluble in aqueous solution, but are capable of degrading within the GI tract by means including enzymatic degradation, surfactant action of bile acids, and/or mechanical erosion.
  • slowly soluble in water refers to materials that are not dissolved in water within a period of 30 minutes.
  • Suitable fats and fatty substances include fatty alcohols (such as lauryl, myristyl stearyl, cetyl or cetostearyl alcohol), fatty acids and derivatives, including but not limited to fatty acid esters, fatty acid glycerides (mono-, di- and tri-glycerides), and hydrogenated fats.
  • fatty alcohols such as lauryl, myristyl stearyl, cetyl or cetostearyl alcohol
  • fatty acids and derivatives including but not limited to fatty acid esters, fatty acid glycerides (mono-, di- and tri-glycerides), and hydrogenated fats.
  • Specific examples include, but are not limited to hydrogenated vegetable oil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenated oils available under the trade name Sterotex®, stearic acid, cocoa butter, and stearyl alcohol.
  • Suitable waxes and wax-like materials include natural or synthetic waxes, hydrocarbons, and normal waxes. Specific examples of waxes include beeswax, glycowax, castor wax, carnauba wax, paraffins and candelilla wax. As used herein, a wax-like material is defined as any material which is normally solid at room temperature and has a melting point of from about 30 to 300°C.
  • rate-controlling (wicking) agents may be formulated along with the fats or waxes listed above.
  • rate-controlling materials include certain starch derivatives (e.g., waxy maltodextrin and drum dried corn starch), cellulose derivatives (e.g., hydroxypropylmethyl-cellulose, hydroxypropylcellulose, methylcellulose, and carboxymethyl- cellulose), alginic acid, lactose and talc.
  • a pharmaceutically acceptable surfactant for example, lecithin may be added to facilitate the degradation of such microparticles.
  • Proteins which are water insoluble can also be used as materials for the formation of drug containing microparticles.
  • proteins, polysaccharides and combinations thereof which are water soluble can be formulated with drug into microparticles and subsequently cross-linked to form an insoluble network.
  • cyclodextrins can be complexed with individual drug molecules and subsequently cross-linked.
  • Encapsulation or incorporation of drug into carrier materials to produce drug containing microparticles can be achieved through known pharmaceutical formulation techniques.
  • the carrier material is typically heated above its melting temperature and the drug is added to form a mixture comprising drug particles suspended in the carrier material, drug dissolved in the carrier material, or a mixture thereof.
  • Microparticles can be subsequently formulated through several methods including, but not limited to, the processes of congealing, extrusion, spray chilling or aqueous dispersion.
  • wax is heated above its melting temperature, drug is added, and the molten wax-drug mixture is congealed under constant stirring as the mixture cools.
  • the molten wax-drug mixture can be extruded and spheronized to form pellets or beads.
  • a solvent evaporation technique to produce drug containing microparticles.
  • drug and carrier material are co-dissolved in a mutual solvent and microparticles can subsequently be produced by several techniques including, but not limited to, forming an emulsion in water or other appropriate media, spray drying or by evaporating off the solvent from the bulk solution and milling the resulting material.
  • drug in a particulate form is homogeneously dispersed in a water- insoluble or slowly water soluble material.
  • the drug powder itself may be milled to generate fine particles prior to formulation. The process of jet milling, known in the pharmaceutical art, can be used for this purpose.
  • drug in a particulate form is homogeneously dispersed in a wax or wax like substance by heating the wax or wax like substance above its melting point and adding the drug particles while stirring the mixture.
  • a pharmaceutically acceptable surfactant may be added to the mixture to facilitate the dispersion of the drug particles.
  • the particles can also be coated with one or more modified release coatings.
  • Solid esters of fatty acids which are hydrolyzed by lipases, can be spray coated onto microparticles or drug particles.
  • Zein is an example of a naturally water-insoluble protein. It can be coated onto drug containing microparticles or drug particles by spray coating or by wet granulation techniques.
  • some substrates of digestive enzymes can be treated with cross-linking procedures, resulting in the formation of non-soluble networks.
  • Many methods of cross-linking proteins initiated by both chemical and physical means, have been reported. One of the most common methods to obtain cross-linking is the use of chemical cross- linking agents.
  • cross-linking agents examples include aldehydes (gluteraldehyde and formaldehyde), epoxy compounds, carbodiimides, and genipin.
  • aldehydes gluteraldehyde and formaldehyde
  • epoxy compounds carbodiimides
  • genipin examples include aldehydes (gluteraldehyde and formaldehyde), epoxy compounds, carbodiimides, and genipin.
  • oxidized and native sugars have been used to cross-link gelatin.
  • Cross-linking can also be accomplished using enzymatic means; for example, transglutaminase has been approved as a GRAS substance for cross-linking seafood products.
  • cross-linking can be initiated by physical means such as thermal treatment, UV irradiation and gamma irradiation.
  • a water soluble protein can be spray coated onto the
  • microparticles and subsequently cross-linked by the one of the methods described above.
  • drug containing microparticles can be microencapsulated within protein by coacervation-phase separation (for example, by the addition of salts) and subsequently cross- linked.
  • suitable proteins for this purpose include gelatin, albumin, casein, and gluten.
  • Polysaccharides can also be cross-linked to form a water-insoluble network. For many polysaccharides, this can be accomplished by reaction with calcium salts or multivalent cations which cross-link the main polymer chains. Pectin, alginate, dextran, amylose and guar gum are subject to cross-linking in the presence of multivalent cations. Complexes between oppositely charged polysaccharides can also be formed; pectin and chitosan, for example, can be complexed via electrostatic interactions. In certain embodiments, it may be desirable to provide continuous delivery of one or more compounds to a patient in need thereof. For intravenous or intraarterial routes, this can be accomplished using drip systems, such as by intravenous administration. For topical
  • repeated application can be done or a patch can be used to provide continuous administration of the compounds over an extended period of time.
  • the compounds described herein can be incorporated into injectable/implantable solid or semi-solid implants, such as polymeric implants.
  • the compounds are incorporated into a polymer that is a liquid or paste at room temperature, but upon contact with aqueous medium, such as physiological fluids, exhibits an increase in viscosity to form a semisolid or solid material.
  • exemplary polymers include, but are not limited to, hydroxyalkanoic acid polyesters derived from the copolymerization of at least one unsaturated hydroxy fatty acid copolymerized with hydroxyalkanoic acids. The polymer can be melted, mixed with the active substance and cast or injection molded into a device.
  • melt fabrication require polymers having a melting point that is below the temperature at which the substance to be delivered and polymer degrade or become reactive.
  • the device can also be prepared by solvent casting where the polymer is dissolved in a solvent and the drug dissolved or dispersed in the polymer solution and the solvent is then evaporated. Solvent processes require that the polymer be soluble in organic solvents.
  • Another method is compression molding of a mixed powder of the polymer and the drug or polymer particles loaded with the active agent.
  • the compounds can be incorporated into a polymer matrix and molded, compressed, or extruded into a device that is a solid at room temperature.
  • the compounds can be incorporated into a biodegradable polymer, such as polyanhydrides, polyhydroalkanoic acids (PHAs), PLA, PGA, PLGA, polycaprolactone, polyesters, polyamides, polyorthoesters, polyphosphazenes, proteins and polysaccharides such as collagen, hyaluronic acid, albumin and gelatin, and combinations thereof and compressed into solid device, such as disks, or extruded into a device, such as rods.
  • PHAs polyhydroalkanoic acids
  • PLA polyhydroalkanoic acids
  • PGA PGA
  • PLGA polycaprolactone
  • polyesters polyamides
  • polyorthoesters polyphosphazenes
  • proteins and polysaccharides such as collagen, hyaluronic acid, albumin and gelatin
  • the release of the one or more compounds from the implant can be varied by selection of the polymer, the molecular weight of the polymer, and/ir modification of the polymer to increase degradation, such as the formation of pores and/or incorporation of hydrolyzable linkages.
  • Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art. Formulations may be prepared using a pharmaceutically acceptable carrier. As generally used herein "carrier” includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.
  • Carrier also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. Delayed release dosage formulations may be prepared as described in standard references. These references provide information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.
  • suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.
  • cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate
  • polyvinyl acetate phthalate acrylic acid polymers and copolymers
  • methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), ze
  • the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.
  • Optional pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants.
  • Diluents also referred to as "fillers,” are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules.
  • Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.
  • Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms.
  • Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid and polyvinylpyrrolidone.
  • Lubricants are used to facilitate tablet manufacture.
  • suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.
  • Disintegrants are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross- linked PVP (Polyplasdone® XL from GAF Chemical Corp).
  • starch sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross- linked PVP (Polyplasdone® XL from GAF Chemical Corp).
  • Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions.
  • Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA).
  • Oral dosage forms such as capsules, tablets, solutions, and suspensions, can for formulated for controlled release.
  • the one or more compounds and optional one or more additional active agents can be formulated into nanoparticles, microparticles, and combinations thereof, and encapsulated in a soft or hard gelatin or non-gelatin capsule or dispersed in a dispersing medium to form an oral suspension or syrup.
  • the particles can be formed of the drug and a controlled release polymer or matrix.
  • the drug particles can be coated with one or more controlled release coatings prior to incorporation in to the finished dosage form.
  • the one or more compounds and optional one or more additional active agents are dispersed in a matrix material, which gels or emulsifies upon contact with an aqueous medium, such as physiological fluids. In the case of gels, the matrix swells entrapping the active agents, which are released slowly over time by diffusion and/or degradation of the matrix material.
  • aqueous medium such as physiological fluids.
  • Such matrices can be formulated as tablets or as fill materials for hard and soft capsules.
  • the one or more compounds, and optional one or more additional active agents are formulated into a sold oral dosage form, such as a tablet or capsule, and the solid dosage form is coated with one or more controlled release coatings, such as a delayed release coatings or extended release coatings.
  • the coating or coatings may also contain the compounds and/or additional active agents.
  • the extended release formulations are generally prepared as diffusion or osmotic systems, which are known in the art.
  • a diffusion system typically consists of two types of devices, a reservoir and a matrix, and is well known and described in the art.
  • the matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form.
  • the three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds.
  • Plastic matrices include, but are not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene.
  • Hydrophilic polymers include, but are not limited to, cellulosic polymers such as methyl and ethyl cellulose, hydroxyalkylcelluloses such as hydroxypropyl-cellulose,
  • Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate and wax-type substances including hydrogenated castor oil or hydrogenated vegetable oil, or mixtures thereof.
  • the plastic material is a pharmaceutically acceptable acrylic polymer, including but not limited to, acrylic acid and methacrylic acid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly( acrylic acid), poly(methacrylic acid), methacrylic acid alkylamine copolymer poly(methyl methacrylate), poly(methacrylic acid)(anhydride), polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), and glycidyl methacrylate copolymers.
  • acrylic acid and methacrylic acid copolymers including but not limited to, acrylic acid and methacrylic acid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl meth
  • the acrylic polymer is comprised of one or more ammonio methacrylate copolymers.
  • Ammonio methacrylate copolymers are well known in the art, and are described in NF XVII as fully polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.
  • the acrylic polymer is an acrylic resin lacquer such as that which is commercially available from Rohm Pharma under the tradename Eudragit®.
  • the acrylic polymer comprises a mixture of two acrylic resin lacquers commercially available from Rohm Pharma under the tradenames Eudragit® RL30D and Eudragit ® RS30D, respectively.
  • Eudragit® RL30D and Eudragit® RS30D are copolymers of acrylic and methacrylic esters with a low content of quaternary ammonium groups, the molar ratio of ammonium groups to the remaining neutral (meth)acrylic esters being 1:20 in Eudragit® RL30D and 1:40 in Eudragit® RS30D.
  • the mean molecular weight is about 150,000.
  • Edragit® S-100 and Eudragit® L-100 are also preferred.
  • the code designations RL (high permeability) and RS (low permeability) refer to the permeability properties of these agents.
  • Eudragit® RL/RS mixtures are insoluble in water and in digestive fluids. However, multiparticulate systems formed to include the same are swellable and permeable in aqueous solutions and digestive fluids.
  • the polymers described above such as Eudragit® RL/RS may be mixed together in any desired ratio in order to ultimately obtain a sustained-release formulation having a desirable dissolution profile. Desirable sustained-release multiparticulate systems may be obtained, for instance, from 100% Eudragit® RL, 50% Eudragit® RL and 50% Eudragit® RS, and 10% Eudragit® RL and 90% Eudragit® RS.
  • Desirable sustained-release multiparticulate systems may be obtained, for instance, from 100% Eudragit® RL, 50% Eudragit® RL and 50% Eudragit® RS, and 10% Eudragit® RL and 90% Eudragit® RS.
  • acrylic polymers may also be used, such as, for example, Eudragit® L.
  • extended release formulations can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form.
  • the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion.
  • the devices with different drug release mechanisms described above can be combined in a final dosage form comprising single or multiple units.
  • multiple units include, but are not limited to, multilayer tablets and capsules containing tablets, beads, or granules.
  • An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using a coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.
  • Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, or dry granulation.
  • diluents include inert powdered substances such as starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders.
  • Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful.
  • Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders.
  • a lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die.
  • the lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.
  • Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method.
  • the congealing method the drug is mixed with a wax material and either spray- congealed or congealed and screened and processed.
  • Delayed release formulations can be created by coating a solid dosage form with a polymer film, which is insoluble in the acidic environment of the stomach, and soluble in the neutral environment of the small intestine.
  • the delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material.
  • the drug-containing composition may be, e.g., a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core” dosage form, or a plurality of drug-containing beads, particles or granules, for incorporation into either a tablet or capsule.
  • Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble, and/or enzymatically degradable polymers, and may be conventional "enteric" polymers.
  • Enteric polymers become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon.
  • Suitable coating materials for effecting delayed release include, but are not limited to, cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, and other methacrylic resins that are commercially available under the tradename Eudragit® (Rohm Pharma; Westerstadt, Germany), including Eudragit® L30D-55 and L100-55 (soluble at pH 5.5 and above), Eudragit® L-100 (soluble at pH
  • the preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials, method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies.
  • the coating composition may include conventional additives, such as plasticizers, pigments, colorants, stabilizing agents, glidants, etc.
  • a plasticizer is normally present to reduce the fragility of the coating, and will generally represent about 10 wt. % to 50 wt. % relative to the dry weight of the polymer.
  • typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil and acetylated monoglycerides.
  • a stabilizing agent is preferably used to stabilize particles in the dispersion.
  • Typical stabilizing agents are nonionic emulsifiers such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying, and will generally represent approximately 25 wt. % to 100 wt. % of the polymer weight in the coating solution.
  • One effective glidant is talc.
  • Other glidants such as magnesium stearate and glycerol monostearates may also be used.
  • Pigments such as titanium dioxide may also be used.
  • Small quantities of an anti-foaming agent such as a silicone (e.g., simethicone), may also be added to the coating composition.
  • Topical Formulations such as a silicone (e.g., simethicone), may also be added to the coating composition.
  • Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, and transdermal patches.
  • the formulation may be formulated for transmucosal, transepithelial, transendothelial, or transdermal administration.
  • the compositions may further contain one or more chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, and combination thereof.
  • “Emollients” are an externally applied agent that softens or soothes skin and are generally known in the art and listed in compendia, such as the "Handbook of Pharmaceutical Excipients", 4th Ed., Pharmaceutical Press, 2003. These include, without limitation, almond oil, castor oil, ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycol palmitostearate, glycerin, glycerin monostearate, glyceryl monooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium-chain triglycerides, mineral oil and lanolin alcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol and combinations thereof. In one embodiment, the emollients are ethyl
  • “Surfactants” are surface-active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading and wetting properties of a product.
  • Suitable non-ionic surfactants include emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof.
  • the non-ionic surfactant is stearyl alcohol.
  • Emmulsifiers are surface active substances which promote the suspension of one liquid in another and promote the formation of a stable mixture, or emulsion, of oil and water.
  • Common emulsifiers are: metallic soaps, certain animal and vegetable oils, and various polar compounds.
  • Suitable emulsifiers include acacia, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate,
  • polyoxyethylene alkyl ethers polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, self-emulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gum and combinations thereof.
  • the emulsifier is glycerol stearate.
  • Suitable classes of penetration enhancers include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithins, cholate salts, enzymes, amines and amides, complexing agents
  • liposomes, cyclodextrins, modified celluloses, and diimides liposomes, cyclodextrins, modified celluloses, and diimides
  • macrocyclics such as macrocylic lactones, ketones, and anhydrides and cyclic ureas
  • surfactants N-methyl pyrrolidones and derivatives thereof, DMSO and related compounds, ionic compounds, azone and related compounds
  • solvents such as alcohols, ketones, amides, polyols (e.g., glycols). Examples of these classes are known in the art.
  • Hydrophilic refers to substances that have strongly polar groups that readily interact with water.
  • Lipophilic refers to compounds having an affinity for lipids.
  • Amphiphilic refers to a molecule combining hydrophilic and lipophilic (hydrophobic) properties
  • Hydrophilic refers to substances that lack an affinity for water; tending to repel and not absorb water as well as not dissolve in or mix with water.
  • a “gel” is a colloid in which the dispersed phase has combined with the continuous phase to produce a semisolid material, such as jelly.
  • An “oil” is a composition containing at least 95% wt of a lipophilic substance.
  • lipophilic substances include but are not limited to naturally occurring and synthetic oils, fats, fatty acids, lecithins, triglycerides and combinations thereof.
  • a “continuous phase” refers to the liquid in which solids are suspended or droplets of another liquid are dispersed, and is sometimes called the external phase. This also refers to the fluid phase of a colloid within which solid or fluid particles are distributed. If the continuous phase is water (or another hydrophilic solvent), water-soluble or hydrophilic drugs will dissolve in the continuous phase (as opposed to being dispersed). In a multiphase formulation (e.g., an emulsion), the discreet phase is suspended or dispersed in the continuous phase.
  • an “emulsion” is a composition containing a mixture of non-miscible components homogenously blended together.
  • the non-miscible components include a lipophilic component and an aqueous component.
  • An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase.
  • oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion
  • water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase
  • water-in-oil emulsion When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase, it is known as a water
  • Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients.
  • Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol.
  • the oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers.
  • An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid.
  • the dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase.
  • oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion
  • water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase
  • the oil phase may consist at least in part of a propellant, such as an HFA propellant.
  • Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients.
  • Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol.
  • the oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers.
  • a sub-set of emulsions are the self-emulsifying systems.
  • These drug delivery systems are typically capsules (hard shell or soft shell) comprised of the drug dispersed or dissolved in a mixture of surfactant(s) and lipophilic liquids such as oils or other water immiscible liquids.
  • capsules hard shell or soft shell
  • surfactant(s) and lipophilic liquids such as oils or other water immiscible liquids.
  • a lotion can contain finely powdered substances that are in soluble in the dispersion medium through the use of suspending agents and dispersing agents.
  • lotions can have as the dispersed phase liquid substances that are immiscible wit the vehicle and are usually dispersed by means of emulsifying agents or other suitable stabilizers.
  • the lotion is in the form of an emulsion having a viscosity of between 100 and 1000 centistokes. The fluidity of lotions permits rapid and uniform application over a wide surface area. Lotions are typically intended to dry on the skin leaving a thin coat of their medicinal components on the skin's surface.
  • a “cream” is a viscous liquid or semi-solid emulsion of either the "oil-in- water” or “water-in-oil type”. Creams may contain emulsifying agents and/or other stabilizing agents. In one embodiment, the formulation is in the form of a cream having a viscosity of greater than 1000 centistokes, typically in the range of 20,000-50,000 centistokes. Creams are often time preferred over ointments as they are generally easier to spread and easier to remove.
  • the difference between a cream and a lotion is the viscosity, which is dependent on the amount/use of various oils and the percentage of water used to prepare the formulations.
  • Creams are typically thicker than lotions, may have various uses and often one uses more varied oils/butters, depending upon the desired effect upon the skin.
  • the water- base percentage is about 60-75 % and the oil-base is about 20-30 % of the total, with the other percentages being the emulsifier agent, preservatives and additives for a total of 100 %.
  • an “ointment” is a semisolid preparation containing an ointment base and optionally one or more active agents.
  • suitable ointment bases include hydrocarbon bases (e.g., petrolatum, white petrolatum, yellow ointment, and mineral oil); absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream); water-removable bases (e.g., hydrophilic ointment), and water-soluble bases (e.g., polyethylene glycol ointments).
  • Pastes typically differ from ointments in that they contain a larger percentage of solids. Pastes are typically more absorptive and less greasy that ointments prepared with the same components.
  • a "gel” is a semisolid system containing dispersions of small or large molecules in a liquid vehicle that is rendered semisolid by the action of a thickening agent or polymeric material dissolved or suspended in the liquid vehicle.
  • the liquid may include a lipophilic component, an aqueous component or both.
  • Some emulsions may be gels or otherwise include a gel component.
  • Some gels, however, are not emulsions because they do not contain a homogenized blend of immiscible components.
  • Suitable gelling agents include, but are not limited to, modified celluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose; Carbopol homopolymers and copolymers; and combinations thereof.
  • Suitable solvents in the liquid vehicle include, but are not limited to, diglycol monoethyl ether; alklene glycols, such as propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl alcohol and ethanol.
  • the solvents are typically selected for their ability to dissolve the drug.
  • Other additives, which improve the skin feel and/or emolliency of the formulation, may also be incorporated. Examples of such additives include, but are not limited, isopropyl myristate, ethyl acetate, C12-C15 alkyl benzoates, mineral oil, squalane, cyclomethicone, capric/caprylic triglycerides, and
  • Foams consist of an emulsion in combination with a gaseous propellant.
  • the gaseous propellant consists primarily of hydrofluoroalkanes (HFAs).
  • HFAs hydrofluoroalkanes
  • Suitable propellants include HFAs such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1, 1,2,3,3, 3-heptafluoropropane (HFA 227), but mixtures and admixtures of these and other HFAs that are currently approved or may become approved for medical use are suitable.
  • the propellants preferably are not hydrocarbon propellant gases which can produce flammable or explosive vapors during spraying.
  • compositions preferably contain no volatile alcohols, which can produce flammable or explosive vapors during use.
  • Buffers are used to control pH of a composition.
  • the buffers buffer the composition from a pH of about 4 to a pH of about 7.5, more preferably from a pH of about 4 to a pH of about 7, and most preferably from a pH of about 5 to a pH of about 7.
  • the buffer is triethanolamine.
  • Preservatives can be used to prevent the growth of fungi and microorganisms.
  • Suitable antifungal and antimicrobial agents include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal.
  • repeated application can be done or a patch can be used to provide continuous administration of the compounds over an extended period of time.
  • the compounds are formulated for pulmonary delivery, such as intranasal administration or oral inhalation.
  • the respiratory tract is the structure involved in the exchange of gases between the atmosphere and the blood stream.
  • the lungs are branching structures ultimately ending with the alveoli where the exchange of gases occurs.
  • the alveolar surface area is the largest in the respiratory system and is where drug absorbtion occurs.
  • the alveoli are covered by a thin epithelium without cilia or a mucus blanket and secrete surfactant phospholipids.
  • the respiratory tract encompasses the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli.
  • the upper and lower airways are called the conducting airways.
  • the terminal bronchioli then divide into respiratory bronchioli which then lead to the ultimate respiratory zone, the alveoli, or deep lung.
  • the deep lung, or alveoli are the primary target of inhaled therapeutic aerosols for systemic drug delivery.
  • Pulmonary administration of therapeutic compositions comprised of low molecular weight drugs has been observed, for example, beta- androgenic antagonists to treat asthma.
  • Other therapeutic agents that are active in the lungs have been administered systemically and targeted via pulmonary absorption.
  • Nasal delivery is considered to be a promising technique for administration of therapeutics for the following reasons: the nose has a large surface area available for drug absorption due to the coverage of the epithelial surface by numerous microvilli, the subepithelial layer is highly vascularized, the venous blood from the nose passes directly into the systemic circulation and therefore avoids the loss of drug by first-pass metabolism in the liver, it offers lower doses, more rapid attainment of therapeutic blood levels, quicker onset of pharmacological activity, fewer side effects, high total blood flow per cm3, porous endothelial basement membrane, and it is easily accessible.
  • aerosol refers to any preparation of a fine mist of particles, which can be in solution or a suspension, whether or not it is produced using a propellant.
  • Aerosols can be produced using standard techniques, such as ultrasonication or high pressure treatment.
  • Carriers for pulmonary formulations can be divided into those for dry powder formulations and for administration as solutions. Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art.
  • the formulation can be formulated into a solution, e.g., water or isotonic saline, buffered or unbuffered, or as a suspension, for intranasal administration as drops or as a spray.
  • solutions or suspensions are isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0.
  • Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers.
  • a representative nasal decongestant is described as being buffered to a pH of about 6.2.
  • a suitable saline content and pH for an innocuous aqueous solution for nasal and/or upper respiratory administration is described.
  • the aqueous solutions is water, physiologically acceptable aqueous solutions containing salts and/or buffers, such as phosphate buffered saline (PBS), or any other aqueous solution acceptable for administration to a animal or human.
  • PBS phosphate buffered saline
  • Such solutions are well known to a person skilled in the art and include, but are not limited to, distilled water, de-ionized water, pure or ultrapure water, saline, phosphate-buffered saline (PBS).
  • Other suitable aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride.
  • Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin.
  • suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth
  • a wetting agent such as lecithin.
  • Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.
  • solvents that are low toxicity organic (i.e. nonaqueous) class 3 residual solvents such as ethanol, acetone, ethyl acetate, tetrahydofuran, ethyl ether, and propanol may be used for the formulations.
  • the solvent is selected based on its ability to readily aerosolize the formulation.
  • the solvent should not detrimentally react with the compounds.
  • An appropriate solvent should be used that dissolves the compounds or forms a suspension of the compounds.
  • the solvent should be sufficiently volatile to enable formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as freons, can be added as desired to increase the volatility of the solution or suspension.
  • compositions may contain minor amounts of polymers, surfactants, or other excipients well known to those of the art.
  • minor amounts means no excipients are present that might affect or mediate uptake of the compounds in the lungs and that the excipients that are present are present in amount that do not adversely affect uptake of compounds in the lungs.
  • Dry lipid powders can be directly dispersed in ethanol because of their hydrophobic character.
  • organic solvents such as chloroform
  • the desired quantity of solution is placed in a vial, and the chloroform is evaporated under a stream of nitrogen to form a dry thin film on the surface of a glass vial.
  • the film swells easily when reconstituted with ethanol.
  • the suspension is sonicated.
  • Nonaqueous suspensions of lipids can also be prepared in absolute ethanol using a reusable PARI LC Jet-i- nebulizer (PARI Respiratory Equipment, Monterey, CA).
  • Dry powder formulations with large particle size have improved flowability characteristics, such as less aggregation, easier aerosolization, and potentially less phagocytosis.
  • Dry powder aerosols for inhalation therapy are generally produced with mean diameters primarily in the range of less than 5 microns, although a preferred range is between one and ten microns in aerodynamic diameter. Large "carrier" particles (containing no drug) have been co- delivered with therapeutic aerosols to aid in achieving efficient aerosolization among other possible benefits.
  • Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervation, interfacial polymerization, and other methods well known to those of ordinary skill in the art.
  • Particles may be made using methods for making microspheres or microcapsules known in the art.
  • the preferred methods of manufacture are by spray drying and freeze drying, which entails using a solution containing the surfactant, spraying to form droplets of the desired size, and removing the solvent.
  • the particles may be fabricated with the appropriate material, surface roughness, diameter and tap density for localized delivery to selected regions of the respiratory tract such as the deep lung or upper airways. For example, higher density or larger particles may be used for upper airway delivery. Similarly, a mixture of different sized particles, provided with the same or different EGS may be administered to target different regions of the lung in one
  • Formulations for pulmonary delivery include unilamellar phospholipid vesicles, liposomes, or lipoprotein particles. Formulations and methods of making such formulations containing nucleic acid are well known to one of ordinary skill in the art. Liposomes are formed from commercially available phospholipids supplied by a variety of vendors including Avanti Polar Lipids, Inc. (Birmingham, Ala.). In one embodiment, the liposome can include a ligand molecule specific for a receptor on the surface of the target cell to direct the liposome to the target cell.
  • Example 1 Screening for compounds maintaining viability of Zebrafish model of Duchenne muscular dystrophy
  • the Prestwick chemical library (Harvard Institute of Chemistry and Cell Biology) was used as the source of small molecules for these experiments.
  • This library contains 1,120 small molecules composed of 90% marketed drugs and 10% bioactive alkaloids or related substances.
  • the active compounds in the library were selected for their high chemical and pharmacological diversity, as well as their known bioavailability and safety in humans.
  • the library was first screened in pooled groups of eight compounds in duplicate. After the identification of pools that decreased the number of affected fish, each individual compound in these pools was screened.
  • Antisense MO targeted to interfere with fish dystrophin translation was designed using the 5' sequence around the putative translation start site of the zebrafish dystrophin mRNA (Binder, et al., N. Engl. J. Med. 273, 1289-1297 (1965).
  • the morpholino sequences for dystrophin were MO: 5 '-TTGAGTCCTTTAATCCTACAATTTT-3 ' (SEQ ID NO: 1); 6 ng morpholinos were injected into the yolk of one-to two-cell stage embryos. At 1 dpf, 20 injected embryos were arrayed in a 24-well plate and were treated with individual chemical. Histology and Immunohisto chemistry .
  • Embryos were incubated separately with anti-dystrophin (1:25; Sigma) or anti-laminin (1:25; Sigma) antibodies at 4 °C overnight.
  • fish were frozen in a cold acetone. Fish muscle samples were sectioned with a cryostat (HM505E; Microm) at a 10- ⁇ thickness. Fish muscle sections were incubated with anti- dystrophin (1:25; Sigma), anti-myosin heavy chain (1:25, F59; Hybridoma Bank), or anti- laminin (1:25; Sigma) antibodies at 4 °C overnight. After washing three times, sections were incubated with secondary antibodies and examined. Frozen section were obtained as for immunohistochemistry and stained with H&E. For H&E staining of frozen muscle sections, sections were stained with Mayer's hematoxylin solution (Fluka) for 10 min and 1% eosin solution (Sigma) for 30 s after washing with water.
  • Mayer's hematoxylin solution Fl
  • Pairs of heterozygous sapje fish were mated, and fertilized eggs were cultured at 28.5 °C.
  • Zebrafish embryos were collected and raised at 28.5 °C according to standard procedures and criteria.
  • mutant fish showing abnormal birefringence were identified under the dissection scope at 4 dpf and placed in a new plate to be treated with candidate chemicals 1-7 from 4 to 30 dpf; 10 affected and 10 unaffected embryos were arrayed in 24-well plates and cultured in 1 mL fish water containing individual chemicals at 28.5 °C starting at 4 dpf.
  • treated fish were cultured at room temperature in 100 mL fish water containing individual chemicals (2.5 ⁇ g/ mL) until 30 dpf at room temperature. The number of surviving fish wawas counted and recorded every other day, and at 30 dpf, their genotypes were determined.
  • Genotyping sapje and sapje-Like Fish Genomic DNA extracted from chemically treated fish was used as the PCR template.
  • primer sets for genotyping the specific mutations in the dystrophin gene of sapje or sapje- like fish (for sapje: forward primer 5 '-CTGGTTAC ATTCTGAGAGACTTTC-3 ' (SEQ ID NO: 2) and reverse primer 5 'AGCCAGCTGAACCAATTAACTCAC-3 ' (SEQ ID NO: 3);for sapje- like: forward primer 5 '-TCTGAGTCAGCTGACCACAGCC-3 ' (SEQ ID NO: 4) and reverse primer 5 '- ATGTGCCTGACATCAAC ATGTGG -3' (SEQ ID NO: 5)), PCR was performed at 52 °C with 35 cycles.
  • Embryos or fish were homogenized in Tris-buffered saline (TBS) containing 4 M Urea, 2% SDS, 2% CHAPS, protease inhibitors, and phosphatase inhibitors (Roche). Proteins were analyzed with Western blot. Blotted proteins were incubated with primary antibody, anti-PKA C (1: 100; Cell Signaling Technology), anti-phospho-PKA C (Thrl97, 1: 100; Cell Signaling Technology), or anti-P-actin (1:500; Sigma). PKA Assay. To assay the activity of PKA, the PepTag Assay for Non-Radioactive Detection of cAMP-Dependent Protein Kinase (Promega) was used following the manufacturer's protocol.
  • the six chemical pools (total of 48 chemicals) that had less than 7.5% of fish showing abnormal birefringence were selected as therapeutic candidates for restoring normal muscle function and were tested further in a secondary screening.
  • the 256 chemicals comprising the 32 pools that caused death of all treated embryos were rescreened individually. However, there was no chemical exhibiting less than 7.5% of mutant fish with abnormal birefringence.
  • an antiinflammatory agent (chemical 1, epirizole), antiallergic agents (chemicals 2 and 3, homochlorcyclizine-dihydrochloride and conessine), a phosphodiesterase (PDE) inhibitor (chemical 4, aminophylline), an estradiol steroid (chemical 5, equilin), a chelating agent (chemical 6, pentetic acid), and a cardiotonic glycoside (chemical 7, proscillaridin A).
  • genotypes were determined using the genomic DNA extracted from heads of the individual fish (40 treated fish with each chemical).
  • the resulting genotype percentages were 30% WT, 45% heterozygous, and 25% homozygous for dystrophin deficiency.
  • All unaffected fish as detected by the birefringence assay were confirmed to be WT or heterozygous fish by genotyping.
  • some homozygous dystrophinnull fish had normal birefringence and thus, were considered phenotypically unaffected.
  • dystrophin was examined by immuno staining individual fish bodies with anti-dystrophin antibodies. WT fish showed positive staining of dystrophin in the myosepta. However, the homozygous dystrophin-null fish that showed no abnormality of birefringence showed no immunoreactivity with anti-dystrophin, and the same lack of staining was found in the nontreated dystrophin-null fish. Those treated dystrophin-null fish with normal birefringence did not restore dystrophin expression.
  • affected fish selected at 4 dpf by birefringence assay
  • the fish treated with chemical 4 were able to survive longer compared with the nontreated dystrophin-null fish (Fig. 3).
  • Fish treated with chemicals 1 or 6 were able to survive for 30 d (Fig. 3).
  • Other chemicals tested had no effect on the survival of affected fish (Fig. 3), and indeed, some were toxic to all fish.
  • the motility of surviving affected fish was not relatively altered compared with that of WT.
  • Aminophylline is known to be a nonselective PDE inhibitor that increases the levels of intercellular cAMP, causing activation of cAMP-dependant PKA.
  • the expression, phosphorylation, and activation of PKA in dystrophin-null fish treated with aminophylline for 25 d were examined.
  • the expression of PKA was detected in all samples. More phosphorylated PKA was detected in aminophylline-treated fish compared with WT and untreated dystrophin- null fish (Fig. 4 C and D, P ⁇ 0.05).
  • the activity of PKA in aminophylline-treated fish was significantly increased compared with WT and untreated dystrophin-null fish (P ⁇ 0.05).
  • Aminophylline is a nonselective PDE inhibitor, and it is among a group of PDE inhibitors with different specificities.
  • PDE inhibitors To test whether other PDE inhibitors might also ameliorate dystrophic symptoms in sapje fish, 20 embryos from matings of heterozygous sapje fish were treated with a series of PDE inhibitors from 1 to 4 dpf (in triplicate). At 4 dpf, the percentage of affected fish was examined by birefringence assay. The results are plotted (Fig.
  • enoximone PDE3 inhibitor
  • milrinone PDE3 inhibitor
  • ibudilast PDE4 inhibitor
  • rolipram PDE4 inhibitor
  • sildenafil citrate salt PDE5 inhibitor
  • dipyridamole PDE5 inhibitor
  • aminophylline nonselective PDE inhibitor
  • DMSO DMSO
  • Each of the seven chemicals increased the percentage of fish with normal birefringence. Genotyping of dystrophin mutations in these treated fish indicated that dystrophin-null fish were among those with normal birefringernce. This suggests that these seven chemicals can prevent the onset of abnormal muscle structure in these dystrophin-null fish and result in apparently normal fish with a dystrophin mutation.
  • the immunohistochemical results of the dystrophin expression showed that the chemical treatment did not restore dystrophin expression.
  • Recent reports propose that some chemicals may cause exon skipping in mutant dystrophin cDNA and recover the expression of truncated dystrophin.
  • the seven chemicals examined here did not restore dystrophin expression, which suggests that expression of other proteins might be influenced by treatment with these seven chemicals and that they may be the potential therapeutic pathways. These pathways may also have additional compounds that influence their expression.
  • PDE inhibitors cause an increase in intracellular cAMP and/or cGMP.
  • PKA pathway One of pathways up-regulated by increasing the amount of cAMP is the PKA pathway (Willoughby, et al. EMBO J. 25:2051-2061 (2006)). The results show that the activity of PKA is clearly up- regulated in aminophylline-treated dystrophin-null fish.
  • PKA has some interesting target proteins for supporting muscle structure, such as cAMP response element-binding (CREB), and
  • skeletal muscle Ca channels Up-regulation of expression of these target proteins by PKA activation might modulate the progression of phenotypes in skeletal muscle.
  • the Prestwick Library 1 a commercially available collection of 1120 compounds, was screened initially. By pooling compounds 6 pools of 8 compounds each of which substantially decreased the number of zebrafish from sapje heterozygous matings showing the abnormal birefringence phenotype at 4 dpf. The pools were broken into individual compounds and again used to screen for those which would decrease the number of affected zebrafish from these matings. This resulted in seven compounds which each decreased the number of zebrafish showing the abnormal birefringence 4 dpf. Each fish in the treated groups was sequenced to confirm that some of the improved zebrafish were indeed dystrophin deficient. This was further confirmed on immunofluorescent assay of dystrophin protein expression.
  • affected fish with the abnormal birefringence phenotype at 4 dpf were isolated and treated only affected fish with these chemicals.
  • Ten affected 4 dpf zebrafish were treated for 26 days with each of the seven compounds in triplicate. Three of the compounds were found to be toxic in longer term treatment of older zebrafish. One compound seemed to have little effect on dystrophin null fish, while two increased the survival of dystrophin deficient zebrafish. The most impressive survival improvement was with chemical #4, aminophylline, the non-selective PDE inhibitor.
  • Example 2 A screen of additional chemical libraries
  • Prestwick Library 1 demonstrated the feasibility and utility of dystrophic zebrafish to identify new therapeutic approaches to DMD. Tis approach was extended by screening two additional chemical libraries, NINDS2 (1040 chemicals) and ICCB Bioactive molecule (480 chemicals). Seven more compounds which decreased the number of fish showing abnormal birefringence were identified. This yielded a total of 14 chemicals from the three screens that showed some potential to restore normal muscle structure (Table 1).
  • PDE inhibitors such as aminophylline can activate PKA via increasing cAMP.
  • PKA can induce various pathways including eNOS and increasing cGMP.
  • drugs for vasodilation like tadalafil and sildenafil, PDE5 inhibitors were effective in restoring muscular function in dystrophic models (Adamo et al., Proc Natl Acad Sci U S A. 2010 Nov 2; 107(44): 19079-83).
  • PKA-heme oxygenase 1 (HMOX1) related pathway indirectly induced vasodilation and inhibition of nuclear factor kappa-light-chain- enhancer of activated B cells (NF-kappa B), an immunoactivator.
  • HMOX1 PKA-heme oxygenase 1
  • NF-kappa B is up- regulated in DMD patients, the inhibition of this pathway may be one of the best potential candidate pathways for DMD therapy.
  • results of the long-term culture of dystrophin null fish has demonstrated that aminophylline, sildenafil, crassin acetate and cerulenin had a substantial influence in the number mutant fish which survive without dystrophin. Analysis of these surviving fish indicated that expression of HMOX1 was up-regulated by treatment with these compounds, which suggests that treatment may induce activation of PKA and HMOX1 expression (Fig. 1).
  • Example 4 Over expression ofHMOXl influences muscle phenotype Injection of HMOX1 mRNA into 1-2 cell stage eggs from the matings of sapje heterozygous fish pairs significantly reduced the number of affected fish compared to those of non-injected sapje fish. Overexpression from the HMOX1 construct was confirmed with anti- myc antibody in the extract of the mRNA injected fish. Immunostaining of wild type, non- treated fish and HMOX mRNA injected fish with anti-myosin heavy chain and anti-dystrophin antibody at 4 dpf showed that non-treated dystrophin-null fish had broken and disturbed structure of skeletal muscle fibers and those dystrophin-null fish which expressed increased HMOX1 had normal skeletal muscle structure. These finding demonstrate that the HMOX overexpression induced the reduction of muscle phenotype in affected fish and modulation of this molecule should have potential for DMD therapy.
  • One advantage of a zebrafish model is that it is possible to quickly produce large numbers of mutant offspring that can then be assayed in multiwell plates and treated with different chemicals to determine if disease progression is modulated.
  • Chemical compounds of relatively small molecular weight can bind to specific proteins and alter their function.
  • “dystrophic” mutants exhibit normal birefringence under polarized light at 2 days post- fertilization (dpf) but decreased birefringence and motility defects in response to touch at 4 dpf. Birefringence measures the rotation of polarized light through the transparent zebrafish embryo at the highly ordered sarcomeric structure of the somitic muscle. While these "dystrophic" mutants all show similar phenotypes, sapje is the only one that carries a mutation of the dystrophin gene.
  • a genetic screen of ENU mutagenized zebrafish identified a second allele of dystrophin deficiency in zebrafish (sapjeAUke with a point mutation in a splice site adjacent to exon 62 which results in a deletion of exon 62 from the mature transcript and a switch in reading frame resulting in a premature stop.
  • Both alleles of dystrophin deficiency are available in the laboratory and both have been documented to not produce dystrophin and have identical phenotypes of markedly reduced mobility and characteristic structural defects manifest as a birefringence phenotype at 4-5 dpf and markedly decreased survival beyond 9 days post fertilization.
  • zebrafish Because of their optical transparency, zebrafish have been used extensively to identify genes critical for development of embryonic structures including muscle. Zebrafish muscle is separated into slow (located just beneath the skin) and fast muscle (located within the main body of the fish). The zebrafish dystrophin protein localizes to the muscle cell membrane in adult fish and the dystrophin gene has been positioned on Linkage Group (LG) 1. In situ hybridization experiments have shown that dystrophin is expressed early in development predominantly at the borders of the muscle somites. Since zebrafish muscle cells stretch across the entire muscle somite, it is reasonable that proteins such as dystrophin involved in connecting the cell membrane with the internal cytoskeleton would be expressed near the myoseptas, the location of the highest amount of mechanical stress.
  • LG Linkage Group
  • zebrafish express dystrophin as part of a large complex (dystrophin- associated protein complex; DAPC) whose stability is dependent on dystrophin expression.
  • DAPC distrophin- associated protein complex
  • Morpholinos, or antisense RNA directed against the translational initiation site of dystrophin block dystrophin expression and lead to embryos that are only 20% as active when compared to embryos injected with control morpholinos, suggesting that the DAPC proteins function similarly in zebrafish and that misexpression of these proteins can result in a muscle specific phenotype that can be scored early in zebrafish development.
  • Zebrafish represent a good model to investigate genes involved in muscle development and degeneration, including human muscular dystrophy.
  • the orthoglogs of many dystrophin- glycoprotine complex (DGC) components are expressed in zebrafish.
  • DGC dystrophin- glycoprotine complex
  • Two fish models of DMD with dystrophin deficiency, sapje and sapje-like, have a muscle degeneration phenotype transmitted in a recessive manner, such that 25% of the offspring show dystrophic features of skeletal muscle after 3 days post-fertilization (dpf).
  • This disorder results in a muscle pathology that can be detected by birefringence under polarizing light and usually results in death by 7-9 dpf.
  • the muscle degeneration phenotype in the dystrophin-null sapje and sapje-Yke, zebrafish is transmitted in a recessive manner such that 25 percent of the offspring show dystrophic symptoms after 4 dpf, as assayed by birefringence and motility.
  • By crossing many fish it is possible to quickly produce large numbers of mutant offspring that can then be arrayed on plates and exposed to different chemicals in their water. This allows robust screens of chemicals that diffuse into the dystrophic mutant and mitigate the symptoms of disease. These are useful for screening for candidate chemicals that restore normal muscle structure.
  • the compounds identified impact a number of different pathways, including the recently reported pathway that is modulated by sildenafil (Viagra) (Seimiya, et al. Eur. J. Immunol. 34, 1322-1332 (2004)), and each has the potential to ameliorate the symptoms of muscular dystrophy.
  • Zebrafish with absent dystrophin exhibit severe skeletal muscle pathology within a few days of fertilization and most die within 7 dpf.
  • the zebrafish is used as a whole animal model of dystrophin deficiency to screen libraries of small molecules for those that might help skeletal muscle compensate for absent dystrophin.
  • This screening activity has highlighted a pivotal pathway converging on increased levels of heme oxygenase I (HMOX1) as means for extending the life of the zebrafish model.
  • HMOX1 heme oxygenase I
  • Both a focused chemogenetics and genetics approach is used to interrogate the dystrophin-deficient zebrafish.
  • the selected compounds are used to further refine the targets, (e.g. selective inhibition of PDE5 vs PDE6).
  • the compounds are initially screened in the 1-4-dpf model, with promising compounds then evaluated in the 30-day survival assay.
  • the pivotal pathway is then genetically manipulated by up and down regulation of components of the pathway starting with HMOX 1 to identify compounds modulating the HMOX1 pathway to restore muscle structure and improve survival of the dystrophin-deficient zebrafish.
  • preliminary screens have identified 14 compounds that increase the survival rate of dystrophin deficient fish from all but 10% surviving to more than 75% of affected fish surviving.
  • PDE inhibitors as well as five other compounds that influence blood flow to skeletal muscle improve outcome measures in affected fish.
  • PDE5 inhibitors like tadalafil and sildenafil have been shown to be effective in restoring normal muscular structure to mouse models of DMD (Asai, A., et al., PLoS One, 2007. 2(8): p. e806; Adamo, CM., et al., Proc Natl Acad Sci U S A, 2010. 107(44): p. 19079-83)
  • Zebrafish mutants with muscular dystrophy are characterized using changes in birefringence to score changes in muscle integrity during the first 9 days of development.
  • birefringence can be monitored as frequently as necessary without causing any harm to the animal so that the effects of the drug can be monitored at numerous times following drug administration. It is possible to manually screen a 48-well plate using birefringence in a matter of minutes scoring the number of fish that exhibit abnormal birefringence and whether there are less affected fish than those expected.
  • zebrafish offer numerous advantages for performing chemical screens including, for example, its permeability to drugs (the drugs can be administered by simply adding them to the water), and the fact that an entire organism is used during the screen (so that any positive effects on muscle can be measured in the context of complete living model organism).
  • the dystrophic mutants show a muscle birefringence phenotype between 3 and 7 dpf, and in some aspects this phenotype is a direct measure of the degeneration of their skeletal muscle.
  • Birefringence is an efficient assay requiring that the animals simply be positioned between two polarizing filters oriented 90 degrees with respect to each other.
  • the chemicals are added to the water.
  • as a normal control e.g., twenty embryos are cultured without chemicals in every 10 wells in duplicate. All plates containing embryos are incubated at e.g., 28.5 °C for 96 hrs. At 4 dpf, the birefringence of all fish is tested using a dissecting microscope.
  • the first assay tests each compound in triplicate assays over 4 different dosage ranges in 48 well plates for 0 to 4 days post-fertilization of the zebrafish. The goal is to find the dose and compound which changes the ratio of affected fish to substantially less than 25%.
  • 4 different compounds can be tested simultaneously and 5 plates can be processed in parallel for a dosage assay of 20 different chemicals. Motility detection assays automated for high through put screening can also be used.
  • administering drugs this early in development has other advantages including that the fish are small enough to put many (-20) in a well of a 48 well plate and the fish are still permeable making it easy to test the chemicals by adding them to the fish water.
  • optimized efficacious compounds can be identified.
  • focused chemical libraries can be used to explore modulation of specific biological mechanisms using the phenotypic zebrafish screen, described herein.
  • cheminformatics and in-silico predictive models are used to increase the efficiency of the experimental approaches. Additional information such as compound - target interactions, target - mechanism of action/pathway relationships, and target - disease
  • associations are mined from publically available external databases.
  • the combination of experimental and predicted compound-target pharmacological profiles are used to prioritize compounds for additional screening and to provide evidence for proposed mechanisms of action.
  • these profiles can be used to retrieve similar compounds for additional testing.
  • Chemogenomics library represents an additional opportunity to identify a biological target.
  • Chemogenomics sets consist of -5,000 compounds covering > 1,000 targets.
  • Compounds screening set is created based on single targets or clustered biology space. These compound sets (10-20 compounds) provide an additional set of tools to confirm the biology space identified by their Chemogenomics screening hits.
  • the selected compounds are used to further refine the targets (e.g.
  • the compounds are screened in the 4-day model, with any promising compounds to then be evaluated in the 30-day survival assay.
  • the pivotal pathway will also be genetically manipulated by up and down regulation of components of the pathway starting with HMOX 1 itself.
  • chemicals identified in addition to those already known to target the pathway lead to additional compounds related in the targets or activity of the known compounds and these can be identified by informatics tools.
  • a significant portion of the screen will be a pathway enriched screen. Screening with compounds of known biological mechanism-of-action reduces transition time from the primary stage to a more focused screen based on improved selectivity and chemical properties.
  • two strategies are employed for compound selection.
  • the first strategy is based on the identification of alternative targets from the bioinformatics screening. Compounds are selected based on their selectivity profile, as well as chemical properties.
  • the second strategy selects compounds following screening of compounds from focused chemical libraries, such as the chemogenomics set. This provides a library of up to 5000 compounds that covers -1000 biological targets for a full phenotypic screen. In combination with the bioinformatics results, appropriate compounds are used for screening in the zebrafish.
  • chemoinformatics and in-silico models are employed to examine data from previous screens conducted, as described above and herein. All of the compound efficacy data from previous screens are mapped to targets and those targets are used for a pathway-enrichment analysis. Component genes from pathways containing a significantly enriched number of screening hits are then used to query a drug library. Compounds that target genes from expanded pathways are then selected for follow-up analysis in the zebrafish model. In some aspects then, the combination of experimental and predicted compound-target pharmacological profiles are used to prioritize compounds for additional screening and to provide evidence for proposed mechanisms of action. In addition, these profiles can be used to retrieve similar compounds for additional testing. As noted, there are a significant number of predictive models available to generate predicted profiles.
  • Example 7 Long-term effects of candidate chemicals in zebrafish model
  • an assay is provided herein which involves exposing affected mutant zebrafish to compounds from day 4 dpf to day 30 dpf. Ratios of affected to unaffected fish are compared to those that were not treated. Once a dose is determined that prevents the onset of abnormal birefringence during development as described above, a second assay is initiated.
  • birefringence are transferred to separate 48 well plates in triplicate and treated continuously with the selected compound until 30 days post fertilization.
  • the fish being treated are observed e.g., every other day, and surviving fish counted. Those fish that survive are tail clipped for a small piece of tissue for DNA isolation and genotyping for dystrophin mutation.
  • fewer compounds are compared in parallel.
  • a conservative estimate will be 4 drugs in one set of parallel experiments.
  • these are staggered to double this number with only 50% increase in experimental time.
  • outcome measures on the surviving fish include muscle immunohistochemistry for both dystrophin expression and for general restoration of skeletal muscle integrity.
  • changes in vascular structure and function may be investigated. Further, options to monitor longitudinal activity of the zebrafish are considered.
  • DNA is extracted from the fish head or from tail clips by protease K digestion.
  • the mutation bearing exon is PCR amplified and the DNA sequenced to ascertain which fish were dystrophic mutants and those that are wild type.
  • the dys -/- mutants and the wild type siblings are sectioned and immunostained with antibodies against dystrophin (to determine whether dystrophin expression has been restored) and sarcomeric actin (to monitor the integrity of the muscle fiber).
  • An effective antibody against zebrafish sarcomeric actin is commercially available through Sigma.
  • surviving fish treated with candidate chemicals and non-treated fish after culture are processed into total RNA which is then converted to cDNA.
  • the cDNA is applied to cDNA expression analysis using zebrafish Affymetrix gene array (15600 genes on the array). The results indicate differences in expression among normal fish, non-treated affected fish and chemical treated fish. This analysis reveals pathways of gene expression affected by these candidate chemicals and the mechanism of chemical action at the gene expression level. Protein analysis in western blot and immunohistochemistry
  • methods to analyze the specific pathways affected by candidate chemicals or compounds are provided.
  • zebrafish cultured in various chemicals are harvested and total protein extracts prepared.
  • Western blot analysis with anti- dystrophin-glycoprotein-complex (anti-dystrophin, ⁇ -, ⁇ -dystroglycan, actin), anti-laminin, anti- HMOX1, or other pathway proteins are used to determine levels of such proteins.
  • the skeletal muscle sections of treated fish is examined.
  • the treated fish are fixed and sectioned at 10-20mm thickness.
  • the sections are immunostained with anti-dystrophin glycoprotein complex (anti-dystrophin, ⁇ -, ⁇ - dystroglycan, actin) antibodies, anti-laminin, and with other antibodies against proteins in the pathways (e.g. Anti-HMOXl or PKA) that might have been revealed in the protein western blots.
  • anti-dystrophin glycoprotein complex anti-dystrophin, ⁇ -, ⁇ - dystroglycan, actin
  • other antibodies against proteins in the pathways e.g. Anti-HMOXl or PKA
  • candidate chemicals are be mixed with one another at various different combinations and placed in the culture of sapje or sapje-like fish. The outcomes of survival and birefringence are compared to each of the compounds used separately.
  • the lead compounds from the zebrafish screens are tested in mouse models of dystrophin deficiency to determine efficacy in a mammalian model of human DMD. In some aspects, this involves outcome measurements such as biochemical analysis of the targeted pathways, improved histology, functional improvement measurements and exercise damage improvement.
  • Hmoxl open reading frame ORF
  • pCMV-3Tag-4 Agilent Technologies
  • the 3 myc tags allow for easy determination of endogenous Hmoxl from the transgene-derived Hmoxl and facilitates future co-immunoprecipitation (CoIP) experiments with recombinant Hmoxl and muscle proteins.
  • the Hmoxl -3x-myc ORF is then subcloned by PCR into the pCAGEN plasmid (Addgene) in which gene expression is driven by the ubiquitous pCAGGS (CMV enhancer with ⁇ -actin promoter) element for subsequent ubiquitous tissue expression.
  • pCAGEN plasmid Additional DNA sequence
  • the linearized transgene plasmid is pro-nuclear injected into 2-cell C57B16/J (Jackson Laboratories) embryos and later implanted into pseduopregnant females.
  • Hmoxl-Tg mice Following the identification of at least 3 founder lines, the F generation of Hmoxl-Tg mice will be mated with homozygous mdx5cv females (same strain) to generate transgenic Hmoxl mice on the mdx5cv background.
  • methods are provided for determining the efficacy of a candidate drug or compound for DMD treatment in a mouse model. In one example, this involves outcomes measurements such as biochemical analysis of the targeted pathways, improved histology, functional improvement measurements and exercise damage improvement.
  • the purpose of performing the chemical screen in zebrafish is to have a high throughput method to identify new drugs with the potential of treating muscular dystrophy in humans.
  • the effectiveness of candidate drugs isolated from the zebrafish screen are evaluated in mammalian models of muscular dystrophy. There is evidence that drugs in fish and mammals will work similarly. For example, the Zon laboratory has identified dmPGE2 as a drug that stimulates stem cells, which was discovered using a zebrafish chemical screen
  • mice show that the drug works in a similar manner, and the drug is now in human clinical trials.
  • the top of the pathway has already been implicated using mouse models of dystrophin deficiency.
  • the relevance of this pathway by overexpression of a number of pathway components such as HMOX1 in the mdx5cv mouse.
  • the most commonly used mammalian model is the mdx mouse with the mdx 5cv allele showing a more severe phenotype with less revertant fibers thereby phenocopying the human disease better than the mdx allele.
  • isolated drugs from the zebrafish chemical screen are be given e.g., orally or injected
  • IP intraperitoneal
  • mdx 5cv dystrophic mouse host
  • these time points will be young mice at a stage where they undergo major muscle degeneration e.g., at 4-6 weeks after birth, at 6 months, and at 1 year where there is more evidence of muscle damage.
  • the administration of these drugs begin at the time skeletal muscle degeneration starts to peak, e.g., at 3 weeks of age, and is continued for 1 year with assay time points just after delivery, at 6 months and at 1 year.
  • biochemical markers of muscle damage as well as behavioral and histological parameters are compared to mice receiving just the vehicle (placebo).
  • Toxicity tests are performed in mice by administering various amounts of the chemical effective at treating dystrophin null fish. Since the effective dose curve is likely to be specific for each drug, the 1 mg drug/kg body weight standard provides a starting point for analyzing doses. To test dosages, logarithmic differences are injected intraperitoneally into adult mice to quickly determine drug toxicity such that 100 fold less, 10 fold less, 1 mg drug/kg body weight, 10 fold more, and 100 fold more drug is used. Toxicity is measured by monitoring the overall health of the mouse by assaying heart rate, respiration, hydration, and body temperature. In one example, the highest dose in which no negative side effects are apparent is selected. Initially, different doses are used to determine toxicity.
  • the initial injections are given via i.p. route, performed daily for 4 weeks but alternative delivery such as oral may also be tested.
  • the following outcome indices are then monitored:
  • mice are weighed daily in the first month of treatment and then weekly to determine body mass (g). This measurement will be used to determine the proper dose of the injections as well as to monitor the health and changes in weight that may result from treatment between groups.
  • mice are placed in a cage warmed using a heat lamp and a plate of glass to achieve vasodilatation. After one minute, the mice are removed and placed on the wire lid of another cage. A shallow cut on the ventral side of the tail using a sterile scalpel or single edge blade is made.
  • CK creatine kinase
  • CK activity is measured spectrophotometrically using a commercial kit (Stanbio Lab) based on the Szasz modification of Rosalki technique, which optimizes the reaction by reactivation of CK activity with N-acetyl-L-cysteine and follows NADH production through the absorbance increase per minute at 340 nm. The resulting data (change in absorption/min) is then be translated into U/L of CK.
  • Measuring locomotor activity is a non-invasive way to monitor the functional capability of muscle in dystrophic versus wild-type mice.
  • the TSE InfraMot System which uses infrared scanners to detect the movement of mice in the x, y, and z planes by monitoring body heat, is used.
  • One of the advantages of this system is that it reduces anxiety-related movements by allowing the mice to be recorded in their home cage. Horizontal and vertical activity as well as total distance and rest time, parameters that have been validated as useful for pre-clinical assessment of drugs in the mdx mouse, is used.
  • mice Functional muscular differences are detected using a grip strength test.
  • a mouse is placed on top of the cage lid, which is then shaken three times to cause the mouse to grip the lid.
  • the lid is then inverted upside-down and the mouse is timed to determine how long it can hold on upside down within a two-minute period.
  • the edges of a standard cage lid are covered with tape to prevent the mouse from crawling on top of the cage.
  • the height of the lid is about 20 cm above a soft landing (either cage bedding, or towels).
  • the mice are tested three times on different days weekly, beginning at three weeks of age to provide habituation, and
  • mice Skeletal muscle physiology of mdx5v mice that are treated with drug or placebo are quantified by a series of muscle endurance treadmill tests. Following an initial acclimation of the mice to the treadmill performed 1 week prior to the examination, mice are placed on the treadmill at a 20° incline and forced to run until exhaustion (mice fall off). These tests are performed in cohorts of 5, and at gradually increasing speeds. Previously, evaluation of the mdx mice have demonstrated that they show significant quantifiable deficits in their muscle endurance on the treadmill test with average falls occurring at 22 min, as compared to wild type littermates.
  • mice In the vertical pole test, a mouse is placed on a pole (2 cm in diameter) wrapped in tape to improve traction. The pole is slowly raised to a vertical position and the latency to fall is measured. Mice with muscle deficits will tend to fall off the pole earlier than wild-type cohort. Histological Analysis At the end of each treatment period, the mice are euthanized and the diaphragm, quadriceps, gastrocnemius and TA muscles are harvested and snap frozen in isopentane chilled in liquid nitrogen. H&E staining of muscle sections are used to visualize areas of necrosis and inflammation as well as the myofibers, their cross sectional area and the number and location of their nuclei.
  • a non-toxic dye, Evan's Blue, is injected (1% dye in a 1% volume relative to body mass) 16-24 hours before a mouse is sacrificed.
  • the dye is absorbed by leaky cell membranes often found in myopathies and dystrophic muscle, and thus is another good marker of muscle damage.
  • frozen muscles are sectioned and examined under fluorescence.
  • the Evan's Blue Dye autofluoresces red in positive myofibers and thus damage can be quantified.
  • muscle sections are stained with immunohistochemical markers for different proteins (i.e. dystrophin, utrophin, follistatin, Ki67, etc.) that are indicative of muscle function
  • Frozen muscle tissue is also subjected to lysis for protein or RNA extraction.
  • Western blots and RT-PCR studies for particular proteins/genes are performed to determine the involvement of suspected mechanisms based on the histological results and from in vitro studies.
  • Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context.
  • the disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
  • any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

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Abstract

L'invention concerne une voie qui peut être ciblée pour le traitement ou l'atténuation de symptômes de la maladie de Duchenne de Boulogne (DMD). Des composés ont été identifiés et testés dans un modèle de poisson zèbre de DMD et se sont avérés être efficaces sur la base d'une augmentation du taux de survie des poissons déficients en dystrophine. Plusieurs de ces composés sont dans la voie de l'inhibiteur de PDE. Des composants supplémentaires de la voie non inhibée par des inhibiteurs de PDE, mais actifs dans l'analyse de poisson zèbre, sont criblés pour leur inhibition de NFK-β et l'augmentation de l'hème oxygénase-1. Dans certains modes de réalisation, ces composés influencent le débit sanguin vers un muscle squelettique. Dans certains modes de réalisation, la voie combinée menant à l'inhibition de NFK-β et à l'augmentation de l'hème oxygénase-1 est importante dans l'identification de composés efficaces dans le traitement ou l'atténuation de symptômes de DMD.
PCT/US2013/025464 2012-02-10 2013-02-09 Inhibition d'une voie ciblée pour améliorer la structure, le fonctionnement et l'activité musculaires dans la dystrophie musculaire WO2013120040A1 (fr)

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