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WO2011146819A2 - METHODS AND COMPOSITIONS FOR TREATMENT NF-κB-MEDIATED AND α7 INTEGRIN-SUPPRESSED DISEASES - Google Patents

METHODS AND COMPOSITIONS FOR TREATMENT NF-κB-MEDIATED AND α7 INTEGRIN-SUPPRESSED DISEASES Download PDF

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Publication number
WO2011146819A2
WO2011146819A2 PCT/US2011/037308 US2011037308W WO2011146819A2 WO 2011146819 A2 WO2011146819 A2 WO 2011146819A2 US 2011037308 W US2011037308 W US 2011037308W WO 2011146819 A2 WO2011146819 A2 WO 2011146819A2
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Prior art keywords
agents
methyl
pair
pairs
kit
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PCT/US2011/037308
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French (fr)
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WO2011146819A3 (en
Inventor
Anne M. Whalen
Jane Staunton
Janine Steiger
Amy B. Wilson
Yang Wu
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Dmd Therapies, Llc
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Publication of WO2011146819A2 publication Critical patent/WO2011146819A2/en
Publication of WO2011146819A3 publication Critical patent/WO2011146819A3/en
Priority to IL223095A priority Critical patent/IL223095A0/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the treatment of diseases mediated by NF- ⁇ or suppressed by a 7 integrin.
  • Nuclear factor kappa-B is a transcription factor of the immune system.
  • Cell stimulation by a variety of extracellular signals such as oxidative stress, cytokines, and lipopolysaccharide induces a network of events that lead to NF- ⁇ activation.
  • Activation of an NF-KB molecule is typically accompanied by its translocation from the cytoplasm into the nucleus.
  • NF-KB activation may be triggered by degradation of ⁇ - ⁇ , which normally sequesters NF- ⁇ in the cytoplasm, or by other less characterized mechanisms.
  • activated NF- ⁇ binds to DNA elements in the promoters of a number of proinflammatory gene families.
  • NF- ⁇ defends against damaging agents such as microbes and free radicals by activating the inflammatory response; however, activation of NF- ⁇ is also associated with a number of disease states promoted by inflammation, including cancer, autoimmune diseases, and neurodegenerative diseases.
  • integrin alpha chain 7 ⁇ al integrin is a constituent of a group of heterodimeric integral membrane proteins collectively called integrins. Highly expressed in muscle cells, a 7 integrin is processed to yield light and heavy chains that join with ⁇ ⁇ integrin to form ⁇ 7 ⁇ 1 integrin, which binds the extracellular matrix protein laminin- 1 and laminin-2. al integrin appears not only to aid appropriate anchorage of muscular tissues, but also to promote differentiation in diverse cell types (e.g., in melanocytes) and to suppress malignant potential when ectopically expressed.
  • the present invention features methods, compositions, and kits for the treatment of NF-KB-mediated diseases and diseases suppressed by a7 integrin.
  • the invention features a method of treating a patient having or at risk for an NF-KB-mediated disease.
  • the method includes administering to the patient a pair of agents selected from the pairs of Table 1, or analogs thereof, in amounts that are effective to treat the patient.
  • NF-KB-mediated diseases include, e.g., inflammatory myopathies and cachexia.
  • the invention features a method of treating a patient having or at risk for a disease suppressed by al integrin. This method includes administering to the patient a pair of agents selected from the pairs of Table 2, or analogs thereof, in amounts that are effective to treat the patient.
  • ct7 integrin Diseases suppressed by ct7 integrin include cancer, vasculoproliferative diseases, and atherosclerosis.
  • the agents of a pair may be administered within 28 days, 21 days, 14 days, 10 days, 7 days, 3 days, 2 days, 24 hours, 12 hours, six hours, two hours, or one hour of each other, or substantially simultaneously. Agents may be
  • the methods of the invention may be performed in conjunction with the administration of additional agents appropriate for the NF-KB-mediated disease or the disease suppressed by al integrin.
  • the patient to be treated may be any animal (e.g., a human).
  • the invention features a composition that includes a pair of agents selected from the pairs of agents of Table 1 or Table 2, or analogs of these agents.
  • the composition optionally contains excipients, while the only active agents in the composition are the pair of agents of the invention (e.g., a pair of agents of Table 1 or Table 2).
  • compositions of the invention are present in amounts that, when administered together to a patient having or at risk for an NF-KB- mediated disease or a disease suppressed by al integrin, are effective to treat the patient.
  • Compositions of the invention may be formulated, e.g., for oral, systemic, parenteral, topical (e.g., ophthalmic, dermatologic), intravenous, inhalational, or intramuscular administration.
  • the invention features a kit including a pair of active agents.
  • the kit contains a pair of agents selected from the pairs of agents of Table 1 or analogs thereof and includes instructions for administering the agents to a patient having or at risk for an NF-KB-mediated disease.
  • the kit contains a pair of agents selected from the pairs of agents of Table 2 or analogs thereof and includes
  • the pair of agents may be included together in a composition or may be formulated separately.
  • kits including a first agent of a pair of agents and instructions for administering the first agent together with the second agent of the pair to a patient having or at risk for a disease.
  • the pair is selected from the pairs of agents of Table 1 or analogs of these agents, and the disease is a disease mediated by NF-KB.
  • the pair is selected from the pairs of agents of Table 2 or analogs of these agents, and the disease is a disease suppressed by al integrin.
  • one or more active agents may be formulated, e.g., for oral, parenteral, systemic, topical, or inhalational administration.
  • compositions, methods, and kits of the invention an analog of any agent listed in Table 1 or Table 2 may be used instead of the agent listed in Table 1 or Table 2.
  • Compounds useful in the invention may also be isotopically labeled compounds.
  • Useful isotopes include hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, (e.g., 2 H, 3 H, 13 C, 14 C, 15 N, 18 0, 17 0, 31 P, 32 P, 35 S, 18 F, and 36 C1).
  • Isotopically-labeled compounds can be prepared by synthesizing a compound using a readily available isotopically-labeled reagent in place of a non-isotopically-labeled reagent.
  • agent is meant a compound, e.g., dipyridamole, or mixture of compounds, e.g., ergoloid mesylates, having a pharmacological activity.
  • agent a compound, e.g., dipyridamole, or mixture of compounds, e.g., ergoloid mesylates, having a pharmacological activity.
  • agent compound, e.g., dipyridamole
  • mixture of compounds e.g., ergoloid mesylates
  • NF- ⁇ comprises a family of dimeric transcription factors containing a class I NF-KB protein, e.g., a protein encoded by the NFKBl or NFKB2 gene in humans.
  • NF-KB-mediated disease any disease or adverse health condition whose pathogenesis is associated with activation of NF- ⁇ .
  • exemplary NF-KB-mediated diseases are provided herein.
  • a disease suppressed by ot7 integrin is meant any disease or adverse health condition whose symptoms or pathogenesis is reduced by expression of a7 integrin.
  • glucocorticoid is meant a synthetic or natural steroid hormone that binds the glucocorticoid receptor, preferably with selectivity over the mineralocorticoid receptor.
  • ENT inhibitor is meant an agent that inhibits the activity of an equilibrative nucleoside transporter, e.g., an equilibrative adenosine transporter, in vitro, in vivo, or both, by at least 5%, e.g., by 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95%.
  • PDE inhibitor an agent that inhibits a phosphodiesterase enzyme in vitro, in vivo, or both, by at least 5%, e.g., by 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95%.
  • a PDE inhibitor may be selective for a particular type of PDE, e.g., PDE type V (PDE5).
  • acetylcholinesterase inhibitor an agent that inhibits the activity of an acetylcholinesterase in vitro, in vivo, or both by at least 5%, e.g., by 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95%.
  • Rho kinase inhibitor an agent that inhibits that activity of Rho kinase in vitro, in vivo, or both by at least 5%, e.g., by 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95%.
  • In vitro kinase assays or cell-based bioassays e.g., using fluorescence microscopy, may be used to detect and measure the Rho kinase inhibitory activity of an agent.
  • mTOR inhibitor a compound that inhibits the activity of mTOR, also known as FK506 binding protein 12-rapamycin associated protein 1 (FRAP1), in vitro, in vivo, or both by at least 5%, e.g., by 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95%.
  • FRAP1 FK506 binding protein 12-rapamycin associated protein 1
  • a calcium channel blocker an agent that directly or indirectly inhibits an activity of a calcium channel, e.g., current frequency, by at least 5%, e.g., by 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95%.
  • Blocking (inhibitory) activity can be measured by methods known in the art.
  • bisphosphonate is meant one of a class of compounds that has two phosphate groups.
  • the generic chemical structure of bisphosphonates is described herein. Many bisphosphonates can prevent the loss of bone mass or lower blood calcium when
  • CoQIO analog an analog of coenzyme Q10, also known as
  • ubiquinone By a "corticosteroid” is meant a natural or synthetic steroid hormone that binds either glucocorticoid receptors, mineralocorticoid receptors, or both.
  • patient any animal, e.g., a human.
  • treating refers to administering a pharmaceutical composition for therapeutic purposes.
  • the therapeutic purpose may be, e.g., to prevent a disease or symptom in a person at risk for the disease or symptom, or to ameliorate or stabilize the condition of a person already suffering from the disease or symptom.
  • treating results in prevention of a disease or symptom, or amelioration of a disease or symptom by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • treatment is observed by a trained physician or other person skilled in the art as an appreciable or substantial relief of symptoms in a patient with an NF-KB-mediated disease or a disease suppressed by a7-integrin.
  • an effective amount is meant the amount of an agent, alone or in combination with another agent, required to treat a patient with an NF-KB-mediated disease or a disease suppressed by a7-integrin in a clinically relevant manner.
  • a sufficient amount of an active agent used to practice the present invention for therapeutic treatment of an NF-KB-mediated disease or a disease suppressed by a7-integrin varies depending upon the manner of administration, the age, body weight, and general health of the patient. Ultimately, the prescriber will decide the appropriate amount and dosage regimen.
  • the effective amount of an agent may less be than the effective amount if the agent were administered in a non-combinatorial (single-agent) therapy.
  • an effective amount may be an amount of an agent in a combination therapy of the invention that is safe and efficacious in the treatment of a patient having an NF-KB-mediated disease or a disease suppressed by a7-integrin over each agent alone as determined and approved by a regulatory authority (such as the U.S. Food and Drug Administration).
  • a treatment exhibits greater efficacy, or is less toxic, safer, more convenient, or less expensive than another treatment with which it is being compared. Efficacy may be measured by a skilled practitioner using any standard method that is appropriate for a given indication.
  • a low dosage is meant at least 5% less (e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) than the lowest standard recommended dosage of a particular compound formulated for a given route of administration for treatment of any human disease or condition.
  • a low dosage of an agent that treats an inflammatory myopathy and that is formulated for administration by intravenous injection will differ from a low dosage of the same agent formulated for oral administration.
  • the number of atoms of a particular type in a substituent group may be given as a range, e.g., an alkyl group containing from 1 to 4 carbon atoms or alkyl. Reference to such a range is intended to include specific references to groups having each of the integer number of atoms within the specified range.
  • an alkyl group from 1 to 4 carbon atoms includes each of Q, C 2 , C 3 , and C 4 .
  • a Q_ 12 heteroalkyl for example, includes from 1 to 12 carbon atoms in addition to one or more heteroatoms.
  • Other numbers of atoms and other types of atoms may be indicated in a similar manner.
  • the term "lower,” when referring to a particular substituent group, e.g., "lower alkyl” or “lower alkoxy,” generally refers to groups containing 5, 4, or fewer carbon atoms.
  • alkyl and the prefix “alk-” are inclusive of both straight chain and branched chain groups and of cyclic groups, i.e., cycloalkyl.
  • Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 12 ring carbon atoms, inclusive.
  • Exemplary cyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups.
  • Cj_4 alkyl is meant a branched or unbranched hydrocarbon group having from 1 to 4 carbon atoms.
  • a alkyl group may be substituted or unsubstituted.
  • substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
  • alkyls include, without limitation, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl, sec- butyl, tert-butyl, and cyclobutyl.
  • alkenyl is meant a branched or unbranched hydrocarbon group containing one or more double bonds and having from 2 to 4 carbon atoms.
  • a C 2 -4 alkenyl may optionally include monocyclic or polycyclic rings, in which each ring desirably has from three to six members.
  • the C 2 _4 alkenyl group may be substituted or unsubstituted.
  • substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
  • C 2 _4 alkenyls include, without limitation, vinyl, allyl, 2-cyclopropyl-l-ethenyl, 1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2 -methyl- 1-propenyl, and 2-methyl-2-propenyl.
  • C 2 _4 alkynyl is meant a branched or unbranched hydrocarbon group containing one or more triple bonds and having from 2 to 4 carbon atoms.
  • a C 2 _4 alkynyl may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has five or six members.
  • the C 2 _4 alkynyl group may be substituted or unsubstituted.
  • substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
  • C 2 _4 alkynyls include, without limitation, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl.
  • C 2 -6 heterocyclyl is meant a stable 5- to 7-membered monocyclic or 7- to 14- membered bicyclic heterocyclic ring which is saturated, partially unsaturated, or unsaturated (aromatic), and which consists of 2 to 6 carbon atoms and 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring.
  • the heterocyclyl group may be substituted or unsubstituted.
  • substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, ( ⁇ substituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
  • the nitrogen and sulfur heteroatoms may optionally be oxidized.
  • the heterocyclic ring may be covalently attached via any heteroatom or carbon atom which results in a stable structure, e.g., an imidazolinyl ring may be linked at either of the ring-carbon atom positions or at the nitrogen atom.
  • a nitrogen atom in the heterocycle may optionally be quatemized.
  • Heterocycles include, without limitation, lH-indazole, 2- pyrrolidonyl, 2H,6H-l,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl, 4-piperidonyl, 4aH- carbazole, 4H-quinolizinyl, 6H-l,2,5-thiadiazinyl, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl, carbazolyl, 4aH- carbazolyl, b-carbol
  • pyridothiazole pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-l,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4- thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl,
  • thienothiazolyl thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4- triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl.
  • Preferred 5 to 10 membered heterocycles include, but are not limited to, pyridinyl, pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, tetrazolyl, benzofuranyl, benzothiofuranyl, indolyl, benzimidazolyl, lH-indazolyl, oxazolidinyl, isoxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, quinolinyl, and isoquinolinyl.
  • Preferred 5 to 6 membered heterocycles include, without limitation, pyridinyl, pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl, piperazinyl, piperidinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, and tetrazolyl.
  • C6- 12 aryl is meant an aromatic group having a ring system comprised of carbon atoms with conjugated ⁇ electrons (e.g., phenyl).
  • the aryl group has from 6 to 12 carbon atoms.
  • Aryl groups may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has five or six members.
  • the aryl group may be substituted or unsubstituted.
  • substituents include alkyl, hydroxy, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, fluoroalkyl, carboxyl, hydroxyalkyl, carboxyalkyl, amino, aminoalkyl, monosubstituted amino, disubstituted amino, and quaternary amino groups.
  • C 7 _i4 alkaryl is meant an alkyl substituted by an aryl group (e.g., benzyl, phenethyl, or 3,4-dichlorophenethyl) having from 7 to 14 carbon atoms.
  • aryl group e.g., benzyl, phenethyl, or 3,4-dichlorophenethyl
  • C3_!o alkheterocyclyl is meant an alkyl substituted heterocyclic group having from 3 to 10 carbon atoms in addition to one or more heteroatoms (e.g., 3-furanylmethyl, 2- furanylmethyl, 3-tetrahydrofuranylmethyl, or 2-tetrahydrofuranylmethyl).
  • Ci_7 heteroalkyl is meant a branched or unbranched alkyl, alkenyl, or alkynyl group having from 1 to 7 carbon atoms in addition to 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, S, and P.
  • Heteroalkyls include, without limitation, tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides.
  • a heteroalkyl may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has three to six members.
  • the heteroalkyl group may be substituted or unsubstituted.
  • substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, hydroxyalkyl,
  • d_ 7 heteroalkyls include, without limitation, methoxymethyl and ethoxyethyl.
  • halide or halogen is meant bromine, chlorine, iodine, or fluorine.
  • fluoroalkyl is meant an alkyl group that is substituted with a fluorine atom.
  • perfluoroalkyl an alkyl group consisting of only carbon and fluorine atoms.
  • R is selected from Q_ 7 alkyl, C 2 _ 7 alkenyl, C 2 _ 7 alkynyl, C 2 _6
  • heterocyclyl C6_i 2 aryl, C 7 _i 4 alkaryl, C 3 _io alkheterocyclyl, or Q_ 7 heteroalkyl.
  • hydroxyalkyl is meant a chemical moiety with the formula -(R)-OH, wherein R is selected from Q_ 7 alkyl, C 2 _ 7 alkenyl, C 2 _ 7 alkynyl, heterocyclyl,
  • alkoxy is meant a chemical substituent of the formula -OR, wherein R is selected from Ci_ 7 alkyl, C 2 _ 7 alkenyl, C 2 _ 7 alkynyl, C 2 _6 heterocyclyl, C6_i 2 aryl, C 7 _ ]4 alkaryl, C 3 _ 10 alkheterocyclyl, or C]_ 7 heteroalkyl.
  • aryloxy is meant a chemical substituent of the formula -OR, wherein R is a C6-12 aryl group.
  • alkylthio is meant a chemical substituent of the formula -SR, wherein R is selected from Q_ 7 alkyl, C 2 _ 7 alkenyl, C 2 - 7 alkynyl, C 2 _6 heterocyclyl, C n aryl, C 7 _ ]4 alkaryl, C 3 _io alkheterocyclyl, or Q_ 7 heteroalkyl.
  • arylthio is meant a chemical substituent of the formula -SR, wherein R is a C ⁇ 3_i 2 aryl group.
  • R, R', R", and R'" are each independently an alkyl, alkenyl, alkynyl, or aryl group.
  • R may be an alkyl group linking the quaternary amino nitrogen atom, as a substituent, to another moiety.
  • the nitrogen atom, N is covalently attached to four carbon atoms of alkyl, heteroalkyl, heteroaryl, and/or aryl groups, resulting in a positive charge at the nitrogen atom.
  • the present invention provides compositions, methods, and kits useful in therapies for diseases treatable by suppression of NF- ⁇ activity (i.e., NF-KB-mediated diseases or by augmentation of integrin al expression (i.e., diseases suppressed by a7 expression).
  • diseases treatable by suppression of NF- ⁇ activity i.e., NF-KB-mediated diseases or by augmentation of integrin al expression (i.e., diseases suppressed by a7 expression.
  • Exemplary diseases amenable to treatment according to the methods of the invention are described below.
  • Treatment methods of the invention include administration of any pair of agents selected from the pairs of Table 1 or Table 2.
  • Compositions of the invention can include a combination including any pair of agents selected from the pairs of Table 1 or Table 2.
  • the invention may function by decreasing NF- ⁇ expression, phosphorylation, nuclear translocation, or transcriptional activity, by promoting the activity of one or more pathways opposing that of NF- ⁇ , or by any other mechanism that suppresses an activity of NF- ⁇ .
  • the invention may function by increasing ITGA7 gene expression via oc7 integrin promoter sequences or by any post-transcriptional mechanism (e.g., RNA stabilization).
  • the patient being treated is administered a combination of two agents listed in Table 1 within 7 days of each other in amounts that together are sufficient to treat the patient having an NF- ⁇ -mediated disease.
  • the patient being treated is administered a combination of two agents listed in Table 2 within 7 days of each other in amounts that together are sufficient to treat the patient having a disease suppressed by l integrin.
  • an effective amount of one or both of the agents is a low dosage relative to the effective amount of the agent when administered singly for treating the disease, or relative to the standard dosage of the agent for treating another disease.
  • NF- ⁇ Diseases mediated by NF- ⁇ (i.e., an NF-KB-mediated disease) include muscle- wasting disease such as an inflammatory myopathy or cachexia.
  • Inflammatory myopathies include without limitation dermatomyositis, polymyositis, inclusion body myositis, juvenile myositis, myopathic dropped head syndrome, infection-associated myopathies (e.g., myopathies caused by viral, bacterial, fungal, protozoal, or helminthic infection), focal myositis, nodular myositis eosinophilic myofasciitis, macrophagic myo fasciitis, ocular myositis, granulomatous myositis, and acute necrotizing myopathy, and myositis associated with connective tissue disease (e.g., polymyalgia rheumatica, rheumatoid arthritis, systemic sclerosis, and tendonitis), and my
  • muscle- wasting diseases that may be treated are muscle injury/trauma (e.g., injury-induced fibrosis and regeneration), sarcopenia, muscle atrophies associated with denervation or immobilization (e.g., myositis caused by
  • Cachexia may occur, e.g., in patients suffering from cancer (cancer cachexia), human
  • immunodeficiency virus and acquired immunodeficiency syndrome
  • congestive heart failure and chronic obstructive pulmonary disease.
  • al integrin has been associated with protection from several disease processes. For example, forced expression of al integrin in a7-null metastatic melanoma cancer cells suppresses cell migration, tumor growth and metastasis. Consistent with this observation, low levels of oc7 integrin are associated with clinical relapse of human
  • al integrin reverses the malignant potential of cancer cells.
  • Loss of al integrin has also been linked to hyperplasia of vascular smooth muscle cells and reduced cell survival resulting from atherosclerosis. Upregulation of al integrin is expected to improve these conditions. Accordingly, upregulation of al integrin expression by the methods of the invention is useful for treating, e.g., cancer, vasculoproliferative diseases (e.g., stenosis, restenosis, and neointimal hyperplasia), asthma, and atherosclerosis.
  • vasculoproliferative diseases e.g., stenosis, restenosis, and neointimal hyperplasia
  • cancers that may be treated using the methods of the invention include, for example, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., hepatocellular carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
  • choriocarcinoma seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
  • lung cancer e.g., small cell lung cancer, non-small cell lung cancer, pleuropulmonary blastoma, and lung carcinoid tumor
  • colorectal cancer ovarian cancer (e.g., ovarian adenocarcinoma), prostate cancer, gastric cancer, esophageal cancer, head and neck cancer, and thyroid cancer.
  • therapeutic agents may be employed. Certain agents and exemplary analogs are discussed in greater detail below. It is to be understood that an analog of any agent of Table 1 or Table 2 can be used instead of the agent of Table 1 or Table 2 in the methods, compositions, and kits of the invention.
  • the methods, compositions, and kits of the invention employ predisolone or deflazacort.
  • Analogs of prednisolone and deflazacort include their respective structural analogs and other glucocorticoids.
  • 2-(4-acetoxyphenyl)-2-chloro-N-methyl- ethylammonium chloride, a compound with glucocorticoid-like anti-inflammatory properties, is also considered herein to be an analog of prednisolone and deflazacort.
  • Prednisolone is described in U.S. Patent Nos. 2,837,464 and 3,134,718 and has the following structure:
  • Analogs of prednisolone include 5-keto-4,5-seco-3-ynes of the estrane, androstane, and pregnane described in U.S. Patent No. 3,835,160; the 17-benzoate of prednisolone described in U.S. Patent No. 3,857,941; compounds of formula I in U.S. Patent No.
  • Deflazacort is described in Belgian Patent No. 679,820, G. B. Patent No. 1,077,393, and U.S. Patent No. 3,436,389. Deflazacort has the structure:
  • glucocorticoids are dexamethasone, betamethasone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, triamcinolone hexacetonide,
  • methylprednisolone methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, clocortolone pivalate, flucinolone, dexamethasone 21 -acetate, betamethasone 17- valerate, isoflupredone, 9-fluorocortisone, 6-hydroxydexamethasone, dichlorisone, meclorisone, flupredidene, doxibetasol, halopredone, halometasone, clobetasone, diflucortolone, isoflupredone acetate, fluorohydroxyandrostenedione, beclomethasone, flumethasone, diflorasone, clobetasol, cortisone, paramethasone, clocortolone, pre
  • prednisolone metasulphobenzoate prednisolone terbutate, triamcinolone acetonide 21- palmitate, flurometholone, medrysone, loteprednol, fluazacort, betamethasone, prednisone, methylprednisolone, triamcinolone, hexacatonide, paramethasone acetate, diflorasone, fluocinolone and fluocinonide.
  • the compounds dipyridamole and dilazep are ENT inhibitors that may be used in the methods, compositions, and kits of the invention.
  • Analogs of dipyridamole and dilazep include other ENT inhibitors, certain calcium channel blockers (e.g., nimodipine, nifedipine, nicardipine, nitrendipine, and felodipine, isradipine, and nioldipine), and structural analogs of dipyridamole and dialazep, e.g., nitrobenzylthioinosine, R75231, S6-(4-nitrobenzyl)- mercaptopurine riboside (NBMPR), and cannabinoids (e.g., cannabidiol; see Carrier et al. (Proc. Nat. Acad. Sci. USA 103 (20):7895-7900 (2006)).
  • Dipyridamole is an ENT inhibitor described in G.B. Patent No. 807,826 and U.S. Patent No. 3,031,450. D
  • substituents Ri through R4 are basic groups, that is, primary, secondary, or tertiary amino groups; and, if only two or three of said substituents are basic groups, the remaining substituent or substituents are hydrogen, halogen, hydroxyl, mercapto, lower akyl, phenyl, phenoxy, lower alkoxy, lower alkoxy-lower alkoxy, (di-lower alkyl-amino)-lower alkoxy, lower alkyl-mercapto, phenyl-mercapto, benzyl-mercapto, or carboxy-lower alkyl-mercapto.
  • Patent No. 4,478,833 e.g., 8-benzylthio-2-(2-hydroxyethyl-amino)-4-(l- oxido-thiomorpholino)-pyrimido[5 ,4-d]pyrimidine, 2-(2-hydroxyethyl-amino)-4-( 1 -oxido- thiomorpholino)-8-(L- 1 -phenylethylamino)-pyrimido[5,4-d]pyrimidine, and 8-benzylamino- 2-(2-hyd ⁇ O yethyl-amino)-4-(l-oxido-thiomo ⁇ holino)-pyrimido[5,4-d]pyrimidine; by formula I of U.S.
  • 4,963,541 e.g., 2,6-bis(2-(methylamino)ethanol)-4,8-bis(N- perhydroazocinyl)pyrimido[5,4-d]pyrimidine.
  • Other analogs include mopidamol, BIBW 22, 2,6-bis(diethylamino)-4-piperidinopyrimido[5,4-d]pyrimidine (Mills et al. Biochem. J.
  • Dilazep is an ENT inhibitor described in G.B. Patent No. 1,107,470 and U.S. Patent No. 3,53
  • the methods, compositions, and kits of the invention may employ the acetylcholinesterase inhibitor donepezil or physostigmine.
  • acetylcholinesterase inhibitor donepezil or physostigmine Exemplary analogs of donepezil and physostigmine include their respective structural analogs.
  • acetylcholinesterase inhibitors including certain organophosphates (e.g., metrifonate), certain carbamates (e.g., physostigmine, neostigmine, pyridostigmine, and rivastigmine), certain phenanthrene derivatives (e.g., galantamine), certain piperidines (e.g., donepezil, also known as E2020), tacrine, ecothiopate, dyflos, ambenonium, demarcarium, and
  • edrophonium are also considered herein to be analogs of donepezil and physostigmine.
  • Donepezil has the structure:
  • CH CH-CO ⁇ NH-(CH 2 ) 2 -, -NH-, -O-, --S-, a dialkylaminoalkylcarbonyl or a lower
  • T is a nitrogen or carbon
  • Q is nitrogen, carbon or / and q is an integer of 1 to 3
  • K is hydrogen, phenyl, a substituted phenyl, an arylalkyl in which the phenyl may have a substituent, cinnamyl, a lower alkyl, pyridylmethyl, a cycloalkylalkyl, adamantanemethyl, furylmenthyl, a cycloalkyl, a lower alkoxycarbonyl or an acyl; and— shows a single bond or a double bond, as described in U.S. Patent No. 4,895,841 , which is herein incorporated by reference.
  • Exemplary compounds of formula II are l-benzyl-4-((5-methoxy-l-indanon)-2- yl)methylpiperidine, 1 -benzyl-4-((5 ,6-diethoxy- 1 -indanon)-2-yl)methylpiperidine, 1 -benzyl- 4-((5,6-methylenedioxy-l-indanon)-2-yl)methylpiperidine, l-(m-nitrobenzyl)-4-((5,6- dimethoxy- 1 -indanon)-2-yl)methylpiperidine, 1 -(m-fluorobenzyl)-4-((5 ,6-dimethoxy- 1 - mdanon)-2- l)methylpiperidine, l-benzyl-4-((5,6-dimethoxy-l-indanon)-2- yl)propylpiperidine, and 1 -benzyl-4-((5-isopropoxy-6-methoxy
  • Exemplary physostigmine analogs are 1-desmethyl eserine, norphysostigmine,
  • X is O or S
  • R is H, loweralkyl, or— c— R 4j where Y is O or S
  • R 2 is alkyl, cycloalkyl, bicycloalkyl, cycloalkenyl, aryl, arylloweralkyl, heteroaryl or
  • R 3 is H or alkyl, or the group ⁇ NR 2 R 3 taken as a whole is 1- pyrrolidinyl, 1-piperidinyl, 4-morpholinyl, 4-thiomo ⁇ holinyl, 1-piperazinyl, 4-methyl-l- piperazinyl or 2-(2,6-dichlorophenylimino)-l-imidazolidinyl) and R 4 is hydrogen, loweralkyl, arylloweralkyl, diarylloweralkyl, aryl or heteroaryl; (c) m is 1 or 2; (d) each Z is
  • Ri is H, loweralkyl, arylloweralkyl, heteroarylloweralkyl, cycloalkylmethyl or loweralkenylmethyl, with the proviso that when X is O, m is 1, Z is H and Rj is methyl, R is not --CONHCH 3 , ⁇ CONHC 6 H 5, hydrogen, methyl or ethyl, and that when X is O, m is 1 and Z and R ! are both hydrogen, R is not hydrogen or methyl, as described in U.S. Patent Nos. 5,541,340,
  • Exemplary compounds of formula III are (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl- pyrrolo[2,3-b]indol-5-ol, octadecyl carbamate ester; 7-chloro-(3aS-cis)-l,2,3,3a,8,8a- hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, methyl carbamate ester; 7-bromo-(3aS- cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethylpyrrolo[2,3-b]indol-5-ol, methyl carbamate ester; (3aS-cis)-l,2,2,3,3a,8,8a-hexahydro-l,3a,8-trimethylpyrrolo[2,3
  • Rho kinase inhibitors
  • a Rho kinase inhibitor can be used in the compositions, methods, and kits of the invention.
  • a Rho kinase inhibitor is meant a compound that inhibits the activity of a Rho kinase by at least 5%, e.g., greater than 10%, 20%, 40%, 60%, 80%o, 90%, or 95%. Inhibition of Rho kinase activity may be measured, e.g., by an in vitro assay with recombinant or purified Rho kinase, or by a cell-based reporter assay known in the art.
  • Rho kinase inhibitors include fasudil, HA 1077 (Calbiochem), hydroxyfasudil, and Y- Fasudil
  • Fasudil is described in European Patent No. 187371 and U.S. Patent No. 4,678,783 and has the following structure:
  • R ⁇ represents a hydrogen atom, a chlorine atom or a hydroxyl group
  • R ( represents a hydrogen atom A represents an ethylene group unsubstituted or substituted with an alkyl group having 1 to 6 carbon atoms, a phenyl group or a benzyl group
  • R 2 and R 3 are directly bonded with each other, thereby forming a trimethylene group unsubstituted or substituted with an alkyl group having 1 to 6 carbon atoms, a phenyl group or a benzyl group
  • Rj represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms
  • Ri represents a chlorine atom or a hydroxyl group
  • A represents an alkylene group having 2 to 6 carbon atoms, said group being unsubstituted or substituted with an alkyl group having 1 to 6 carbon atoms
  • R 2 and R 3 are not bonded with each other and each independently represent a hydrogen atom or an alkyl group
  • R4 represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or an amidino group, as described in U.S. Patent No. 4,678,783.
  • Exemplary compounds of formula (IV) are l-(5- isoquinolinesulfonyl)homopiperazine, l-(5-isoquinolinesulfonyl)-2-methylhomopiperazine, 1 -(5-isoquinolinesulfonyl)-3-methylhomopiperazine, 1 -(5-isoquinolinesulfonyl)-6- methylhomopiperazine, 1 -(5-isoquinolinesulfonyl)-2,3-dimethylhomopiperazine, 1 -(5- isoquinolinesulfonyl)-3 ,3-dimethylhomopiperazine, 1 -(5-isoquinolinesulfonyl)-3- ethylhomopiperazine, l-(5-isoquinolinesulfonyl)-3-propylhomopiperazine, and l-(5- isoquinolinesulfonyl)-3-isobutylhomopiperazine.
  • the FKBP/mTOR inhibitor everolimus or an everolimus analog may be employed.
  • Analogs of everolimus include compounds structurally related to everolimus and other FKBP/mTOR inhibitors, e.g., temsirolimus, rapamycin, ascomycin, AP23573 (Ariad Pharmaceuticals), NVP-BEZ235, sirolimus, tacrolimus (FK 506), zotarolimus, and pimecrolimus.
  • Everolimus has the following structure:
  • everolimus analogs e.g., pimecrolimus
  • Other analogs of everolimus include mono- and diacylated rapamycin derivatives (U.S. Patent No. 4,316,885); rapamycin water-soluble prodrugs (U.S. Patent No. 4,650,803); carboxylic acid esters (PCT Publication No. WO 92/05179);
  • carbamates U.S. Patent No. 5,118,678; amide esters (U.S. Patent No. 5,118,678); biotin esters (U.S. Patent No. 5,504,091); fluorinated esters (U.S. Patent No. 5,100,883); acetals (U.S. Patent No. 5,151,413); silyl ethers (U.S. Patent No. 5,120,842); bicyclic derivatives (U.S. Patent No. 5,120,725); rapamycin dimers (U.S. Patent No. 5,120,727); O-aryl, O-alkyl, O-alkyenyl and O-alkynyl rapamycin derivatives (U.S. Patent No.
  • the methods, compositions, and kits of the invention may employ ethaverine, drotaverine, papaverine, zardaverine, tetrahydropapaveroline, trequinsin, MBCQ (4-[[3,4- (methylenedioxy)benzyl]amino]-6-chloroquinazoline), or dipyridamole or an analog of one of these.
  • ethaverine, drotaverine, papaverine, zardaverine tetrahydropapaveroline
  • MBCQ 4-[[3,4- (methylenedioxy)benzyl]amino]-6-chloroquinazoline
  • tetrahydropapaveroline, trequinsin, MBCQ, or dipyridamole include their structural analogs and other phosphodiesterase (PDE) inhibitors.
  • PDE phosphodiesterase
  • the PDE inhibitor selectively inhibits a particular type of PDE relative to other types.
  • a selective inhibitor may inhibit PDE type 5 (PDE5) at least 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, or 100-fold more effectively than it inhibits another particular phosphodiesterase, e.g., PDE type II, type III, type IV, type VII, or type VIII, also known as PDE2, 3, 4, 7, and 8, respectively).
  • a PDE inhibitor may also be non-selective or exhibit weak selectivity.
  • Exemplary non-selective or weakly selective PDE inhibitors are theophylline, theobromine, IBMX, pentoxifylline and papaverine.
  • the selectivity of a PDE inhibitor may be determined by measuring its IC50 (the concentration required to achieve 50% inhibition of an enzyme) against at least two different phosphodiesterases.
  • a PDE inhibitor selective for PDE5 is employed.
  • Inhibitors of PDE5 may include griseolic acid derivatives, 2-phenylpurinones,
  • phenylpyridones fused and condensed pyrimidines, pynmidopyrimidines, purine compounds, quinazoline compounds, phenylpyrimidinones, and imidazoquinoxalinones.
  • PDE5 inhibitors are dipyridamole, MBCQ, zaprinast, MY-5445, vinpocetine, FR229934, l-methyl-3-isobutyl-8-methylamino)xanthine, IC-351, vardenafil, GF-196960, Sch-51866,and sodium- l-[6-chloro-4-(3 ,4-methylenedioxybenzyl)-aminoquinazolin-2- yl]piperidine-4 -carboxylate sesquihydrate.
  • a PDE inhibitor has an IC 50 of 100 ⁇ or lower for a phosphodiesterase.
  • the IC 50 of a phosphodiesterase inhibitor is 40, 20, or 10 ⁇ or lower.
  • a phosphodiesterase inhibitor has an IC 50 of 40 ⁇ , 20 ⁇ , 10 ⁇ , 5 ⁇ , 1 ⁇ , 100 ⁇ , 10 ⁇ , or lower for a particular type of phosphodiesterase.
  • the inhibitor may also have activity against other types, unless otherwise stated.
  • PDE inhibitors are theophylline(l,3-dimethylxanthine), caffeine, quercetin dihydrate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one,
  • propentofylline 3-methyl- 1 -(5-oxohexyl)-7-propylxanthine), 3-isobutyl- 1 -methylxanthine
  • IBMX 3-isobutyl-l-methyl-2,6(lH,3H)-purine-dione, l-methyl-3-isobutylxanthine, 8- methoxymethyl-3-isobutyl-l -methylxanthine, enoximone, papaverine hydrochloride, calmidazolium chloride, imidazolium chloride, l-[bis(4-chlorophenyl)methyl]-3-[2-(2,4- dichlorophenyl)-2-(2,4-dichlorobenzyloxy)ethyl]-lH-imidazolium chloride, SKF 94836, neuropeptide Y fragment 22-36, aminophylline hydrate, butein, etazolate hydrochloride, trifluopera
  • arofylline, atizoram A WD- 12-281 (N-(3 , 5-dichloro-4-pyridinyl)-2-[ 1 -(4-fluorobenzyl)-5-hydroxy- 1 H-indol-3- yl]-2-oxoacetamide), BAY- 19-8004 (ethanesulfonic acid 2-(2, 4-dichlorophenylcarbonyl)-3- ureido-benzofuran-6-yl ester), benafentrine, CC-1088, CDC-801 (p-[3-(cyclopentyloxy)-4- methoxyphenyl]- 1 ,3-dihydro- 1 ,3-dioxo-2H-isoindole-2-propanamide), CDC-998, CI- 1018, cilomilast (cis-[4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclo
  • PDE inhibitors that may be useful in the compositions, methods, and kits provided herein are disclosed in U.S. Patent No. 6,818,651, U.S. Patent No. 6,737,436, U.S. Patent No. 6,613,778, U.S. Patent No. 6,617,357, U.S. Patent No.
  • Patent No. 6,372,777 U.S. Patent No. 6,362,213, U.S. Patent No. 6,313,156, U.S. Patent No. 6,294,561, U.S. Patent No. 6,258,843, U.S. Patent No. 6,258,833, U.S. Patent No. 6,043,263, U.S. Patent No. 6,297,257, U.S. Patent No. 6,251,923, U.S. Patent No. 6,613,794, U.S.
  • Patent No. 6,407,108 U.S. Patent No. 6,107,295, U.S. Patent No. 6,103,718, U.S. Patent No. 6,479,494, U.S. Patent No. 6,545,158, U.S. Patent No. 6,545,025, U.S. Patent No. 6,498,160, U.S. Patent No. 6,743,802, U.S. Patent No. 6,787,554, U.S. Patent No. 6,828,333, U.S.
  • Patent No. 5,716,967 U.S. Patent No. 5,686,434, U.S. Patent No. 5,502,072, U.S. Patent No. 5,116,837, U.S. Patent No. 5,091,431, U.S. Patent No. 4,670,434, U.S. Patent No. 4,490,371 , U.S. Patent No. 5,710,160, U.S. Patent No. 5,710,170, U.S. Patent No. 6,384,236, U.S.
  • Patent No. 3,941,785 in U.S. Patent publications 2005/0119225, 2005/0026913,
  • YM 976 4-(3-chlorophenyl)-l -diethylpyrido(2,3-d)pyrimidin-2(lH)-one 4
  • PDE4 inhibitors e.g., ibudilast
  • examples of PDE4 inhibitors include pyrrolidinones, such
  • benzoxazoles such as those disclosed in U.S. Patent No. 6,166,041 and U.S. Patent No.
  • the compound 1 -ethyl-4-(( 1 -methylethylidene)hydrazino)- 1 H-pyrazolo(3 ,4- b)pyridine-5-carboxylic acidethyl ester, also known as etazolate, is a PDE inhibitor having the structure:
  • Exemplary analogs of etazolate are ethyl l-methyl-4-(2-propan-2- ylidenehydrazinyl)pyrazolo[3 ,4-b]pyridine-5-carboxylate, butyl 1 -ethyl-4-(2-propan-2- ylidenehydrazinyl)pyrazolo[3 ,4-b]pyridine-5-carboxylate, ethyl 1 -propan-2-yl-4-(2-propan-2- ylidenehydrazinyl)pyrazolo[3 ,4-b]pyridine-5-carboxylate, ethyl 4-(2- cyclohexylidenehydrazinyl)- 1 -ethylpyrazolo[3 ,4-b]pyridine-5-carboxylate, ethyl 1 -ethyl-4-(2- nonan-5-ylidenehydrazinyl)pyrazolo[3,4-
  • Analogs of papaverine include 3-(3,4-dirnethoxyphenyl)-6,7-dimethoxyisoquinoline, 1 -[ 1 -(3 ,4-dimethoxyphenyl)ethenyl]-6,7-dimethoxyisoquinoline, 1 -(3 ,4-dimethoxyphenyl)- 6,7-dimethoxyisoquinoline, 1 -[ 1 -(3 ,4-dimethoxyphenyl)ethyl]-6,7-dimethoxyisoquinoline, 1 - [ 1 -(3 ,4-dimethoxyphenyl)ethyl]-6,7-dimethoxyisoquinoline, 6,7-dimethoxy- 1 -[(4- methoxyphenyl)methyl]isoquinoline, 6,7-dimethoxy- 1 -[(3- methoxyphenyl)methyl]isoquinoline, 6,7-dimethoxy-3-(4-meth
  • Ethaverine is the tetraethoxy analogue of papaverine and is described in U.S. Patent No. 1,962,224. Ethav
  • Analogs of ethaverine include without limitation l-(3,4-diethoxyphenyl)-6,7- diethoxyisoquinoline, 6,7-dimethoxy-l-[(3-methoxy-4-propoxyphenyl)methyl]isoquinoline, 1 - [(4-ethoxy-3 -methoxyphenyl)methyl] -6,7-dimethoxyisoquinoline, 6,7-dimethoxy- 1 - [(3 - methoxy-4-propoxyphenyl)methyl] isoquinoline hydrochloride, 1 - [(3 ,4- diethoxyphenyl)methyl]-6,7-diethoxyisoquinoline hydrochloride, l-[(3,4- diethoxyphenyl)methyl]-6,7-di(propan-2-yloxy)isoquinoline, l-[(2,3- dimethoxyphenyl)methyl]-5,6-diethoxyisoquinoline, l-[(3,4-
  • EHNA (9-(2-hydroxy-3-nonyl)adenine) is a PDE2-selective inhibitor having following structure:
  • EHNA Exemplary analogs of EHNA are described by formula I of U.S. Patent No. 7,022,709 and by formula I of U.S. Patent No. 5,861,396.
  • Other analogs of EHNA include 1,3-dideaza- EHNA, 7-deaza-EHNA, 1-deaza-EHNA, 3-deaza-EHNA, and erythro-(3-nonyl-p- aminobenzyl-adenine) .
  • Drotaverine (l-benzyl-3',4',6,7-tetraethoxy-l,2,3,4- tetrahydroisoquinoline) is a PDE4- selective PDE inhibitor structurally related to papaverine. Drotaverine is described in
  • Trequinsin (9, 10-dimethoxy-3-methyl-2-(2,4,6-trimethylphenyl)imino-6,7- dihydropyrimido[6,l-a]isoquinolin-4-one) is an inhibitor of PDE3.
  • the structure of trequinsin is:
  • Analogs of trequinsin include 3-ethyl-9,10-dimethoxy-2-(2,4,6- trimethylphenyl)imino-6,7-dihydropyrimido[6, 1 -a]isoquinolin-4-one, (7)-9, 10-dimethoxy- 3 ,7-dimethyl-2-(2,4,6-trimethylphenyl)imino-6,7-dihydropyrimido[6, 1 -a]isoquinolin-4-one, 9,10-dimethoxy-3,7-dimethyl-2-(2,4,6-trime
  • Tadalafil Tetrahydropapaveroline MBCQ Zardaverine
  • the methods, compositions, and kits of the invention employ idebenone, a CoQIO (ubiquinone) analog.
  • idebenone a CoQIO (ubiquinone) analog.
  • Analogs of idebenone include other CoQIO analogs, e.g., MitoQIO, decyl-ubiquinone and atovaquone.
  • 4,985,447 e.g., 3,5,6-trimethyl-2-(3-pyridyl-2-thienylmethyl)-l,4-benzoquinone hydrochloride and 7-(3,5,6- trimethyl-l,4-benzoquinon-2-yl)-7-(3-pyridyl)heptanoic acid; by formula I of U.S. Patent No.
  • Patent No. 5,304,658 e.g., 7-(3,5,6-trimethyl-l,4- benzoquinon-2-yl)-7-phenylheptanol, 7-(3,5,6-trimethyl-l,4-benzoquinon-2-yl)-7- phenylheptanamide, 7-(3,5,6-trimethyl-l ,4-benzoquinon-2-yl)-7-phenylheptanoglycine, and
  • Verapamil may be used in the methods, compositions, and kits of the invention.
  • Analogs of verapamil include structural analogs of verapamil and other calcium channel blockers, e.g., dihydropyridines (e.g., amlodipine, aranidipine, azelnidipine, bamidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, and pranidipine), phenylalkylamines(e.g., gallopamil), and benzothiazepines (e.g., diltiazem).
  • dihydropyridines e.g., amlodipine, aranidipine, azelnidipine, bamidipine, benidipine,
  • Analogs of verapamil include 4-desmethoxy- verapamil, 2-(3,4-dimethoxyphenyl)-5- amino-2-isopropylvaleronitrile, alpha-(3 -aminopropyl)-3 ,4-dimethoxy-alpha-( 1 - methylethyl)benzeneacetonitrile, carboxyverapamil, devapamil, norgallopamil, and nexopamil.
  • a bisphosonate also called a diphosphonate
  • Bisphosphonates are a class of drugs that inhibits bone resporption. Examples of bisphonates are described below.
  • Pamidronic acid is described in German Patent No. 2,130,794 and U.S. Patent No. 4,327,039, and alendronic acid is described in Belgian Patent No. 903,519 and U.S. Patent No. 4,705,651.
  • the structures of alendronate and pamidronate are:
  • Exemplary analogs of alendronate and pamidronate are etidronate, clodronate, tiludronate, risedronate, ibandronate, EB-1053 (l-hydroxy-3-(l-pyrrolidinyl)-propylidene- 1,1 -bisphosphonate), olpadronate, amino-olpadronate, 6-ammo-l-hydroxyhexylidene- bisphosphonate, cimadronate, neridronate, piridronate, zoledronate, and 1-hydroxy- 3(methylpentylamino)-propylidene bisphosphonate.
  • Other exemplary analogs are described by the general formula of U.S. Patent No. 4,327,039; by formula I of U.S. Patent No.
  • 4,407,761 e.g., 6-amino-l-hydroxyhexylidene-l,l-bisphosphonic acid; by formula I of U.S. Patent No. 4,536,348, e.g., l,3-dihydroxypropane-l,l-diphosphonic acid and 1,6- dihydroxyhexane-l,l-diphosphonic acid; by formula I of U.S. Patent No.
  • perfluorohexylthio-methylene-disphosphonate (SR 42327), tri-(tertiary butylamine) salt of methylthio-methylene-diphosphonic acid (SR 41036), di-(tertiary butylamine) salt of (4- chlorophenyl)thiomethylene-diphosphonic acid (SR 41319), tertiary butylamine salt of 3- methylthio-propylidene- 1,1 -diphosphonic acid (SR 41273), di-(tertiary butylamine salt) of 4- phenylthio-butylidene- 1 , 1 -diphosphonic acid (SR 41342), monoammonium
  • hexadecylthiomethylene-diphosphonate (SR 41453), di-(tertiary butylamine) salt of (2- hydroxyethylthio)methylene-diphosphonic acid (SR 41318), disodium methylthiomethylene- diphosphonate (SR 41553), tri-(tertiary butylamine) salt of benzothiazol-2-yl-thiomethylene- diphosphonic acid (SR 41481), tertiary-butylammonium 4-(methylthio)-butylidene-l,l- diphosphonate (SR 41177), di-(tertiary butylalmine) salt of 5-mercapto-pentylidene-l,l- diphosphonic acid (SR 41527), di-(tertiary butylamine) salt of 7-(l-methyl-imidazol-2-yl- thio)-heptylidene- 1,1 -diphosphonic acid (SR
  • agents used in any of the combinations described herein may be covalently attached to one another to form a conjugate of formula I.
  • (A) is a Compound A and (B) is Compound B of a pair of agents from e.g., Table 1, and L is a covalent linker that tethers (A) to (B).
  • Conjugates of the invention can be administered to a subject by any route and for the treatment of an NF- B-mediated disease or a disease suppressed by oc7 integrin.
  • the conjugates of the invention can be prodrugs, releasing drug (A) and drug (B) upon, for example, cleavage of the conjugate by intracellular and extracellular enzymes (e.g., amidases, esterases, and phosphatases).
  • the conjugates of the invention can also be designed to largely remain intact in vivo, resisting cleavage by intracellular and extracellular enzymes. The degradation of the conjugate in vivo can be controlled by the design of linker (L) and the covalent bonds formed with drug (A) and drug (B) during the synthesis of the conjugate.
  • Conjugates can be prepared using techniques familiar to those skilled in the art.
  • the conjugates can be prepared using the methods disclosed in G. Hermanson,
  • conjugates may involve the selective protection and deprotection of alcohols, amines, ketones, sulfhydryls or carboxyl functional groups of drug (A), the linker, and/or drug (B).
  • protecting groups for amines include carbamates, such as tert-butyl, benzyl, 2,2,2- trichloroethyl, 2-trimethylsilylethyl, 9-fluorenylmethyl, allyl, and m-nitrophenyl.
  • Other commonly used protecting groups for amines include amides, such as formamides, acetamides, trifluoroacetamides, sulfonamides, trifluoromethanesulfonyl amides,
  • trimethylsilylethanesulfonamides and tert-butylsulfonyl amides.
  • Examples of commonly used protecting groups for carboxyls include esters, such as methyl, ethyl, tert-butyl, 9- fluorenylmethyl, 2-(trimethylsilyl)ethoxy methyl, benzyl, diphenylmethyl, O-nitrobenzyl, ortho-esters, and halo-esters.
  • Examples of commonly used protecting groups for alcohols include ethers, such as methyl, methoxymethyl, methoxyethoxymethyl, methylthiomethyl, benzyloxymethyl, tetrahydropyranyl, ethoxyethyl, benzyl, 2-napthylmethyl, O-nitrobenzyl, P- nitrobenzyl, P-methoxybenzyl, 9-phenylxanthyl, trityl (including methoxy-trityls), and silyl ethers.
  • Examples of commonly used protecting groups for sulfhydryls include many of the same protecting groups used for hydroxyls.
  • sulfhydryls can be protected in a reduced form (e.g., as disulfides) or an oxidized form (e.g., as sulfonic acids, sulfonic esters, or sulfonic amides).
  • Protecting groups can be chosen such that selective conditions (e.g., acidic conditions, basic conditions, catalysis by a nucleophile, catalysis by a lewis acid, or hydrogenation) are required to remove each, exclusive of other protecting groups in a molecule.
  • the conditions required for the addition of protecting groups to amine, alcohol, sulfhydryl, and carboxyl functionalities and the conditions required for their removal are provided in detail in T.W. Green and P.G.M. Wuts, Protective Groups in Organic Synthesis (2 nd Ed.), John Wiley & Sons, 1991 and P.J. Kocienski, Protecting Groups, Georg Thieme Verlag, 1994. Additional synthetic details are provided below.
  • the linker component of the invention is, at its simplest, a bond between drug (A) and drug (B), but typically provides a linear, cyclic, or branched molecular skeleton having pendant groups covalently linking drug (A) to drug (B).
  • linking of drug (A) to drug (B) is achieved by covalent means, involving bond formation with one or more functional groups located on drug (A) and drug (B).
  • functional groups located on drug (A) and drug (B).
  • chemically reactive functional groups include, without limitation, amino, hydroxyl, sulfhydryl, carboxyl, carbonyl, carbohydrate groups, vicinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl, and phenolic groups.
  • the covalent linking of drug (A) and drug (B) may be effected using a linker that contains reactive moieties capable of reaction with such functional groups present in drug (A) and drug (B).
  • a linker that contains reactive moieties capable of reaction with such functional groups present in drug (A) and drug (B).
  • an amine group of drug (A) may react with a carboxyl group of the linker, or an activated derivative thereof, resulting in the formation of an amide linking the two.
  • N-Maleimide derivatives are also considered selective towards sulfhydryl groups, but may additionally be useful in coupling to amino groups under certain conditions.
  • Reagents such as 2- iminothiolane (Traut et al., Biochemistry 12:3266 (1973)), which introduce a thiol group through conversion of an amino group, may be considered as sulfhydryl reagents if linking occurs through the formation of disulfide bridges.
  • reactive moieties capable of reaction with amino groups include, for example, alkylating and acylating agents.
  • Representative alkylating agents include:
  • N-maleimide derivatives which may react with amino groups either through a Michael type reaction or through acylation by addition to the ring carbonyl group, for example, as described by Smyth et al., J. Am. Chem. Soc. 82:4600 (1960) and Biochem. J. 91:589 (1964);
  • epoxide derivatives such as epichlorohydrin and bisoxiranes, which may react with amino, sulfhydryl, or phenolic hydroxyl groups
  • epoxide derivatives such as epichlorohydrin and bisoxiranes, which may react with amino, sulfhydryl, or phenolic hydroxyl groups
  • chlorine-containing derivatives of s-triazines which are very reactive towards nucleophiles such as amino, sufhydryl, and hydroxyl groups
  • Representative amino-reactive acylating agents include:
  • active esters such as nitrophenylesters or N-hydroxysuccinimidyl esters
  • acylazides e.g., wherein the azide group is generated from a preformed hydrazide derivative using sodium nitrite, as described by Wetz et al., Anal. Biochem. 58:347 (1974); and
  • Aldehydes and ketones may be reacted with amines to form Schiff s bases, which may advantageously be stabilized through reductive amination.
  • Alkoxylamino moieties readily react with ketones and aldehydes to produce stable alkoxamines, for example, as described by Webb et al., in Bioconjugate Chem. 1:96 (1990).
  • reactive moieties capable of reaction with carboxyl groups include diazo compounds such as diazoacetate esters and diazoacetamides, which react with high
  • ester groups for example, as described by Herriot, Adv. Protein Chem. 3:169 (1947).
  • Carboxyl modifying reagents such as carbodiimides, which react through O- acylurea formation followed by amide bond formation, may also be employed.
  • So-called zero-length linkers involving direct covalent joining of a reactive chemical group of drug (A) with a reactive chemical group of drug (B) without introducing additional linking material may, if desired, be used in accordance with the invention.
  • the linker will include two or more reactive moieties, as described above, connected by a spacer element.
  • the presence of such a spacer permits bifunctional linkers to react with specific functional groups within drug (A) and drug (B), resulting in a covalent linkage between the two.
  • the reactive moieties in a linker may be the same (homobifunctional linker) or different (heterobifunctional linker, or, where several dissimilar reactive moieties are present, heteromultifunctional linker), providing a diversity of potential reagents that may bring about covalent attachment between drug (A) and drug (B).
  • Spacer elements in the linker typically consist of linear or branched chains and may include a C ⁇ 0 alkyl, C 2 _io alkenyl, C 2 _i 0 alkynyl, C 2 -6 heterocyclyl, C6_i 2 aryl, C 7 _i 4 alkaryl, C 3 _ 10 alkheterocyclyl, or C)_ 10 heteroalkyl.
  • linker is described by formula (V):
  • G 1 is a bond between drug (A) and the linker;
  • G 2 is a bond between the linker and drug (B);
  • Z 1 , Z 2 , Z 3 , and Z 4 each, independently, is selected from O, S, and NR 31 ;
  • R 31 is hydrogen, alkyl, C 2 _4 alkenyl, C 2 _4 alkynyl, C 2 _ 6 heterocyclyl, C6_ 12 aryl, C 7 _ i 4 alkaryl, C 3 _ 10 alkheterocyclyl, or C]_ 7 heteroalkyl;
  • Y 1 and Y 2 are each, independently, selected from carbonyl, thiocarbonyl, sulphonyl, or phosphoryl;
  • o, p, s, t, u, and v are each, independently, 0 or 1;
  • R 30 is a C]_ 10 alkyl, C 2 _ 10 alkenyl, C 2 _ 10 alkyn
  • homobifunctional linkers useful in the preparation of conjugates of the invention include, without limitation, diamines and diols selected from ethylenediamine, propylenediamine and hexamethylenediamine, ethylene glycol, diethylene glycol, propylene glycol, 1,4-butanediol, 1,6-hexanediol, cyclohexanediol, and polycaprolactone diol.
  • compositions, methods, and kits of the invention can include formulation(s) of compound(s) that, upon administration to a subject, result in a concentration of the compound(s) that treats an NF-KB-mediated or oc7 integrin-suppressed disease.
  • the compound(s) may be contained in any appropriate amount in any suitable carrier substance, and are generally present in an amount of 1-95% by weight of the total weight of the composition.
  • the composition may be provided in a dosage form that is suitable for the oral, parenteral (e.g., intravenously or intramuscularly), rectal, dermatological, cutaneous, nasal, vaginal, inhalant, skin (patch), ocular, intrathecal, or intracranial administration route.
  • the composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols.
  • the pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 20th edition, 2000, ed. A.R. Gennaro, Lippincott Williams & Wilkins,
  • compositions according to the invention or used in the methods of the invention may be formulated to release the active compound immediately upon
  • compositions are generally known as controlled release formulations, which include (i) formulations that create substantially constant concentrations of the agent(s) of the invention within the body over an extended period of time; (ii) formulations that after a predetermined lag time create substantially constant concentrations of the agent(s) of the invention within the body over an extended period of time; (iii) formulations that sustain the agent(s) action during a predetermined time period by maintaining a relatively constant, effective level of the agent(s) in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the agent(s) (sawtooth kinetic pattern); (iv) formulations that localize action of agent(s), e.g., spatial placement of a controlled release composition adjacent to or in the diseased tissue or organ; (v) formulations that achieve convenience of dosing, e.g., administering the composition once per week or once every two weeks; and (vi) formulations that target the action of the
  • controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings.
  • the compound(s) are formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the compound(s) in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, molecular complexes, microspheres, nanoparticles, patches, and liposomes.
  • a first agent is delivered orally, and a second agent is delivered intravenously.
  • the dosage of a compound or a combination of compounds depends on several factors, including: the administration method, the type of disease to be treated, the severity of the symptoms, whether administration first occurs at an early or late stage of disease progression, and the age, weight, and health of the patient to be treated.
  • the recommended dosage for the agent can be less than or equal to the recommended dose as given in the Physician 's Desk Reference, 60 th Edition (2006).
  • the compound(s) in question may be administered orally in the form of tablets, capsules, elixirs or syrups, or rectally in the form of suppositories.
  • Parenteral administration of a compound is suitably performed, for example, in the form of saline solutions or with the compound(s) incorporated into liposomes.
  • a solubilizer such as ethanol can be applied.
  • the correct dosage of a compound can be determined by examining the efficacy of the compound in reporter assays, e.g., one described herein, as well as toxicity in humans.
  • a therapeutic agent is usually given by the same route of administration that is known to be effective for delivering it as a monotherapy.
  • an agent of Table 1 or Table 2, or an analog thereof is dosed in amounts and frequencies equivalent to or less than those that result in its effective monotherapeutic use if the agent is used monotherapeutically for the treatment of an NF-KB-mediated disease,an al integrin-suppressed disease, or another indication.
  • the compounds of the invention may be employed in mechanistic assays to determine whether other combinations, or single agents, are as effective as the combinations of the invention treating an NF-KB-mediated or al integrin-suppressed disease (e.g., a disease listed herein) using assays known in the art or described herein.
  • candidate compounds may be tested, alone or in combination with other agents and applied to cells (e.g., the ct7 +/" -P-gal mouse myoblast or C2C12 NF-KB-Luciferase cell lines described herein). After a suitable time, reporter gene activity can be measured. Reporter assays such as those described herein can be used to identify additional combination of agents as effective agent for treating an NF-KB-mediated or al integrin-suppressed disease.
  • the agents of the invention are also useful tools in elucidating mechanistic
  • NF-KB-mediated diseases information about the biological pathways involved in NF-KB-mediated diseases and al integrin-suppressed diseases. Such information can lead to the development of new combinations or single agents for treating NF-KB-mediated diseases and oc7 integrin- suppressed diseases.
  • Methods known in the art to determine biological pathways can be used to determine the pathway, or network of pathways affected by contacting cells (e.g., the a7 + ⁇ - ⁇ -gal mouse myoblast or C2C12 NF-KB-Luciferase cell lines described herein) with the compounds of the invention. Such methods can include analyzing cellular constituents that are expressed or repressed after contact with the compounds of the invention as compared to untreated, positive or negative control compounds, and/or new single agents and
  • Cellular components analyzed can include gene transcripts, protein expression, and DNA digestion. Suitable methods can include standard biochemistry techniques, radiolabeling the compounds of the invention (e. g., ,4 C or 3 ⁇ 4 labeling), and observing the compounds binding to proteins, e.g., using 2D gels, and gene expression profiling. Once identified, such compounds can be used in in vivo models (e.g., knockout or mutant mice) to further validate the tool or develop new agents or strategies to treat NF-KB-mediated diseases and al integrin-suppressed diseases.
  • in vivo models e.g., knockout or mutant mice
  • Peptides, peptide mimetics, and peptide fragments are suitable for use in the methods of the invention.
  • exemplary inhibitors include compounds that reduce the amount of a target protein or RNA levels (e.g., antisense compounds, dsRNA, ribozymes) and compounds that increase the amount of a target protein or RNA levels.
  • Other agents may influence the intraceullar modification or trafficking of a molecule, e.g., NF- ⁇ (e.g., dominant negative proteins or polynucleotides encoding the same).
  • RNA secondary structure folding program such as MFOLD (M. Zuker, D. H. Mathews & D. H. Turner, Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide. In: RNA Biochemistry and Biotechnology, J. Barciszewski & B. F. C.
  • Sub-optimal folds with a free energy value within 5% of the predicted most stable fold of the mRNA are predicted using a window of 200 bases within which a residue can find a complimentary base to form a base pair bond. Open regions that do not form a base pair are summed together with each suboptimal fold and areas that are predicted as open are considered more accessible to the binding to antisense nucleobase oligomers.
  • Other methods for antisense design are described, for example, in U.S. Pat. No. 6,472,521 , Antisense Nucleic Acid Drug Dev. 1997 7:439-444, Nucleic Acids Res. 28:2597-2604, 2000, and Nucleic Acids Res. 31 :4989-4994, 2003.
  • RNA interference employing, e.g., a double stranded RNA (dsRNA) or small interfering RNA (siRNA) directed to the signaling molecule in question (see, e.g., Miyamoto et al., Prog. Cell Cycle Res. 5:349-360, 2003; U.S. Pat. Application Publication No. 20030157030).
  • dsRNA double stranded RNA
  • siRNA small interfering RNA
  • Methods for designing such interfering RNAs are known in the art. For example, software for designing interfering RNA is available from Oligoengine (Seattle, WA).
  • NF- B activation has been positively correlated with disease states including muscle wasting diseases.
  • a screen was performed using a cell line
  • the cells line called C2C12 NF- ⁇ -Luciferase (Luc) was derived from mouse myoblast C2C12 cells by chromosomal integration of a construct encoding the luciferase gene and a regulatory element containing 6- copies of the NF- ⁇ response element, a minimal TA promoter, and the TATA box from the thymidine kinase promoter. In this assay, the intensity of luminescence is proportional to the level of reporter gene expression.
  • C2C12 NF-KB-LUC cells (Panomics, Catalog No. RC0016) cultured in growth medium) were cultured in T-175 flasks or HYPERFlasks (Fisher Scientific) in DMEM growth media containing hygromycin B, 1% penicillin-streptomycin, and 10% fetal bovine serum. Cells were passaged once 90% confluence was achieved at a ratio of approximately 1:8. Briefly, cells were rinsed with PBS (10 mL for a T-175 flask and 50 mL for a
  • HYPERFlask Trypsin-EDTA was added to the cells (2.5 mL for a T-175 flask and 55 mL for a HYPERFlask), and the cells were incubated at 37°C and 5% CO 2 for three minutes.
  • Cell growth medium (10 mL for a T-175 flask and 55 mL for a HYPERFlask) was added to neutralize the trypsin and cells were triturated to break apart clumps. For seeding cells in 384- well assay plates, cell suspensions were combined and cell density was calculated.
  • Cells were spun down at 1000 rpm for five minutes and resuspended in Phenol red-free DMEM containing 2mM L-glutamine, hygromycin B, 1% penicillin-streptomycin, and 10% fetal bovine serum. Additional assay medium was added to dilute the cell suspension to a concentration of 2.5 x 10 5 cells per mL. Cells were plated at 10,000 cells in 40 ⁇ , per well of a 384- well plate (Matrix Technologies, Custom Order No. BC30316), and incubated at 37°C and 5% CO 2 for 24 hours. On the following day, compounds were diluted 1 : 100 in assay medium containing 40 ng/mL TNFa.
  • the compounds and TNFa (4ng/mL final concentration) were then simultaneously added at a ratio of 1 : 10 to each well.
  • the plates were incubated at 37°C and 5% CO 2 for an additional 18 hours. On the following day, the plates were brought to room temperature for 20 minutes before adding 40 of SteadyLite reagent (Perkin Elmer, Catalog No. 6016989) to each well. After incubation for 15 minutes at room temperature, luminescence was read on a plate reader.
  • Synergy Score log ⁇ log/ y ⁇ max(0,l data ) (Ida t a-lLoewe) is the positive- gated, inhibition- weighted volume over Loewe additivity.
  • Example 2 Identification of therapeutic agents using a beta-galactosidase screen based on expression of alpha 7 integrin
  • Overexpression of the al integrin gene can promote cell attachment and
  • the a7 +/ ⁇ - ⁇ -gal mouse myoblast cells were cultured in T-175 flasks (Corning, Catalog
  • the highest single agent model IH S A(CX,C y ) max(I x ,I Y ) is a simple reference model where CX , Y are the coentrations of the X and Y coumpound, and I X Y are the inductions of the single agents at Cx, Y .

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Abstract

The invention features compositions, methods, and kits useful for the treatment of diseases mediated by NF-κΒ and diseases suppressed by α7 integrin.

Description

METHODS AND COMPOSITIONS FOR TREATMENT OF NF-KB-MEDIATED
AND al INTEGRIN-SUPPRESSED DISEASES
Background of the Invention
The invention relates to the treatment of diseases mediated by NF- Β or suppressed by a 7 integrin.
Nuclear factor kappa-B (NF-κΒ) is a transcription factor of the immune system. Cell stimulation by a variety of extracellular signals such as oxidative stress, cytokines, and lipopolysaccharide induces a network of events that lead to NF-κΒ activation. Activation of an NF-KB molecule is typically accompanied by its translocation from the cytoplasm into the nucleus. NF-KB activation may be triggered by degradation of Ι-κΒ, which normally sequesters NF-κΒ in the cytoplasm, or by other less characterized mechanisms. In the nucleus, activated NF-κΒ binds to DNA elements in the promoters of a number of proinflammatory gene families. In its beneficial role, NF-κΒ defends against damaging agents such as microbes and free radicals by activating the inflammatory response; however, activation of NF-κΒ is also associated with a number of disease states promoted by inflammation, including cancer, autoimmune diseases, and neurodegenerative diseases.
Encoded by the ITGA7 gene, integrin alpha chain 7 {al integrin) is a constituent of a group of heterodimeric integral membrane proteins collectively called integrins. Highly expressed in muscle cells, a 7 integrin is processed to yield light and heavy chains that join with β ΐ integrin to form α7β 1 integrin, which binds the extracellular matrix protein laminin- 1 and laminin-2. al integrin appears not only to aid appropriate anchorage of muscular tissues, but also to promote differentiation in diverse cell types (e.g., in melanocytes) and to suppress malignant potential when ectopically expressed. Based on the results of a screen identifying compounds and combinations of compounds having (1) inhibitory activity toward NF-KB-mediated gene activation, or (2) stimulatory activity toward an a7 integrin promoter element, the present invention features methods, compositions, and kits for the treatment of NF-KB-mediated diseases and diseases suppressed by a7 integrin.
In a first aspect, the invention features a method of treating a patient having or at risk for an NF-KB-mediated disease. The method includes administering to the patient a pair of agents selected from the pairs of Table 1, or analogs thereof, in amounts that are effective to treat the patient.
Table 1. Agent pairs for the treatment of NF-KB-mediated diseases
Combination Agent Pairs Combination Agent Pairs
Dilazep dihydrochloride and Dexamethasone Mitoxantrone and Demecarium Bromide
Etonogestrel and Dilazep NKH 477 and MBCQ
Prednisolone and Dilazep Epiandrosterone and Dexamethasone
Prednisolone and Ergoloid MBCQ and Ethaverine
Mitoxantrone and Etonogestrel Rosuvastatin calcium and Mitoxantrone
Prednisolone and Ethaverine Quinidine and Prednisolone
Prednisolone and Dihydroergotamine N-Methyl-Paroxetine and Mitoxantrone
Trequinsin and Mitoxantrone Bromocriptine and Bethanechol Chloride
NKH 477 and Ethaverine Prednisolone and MBCQ
Ergoloid and Dexamethasone Dexamethasone and Cilobradine
Tretinoin and Etonogestrel Otilonium Bromide and MBCQ
Prednisolone and Bromocriptine Mitoxantrone and Drotaverine
NKH 477 and Mitoxantrone Drotaverine and Calcitriol
Prednisolone and Cilobradine Dilazep and Demecarium Bromide
Verapamil and Prednisolone Prednisolone and Piperacetazine
MS-275 and Mitoxantrone MS-275 and Dexamethasone
Mitoxantrone and Ethaverine Trequinsin and Mivacurium Chloride
MS-275 and Dilazep NKH 477 and Calcitriol
Prednisolone and Dilazep NKH 477 and Mivacurium Chloride
Verapamil and Mitoxantrone Etonogestrel and Bexarotene
Trequinsin and Dexamethasone Salmeterol Xinafoate and MS-275
Etonogestrel and Calcitriol MS-275 and Etonogestrel
NKH 477 and MS-275 Vitamin A Acetate and Trequinsin
Ethaverine and Dexamethasone Otilonium Bromide and Etonogestrel
NKH 477 and Etonogestrel Verapamil and NKH 477
Mitoxantrone and Bromocriptine MS-275 and MBCQ
Mitoxantrone and Dilazep Demecarium Bromide and Calcitriol
Prednisolone and Drotaverine Mitoxantrone and Methoxsalen
Mitoxantrone and Dihydroergotamine NKH 477 and Dilazep
Etonogestrel and Dihydroergotamine Vinburnine and Prednisolone
Mitoxantrone and Calcitriol Dihydroergotamine and Calcitriol
Mivacurium Chloride and Mitoxantrone MS-275 and Ethaverine
Etonogestrel and Demecarium Bromide K-252a and Ethaverine
Prednisolone and NKH 477 Mivacurium Chloride and K-252a
Mitoxantrone and Cilobradine Procaterol and Dilazep Combination Agent Pairs Combination Agent Pairs
Mivacurium Chloride and Etonogestrel Tretinoin and Ethaverine
Prednisolone and Calcitriol Mivacurium Chloride and Calcitriol
Mitoxantrone and Deflazacort NKH 477 and Deflazacort
Trequinsin and Prednisolone Ergoloid and Calcitriol
Sulforaphane and Dihydroergotamine MS-275 and Dilazep
Trequinsin and Dilazep Dilazep and Calcitriol
Verapamil and Dexamethasone MBCQ and Deflazacort
Mitoxantrone and Ergoloid Dihydroergotamine and Deflazacort
Prednisolone and MS-275 Dilazep and Deflazacort
Dilazep and Dexamethasone Ergoloid and Deflazacort
Dihydroergotamine and Dexamethasone Ethaverine and Deflazacort
Prednisolone and Epiandrosterone MBCQ and Dilazep
Etonogestrel and Dilazep Trequinsin and Dilazep
Etonogestrel and Ethaverine Dilazep and Deflazacort
Procaterol and Etonogestrel Drotaverine and Deflazacort
Prednisolone and Mitoxantrone Dipyridamole and Dexamethasone
Trequinsin and MS-275 Prednisolone and Dipyridamole
Sulforaphane and Mitoxantrone Dipyridamole and Deflazacort
Trequinsin and NKH 477 Papaverine and Prednisolone
MBCQ and Etonogestrel Dexamethasone and Papaverine
Drotaverine and Dexamethasone AL-438 and Dilazep
Prednisolone and Mivacurium Chloride AL-438 and Papaverine
Efavirenz and Dilazep AL-438 and Ergoloid
Mitoxantrone and MBCQ Dexamethasone and Zardaverine
Levalbuterol and Dilazep AL-438 and Dihydroergotamine
Unithiol Monohydrate and Etonogestrel Deflazacort and Zardaverine
Verapamil and Etonogestrel Prednisolone and Zardaverine
MS-275 and Deflazacort Deflazacort and Papaverine
Vinburnine and Mitoxantrone 2-(4-acetoxyphenyl)-2-chloro-N-methyl- ethylammonium chloride and Dilazep
Vitamin A Acetate and Mitoxantrone Dipyridamole and Mitoxantrone
Prednisolone and N-Methyl-Paroxetine AL-438 and Ethaverine
NKH 477 and Dilazep Mitoxantrone and Papaverine
Tretinoin and MS-275 2-(4-acetoxyphenyl)-2-chloro-N-methyI- ethylarnmonium chloride and Ergoloid
Mitoxantrone and Dexamethasone 2-(4-acetoxyphenyl)-2-chIoro-N-methyl- ethylammonium chloride and Papaverine
Dexamethasone and Bromocriptine Mitoxantrone and Tetrahydropapaveroline
Procaterol and Mivacurium Chloride Dexamethasone and Tetrahydropapaveroline
Trequinsin and Deflazacort Prednisolone and Tetrahydropapaveroline
Prednisolone and Amoxapine Deflazacort and Tetrahydropapaveroline
Etonogestrel and Bromocriptine 2-(4-acetoxyphenyl)-2-chloro-N-methyl- ethylammonium chloride and Ethaverine
NKH 477 and Ergoloid 2-(4-acetoxyphenyl)-2-chloro-N-methyl- ethylammonium chloride and Dihydroergotamine
Dilazep and Dihydroergotamine
NF-KB-mediated diseases include, e.g., inflammatory myopathies and cachexia. In another aspect, the invention features a method of treating a patient having or at risk for a disease suppressed by al integrin. This method includes administering to the patient a pair of agents selected from the pairs of Table 2, or analogs thereof, in amounts that are effective to treat the patient.
Table 2. Agent pairs for the treatment of ITGA7-suppressed diseases
Combination Agent Pairs Combination Agent Pairs
Dipyridamole and MBCQ MS-275 and Nl N 12-diethylspermine 4HCL
Everolimus and N-(2-Aminoethyl)-5- LY 294002 and MBCQ
Isoquinolinesulfonamide
Ethaverine and MBCQ Methyldopa and Tretinoin
EHNA and Everolimus Donepezil and Tretinoin
Everolimus and Fasudil Berberine and Florfenicol
Dipyridamole and Everolimus Dopamine and MBCQ
Dilazep and MBCQ Levalbuterol and MBCQ
MBCQ and Nl Nl 2-diethylspermine 4HCL Antimycin A and LY 294002
Berberine and Papaverine Dilazep and MS-275
Fasudil and LY 294002 Alendronate and Ergoloid
Berberine and MBCQ LY 294002 and N-(2-Aminoethyl)-5- Isoquinolinesulfonamide
Adefovir Dipivoxil and LY 294002 Berberine and Everolimus
Antimycin A and MBCQ Ergoloid and N-(2-Aminoethyl)-5- Isoquinolinesulfonamide
10-Hydroxycamptothecin and MBCQ Adefovir Dipivoxil and Fasudil
Berberine and Fasudil Ethaverine and N-(2-Aminoethyl)-5- Isoquinolinesulfonamide
Dipyridamole and S-Petasin Adefovir Dipivoxil and Droxidopa
Everolimus and MBCQ Fasudil and Physostigmine
MS-275 and N-(2-Aminoethyl)-5-Isoquinolinesulfonamide Ergoloid and Pamidronate
Idebenone and Tretinoin Dipyridamole and N-(2-Aminoethyl)-5- Isoquinolinesulfonamide
10-Hydroxycamptothecin and Idebenone Antimycin A and MS-275
Adefovir Dipivoxil and MBCQ EHNA and Idebenone
MBCQ and Simvastatin 10-Hydroxycamptothecin and Donepezil
MBCQ and Suberoylanilide Hydroxamic Acid EHNA and S-Petasin
2-(p-Hydroxyanilino)-4-(p-chlorophenyl) thiazole and Physostigmine and S-Petasin
Everolimus
MBCQ and PDTC, NH4 Dipyridamole and Florfenicol
MBCQ and Tretinoin Berberine and Fumagillin
Dilazep and Everolimus Fasudil and MBCQ
Deguelin and Fasudil Fumagillin and Physostigmine
LY 294002 and Physostigmine 10-Hydroxycamptothecin and Fumagillin
10-Hydroxycamptothecin and LY 294002 Berberine and MS-275
Adefovir Dipivoxil and Physostigmine Dipyridamole and Ergoloid
Everolimus and Physostigmine EHNA and MS-275
Ethaverine and Tadalafil Levalbuterol and Physostigmine
Deguelin and MBCQ Adefovir Dipivoxil and Ergoloid
Deguelin and MS-275 PDTC, NH4 and Physostigmine
Idebenone and MS-275 Alendronate and N-(2-Aminoethyl)-5- Isoquinolinesulfonamide
EHNA and MBCQ Adefovir Dipivoxil and Fumagillin
EHNA and Fumagillin EHNA and Nl Nl 2-diethylspermine 4HCL
Deguelin and N-(2-Aminoethyl)-5- Ergoloid and Methyldopa
Isoquinolinesulfonamide
Fasudil and N-(2-Aminoethyl)-5-Isoquinolinesulfonamide Andrographis and Fumagillin
10-Hydroxycamptothecin and EHNA Deguelin and Fumagillin
MBCQ and Physostigmine EHNA and Ergoloid Combination Agent Pairs Combination Agent Pairs
MBCQ and N-(2-Aminoethyl)-5-Isoquinolinesulfonamide 10-Hydroxycamptothecin and Everolimus
MBCQ and S-Petasin Everolimus and Methyldopa
MBCQ and Pamidronate Methyldopa and Simvastatin
Dipyridamole and Methyldopa Everolimus and Florfenicol
Fasudil and Nl 12-diethylspermine 4HCL Berberine and Etazolate
Everolimus and Tretinoin Droxidopa and Fasudil
Florfenicol and MBCQ Adefovir Dipivoxil and Tadalafil
Fumagillin and Nl Nl 2-diethylspermine 4HCL Alendronate and MS-275
Fumagillin and N-(2-Aminoethyl)-5- Adefovir Dipivoxil and Methyldopa
Isoquinolinesulfonamide
N-(2-Aminoethyl)-5-Isoquinolinesulfonamide and Alendronate and Idebenone
Nl N 12-diethylspermine 4HCL
EHNA and N-(2-Aminoethyl)-5-Isoquinolinesulfonamide Berberine and S-Petasin
Physostigmine and Tretinoin Ergoloid and Isoetharine
EHNA and Florfenicol Adefovir Dipivoxil and Ethaverine
Dipyridamole and Fumagillin Fumagillin and PDTC, NH4
Deguelin and Droxidopa 10-Hydroxycamptothecin and Droxidopa
Fumagillin and MBCQ Fasudil and Simvastatin
Ethaverine and Fasudil Physostigmine and Tadalafil
Ethaverine and Everolimus 10-Hydroxycamptothecin and Berberine
10-Hydroxycamptothecin and Etazolate Methyldopa and Nl Nl 2-diethylspermine 4HCL
N-(2-Aminoethyl)-5-Isoquinolinesulfonamide and Florfenicol and S-Petasin
Pamidronate
Deguelin and Simvastatin EHNA and Physostigmine
Deguelin and Everolimus 10-Hydroxycamptothecin and Fasudil
Ergoloid and Everolimus MBCQ and Tadalafil
Ethaverine and LY 294002 Berberine and Ethaverine
Ethaverine and MS-275 EHNA and Methyldopa
Physostigmine and Simvastatin Berberine and EHNA
Deguelin and LY 294002 Droxidopa and MBCQ
Dilazep and Ergoloid 10-Hydroxycamptothecin and Pamidronate
Dipyridamole and MS-275 Andrographis and MBCQ
10-Hydroxycamptothecin and Dipyridamole MS-275 and PDTC, NH4
Idebenone and MBCQ Berberine and Ergoloid
Berberine and N-(2-Aminoethyl)-5- 10-Hydroxycamptothecin and Methyldopa
Isoquinolinesulfonamide
Nl Nl 2-diethylspermine 4HCL and Pamidronate 10-Hydroxycamptothecin and Florfenicol
EHNA and Pamidronate MBCQ and Physostigmine
Droxidopa and Simvastatin Fasudil and Methyldopa
Droxidopa and Tretinoin
Diseases suppressed by ct7 integrin include cancer, vasculoproliferative diseases, and atherosclerosis.
In the methods of the invention, the agents of a pair may be administered within 28 days, 21 days, 14 days, 10 days, 7 days, 3 days, 2 days, 24 hours, 12 hours, six hours, two hours, or one hour of each other, or substantially simultaneously. Agents may be
administered by any acceptable route (e.g., by oral, systemic, parenteral, topical (e.g., ophthalmic, dermatologic), intravenous, inhalational, or intramuscular administration). Optionally, the methods of the invention may be performed in conjunction with the administration of additional agents appropriate for the NF-KB-mediated disease or the disease suppressed by al integrin. The patient to be treated may be any animal (e.g., a human).
In another aspect, the invention features a composition that includes a pair of agents selected from the pairs of agents of Table 1 or Table 2, or analogs of these agents. In one particular embodiment, the composition optionally contains excipients, while the only active agents in the composition are the pair of agents of the invention (e.g., a pair of agents of Table 1 or Table 2).
Desirably, in any of the compositions of the invention, the two agents are present in amounts that, when administered together to a patient having or at risk for an NF-KB- mediated disease or a disease suppressed by al integrin, are effective to treat the patient. Compositions of the invention may be formulated, e.g., for oral, systemic, parenteral, topical (e.g., ophthalmic, dermatologic), intravenous, inhalational, or intramuscular administration.
In another aspect, the invention features a kit including a pair of active agents. In one embodiment, the kit contains a pair of agents selected from the pairs of agents of Table 1 or analogs thereof and includes instructions for administering the agents to a patient having or at risk for an NF-KB-mediated disease. In another embodiment, the kit contains a pair of agents selected from the pairs of agents of Table 2 or analogs thereof and includes
instructions for administering the agents to a patient having or at risk for a disease suppressed by al integrin. The pair of agents may be included together in a composition or may be formulated separately.
Related embodiments of the invention are kits including a first agent of a pair of agents and instructions for administering the first agent together with the second agent of the pair to a patient having or at risk for a disease. In one embodiment, the pair is selected from the pairs of agents of Table 1 or analogs of these agents, and the disease is a disease mediated by NF-KB. In another embodiment, the pair is selected from the pairs of agents of Table 2 or analogs of these agents, and the disease is a disease suppressed by al integrin. In any of the kits of the invention, one or more active agents may be formulated, e.g., for oral, parenteral, systemic, topical, or inhalational administration.
In any of the compositions, methods, and kits of the invention, an analog of any agent listed in Table 1 or Table 2 may be used instead of the agent listed in Table 1 or Table 2.
Compounds useful in the invention include those described herein in any of their pharmaceutically acceptable forms, including isomers such as diastereomers and
enantiomers, salts, solvates, and polymorphs thereof, as well as racemic mixtures.
Compounds useful in the invention may also be isotopically labeled compounds. Useful isotopes include hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, (e.g., 2H, 3H, 13C, 14C, 15N, 180, 170, 31P, 32P, 35S, 18F, and 36C1). Isotopically-labeled compounds can be prepared by synthesizing a compound using a readily available isotopically-labeled reagent in place of a non-isotopically-labeled reagent.
By "agent" is meant a compound, e.g., dipyridamole, or mixture of compounds, e.g., ergoloid mesylates, having a pharmacological activity. The terms "agent," "compound," and "drug" are used interchangeably herein.
The term "NF-κΒ" comprises a family of dimeric transcription factors containing a class I NF-KB protein, e.g., a protein encoded by the NFKBl or NFKB2 gene in humans.
By an "NF-KB-mediated disease" is meant any disease or adverse health condition whose pathogenesis is associated with activation of NF-κΒ. Exemplary NF-KB-mediated diseases are provided herein.
By a "disease suppressed by ot7 integrin" is meant any disease or adverse health condition whose symptoms or pathogenesis is reduced by expression of a7 integrin.
Exemplary diseases suppressed by ! integrin are provided herein.
By "glucocorticoid" is meant a synthetic or natural steroid hormone that binds the glucocorticoid receptor, preferably with selectivity over the mineralocorticoid receptor. By "ENT inhibitor" is meant an agent that inhibits the activity of an equilibrative nucleoside transporter, e.g., an equilibrative adenosine transporter, in vitro, in vivo, or both, by at least 5%, e.g., by 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95%.
By "PDE inhibitor" is meant an agent that inhibits a phosphodiesterase enzyme in vitro, in vivo, or both, by at least 5%, e.g., by 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95%. A PDE inhibitor may be selective for a particular type of PDE, e.g., PDE type V (PDE5).
By an "acetylcholinesterase inhibitor" is meant an agent that inhibits the activity of an acetylcholinesterase in vitro, in vivo, or both by at least 5%, e.g., by 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95%.
By a "Rho kinase inhibitor" is meant an agent that inhibits that activity of Rho kinase in vitro, in vivo, or both by at least 5%, e.g., by 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95%. In vitro kinase assays or cell-based bioassays, e.g., using fluorescence microscopy, may be used to detect and measure the Rho kinase inhibitory activity of an agent.
By an "mTOR inhibitor" is meant a compound that inhibits the activity of mTOR, also known as FK506 binding protein 12-rapamycin associated protein 1 (FRAP1), in vitro, in vivo, or both by at least 5%, e.g., by 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95%.
By a "calcium channel blocker" is meant an agent that directly or indirectly inhibits an activity of a calcium channel, e.g., current frequency, by at least 5%, e.g., by 10%, 25%, 50%, 60%, 70%, 80%, 90%, or 95%. Blocking (inhibitory) activity can be measured by methods known in the art.
By a "bisphosphonate" is meant one of a class of compounds that has two phosphate groups. The generic chemical structure of bisphosphonates is described herein. Many bisphosphonates can prevent the loss of bone mass or lower blood calcium when
administered to a patient.
By a "CoQIO analog" is meant an analog of coenzyme Q10, also known as
ubiquinone. By a "corticosteroid" is meant a natural or synthetic steroid hormone that binds either glucocorticoid receptors, mineralocorticoid receptors, or both.
By "patient" is meant any animal, e.g., a human.
As used herein, the term "treating" refers to administering a pharmaceutical composition for therapeutic purposes. The therapeutic purpose may be, e.g., to prevent a disease or symptom in a person at risk for the disease or symptom, or to ameliorate or stabilize the condition of a person already suffering from the disease or symptom. Desirably, treating results in prevention of a disease or symptom, or amelioration of a disease or symptom by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
Alternatively, in certain embodiments, treatment is observed by a trained physician or other person skilled in the art as an appreciable or substantial relief of symptoms in a patient with an NF-KB-mediated disease or a disease suppressed by a7-integrin.
By "an effective amount" is meant the amount of an agent, alone or in combination with another agent, required to treat a patient with an NF-KB-mediated disease or a disease suppressed by a7-integrin in a clinically relevant manner. A sufficient amount of an active agent used to practice the present invention for therapeutic treatment of an NF-KB-mediated disease or a disease suppressed by a7-integrin varies depending upon the manner of administration, the age, body weight, and general health of the patient. Ultimately, the prescriber will decide the appropriate amount and dosage regimen. In a combination therapy of the invention, the effective amount of an agent may less be than the effective amount if the agent were administered in a non-combinatorial (single-agent) therapy. Additionally, an effective amount may be an amount of an agent in a combination therapy of the invention that is safe and efficacious in the treatment of a patient having an NF-KB-mediated disease or a disease suppressed by a7-integrin over each agent alone as determined and approved by a regulatory authority (such as the U.S. Food and Drug Administration).
By "more effective" is meant that a treatment exhibits greater efficacy, or is less toxic, safer, more convenient, or less expensive than another treatment with which it is being compared. Efficacy may be measured by a skilled practitioner using any standard method that is appropriate for a given indication.
By a "low dosage" is meant at least 5% less (e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) than the lowest standard recommended dosage of a particular compound formulated for a given route of administration for treatment of any human disease or condition. For example, a low dosage of an agent that treats an inflammatory myopathy and that is formulated for administration by intravenous injection will differ from a low dosage of the same agent formulated for oral administration.
In the generic descriptions of compounds of this invention, the number of atoms of a particular type in a substituent group may be given as a range, e.g., an alkyl group containing from 1 to 4 carbon atoms or alkyl. Reference to such a range is intended to include specific references to groups having each of the integer number of atoms within the specified range. For example, an alkyl group from 1 to 4 carbon atoms includes each of Q, C2, C3, and C4. A Q_12 heteroalkyl, for example, includes from 1 to 12 carbon atoms in addition to one or more heteroatoms. Other numbers of atoms and other types of atoms may be indicated in a similar manner. The term "lower," when referring to a particular substituent group, e.g., "lower alkyl" or "lower alkoxy," generally refers to groups containing 5, 4, or fewer carbon atoms.
As used herein, the terms "alkyl" and the prefix "alk-" are inclusive of both straight chain and branched chain groups and of cyclic groups, i.e., cycloalkyl. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 12 ring carbon atoms, inclusive. Exemplary cyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups.
By "Cj_4 alkyl" is meant a branched or unbranched hydrocarbon group having from 1 to 4 carbon atoms. A alkyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. alkyls include, without limitation, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl, sec- butyl, tert-butyl, and cyclobutyl.
By alkenyl" is meant a branched or unbranched hydrocarbon group containing one or more double bonds and having from 2 to 4 carbon atoms. A C2-4 alkenyl may optionally include monocyclic or polycyclic rings, in which each ring desirably has from three to six members. The C2_4 alkenyl group may be substituted or unsubstituted.
Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. C2_4 alkenyls include, without limitation, vinyl, allyl, 2-cyclopropyl-l-ethenyl, 1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2 -methyl- 1-propenyl, and 2-methyl-2-propenyl.
By "C2_4 alkynyl" is meant a branched or unbranched hydrocarbon group containing one or more triple bonds and having from 2 to 4 carbon atoms. A C2_4 alkynyl may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has five or six members. The C2_4 alkynyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. C2_4 alkynyls include, without limitation, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl.
By "C2-6 heterocyclyl" is meant a stable 5- to 7-membered monocyclic or 7- to 14- membered bicyclic heterocyclic ring which is saturated, partially unsaturated, or unsaturated (aromatic), and which consists of 2 to 6 carbon atoms and 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, (^substituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. The nitrogen and sulfur heteroatoms may optionally be oxidized. The heterocyclic ring may be covalently attached via any heteroatom or carbon atom which results in a stable structure, e.g., an imidazolinyl ring may be linked at either of the ring-carbon atom positions or at the nitrogen atom. A nitrogen atom in the heterocycle may optionally be quatemized. Preferably when the total number of S and O atoms in the heterocycle exceeds 1, then these heteroatoms are not adjacent to one another. Heterocycles include, without limitation, lH-indazole, 2- pyrrolidonyl, 2H,6H-l,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl, 4-piperidonyl, 4aH- carbazole, 4H-quinolizinyl, 6H-l,2,5-thiadiazinyl, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl, carbazolyl, 4aH- carbazolyl, b-carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-
1.5.2- dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,
isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,
1.2.3- oxadiazolyl, 1,2,4-oxadiazolyl, 1,2, 5 -oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinylperimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole,
pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-l,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4- thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl,
thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4- triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl. Preferred 5 to 10 membered heterocycles include, but are not limited to, pyridinyl, pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, tetrazolyl, benzofuranyl, benzothiofuranyl, indolyl, benzimidazolyl, lH-indazolyl, oxazolidinyl, isoxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, quinolinyl, and isoquinolinyl. Preferred 5 to 6 membered heterocycles include, without limitation, pyridinyl, pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl, piperazinyl, piperidinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, and tetrazolyl.
By "C6-12 aryl" is meant an aromatic group having a ring system comprised of carbon atoms with conjugated π electrons (e.g., phenyl). The aryl group has from 6 to 12 carbon atoms. Aryl groups may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has five or six members. The aryl group may be substituted or unsubstituted. Exemplary substituents include alkyl, hydroxy, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, fluoroalkyl, carboxyl, hydroxyalkyl, carboxyalkyl, amino, aminoalkyl, monosubstituted amino, disubstituted amino, and quaternary amino groups.
By "C7_i4 alkaryl" is meant an alkyl substituted by an aryl group (e.g., benzyl, phenethyl, or 3,4-dichlorophenethyl) having from 7 to 14 carbon atoms.
By "C3_!o alkheterocyclyl" is meant an alkyl substituted heterocyclic group having from 3 to 10 carbon atoms in addition to one or more heteroatoms (e.g., 3-furanylmethyl, 2- furanylmethyl, 3-tetrahydrofuranylmethyl, or 2-tetrahydrofuranylmethyl).
By "Ci_7 heteroalkyl" is meant a branched or unbranched alkyl, alkenyl, or alkynyl group having from 1 to 7 carbon atoms in addition to 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. Heteroalkyls include, without limitation, tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. A heteroalkyl may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has three to six members. The heteroalkyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, hydroxyalkyl,
carboxyalkyl, and carboxyl groups. Examples of d_7 heteroalkyls include, without limitation, methoxymethyl and ethoxyethyl.
By "halide" or "halogen" is meant bromine, chlorine, iodine, or fluorine.
By "fluoroalkyl" is meant an alkyl group that is substituted with a fluorine atom.
By "perfluoroalkyl" is meant an alkyl group consisting of only carbon and fluorine atoms.
By "carboxyalkyl" is meant a chemical moiety with the formula
-(R)-COOH, wherein R is selected from Q_7 alkyl, C2_7 alkenyl, C2_7 alkynyl, C2_6
heterocyclyl, C6_i2 aryl, C7_i4 alkaryl, C3_io alkheterocyclyl, or Q_7 heteroalkyl.
By "hydroxyalkyl" is meant a chemical moiety with the formula -(R)-OH, wherein R is selected from Q_7 alkyl, C2_7 alkenyl, C2_7 alkynyl, heterocyclyl,
C6-i2 aryl, C7_14 alkaryl, C3_10 alkheterocyclyl, or Ci_7 heteroalkyl.
By "alkoxy" is meant a chemical substituent of the formula -OR, wherein R is selected from Ci_7 alkyl, C2_7 alkenyl, C2_7 alkynyl, C2_6 heterocyclyl, C6_i2 aryl, C7_]4 alkaryl, C3_10 alkheterocyclyl, or C]_7 heteroalkyl.
By "aryloxy" is meant a chemical substituent of the formula -OR, wherein R is a C6-12 aryl group.
By "alkylthio" is meant a chemical substituent of the formula -SR, wherein R is selected from Q_7 alkyl, C2_7 alkenyl, C2-7 alkynyl, C2_6 heterocyclyl, C n aryl, C7_]4 alkaryl, C3_io alkheterocyclyl, or Q_7 heteroalkyl.
By "arylthio" is meant a chemical substituent of the formula -SR, wherein R is a C<3_i2 aryl group.
By "quaternary amino" is meant a chemical substituent of the formula
-(R)-N(R')(R")(R"')+, wherein R, R', R", and R'" are each independently an alkyl, alkenyl, alkynyl, or aryl group. R may be an alkyl group linking the quaternary amino nitrogen atom, as a substituent, to another moiety. The nitrogen atom, N, is covalently attached to four carbon atoms of alkyl, heteroalkyl, heteroaryl, and/or aryl groups, resulting in a positive charge at the nitrogen atom.
Other features and advantages of the invention will be apparent from the following Detailed Description and the claims.
Detailed Description of the Invention
Using cell lines engineered to contain a reporter of either NF-KB-mediated gene activation or transcriptional activation via a7 integrin promoter elements, we have identified compounds that inhibit the activation of NF-κΒ or that enhance expression of integrin ct7. Accordingly, the present invention provides compositions, methods, and kits useful in therapies for diseases treatable by suppression of NF-κΒ activity (i.e., NF-KB-mediated diseases or by augmentation of integrin al expression (i.e., diseases suppressed by a7 expression). Exemplary diseases amenable to treatment according to the methods of the invention are described below.
Treatment methods of the invention include administration of any pair of agents selected from the pairs of Table 1 or Table 2. Compositions of the invention can include a combination including any pair of agents selected from the pairs of Table 1 or Table 2.
Optionally, in any of the compositions, methods, and kits of the invention, functional or structural analogs (e.g., an analog described herein) of these agents may be employed instead of an agent listed in Table 1 or Table 2. In one aspect, the invention may function by decreasing NF-κΒ expression, phosphorylation, nuclear translocation, or transcriptional activity, by promoting the activity of one or more pathways opposing that of NF-κΒ, or by any other mechanism that suppresses an activity of NF-κΒ. In another aspect, the invention may function by increasing ITGA7 gene expression via oc7 integrin promoter sequences or by any post-transcriptional mechanism (e.g., RNA stabilization). In one particular example, the patient being treated is administered a combination of two agents listed in Table 1 within 7 days of each other in amounts that together are sufficient to treat the patient having an NF-κΒ -mediated disease. In a second example, the patient being treated is administered a combination of two agents listed in Table 2 within 7 days of each other in amounts that together are sufficient to treat the patient having a disease suppressed by l integrin. In either example, desirably, an effective amount of one or both of the agents is a low dosage relative to the effective amount of the agent when administered singly for treating the disease, or relative to the standard dosage of the agent for treating another disease.
Indications
Diseases mediated by NF-κΒ (i.e., an NF-KB-mediated disease) include muscle- wasting disease such as an inflammatory myopathy or cachexia. Inflammatory myopathies include without limitation dermatomyositis, polymyositis, inclusion body myositis, juvenile myositis, myopathic dropped head syndrome, infection-associated myopathies (e.g., myopathies caused by viral, bacterial, fungal, protozoal, or helminthic infection), focal myositis, nodular myositis eosinophilic myofasciitis, macrophagic myo fasciitis, ocular myositis, granulomatous myositis, and acute necrotizing myopathy, and myositis associated with connective tissue disease (e.g., polymyalgia rheumatica, rheumatoid arthritis, systemic sclerosis, and tendonitis), and myopathies caused by sepsis, diabetes, acute uremia, starvation, and end-stage kidney disease. Other muscle- wasting diseases that may be treated are muscle injury/trauma (e.g., injury-induced fibrosis and regeneration), sarcopenia, muscle atrophies associated with denervation or immobilization (e.g., myositis caused by
neuromuscular or neurological diseases, e.g., amyotrophic lateral sclerosis, myasthenia gravis, multiple sclerosis, Guillain-Barre syndrome, and motor neuron disease). Cachexia may occur, e.g., in patients suffering from cancer (cancer cachexia), human
immunodeficiency virus (and acquired immunodeficiency syndrome), congestive heart failure, and chronic obstructive pulmonary disease.
Expression of al integrin has been associated with protection from several disease processes. For example, forced expression of al integrin in a7-null metastatic melanoma cancer cells suppresses cell migration, tumor growth and metastasis. Consistent with this observation, low levels of oc7 integrin are associated with clinical relapse of human
malignancies including prostate cancers, hepatocellular carcinomas, and leiomyosarcoma. Thus, over-expression of al integrin reverses the malignant potential of cancer cells. Loss of al integrin has also been linked to hyperplasia of vascular smooth muscle cells and reduced cell survival resulting from atherosclerosis. Upregulation of al integrin is expected to improve these conditions. Accordingly, upregulation of al integrin expression by the methods of the invention is useful for treating, e.g., cancer, vasculoproliferative diseases (e.g., stenosis, restenosis, and neointimal hyperplasia), asthma, and atherosclerosis.
Cancers that may be treated using the methods of the invention include, for example, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., hepatocellular carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, pleuropulmonary blastoma, and lung carcinoid tumor), colorectal cancer, ovarian cancer (e.g., ovarian adenocarcinoma), prostate cancer, gastric cancer, esophageal cancer, head and neck cancer, and thyroid cancer.
Agents of the invention
In various embodiments of the present invention, particular therapeutic agents may be employed. Certain agents and exemplary analogs are discussed in greater detail below. It is to be understood that an analog of any agent of Table 1 or Table 2 can be used instead of the agent of Table 1 or Table 2 in the methods, compositions, and kits of the invention.
Glucocorticoids
In certain embodiments, the methods, compositions, and kits of the invention employ predisolone or deflazacort. Analogs of prednisolone and deflazacort include their respective structural analogs and other glucocorticoids. 2-(4-acetoxyphenyl)-2-chloro-N-methyl- ethylammonium chloride, a compound with glucocorticoid-like anti-inflammatory properties, is also considered herein to be an analog of prednisolone and deflazacort.
Prednisolone
Prednisolone is described in U.S. Patent Nos. 2,837,464 and 3,134,718 and has the following structure:
Figure imgf000020_0001
Analogs of prednisolone include 5-keto-4,5-seco-3-ynes of the estrane, androstane, and pregnane described in U.S. Patent No. 3,835,160; the 17-benzoate of prednisolone described in U.S. Patent No. 3,857,941; compounds of formula I in U.S. Patent No.
7,498,321, e.g., 6a,9a-difluoro-l i p-hydroxy-17a-[(isoxazole-5-carbonyl)oxy]-16a-methyl- 3-oxo-androsta-l,4-diene-^-carbothioic acid S-fluoromethyl ester; 17a-[(5- chlorothiophene-2-carbonyl)oxy]-6a,9a-difluoro-methyl ester; 1 i -hydroxy-16 -methyl-3- oxo-androsta-l,4-diene-17 -carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro-17a- [(3,5-dimethylisoxazole-4-carbonyl)oxy]-l 1 -hydroxy-16a-methyl-3-oxo-androsta-l ,4- diene- 17p-carbothioic acid S-fluoromethyl ester; 17a-[(5-chloro-4-methoxy-thiophene-3- carbonyl)oxy]-6a,9a-difluoro- 11 β-hydroxy- 16a-methyl-3-oxo-androsta- 1 ,4-diene- 17β- carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro-l l -hydroxy-16a-methyl-17a-[(4- methyl- 1 ,2,3-thiadiazole-5-carbonyl)oxy]-3-oxo-androsta- 1 ,4-diene- 17P-carbothioic acid S- fluoromethyl ester; 17a-[(3-bromothiophene-2-carbonyl)oxy]-6a,9a-difluoro-l-l β-hydroxy- 16a-methyl-3-oxo-androsta-l,4-diene-17p-carbothioic acid S-fluoromethyl ester; 17a-[(2,5- dichlorothiophene-3-carbonyl)oxy]-6a,9a-difluoro- 11 β-hydroxy- 16a-methyl-3-oxo- androsta-l,4-diene-17β-carbothioic acid S-fluoromethyl ester; 17a-[(3-chlorothiophene-2- carbonyl)oxy]-6a,9a-difluoro- 11 β-hydroxy- 16a-methyl-3-oxo-androsta- 1 ,4-diene- 17β- carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro-l ^-hydroxy-16a-methyl-17a-[(-5- methylisoxazole-4-carbonyl)oxy]-3-oxo-androsta- 1 ,4-diene- 17β^Λοί1ιϊοΐο acid S- fluoromethyl ester; 6a,9a-difluoro-l ^-hydroxy-16a-methyl-17a-[(-l-methyl-lH-pyrrole-2- carbonyl)oxy]-3-oxo-androsta-l,4-diene-17 -carbothioic acid S-fluoromethyl ester; 6α,9α- difluoro- 11 β-hydroxy- 16a-methyl-3-oxo- 17a-[(l ,3-thiazole-4-carbonyl)oxy]-androsta- 1 ,4- diene- 17 β-carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro-17a-[(2,4-dimethyl-l,3- thiazole-5-carbonyl)oxy]- 11 β-hydroxy- 16a-methyl-3-oxo-androsta- 1 ,4-diene- 17 β- carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro-l i -hydroxy-16a-methyl-17a-[(-5- methylisoxazole-3-carbonyl)oxy]-3-oxo-androsta-l ,4-diene- 17 -carbothioic acid S- fluoromethyl ester; 6a,9a-difluoro-l i -hydroxy-16 -methyl-17a-[(3-methylisoxazole-5- carbonyl)oxy]-3-oxo-androsta-l,4-diene-17p-carbothioic acid S-fluoromethyl ester; 6α,9α- difluoro- 17 a- [(1 ,3 -dimethyl- lH-pyrazole-5-carbonyl)oxy]- 11 β-hydroxy- 16a-methyl-3-oxo- androsta-l,4-diene-17β-carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro-l ^-hydroxy- 17a-[(isoxazole-3-carbonyl)oxy]- 16a-methyl-3-oxo-androsta- 1 ,4-diene- 17p-carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro-l ^-hydroxy-17a-[(4-methoxy-thiophene-3- carbonyl)oxy]- 16a-methyl-3-oxo-androsta- 1 ,4-diene- 17P-carbothi()ic acid S-fluoromethyl ester; 6a,9a-difluoro- 11 β-hydroxy- 16a-methyl- 17a-[(2-methyl- 1 ,3-thiazole-4- carbonyl)oxy]-3-oxo-androsta-l,4-diene-^-carbothioic acid S-fluoromethyl ester; 6α,9α- difluoro- 17a-[(3-ethoxy-thiophene-2-carbonyl)oxy]- 11 β-hydroxy- 16a-methyl-3-oxo- androsta-l,4-diene-17β-carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro-l ^-hydroxy- 16a-methyl-3-oxo- 17a-[(l ,2,3-thiadiazole-4-carbonyl)oxy]-androsta- 1 ,4-diene- 17β- carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro-l ^-hydroxy-16a-methyl-3-oxo-17a- [(lH-pyrrole-2-carbonyl)oxy]-androsta-l,4-diene-17β-carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro- 11 β-hydroxy- 16a-methyl-3-oxo- 17a-[(l ,3-thiazole-5-carbonyl)oxy]- androsta-l,4-diene-17β-carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro-l ^-hydroxy- 16a-methyl-3-oxo- 17a-[( 1 ,2,5-thiadiazole-3-carbonyl)oxy]-androsta- 1 ,4-diene- 17β- carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro-l i -hydroxy-17a-[(isothiazole-3- carbonyl)oxy]- 16a-methyl-3-oxo-androsta- 1 ,4-diene- 17β-θ3Λοώίοίο acid S-fluoromethyl ester; 6a,9a-difluoro- 11 β-hydroxy- 17a-[(isothiazole-5-carbonyl)oxy]- 16a-methyl-3-oxo- androsta-l,4-diene-17P-carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro-l i p-hydroxy- 16a-methyl- 17a-[(5-methylthiophene-2-carbonyl)oxy]-3-oxo-androsta- 1 ,4-diene- 17β- carbothioic acid S-fluoromethyl ester; 6a,9a-difluoro-l i -hydroxy-16a-methyl-17a-[(-3- methylthiophene-2-carbonyl)oxy]-3-oxo-androsta-l,4-diene-17 -carbothioic acid S- fluoromethyl ester; 6a,9a-difluoro-l i -hydroxy-16a-methyl-17a-[(-4-methyl-l,3-thiazole- 5-carbonyl)oxy]-3-oxo-androsta-l,4-diene-17 -carbothioic acid S-fluoromethyl ester; 17a- [( 1 -ethyl-3 -methyl- lH-pyrazole-5-carbonyl)oxy]-6a,9a-difluoro- 11 β-hydroxy- 16a-methyl- 3-oxo-androsta-l,4-diene-17P-carbothioic acid 5-fluoromethyl ester; 6a,9a-difiuoro-17a- [( 1 -methyl- lH-imidazole-5-carbonyl)oxy]- 11 β-hydroxy- 16a-methyl-3-oxo-androsta- 1 ,4- diene- 17p-carbothioic acid S-fluoromethyl ester; and 6a,9a-difluoro-l ip-hydroxy-16a- methyl-3-oxo- 17a-[( 1 ,2,3-thiadiazole-5-carbonyl)oxy]-androsta- 1 ,4-diene- 17p-carbothioic acid S-fluoromethyl ester. Other exemplary analogs of prednisolone are described in U.S. Patent Nos. 3,857,941, 3,956,349, 4,035,236, 4,041,055 and 5,225,335.
Deflazacort
Deflazacort is described in Belgian Patent No. 679,820, G. B. Patent No. 1,077,393, and U.S. Patent No. 3,436,389. Deflazacort has the structure:
Figure imgf000022_0001
Analogs of deflazacort are described by the generic formula of U.S. Patent No.
3,624,077, e.g., pregna-l,4-diene-l lp,21-diol-3,20-dione-[17a,16a-d]-2'-phenyloxazoline
21-acetate; by formula I of U.S. Patent No. 4,412,953, e.g., 3 -hydroxy-5-pregnen-20-one-
[17a,16a-d]-2'-methyloxazoline; and by formula I of U.S. Patent No. 4,440,764, e.g., 11 β ,21 -dihydroxy-2 '-methyl-5 Ή-pregna- 1 ,4-dieno[ 17,16-d]-oxazole-3 ,20-dione-21 - hemisuccinate.
Other exemplary glucocorticoids are dexamethasone, betamethasone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, triamcinolone hexacetonide,
beclomethasone, dipropionate, beclomethasone dipropionate monohydrate, flumethasone pivalate, diflorasone diacetate, fluocinolone acetonide, fluorometholone, fluorometholone acetate, clobetasol propionate, desoximethasone, fluoxymesterone, fluprednisolone, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone cypionate, hydrocortisone probutate, hydrocortisone valerate, cortisone acetate, paramethasone acetate,
methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, clocortolone pivalate, flucinolone, dexamethasone 21 -acetate, betamethasone 17- valerate, isoflupredone, 9-fluorocortisone, 6-hydroxydexamethasone, dichlorisone, meclorisone, flupredidene, doxibetasol, halopredone, halometasone, clobetasone, diflucortolone, isoflupredone acetate, fluorohydroxyandrostenedione, beclomethasone, flumethasone, diflorasone, clobetasol, cortisone, paramethasone, clocortolone, prednisolone 21 -hemisuccinate free acid,
prednisolone metasulphobenzoate, prednisolone terbutate, triamcinolone acetonide 21- palmitate, flurometholone, medrysone, loteprednol, fluazacort, betamethasone, prednisone, methylprednisolone, triamcinolone, hexacatonide, paramethasone acetate, diflorasone, fluocinolone and fluocinonide.
Equilibrative nucleoside transporter inhibitors
The compounds dipyridamole and dilazep are ENT inhibitors that may be used in the methods, compositions, and kits of the invention. Analogs of dipyridamole and dilazep include other ENT inhibitors, certain calcium channel blockers (e.g., nimodipine, nifedipine, nicardipine, nitrendipine, and felodipine, isradipine, and nioldipine), and structural analogs of dipyridamole and dialazep, e.g., nitrobenzylthioinosine, R75231, S6-(4-nitrobenzyl)- mercaptopurine riboside (NBMPR), and cannabinoids (e.g., cannabidiol; see Carrier et al. (Proc. Nat. Acad. Sci. USA 103 (20):7895-7900 (2006)). Other exemplary analogs of dipyrimadole and dilazep are described below.
Dipyridamole
Dipyridamole is an ENT inhibitor described in G.B. Patent No. 807,826 and U.S. Patent No. 3,031,450. D
Figure imgf000024_0001
Certain dipyridamole analogs are described by formula (I) of U.S. Patent No.
3,031,450:
Figure imgf000024_0002
wherein two, three, or all four of the substituents Ri through R4 are basic groups, that is, primary, secondary, or tertiary amino groups; and, if only two or three of said substituents are basic groups, the remaining substituent or substituents are hydrogen, halogen, hydroxyl, mercapto, lower akyl, phenyl, phenoxy, lower alkoxy, lower alkoxy-lower alkoxy, (di-lower alkyl-amino)-lower alkoxy, lower alkyl-mercapto, phenyl-mercapto, benzyl-mercapto, or carboxy-lower alkyl-mercapto.
Other analogs of dipyridamole are described by the chemical formula I of U.S. Patent
No. 3,687,950, e.g., 2-diethanol-amino-6-diethanolaminosulfonyl-4,8- dipiperidinopyrimido[5,4-d]pyrimidine, 2-diethanolamino-6- diemylaminoethylaminosulfonyl-4,8-dipiperidinopy^ 2- diethanolamino 16-moφholinosulfonyl-4,8-dip^peridinopyrimido[5,4-d]pyrimidine, 2-(N- methyl)ethanolamino-6-(N-methyl)ethanolaminosulfo^
d]pyrimidine, 2-diethanolamino-6-dimethylaminosulfonyl-4,8-dipiperidinopyrimido[5,4- djpyrimidine, 2-diethanolamino-6-(N-methyl)ethanolaminosulfonyl-4,8- dipiperidinopyrimido[5,4-d]pyrimidine, 2-diethanolaminosulfonyl-6-methylsulfonyl-4,8- dipiperidinopyrimido[5,4-d], 2,6-bis-(diethanolaminosulfonyl)-4,8-dipiperidinopyrimido[5,4- d]pyrimidine, and 2,6-bis(methylsulfonyl)-4,8-dipiperidinopyrimido[5,4-d]pyrimidine; by formula I of U.S. Patent No. 4,478,833 e.g., 8-benzylthio-2-(2-hydroxyethyl-amino)-4-(l- oxido-thiomorpholino)-pyrimido[5 ,4-d]pyrimidine, 2-(2-hydroxyethyl-amino)-4-( 1 -oxido- thiomorpholino)-8-(L- 1 -phenylethylamino)-pyrimido[5,4-d]pyrimidine, and 8-benzylamino- 2-(2-hydΓO yethyl-amino)-4-(l-oxido-thiomoφholino)-pyrimido[5,4-d]pyrimidine; by formula I of U.S. Patent No. 4,690,923, e.g., 4-(l-oxido-thiomoφholino)-8-(2-phenylethyl- mercapto)-2-piperazino-pyrimidino-[5,4-d]pyrimidine and 8-benzylthio-4-(l-oxido- thiomoφholino)-2-piperazino-pyrimido[5,4-d]pyrimidine; by formula I of U.S. Patent No. 4,714,698, e.g., l,8-methylthio-2-piperazino-4-pyrrolidino-pyrimido[5,4-d]pyrimidine; and by formula I of U.S. Patent No. 4,963,541, e.g., 2,6-bis(2-(methylamino)ethanol)-4,8-bis(N- perhydroazocinyl)pyrimido[5,4-d]pyrimidine. Other analogs include mopidamol, BIBW 22, 2,6-bis(diethylamino)-4-piperidinopyrimido[5,4-d]pyrimidine (Mills et al. Biochem. J.
121 : 185 (1971)), RX-RA85, R-E 244 (4-(ethanolisopropanolamino)-2,7-di-(2'- methylmoφholino)-6-phenylpterine), 4-( 1 -oxidothiomoφholino)-8-phenethylthio-2- piperazino-pyrimido[5,4-d]pyrimidine, NU3026 (2,6-di-(2,2-dimethyl- 1 ,3-dioxolan-4-yl)- methoxy-4,8-di-piperidmopyrimidopyrimidine), NU3059 (2,6-bis-(2,3-dimethyoxypropoxy)- 4,8-di-piperidinopyrimidopyrimidine), NU3060 (2,6-bis[N,N-di(2-methoxy)ethyl]-4,6-di- piperidinopyrimidopyrimidine), NU3076, NU3084, NU3108, and NU3121 (Smith et al. Clin. Cancer Res. 7:2105-2113 (2001)). Dilazep
Dilazep is an ENT inhibitor described in G.B. Patent No. 1,107,470 and U.S. Patent No. 3,53
Figure imgf000026_0001
Exemplary analogs of dilazep are described by formula I of U.S. Patent No.
4,035,494, e.g., 4' ,4" '-[(Ν,Ν'-dimethyl- 1 ,2-ethanediyldiimino)dimethylene]
bis(pivalophenone) dihydrochloride, 4' ,4' "-[(1 ,4-piperazinediyl)dimethylene]bis
(pivalophenone), and 4',4' ' '-[(1 ,4-homopiperazinediyl)dimethylene]bis(pivolophenone) dihydrochloride, and by formula I of U.S. Patent No. 4,751,298, e.g., l,4-bis-(3-hydroxy- propyl)-l,4-diazepane dihydrochloride and l,4-bis-[3-(3,4,5-trimethoxybenzoyloxy)propyl]- diazepane dihydrochloride. Other exemplary analogs are hexobendine, 3-[methyl-[2- [methyl-[3-(3,4,5-trimethoxybenzoyl)oxypropyl]amino]ethyl]amino]propyl 3,4,5- trimethoxybenzoate hydrochloride, 3-[methyl-[2-[methyl-[3-[(E)-3-(3,4,5- trimethoxyphenyl)prop-2-enoyl]oxypropyl]amino]ethyl]amino]propyl 3,4,5- trimethoxybenzoate, ST-7092, [(2S)-2-[methyl-[2-[methyl-[(2S)- 1 -(3 ,4,5- trimethoxybenzoyl)oxybutan-2-yl]amino]ethyl]amino]butyl] 3,4,5-trimethoxybenzoate, 3-[4- [3-[(E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl]oxypropyl]piperazin-l-yl]propyl 3,4,5- trimethoxybenzoate, 3-[4-[3-(3,4,5-trimethoxybenzoyl)oxypropyl]-l,4-diazepan-l-yl]propyl 3-formyl-4,5-dimethoxybenzoate, LS-187114 ([(2S)-l-[methyl-[2-[methyl-[(2S)-2-(3,4,5- trimethoxybenzoyl)oxybutyl]amino]ethyl]amino]butan-2-yl]3,4,5- trimethoxybenzoate), buthobendin, LS-47408, KbioGR 000223 (7-(diethylamino)heptyl 3,4,5- trimethoxybenzoate), BRN 2708434 (3-[ethyl(2-phenylethyl)amino]propyl 3,4,5- trimethoxybenzoate), TMB-6 (6-(diethylamino)hexyl 3,4,5-trimethoxybenzoate), STK034691 (3-(dimethylamino)propyl 3,4,5-trimethoxybenzoate), BRN 2791312 (3-[ethyl-[l-(4- methoxyphenyl)propan-2-yl]amino]propyl 3,4,5-trimethoxybenzoate), STK182801 (l-(4- methylpiperazin-l-yl)propan-2-yl 3,4,5-trimethoxybenzoate), BRN 2676106 (4-[ethyl(2- phenylethyl)amino]butyl 3,4,5-trimethoxybenzoate), BRN 2678814 (4-[2- phenylethyl(propyl)amino]butyl 3,4,5-trimethoxybenzoate), BRN 0867091 (2-[4-[(2E,6E)- 3,7,1 l-trimethyldodeca-2,6,10-trienyl]piperazin-l-yl]ethyl 3,4,5-trimethoxybenzoate), BRN 2671634 (4-(diethylamino)butyl 3,4,5-trimethoxybenzoate), and 3-[4- (phenylmethyl)piperazin-l-yl]propyl 3,4,5-trimethoxybenzoate. Other analogs of dilazep include andiamine and K-7259 (Hoque et al., Pharmacology 277:207 (1996)).
Acetylcholinesterase inhibitors
In certain embodiments, the methods, compositions, and kits of the invention may employ the acetylcholinesterase inhibitor donepezil or physostigmine. Exemplary analogs of donepezil and physostigmine include their respective structural analogs. Other
acetylcholinesterase inhibitors, including certain organophosphates (e.g., metrifonate), certain carbamates (e.g., physostigmine, neostigmine, pyridostigmine, and rivastigmine), certain phenanthrene derivatives (e.g., galantamine), certain piperidines (e.g., donepezil, also known as E2020), tacrine, ecothiopate, dyflos, ambenonium, demarcarium, and
edrophonium, are also considered herein to be analogs of donepezil and physostigmine.
Donepezil
Donepezil has the structure:
Figure imgf000027_0001
Exemplary analogs of donepezil are described in U. S. Patent No. 7,105,540, e.g., 1- benzyl-4-((5,6-dimethoxy- 1 -indanon)-2-yl)methylpiperidine, l-benzyl-4-((5,6-dimethoxy- 1 - indanon)-2-ylidenyl)methylpiperidine, 1 -benzyl-4-((5-methoxy- 1 -indanon)-2- yl)methylpiperidine, 1 -benzyl-4-((5,6-diethoxy- 1 -indanon)-2-yl)methylpiperidine, 1 -benzyl- 4-((5,6-methnylenedioxy- 1 -indanon)-2-yl)methylpiperidine, 1 -(m-nitrobenzyl)-4-((5,6- dimethoxy- 1 -indanon)-2-yl)methylpiperidine, 1 -cyclohexylmethyl-4-((5 ,6-dimethoxy- 1 - indanon)-2-yl)methylpiperidine, 1 -(m-fluorobenzyl)-4-((5 ,6-dimethoxy- 1 -indanon)-2- yl)methylpiperidine, 1 -benzyl-4-((5 ,6-dimethoxy- 1 -indanon)-2-yl)propylpiperidine, 1 -benzyl - 4-((5-isopropoxy-6-methoxy- 1 -indanon)-2-yl)methylpiperidine, and 1 -benzyl-4-((5,6- dimethoxy- 1 -oxoindanon)-2-yl)propenylpiperidine.
Some analogs of donepezil are described by formula (II):
Figure imgf000028_0001
wherein J is (a) a group, substituted or unsubstituted, selected from the group consisting of (1) phenyl, (2) pyridyl, (3) pyrazyl, (4) quinolyl, (5) cyclohexyl, (6) quinoxalyl and (7) furyl; (b) a monovalent or divalent group, in which the phenyl may have a substituent(s), selected from the group consisting of (1) indanyl, (2) indanonyl, (3) indenyl, (4) indenonyl, (5) indanedionyl, (6) tetralonyl, (7) benzosuberonyl, (8) indanolyl and (9) C6 H5 --CO- CH(CH3)-; (c) a monovalent group derived from a cyclic amide compound; (d) a lower alkyl or (e) a group of R21 -CH=CH~ in which R21 is hydrogen or a lower alkoxycarbonyl; B is ~ (CHR22)r --, -CO-(CHR22)r --, -NR4-(CHR22)r --, R4 being hydrogen, a lower alkyl, an acyl, a lower alkylsulfonyl, phenyl, a substituted phenyl, benzyl or a substituted benzyl, - CO-NR5 ~(CHR22)r-, R5 being hydrogen, a lower alkyl or phenyl, -CH= CH-(CHR22)r-, - OCOO-(CHR22)r-, -OOC-NH~(CHR22)r-, -NH-CO-(CHR22)r-, -CH2-CO-NH- (CHR22)r «, -(CH2)2-NH-(CHR22)r-, -CH(OH (CHR22)r-, r being zero or an integer of 1 to
10, R 22 being hydrogen or methyl so that one alkylene group may have no methyl branch or one or more methyl branch, =(CH— CH=CH)b~, b being an integer of 1 to 3, =CH~(CH2)C~, c being zero or an integer of 1 to 9, =(CH~CH)d=, d being zero or an integer of 1 to 5; -- CO— CH=CH»CH2~, ~CO~CH2~CH(OH)-CH2~, ~CH(CH3)-CO-NH-CH2-, -
CH=CH-CO~NH-(CH2)2-, -NH-, -O-, --S-, a dialkylaminoalkylcarbonyl or a lower
\
N ►O
alkoxycarbonyl; T is a nitrogen or carbon; Q is nitrogen, carbon or / and q is an integer of 1 to 3; K is hydrogen, phenyl, a substituted phenyl, an arylalkyl in which the phenyl may have a substituent, cinnamyl, a lower alkyl, pyridylmethyl, a cycloalkylalkyl, adamantanemethyl, furylmenthyl, a cycloalkyl, a lower alkoxycarbonyl or an acyl; and— shows a single bond or a double bond, as described in U.S. Patent No. 4,895,841 , which is herein incorporated by reference.
Exemplary compounds of formula II are l-benzyl-4-((5-methoxy-l-indanon)-2- yl)methylpiperidine, 1 -benzyl-4-((5 ,6-diethoxy- 1 -indanon)-2-yl)methylpiperidine, 1 -benzyl- 4-((5,6-methylenedioxy-l-indanon)-2-yl)methylpiperidine, l-(m-nitrobenzyl)-4-((5,6- dimethoxy- 1 -indanon)-2-yl)methylpiperidine, 1 -(m-fluorobenzyl)-4-((5 ,6-dimethoxy- 1 - mdanon)-2- l)methylpiperidine, l-benzyl-4-((5,6-dimethoxy-l-indanon)-2- yl)propylpiperidine, and 1 -benzyl-4-((5-isopropoxy-6-methoxy- 1 -indanon)-2- yl)methylpiperidine.
Physostigmine
Physostigmine has
Figure imgf000029_0001
Exemplary physostigmine analogs are 1-desmethyl eserine, norphysostigmine,
[(3aS,8bS)-3,4,8b-trimethyl-l,2,3,3a-tetrahydropyrrolo[2,3-b]indol-3-ium-7-yl] N- methylcarbamate,LS- 190647 ([(3a,8b)-3,4,8b-trimethyl-2,3,3a,4-tetrahydro-lH-pyrrolo[2,3- b]indole-3,4-diium-7-yl] N-methylcarbamate), bisnorphysostigmine, eptastigmine, eseroline heptacarbamate, eseroline octylcarbamate, benzylnoφhysostigmine, heptylstigmine, geneserine, eseroline, LS-139389 ([(3a,8b)-3,4,8b-trimethyl-2,3a-dihydro-lH-pyrrolo[2,3- b]indol-7-yl]N-octylcarbamate), MF247, MF 256, physostigmine analog 33, and N1,N8- bisbenzylnoφhysostigmine .
Certain analogs of physostigmine are desribed by the formula (III):
Figure imgf000030_0001
where (a) X is O or S; (b) R is H, loweralkyl,
Figure imgf000030_0002
or— c— R4j where Y is O or S; R2 is alkyl, cycloalkyl, bicycloalkyl, cycloalkenyl, aryl, arylloweralkyl, heteroaryl or
heteroarylloweralkyl, R3 is H or alkyl, or the group ~NR2R3 taken as a whole is 1- pyrrolidinyl, 1-piperidinyl, 4-morpholinyl, 4-thiomoφholinyl, 1-piperazinyl, 4-methyl-l- piperazinyl or 2-(2,6-dichlorophenylimino)-l-imidazolidinyl) and R4 is hydrogen, loweralkyl, arylloweralkyl, diarylloweralkyl, aryl or heteroaryl; (c) m is 1 or 2; (d) each Z is
independently H, loweralkyl, halogen, nitro, ~NH2, loweralkylcarbonylamino,
arylcarbonylamino, loweralkoxycarbonylamino or loweralkylamino, and (e) Ri is H, loweralkyl, arylloweralkyl, heteroarylloweralkyl, cycloalkylmethyl or loweralkenylmethyl, with the proviso that when X is O, m is 1, Z is H and Rj is methyl, R is not --CONHCH3, ~ CONHC6H 5, hydrogen, methyl or ethyl, and that when X is O, m is 1 and Z and R! are both hydrogen, R is not hydrogen or methyl, as described in U.S. Patent Nos. 5,541,340,
4,900,748, 4,831,155, and 5,547,977, each of which is herein inco orated by reference. Exemplary compounds of formula III are (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl- pyrrolo[2,3-b]indol-5-ol, octadecyl carbamate ester; 7-chloro-(3aS-cis)-l,2,3,3a,8,8a- hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, methyl carbamate ester; 7-bromo-(3aS- cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethylpyrrolo[2,3-b]indol-5-ol, methyl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, N,N- diethyl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3- b]indol-5-ol, cyclopentylmethyl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8- trimethyl-pyrrolo[2,3-b]indol-5-ol, (thien-3-yl)methyl carbamate ester; (3aS-cis)- l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, benzyl carbamate ester; (3aS-cis)- 1 ,2,3,3a,8,8a-hexahydro- 1 ,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, (2-phenyl)ethyl carbamate ester; 3aS-[3aa,5(R*),8aa]]-l,2,3,3a,8,8a-hexahydro-l ,3a,8-trimethyl-pyrrolo[2,3- b]indol-5-ol, (l-phenyl)ethyl carbamate ester; [3aS-[3aoc,5(S*),8aa]]-l,2,3,3a,8,8a- hexahydro-l,3a,8-trimethylpyrrolo[2,3-b]indol-5-ol, (l-phenyl)ethyl carbamate ester, 7- chloro-[3aa,5(R*),8aa]-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, (l-phenyl)ethyl carbamate ester; 7-bromo-[3aa,5(R*),8aa]-l,2,3,3a,8,8a-hexahydro-l,3a,8- trimethy l-pyrrolo[2,3-b]indol-5-ol; (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl- pyrrolo[2,3-b]indol-5-ol, [l-(l-naphthyl)ethyl] carbamate ester; (3aS-cis)-l,2,3,3a,8,8a- hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, cyclohexyl carbamate ester; 7-chloro- (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, cyclohexyl carbamate ester; 7-bromo-(3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3- b]indol-5-ol, cyclohexyl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl- pyrrolo[2,3-b]indol-5-ol, 4,4-dimethylcyclohexyl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a- hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, 4-ethylcyclohexyl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol,
spiro[5.5]undecan-3-yl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl- pyrrolo[2,3-b]indol-5-ol, cycloheptyl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a-hexahydro- l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, l,2-dimethylcyclohexen-4-yl carbamate ester; (3aS-cis)- 1 ,2,3,3a,8,8a-hexahydro- 1 ,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, cyclohexen- 1 -yl carbamate ester; (3aS-cis)-l ,2,3,3a,8,8a-hexahydro-l ,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, bicyclo[2.2.1]heptan-2-yl carbamate ester; (3aS-cis)- 1,2,3 ,3a,8,8a-hexahydro-l,3a,8- trimethyl-pyrrolo[2,3-b]indol-5-ol, 3-chlorophenyl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a- hexahydro-l,3a,8-trimethyl-pyrrolo[[2,3-b]indol-5-ol, 4-chlorophenyl carbamate ester; (3aS- cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, 2,6-dimethylphenyl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, 4-nitrophenyl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl- pyrrolo[2,3-b]indol-5-ol, 4-pyridinyl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a-hexahydro- l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, 4-methyl-piperazin-l-yl carbamate ester; (3aS-cis)- 1 ,2,3,3a,8,8a-hexahydro-l ,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, 4-morpholinyl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, 4- morpholinyl thiocarbamate ester; (3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl- pyrrolo[2,3-b]indol-5-ol, 2-(2,6-dichlorophenylimino)-l-imidazolidinyl carbamate ester; (3aS-cis)-l,2,3,3a,8,8a-hexahydro-7-nitro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, 3- chlorophenyl carbamate ester; 7-acetylamino-(3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8- trimethylpyrrolo[2,3-b]indol-5-ol, 3-chlorophenyl carbamate ester; 6-bromo-(3aS-cis)- 1 ,2,3,3a,8,8a-hexahydro-l ,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, 3-chlorophenyl carbamate ester; 7-bromo-(3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol, n-heptyl carbamate ester; [3aS-[3aa,5(S*),8aa]]-l,2,3,3a,8,8a-hexahydro-7-nitro-l,3a,8- trimethylpyrrolo[2,3-b]indol-5-ol, (l-phenyl)ethyl carbamate ester; 7-bromo-(3aS-cis)- l,2,3,3a,8,8a-hexahydro-5-methoxy-l,3a,8-trimethylpyrrolo[2,3-b]indole; 7-chloro-(3aS-cis)- l,2,3,3a,8,8a-hexahydro-5-methoxy-l,3a,8-trimethylpyrrolo[2,3-b]indole; 7-acetylamino- (3aS-cis)-l,2,3,3a,8,8a-hexahydro-5-methoxy-l,3a,8-trimethylpyrrolo[2,3-b]indole; (3aS- cis)-l,2,3,3a,8,8a-hexahydro-5-methoxy-7-nitro-l,3a,8-trimethylpyrrolo [2,3-b]indole; 7- bromo-(3aS-cis)-l-cyclopropylmethyl-l,2,3,3a,8,8a-hexahydro-5-methoxy-3a,8- dimethylpyrrolo[2,3-b]indole; 7-bromo-(3aS-cis)- 1 ,2,3,3a,8,8a-hexahydro-5-methoxy- 1 -(2- phenylethyl)-3a,8-dimethylpyrrolo[2,3-b]indole; 7-bromo-(3aS-cis)- 1 ,2,3,3a,8,8a-hexahydro- 5-methoxy-l-(2-propenyl)-3,8a-dimethylpyrrolo[2,3-b]indole; 7-bromo-l-(2-butenyl)-(3aS- cis)-l,2,3,3a,8,8a-hexahydro-5-methoxy-3,8a-dimethylpyrrolo[2,3-b]indole; 7-bromo-(3aS- cis)-l-cyclopropylmethyl-l,2,3,3a,8,8a-hexahydro-3a,8-dimethyl pyrrolo[2,3-b]indol-5-ol; 7- bromo-(3aS-cis)-l,2,3,3a,8,8a-hexahydro-l-(2-phenylethyl)-3a,8-dimethylpyrrolo[2,3- bjindol-5-ol; 7-bromo-(3aS-cis)-l,2,3,3a,8,8a-hexahydro-l-(2-propenyl)-3a,8-dimethylpyrro lo[2,3-b]indol-5-ol; 7-bromo-l-(2-butenyl)-(3aS-cis)-l,2,3,3a,8,8a-hexahydro-3a,8- dimethylpyrrolo[2,3-b]indol-5-ol; (3aS-cis)-l-cyclopropylmethyl-l,2,3,3a,8,8a-hexahydro-7- nitro-3a,8-dimethyl pyrrolo[2,3-b]indol-5-ol; (3aS-cis)- 1 ,2,3,3a,8,8a-hexahydro-7-nitro- 1 -(2- propenyl)-3a,8-dimethylpyrrolo[2,3-b]indol-5-ol; l-(2-butenyl)-(3aS-cis)-l,2,3,3a,8,8a- hexahydro-7-nitro-3a,8-dimethylpyrrolo[2,3-b]indol-5-ol; (3aS-cis)- 1 ,2,3,3a,8,8a-hexahydro- 7-nitro- 1 -(2-phenylethyl)-3a,8-dimethylpyrrolo[2,3-b]indol-5-ol; 7-bromo-(3aS-cis)- l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol acetate; (3aS-cis)- l,2,3,3a,8,8a-hexahydro-7-nitro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol acetate; (3aS-cis)- l,2,3,3a-8,8a-hexahydro-7-nitro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol trimethylacetate; 7-bromo-(3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5-ol heptanoate; (3aS-cis)-l,2,3,3a,8,8a-hexahydro-7-nitro-l,3a,8-trimethyl-pyrrolo[2,3-b]indol-5- ol heptanoate; and 7-amino-(3aS-cis)-l,2,3,3a,8,8a-hexahydro-l,3a,8-trimethyl-pyrrolo[2,3- b]-5-ol, methyl carbamate ester.
Rho kinase inhibitors
In certain embodiments, a Rho kinase inhibitor can be used in the compositions, methods, and kits of the invention. By a "Rho kinase inhibitor" is meant a compound that inhibits the activity of a Rho kinase by at least 5%, e.g., greater than 10%, 20%, 40%, 60%, 80%o, 90%, or 95%. Inhibition of Rho kinase activity may be measured, e.g., by an in vitro assay with recombinant or purified Rho kinase, or by a cell-based reporter assay known in the art. Rho kinase inhibitors include fasudil, HA 1077 (Calbiochem), hydroxyfasudil, and Y- Fasudil
Fasudil is described in European Patent No. 187371 and U.S. Patent No. 4,678,783 and has the following structure:
Figure imgf000034_0001
Certain analogs of fasudil are described by the formula (IV):
Figure imgf000034_0002
wherein R{ represents a hydrogen atom, a chlorine atom or a hydroxyl group; and when R( represents a hydrogen atom, A represents an ethylene group unsubstituted or substituted with an alkyl group having 1 to 6 carbon atoms, a phenyl group or a benzyl group, R2 and R3 are directly bonded with each other, thereby forming a trimethylene group unsubstituted or substituted with an alkyl group having 1 to 6 carbon atoms, a phenyl group or a benzyl group, and Rj represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; and when Ri represents a chlorine atom or a hydroxyl group, A represents an alkylene group having 2 to 6 carbon atoms, said group being unsubstituted or substituted with an alkyl group having 1 to 6 carbon atoms, R2 and R3 are not bonded with each other and each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, or R2 and R3 are directly bonded with each other, thereby forming an ethylene group unsubstituted or substituted with an alkyl group having 1 to 6 carbon atoms or a trimethylene group
unsubstituted or substituted with an alkyl group having 1 to 6 carbon atoms, and R4 represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or an amidino group, as described in U.S. Patent No. 4,678,783. Exemplary compounds of formula (IV) are l-(5- isoquinolinesulfonyl)homopiperazine, l-(5-isoquinolinesulfonyl)-2-methylhomopiperazine, 1 -(5-isoquinolinesulfonyl)-3-methylhomopiperazine, 1 -(5-isoquinolinesulfonyl)-6- methylhomopiperazine, 1 -(5-isoquinolinesulfonyl)-2,3-dimethylhomopiperazine, 1 -(5- isoquinolinesulfonyl)-3 ,3-dimethylhomopiperazine, 1 -(5-isoquinolinesulfonyl)-3- ethylhomopiperazine, l-(5-isoquinolinesulfonyl)-3-propylhomopiperazine, and l-(5- isoquinolinesulfonyl)-3-isobutylhomopiperazine.
Other analogs of fasudil may be described by Formula I of U. S. Patent No. 5,733,904; by Formulae II and IV of U.S. Patent No. 4,798,897; by structural formula I in U.S. Patent No. 4,857,301; by formula I of U.S. Patent No. 5,081,246, e.g., N-[2-(4-benzyloxycarbonyl- piperazinyl)-l-(p-methoxybenzyl)ethyl]-N-methyl-5-isoquinolinesulfonamide; by formula I of U.S. Patent No. 5,244,895, e.g., N(l-(p-hydroxybenzyl)-2-(4-phenylpiperazinyl)ethyl)-5- isoquinoline sulfonamide, N-(2-(4-(m-chlorophenyl)piperazinyl)- l-(p-hydroxybenzyl)ethyl)- N-methyl-5-isoquinoline sulfonamide, N-(2-(4-benzyloxycarbonylpiperazinyl)- 1 -(p- hydroxybenzyl)ethyl)-N-methyl-5-isoquinoline sulfonamide, N-(2-(4- benzyloxycarbonylpiperazinyl)-l-(p-methoxybenzyl)ethyl)-N-methyl-5-isoquinoline sulfonamide, N-(p-hydroxybenzyl)-2-(4-phenylhomopiperazinyl)ethyl)-5-isoquinoline sulfonamide, N-( 1 -(p-hydroxybenzyl)-2-(4-(3-dichlorobenzyloxy)piperidino)ethyl)-5-isoqu inoline sulfonamide, N-( 1 -(p-hydroxybenzyl)-2-(4-(3 ,4-dichlorobenzyloxy)piperidino)ethyl)- N-met hyl-5-isoquinoline sulfonamide, N-(l-(p-methoxybenzyl)-2-(4-(3,4- dichlorobenzyloxy)piperidino)ethyl)-N-met hyl-5-isoquinoline sulfonamide, N-(l-(p- hydroxybenzyl)-2-(4-phenylpiperidino)ethyl)-N-methyl-5-isoquinolin e sulfonamide, N-(2- (4-benzyloxycarbonylhomopiperazinyl)-l-(p-hydroxybenzyl)ethyl)-N-methyl-5-isoquinoline sulfonamide, N-(2-(4-benzyloxycarbonylhomopiperazinyl)- 1 -(p-methoxybenzyl)ethyl)-N-(2- aminoethyl)-5-isoquinoline sulfonamide, and N-(2-(4-benzyloxycarbonylhomopiperazinyl)- 1 -(p-methoxybenzyl)ethyl)-N-(2-dimethylaminoethyl)-5-isoquinoline sulfonamide; by formula I of U.S. Patent No. 5,245,034, e.g., N-anisyl-N-[2-(4-chlorocinnamylamino) ethyl]- 5-isoquinolinesulfonamide; by formula I of U.S. Patent No. 5,340,811, e.g., l-(5- isoquinoline-sulfonylaminoethyl)-4-(3,4-methylenedioxybenzyl)piperazine; by formula I of U.S. Patent No. 5,663,174; by Formula I of U. S. Patent No. 5,747,507; by formula I of U.S. Patent No. 5,942,505; and by formula I of U.S. Patent No. 6,153,608, e.g., hexahydro-l-[(4- methyl-5-isoquinolinyl)sulfonyl]- 1H- 1 ,4-diazepine dihydrochloride, (S)-(+)-hexahydro-2- methyl-l-[(4-methyl-5-isoquinolinyl)sulfonyl]-lH-l,4-diazepine hydrochloride, hexahydro-7- methyl- 1 -[(4-methyl-5-isoquinolinyl)sulfonyl]- 1 H- 1 ,4-diazepine dihydrochloride, hexahydro- 5-methyl- 1 -[(4-methyl-5-isoquinolinyl)sulfonyl]- 1 H- 1 ,4-diazepine dihydrochloride, hexahydro-2-methyl- 1 -[(4-methyl-5-isoquinolinyl)sulfonyl]- 1 H- 1 ,4-diazepine hydrochloride, (R)-(-)-hexahydro-2-methyl- 1 -[(4-methyl-5-isoquinolinyl)sulfonyl]- 1 H- 1 ,4-diazepine hydrochloride and (R)-(+)-hexahydro-5-methyl- 1 -[(4-methyl-5-isoquinolinyl)sulfonyl]- 1 H- 1,4-diazepine hydrochloride.
FKBP/mTOR inhibitors
In certain embodiments of the methods, compositions, and kits of the invention, the FKBP/mTOR inhibitor everolimus or an everolimus analog may be employed. Analogs of everolimus include compounds structurally related to everolimus and other FKBP/mTOR inhibitors, e.g., temsirolimus, rapamycin, ascomycin, AP23573 (Ariad Pharmaceuticals), NVP-BEZ235, sirolimus, tacrolimus (FK 506), zotarolimus, and pimecrolimus.
Everolimus
Everolimus has the following structure:
Figure imgf000037_0001
Exemplary analogs of everolimus are described by the general formula of U.S. Patent No. 5,118,677, e.g., rapamycin-42-ester with 4-[[l-(4-chlorophenyl)methyl]amino]-4- oxobutanoic acid; by the general formula of U.S. Patent No. 5,118,678, e.g., rapamycin 42- ester with (4-fluorophenyl)carbamic acid.; by formula I of U.S. Patent No. 5,120,725, e.g., rapamycin-31,42-cyclic diester with hexanedioic acid; by formula I of U.S. Patent No.
5,138,051, e.g., 33-Deoxy-33-hydroxyrapamycin; by the general structure of U.S. Patent No. 5,194,447, e.g., rapamycin-31 -ester with phenylsulfonylcarbamic acid; by the general structure of U.S. Patent No. 5,378,836, e.g., rapamycin 27-oxime, 42-ester with 8- quinolinesulfonic acid ; by the general structures of U.S. Patent No. 5,387,680, e.g., C-22- methyl-rapamycin; and by formula I of U.S. Patent No. 6,200,985, e.g., 16-pent-2-ynyloxy- 32(S)-dihydro-rapamycin or 16-pent-2-ynyloxy-32(S)-dihydro-40-O-(2-hydroxyethyl)- rapamycin, 32-deoxo-rapamycin or 16-pent-2-ynyloxy-32-deoxo-rapamycin, 16-pent-2- ynyloxy-32(S)-dihydro-rapamycin or 16-pent-2-ynyloxy-32(S)-dihydro-40-O-(2- hydroxyethyl)-rapamycin, 32-deoxo-rapamycin or 16-pent-2-ynyloxy-32-deoxo-rapamycin, 16-pent-2-ynyloxy-32(S)-dihydro-rapamycin or 16-pent-2-ynyloxy-32(S)-dihydro-40-O-(2- hydroxyethyl)-rapamycin, 32-deoxo-rapamycin or 16-pent-2-ynyloxy-32-deoxo-rapamycin, 16-pent-2-ynyloxy-32(S)-dihydrorapamycin or 16-pent-2-ynyloxy-32(S)-dihydro-40-O-(2- hydroxyethyl)-rapamycin, 32-deoxo-rapamycin or 16-pent-2-ynyloxy-32-deoxo-rapamycin, 16-pent-2-ynyloxy-32(S)-dihydro-rapamycin or 16-pent-2-ynyloxy-32(S)-dihydro-40-O-(2- hydroxyethyl)-rapamycin, and 32-deoxo-rapamycin or 16-pent-2-ynyloxy-32-deoxo- rapamycin.
Certain everolimus analogs, e.g., pimecrolimus, are described by the formula I of U.S. Patent No. 5,912,238. Other analogs of everolimus include mono- and diacylated rapamycin derivatives (U.S. Patent No. 4,316,885); rapamycin water-soluble prodrugs (U.S. Patent No. 4,650,803); carboxylic acid esters (PCT Publication No. WO 92/05179);
carbamates (U.S. Patent No. 5,118,678); amide esters (U.S. Patent No. 5,118,678); biotin esters (U.S. Patent No. 5,504,091); fluorinated esters (U.S. Patent No. 5,100,883); acetals (U.S. Patent No. 5,151,413); silyl ethers (U.S. Patent No. 5,120,842); bicyclic derivatives (U.S. Patent No. 5,120,725); rapamycin dimers (U.S. Patent No. 5,120,727); O-aryl, O-alkyl, O-alkyenyl and O-alkynyl rapamycin derivatives (U.S. Patent No. 5,258,389); and deuterated rapamycin (U.S. Patent No. 6,503,921). Yet other exemplary analogs of everolimus are described in U.S. Patent Nos. 4,316,885, 5,023,262, 5,023,263, 5,023,264, 5,091,389, 5,202,332, and 5,169,851.
Phosphodiesterase inhibitors
The methods, compositions, and kits of the invention may employ ethaverine, drotaverine, papaverine, zardaverine, tetrahydropapaveroline, trequinsin, MBCQ (4-[[3,4- (methylenedioxy)benzyl]amino]-6-chloroquinazoline), or dipyridamole or an analog of one of these. Analogs of ethaverine, drotaverine, papaverine, zardaverine,
tetrahydropapaveroline, trequinsin, MBCQ, or dipyridamole include their structural analogs and other phosphodiesterase (PDE) inhibitors.
Optionally, the PDE inhibitor selectively inhibits a particular type of PDE relative to other types. For example, a selective inhibitor may inhibit PDE type 5 (PDE5) at least 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, or 100-fold more effectively than it inhibits another particular phosphodiesterase, e.g., PDE type II, type III, type IV, type VII, or type VIII, also known as PDE2, 3, 4, 7, and 8, respectively). A PDE inhibitor may also be non-selective or exhibit weak selectivity. Exemplary non-selective or weakly selective PDE inhibitors are theophylline, theobromine, IBMX, pentoxifylline and papaverine. The selectivity of a PDE inhibitor may be determined by measuring its IC50 (the concentration required to achieve 50% inhibition of an enzyme) against at least two different phosphodiesterases.
In one embodiment of the invention, a PDE inhibitor selective for PDE5 is employed. Inhibitors of PDE5 may include griseolic acid derivatives, 2-phenylpurinones,
phenylpyridones, fused and condensed pyrimidines, pynmidopyrimidines, purine compounds, quinazoline compounds, phenylpyrimidinones, and imidazoquinoxalinones. Specific exemplary PDE5 inhibitors are dipyridamole, MBCQ, zaprinast, MY-5445, vinpocetine, FR229934, l-methyl-3-isobutyl-8-methylamino)xanthine, IC-351, vardenafil, GF-196960, Sch-51866,and sodium- l-[6-chloro-4-(3 ,4-methylenedioxybenzyl)-aminoquinazolin-2- yl]piperidine-4 -carboxylate sesquihydrate.
In preferred embodiments, a PDE inhibitor has an IC50 of 100 μΜ or lower for a phosphodiesterase. In more preferred embodiments, the IC50 of a phosphodiesterase inhibitor is 40, 20, or 10 μΜ or lower. In some embodiments, a phosphodiesterase inhibitor has an IC50 of 40 μΜ, 20 μΜ, 10 μΜ, 5 μΜ, 1 μΜ, 100 ηΜ, 10 ηΜ, or lower for a particular type of phosphodiesterase. When a phosphodiesterase inhibitor is described herein as having activity against a particular type of phosphodiesterase, the inhibitor may also have activity against other types, unless otherwise stated.
Non-limiting examples of PDE inhibitors are theophylline(l,3-dimethylxanthine), caffeine, quercetin dihydrate, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one,
propentofylline, 3-methyl- 1 -(5-oxohexyl)-7-propylxanthine), 3-isobutyl- 1 -methylxanthine, IBMX, 3-isobutyl-l-methyl-2,6(lH,3H)-purine-dione, l-methyl-3-isobutylxanthine, 8- methoxymethyl-3-isobutyl-l -methylxanthine, enoximone, papaverine hydrochloride, calmidazolium chloride, imidazolium chloride, l-[bis(4-chlorophenyl)methyl]-3-[2-(2,4- dichlorophenyl)-2-(2,4-dichlorobenzyloxy)ethyl]-lH-imidazolium chloride, SKF 94836, neuropeptide Y fragment 22-36, aminophylline hydrate, butein, etazolate hydrochloride, trifluoperazine dihydrochloride, and milrinone. Yet other examples are arofylline, atizoram, A WD- 12-281 (N-(3 , 5-dichloro-4-pyridinyl)-2-[ 1 -(4-fluorobenzyl)-5-hydroxy- 1 H-indol-3- yl]-2-oxoacetamide), BAY- 19-8004 (ethanesulfonic acid 2-(2, 4-dichlorophenylcarbonyl)-3- ureido-benzofuran-6-yl ester), benafentrine, CC-1088, CDC-801 (p-[3-(cyclopentyloxy)-4- methoxyphenyl]- 1 ,3-dihydro- 1 ,3-dioxo-2H-isoindole-2-propanamide), CDC-998, CI- 1018, cilomilast (cis-[4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexane- 1 -carboxylic acid), cilostazol, cipamfylline (8-amino-l,3- bis(cyclopropylmethyl)xanthine), D-4396, D- 4418 (N-(2,5-dichloro-3-pyridinyl)-8-methoxy-5-quinoline-carboxamide), darbufelone, denbufylline, ER-21355, filaminast, IC-485, indolidan, laprafylline, lixazinone, mesopram (5-(methoxy-3-propoxyphenyl)-5-methyl-2-oxazolidinone), nitraquazone, NM-702, olprinone, ORG-20241 (4-(3, 4-dimethoxyphenyl)-N2-hydroxythiazole-2-carboxamidine), piclamilast, pumafentrine ((-)-cis-9-ethoxy-8-methoxy-2-methyl- 1 ,2,3 ,4,4a, 1 Ob-hexahydro-6- (4-diisopro-pylaminocarbonylphenyl)benzo[c] [ 1 ,6]-naphthyridine), quazinone, RO- 15-2041 , roflumilast (3-(cyclopropl[methoxy)-N-(3, 5-dichloro-4-pyridyl)-4-(difluoromethoxy)- benzamide), rolipram, SCH-351591, SH-636, tibenelast (5,6-diethoxybenzo[b]thiophene-2- carboxylic acid), tolafentrine, V-11294A (3-[[3-(cyclopentyloxy)-4-methoxyphenyl]methyl]- N-ethyl-8-( 1 -methylethyl)-3H-purin-6-amine), YM-58997 (4-(3-bromophenyl)- 1 -ethyl-7- methyl-l,8-naphthyridin-2(l H)-one), YM-976 (4-(3-chloro-phenyl)-l,7-diethylpyrido[2,3-d] pyrimidin-2(l H)-one), zardaverine, UK 66838, vasotrope, methyl 3-[6-(2H-3,4,5,6- tetrahydropyran-2-yloxy)-2-(3-thienylcarbonyl)benzo[ ]furan-3-yl]propanoate, 4-[4- methoxy-3-(5-phenylpentyloxy)phenyl]-2-methylbenzoic acid, methyl 3-[2-[(4- chlorophenyl)carbonyl]-6-hydroxybenzo[p]furan-3-yl]propanoate, (R*,R*)-(±)-methyl 3- acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3-methyl- 1 -pyrrolidinecar-boxylate, and 4- (3-bromophenyl)-l-ethyl-7-methylhydropyridmo[2,3- ]pyridin-2-one.
Additional examples of PDE inhibitors that may be useful in the compositions, methods, and kits provided herein are disclosed in U.S. Patent No. 6,818,651, U.S. Patent No. 6,737,436, U.S. Patent No. 6,613,778, U.S. Patent No. 6,617,357, U.S. Patent No.
6,146,876, U.S. Patent No. 6,838,559, U.S. Patent No. 6,884,800, U.S. Patent No. 6,716,987, U.S. Patent No. 6,514,996, U.S. Patent No. 6,740,655, U.S. Patent No. 6,559,168, U.S.
Patent No. 6,069,151, U.S. Patent No. 6,365,585, U.S. Patent No. 6,313,116, U.S. Patent No. 6,245,774, U.S. Patent No. 6,011,037, U.S. Patent No. U.S. Patent No. 6,423,710, U.S.
Patent No. 6,372,777, U.S. Patent No. 6,362,213, U.S. Patent No. 6,313,156, U.S. Patent No. 6,294,561, U.S. Patent No. 6,258,843, U.S. Patent No. 6,258,833, U.S. Patent No. 6,043,263, U.S. Patent No. 6,297,257, U.S. Patent No. 6,251,923, U.S. Patent No. 6,613,794, U.S.
Patent No. 6,407,108, U.S. Patent No. 6,107,295, U.S. Patent No. 6,103,718, U.S. Patent No. 6,479,494, U.S. Patent No. 6,545,158, U.S. Patent No. 6,545,025, U.S. Patent No. 6,498,160, U.S. Patent No. 6,743,802, U.S. Patent No. 6,787,554, U.S. Patent No. 6,828,333, U.S.
Patent No. 6,869,945, U.S. Patent No. 6,894,041, U.S. Patent No. 6,924,292, U.S. Patent No. 6,949,573, U.S. Patent No. 6,953,810, U.S. Patent No. 6,156,753, U.S. Patent No. 5,972,927, U.S. Patent No. 5,962,492, U.S. Patent No. 5,814,651, U.S. Patent No. 5,723,460, U.S.
Patent No. 5,716,967, U.S. Patent No. 5,686,434, U.S. Patent No. 5,502,072, U.S. Patent No. 5,116,837, U.S. Patent No. 5,091,431, U.S. Patent No. 4,670,434, U.S. Patent No. 4,490,371 , U.S. Patent No. 5,710,160, U.S. Patent No. 5,710,170, U.S. Patent No. 6,384,236, U.S.
Patent No. 3,941,785; in U.S. Patent publications 2005/0119225, 2005/0026913,
2005/0059686, 2004/0138279, 2005/0222138, 2004/0214843, 2004/0106631, 2003/0045557, 2002/0198198, 2003/0162802, 2003/0092908, 2003/0104974, 2003/0100571, 2003/0092721, and 2005/0148604; and in PCT publications WO 99/65880, WO 00/26201, WO 98/06704, WO 00/59890, WO9907704, WO9422852, WO 98/20007, WO 02/096423, WO 98/18796, WO 98/02440, WO 02/096463, WO 97/44337, WO 97/44036, and WO 97/44322.
Additional PDE inhibitors are shown in Table 3. Table 3. Phosphodiesterase inhibitors
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
lH-irnidazo[4,5-glquinazoline-2-thione
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
PDE Inhibitor Alternative Identifying Information PDE
Inhibitory Activity
Y 20487 6-(3,6-dihydro-2-oxo-2H-l,3,4-thiadiazin-5-yl)-3,4-dihydro-2(lH)- 3
quinolinone
YM 58997 4-(3-bromophenyl)-l ,7-diethylpyrido[2,3-d]pyrimidin-2(lH)-one 4
YM 976 4-(3-chlorophenyl)-l -diethylpyrido(2,3-d)pyrimidin-2(lH)-one 4
Z 15370A 4
Zaprinast 1 ,4-dihydro-5-(2-propoxyphenyl)-7H- 1 ,2,3-triazolof 4,5-d]pyrimidine-7- 5
one
Zaprinast 2-o-propoxyphenyl-8-azapurine-6-one 1, 5
Zardaverine 6-(4-(difluoromethoxy)-3-methoxyphenyl)-3(2H)-Pyridazinone 3, 4
Zindotrine 8-methyl-6-( 1 -piperidinyl)- 1 ,2,4-triazolo(4,3-b)pyridazine
Examples of PDE4 inhibitors, e.g., ibudilast, include pyrrolidinones, such
compounds disclosed in U.S. Patent No. 5,665,754, US20040152754 and US20040023945; quinazolineones, such as the compounds disclosed in U.S. Patent No. 6,747,035, U.S. Patent
No. 6,828,315, PCT publications WO 97/49702 and WO 97/42174; xanthine derivatives; phenylpyridines, such as the compounds disclosed in U.S. Patent No. 6,410,547, U.S. Patent
No. 6,090,817, and PCT publication WO 97/22585; diazepine derivatives, such as the compounds disclosed in WO 97/36905; oxime derivatives, such as the compounds disclosed in U.S.P.N.. 5,693,659 and PCT publication WO 96/00215; naphthyridines, such as the compounds described in U.S. Patent No. 5,817,670, U.S. Patent No. 6,740,662, U.S. Patent
No. U.S. Patent No. 6,136,821, U.S. Patent No. 6,331,548, U.S. Patent No. 6,297,248, U.S.
Patent No. 6,541,480, U.S. Patent No. 6,642,250, U.S. Patent No. 6,900,205, Trifilieff et al.
(Pharmacology 301 : 241-248 (2002)) and Hersperger et al. (J. Med. Chem. 43:675-82(2000); benzofurans, such as the compounds disclosed in U.S. Patent No. 5,902,824, U.S. Patent No.
6,211,203, U.S. Patent No. 6,514,996, U.S. Patent No. 6,716,987, U.S. Patent No. 6,376,535,
U.S. Patent No. 6,080,782, U.S. Patent No. 6,054,475, EP 819688, EP 685479, and Perrier et al. (Bioorg. Med. Chem. Lett. 9:323-326 (1999)); phenanthridines, such as those disclosed in
U.S. Patent No. 6,191,138, U.S. Patent No. 6,121,279, and U.S. Patent No. 6,127,378;
benzoxazoles, such as those disclosed in U.S. Patent No. 6,166,041 and U.S. Patent No.
6,376,485; purine derivatives, such as the compounds disclosed in U.S. Patent No. 6,228,859; benzamides, such as the compounds described in U.S. Patent No. 5,981,527, U.S. Patent No. 5,712,298, PCT publications WO95/01338 and WO 97/48697, and Ashton et al. (J. Med. Chem. 37: 1696-1703 (1994)); substituted phenyl compounds, such as the compounds disclosed in U.S. Patent No. 6,297,264, U.S. Patent No. 5,866,593, U.S. Patent No.
655,859,034, U.S. Patent No. 6,245,774, U.S. Patent No. 6,197,792, U.S. Patent No.
6,080,790, U.S. Patent No. 6,077,854, U.S. Patent No. 5,962,483, U.S. Patent No. 5,674,880, U.S. Patent No. 5,786,354, U.S. Patent No. 5,739,144, U.S. Patent No. 5,776,958, U.S.
Patent No. 5,798,373, U.S. Patent No. 5,891,896, U.S. Patent No. 5,849,770, U.S. Patent No. 5,550,137, U.S. Patent No. 5,340,827, U.S. Patent No. 5,780,478, U.S. Patent No. 5,780,477, U.S. Patent No. 5,633,257, and PCT publication WO 95/35283; substituted biphenyl compounds, such as those disclosed in U.S. Patent No. 5,877,190; and quinilinones, such as the compounds described in U.S. Patent No. 6,800,625 and PCT publication WO 98/14432.
Yet other inhibitors of PDE4 are disclosed in U.S. Patent No. 6,716,987, U.S. Patent No. 6,514,996, U.S. Patent No. 6,740,655, U.S. Patent No. 6,559,168, U.S. Patent No.
6,069,151, U.S. Patent No. 6,365,585, U.S. Patent No. 6,313,116, U.S. Patent No. 6,245,774, U.S. Patent No. 6,011,037, U.S. Patent No. 6,127,363, U.S. Patent No. 6,303,789, U.S.
Patent No. 6,316,472, U.S. Patent No. 6,348,602, U.S. Patent No. 6,331,543, U.S. Patent No. 6,333,354, U.S. Patent No. 5,491,147, U.S. Patent No. 5,608,070, U.S. Patent No. 5,622,977, U.S. Patent No. 5,580,888, U.S. Patent No. 6,680,336, U.S. Patent No. 6,569,890, U.S.
Patent No. 6,569,885, U.S. Patent No. 6,500,856, U.S. Patent No. 6,486,186, U.S. Patent No. 6,458,787, U.S. Patent No. 6,455,562, U.S. Patent No. 6,444,671, U.S. Patent No. 6,423,710, U.S. Patent No. 6,376,489, U.S. Patent No. 6,372,777, U.S. Patent No. 6,362,213, U.S.
Patent No. 6,313,156, U.S. Patent No. 6,294,561, U.S. Patent No. 6,258,843, U.S. Patent No. 6,258,833, U.S. Patent No. 6,121,279, U.S. Patent No. 6,043,263, U.S. Patent No. 6,297,257, U.S. Patent No. 6,251,923, U.S. Patent No. 6,613,794, U.S. Patent No. 6,407,108, U.S.
Patent No. 6,107,295, U.S. Patent No. 6,103,718, U.S. Patent No. 6,479,494, U.S. Patent No. 6,602,890, U.S. Patent No. 6,545,158, U.S. Patent No. 6,545,025, U.S. Patent No. 6,498,160, U.S. Patent No. 6,743,802, U.S. Patent No. 6,787,554, U.S. Patent No. 6,828,333, U.S. Patent No. 6,869,945, U.S. Patent No. 6,894,041, U.S. Patent No. 6,924,292, U.S. Patent No. 6,949,573, U.S. Patent No. 6,953,810, U.S. Patent No. 5,972,927, U.S. Patent No. 5,962,492, U.S. Patent No. 5,814,651, U.S. Patent No. 5,723,460, U.S. Patent No. 5,716,967, U.S.
Patent No. 5,686,434, U.S. Patent No. 5,502,072, U.S. Patent No. 5,116,837, U.S. Patent No. 5,091,431; U.S. Patent No. 4,670,434; U.S. Patent No. 4,490,371, U.S. Patent No. 5,710,160, U.S. Patent No. 5,710,170, U.S. Patent No. 6,384,236; in U.S. Patent publications
2005/0119225 and 2005/0026913; in PCT publications WO 99/65880, WO 00/26201, WO 98/06704, WO 00/59890, WO9907704, WO9422852, WO 98/20007, WO 02/096423, WO 98/18796, WO 98/02440, WO 02/096463, WO 97/44337, WO 97/44036, and WO 97/44322; in European patent EP 0763534; and in Aoki et al. (J. Pharmacol. Exp. Ther. 295:255-60 (2000)), Del Piaz et al. (Eur. J. Med. Chem.35:463-480 (2000)), and Bamette et al.
(Pharmacol. Rev. Commun. 8:65-73 (1997)).
Certain PDE inhibitors are described in more detail below.
Etazolate
The compound 1 -ethyl-4-(( 1 -methylethylidene)hydrazino)- 1 H-pyrazolo(3 ,4- b)pyridine-5-carboxylic acidethyl ester, also known as etazolate, is a PDE inhibitor having the structure:
Figure imgf000051_0001
Exemplary analogs of etazolate are ethyl l-methyl-4-(2-propan-2- ylidenehydrazinyl)pyrazolo[3 ,4-b]pyridine-5-carboxylate, butyl 1 -ethyl-4-(2-propan-2- ylidenehydrazinyl)pyrazolo[3 ,4-b]pyridine-5-carboxylate, ethyl 1 -propan-2-yl-4-(2-propan-2- ylidenehydrazinyl)pyrazolo[3 ,4-b]pyridine-5-carboxylate, ethyl 4-(2- cyclohexylidenehydrazinyl)- 1 -ethylpyrazolo[3 ,4-b]pyridine-5-carboxylate, ethyl 1 -ethyl-4-(2- nonan-5-ylidenehydrazinyl)pyrazolo[3,4-b]pyridine-5-carboxylate, butyl 1 -ethyl-4- hydrazinylpyrazolo[3,4-b]pyridine-5-carboxylate, ethyl 4-hydrazinyl- l-propan-2- ylpyrazolo[3 ,4-b]pyridine-5-carboxylate, ethyl 1 -ethyl-4-hydrazinylpyrazolo[3 ,4-b]pyridine- 5-carboxylate, ethyl 4-hydrazinyl- l-methylpyrazolo[3,4-b]pyridine-5-carboxylate, ethyl 4- amino- 1 -methylpyrazolo[3,4-b]pyridine-5-carboxylate, ethyl 4-[(2E)-2-(4,4-dimethoxybutan- 2-ylidene)hydrazinyl]-l-ethylpyrazolo[3,4-b]pyridine-5-carboxylate, ethyl 4-[2-(l,3- dihydroxypropan-2-ylidene)hydrazinyl]-l-ethylpyrazolo[3,4-b]pyridine-5-carboxylate, and ethyl 4-(butylamino)- 1 -methylpyrazolo[3 ,4-b]pyridine-5-carboxylate.
Papaverine
The PDE inhibitor papav cture:
Figure imgf000052_0001
Analogs of papaverine include 3-(3,4-dirnethoxyphenyl)-6,7-dimethoxyisoquinoline, 1 -[ 1 -(3 ,4-dimethoxyphenyl)ethenyl]-6,7-dimethoxyisoquinoline, 1 -(3 ,4-dimethoxyphenyl)- 6,7-dimethoxyisoquinoline, 1 -[ 1 -(3 ,4-dimethoxyphenyl)ethyl]-6,7-dimethoxyisoquinoline, 1 - [ 1 -(3 ,4-dimethoxyphenyl)ethyl]-6,7-dimethoxyisoquinoline, 6,7-dimethoxy- 1 -[(4- methoxyphenyl)methyl]isoquinoline, 6,7-dimethoxy- 1 -[(3- methoxyphenyl)methyl]isoquinoline, 6,7-dimethoxy-3-(4-methoxyphenyl)isoquinoline, 1 - [(2,3-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline, l-[(3,4- dimethoxyphenyl)methyl]-6,7-di and l-[(3,4- dimethoxyphenyl)methyl]-5,6-dimethoxyisoquinoline. Other papaverine analogs are described in Shepard and Noth (J. Org. Chem. 19:415-418 (1954)).
Ethaverine
Ethaverine is the tetraethoxy analogue of papaverine and is described in U.S. Patent No. 1,962,224. Ethav
Figure imgf000053_0001
Analogs of ethaverine include without limitation l-(3,4-diethoxyphenyl)-6,7- diethoxyisoquinoline, 6,7-dimethoxy-l-[(3-methoxy-4-propoxyphenyl)methyl]isoquinoline, 1 - [(4-ethoxy-3 -methoxyphenyl)methyl] -6,7-dimethoxyisoquinoline, 6,7-dimethoxy- 1 - [(3 - methoxy-4-propoxyphenyl)methyl] isoquinoline hydrochloride, 1 - [(3 ,4- diethoxyphenyl)methyl]-6,7-diethoxyisoquinoline hydrochloride, l-[(3,4- diethoxyphenyl)methyl]-6,7-di(propan-2-yloxy)isoquinoline, l-[(2,3- dimethoxyphenyl)methyl]-5,6-diethoxyisoquinoline, l-[(3,4-diethoxyphenyl)methyl]-6,7- diethoxy-2-methylisoquinolin-2-ium, l-[(2,3-dimethoxyphenyl)methyl]-5,6- diethoxyisoquinoline hydrochloride, and l-(2,3-dimethoxyphenyl)-5,6-diethoxyisoquinoline.
EHNA
EHNA (9-(2-hydroxy-3-nonyl)adenine) is a PDE2-selective inhibitor having following structure:
Figure imgf000054_0001
Exemplary analogs of EHNA are described by formula I of U.S. Patent No. 7,022,709 and by formula I of U.S. Patent No. 5,861,396. Other analogs of EHNA include 1,3-dideaza- EHNA, 7-deaza-EHNA, 1-deaza-EHNA, 3-deaza-EHNA, and erythro-(3-nonyl-p- aminobenzyl-adenine) .
Drotaverine
Drotaverine (l-benzyl-3',4',6,7-tetraethoxy-l,2,3,4- tetrahydroisoquinoline) is a PDE4- selective PDE inhibitor structurally related to papaverine. Drotaverine is described in
Belgium Patent No. 621,9
Figure imgf000054_0002
Trequinsin
Trequinsin (9, 10-dimethoxy-3-methyl-2-(2,4,6-trimethylphenyl)imino-6,7- dihydropyrimido[6,l-a]isoquinolin-4-one) is an inhibitor of PDE3. The structure of trequinsin is:
Analogs of trequinsin include 3-ethyl-9,10-dimethoxy-2-(2,4,6- trimethylphenyl)imino-6,7-dihydropyrimido[6, 1 -a]isoquinolin-4-one, (7)-9, 10-dimethoxy- 3 ,7-dimethyl-2-(2,4,6-trimethylphenyl)imino-6,7-dihydropyrimido[6, 1 -a]isoquinolin-4-one, 9,10-dimethoxy-3,7-dimethyl-2-(2,4,6-trime
a]isoquinolin-4-one, 9, 10-dimethoxy-3-propan-2-yl-2-(2,4,6-trimethylphenyl)imino-6,7- dihydropyrimido[6, 1 -a]isoquinolin-4-one, (6)-6-ethyl-9, 10-dimethoxy-3-methyl-2-(2,4,6- trimethylphenyl)imino-6 -dihydropyrimido[6,l-a]isoquinolin-4-one, 9,10-dimethoxy-3,7,7- trimethyl-2-(2,4,6-trimethylphenyl)imino-6H-pyrimido[6,l-a]isoquinoli 6-ethyl-9,10- dimethoxy-3-methyl-2-(2,4,6-trimethylphenyl)imino-6,7-dihydropyrimido
4-one, 9, 10-dimethoxy-3 ,6,7-trimethyl-2-(2,4,6-trimethylphenyl)imino-6,7- dihydropyrimido[6, 1 -a]isoquinolin-4-one, 9, 10-dimethoxy-7-methyl-3-propyl-2-(2,4,6- trimethylphenyl)imino-6,7-dihydropyrimido[6, 1 -a]isoquinolin-4-one, 2-(2,4- dimethylphenyl)imino-9,10-dimethoxy-3,7-dimethyl-6,7-dihydropyrimido[64
4-one, 7,7-diethyl-9,10-dimethoxy-3-met yl-2-(2,4,6-trimethylphenyl)imino-6H- pyrimido[6, 1 -a]isoquinolin-4-one, (6,7)-9, 10-dimethoxy-3 ,6,7-trimethyl-2-(2,4,6- trimethylphenyl)imino-6,7-dihydropyrimido[6, 1 -a]isoquinolin-4-one, 9, 10-dimethoxy- 1,3- dimethyl-2-(2,4,6-trimethylphenyl)imino-6,7-dihydropyrimido[6, 1 -a]isoquinolin-4-one, 7,7- diethyl-9, 10-dimethoxy-3-methyl-2-(2,4,6-trimethylphenyl)imino-6H-pyrimido[6, 1 - a]isoquinolin-4-one hydrochloride, 9,10-dimethoxy-3,7,7-trimethyl-2-(2,4,6- trimethylphenyl)imino-6H-pyrimido[6, 1 -a]isoquinolin-4-one hydrochloride, 2-(2,6- dimethylphenyl)imino-9, 10-dimethoxy-3,7-dimethyl-6,7-dihydropyrimido[6, 1 -a]isoquinolin- 4-one, and 2-(2,6-diethylphenyl)imino-9,10-dimethoxy-3,7-dimethyl-6,7- dihydropyrimido[6, 1 -a]isoquinolin-4-one.
The structures of additional PDE inhibitors that may be particularly useful are shown below.
Figure imgf000056_0001
Tadalafil Tetrahydropapaveroline MBCQ Zardaverine
CoQIO analogs
In certain embodiments, the methods, compositions, and kits of the invention employ idebenone, a CoQIO (ubiquinone) analog. Analogs of idebenone include other CoQIO analogs, e.g., MitoQIO, decyl-ubiquinone and atovaquone.
Idebenone
Idebenone is described in German Patent No. 2,130,794 and U.S. Patent No.
4,271,083 and has the structure:
Figure imgf000056_0002
Analogs of idebenone are described by formulae I-IV of U.S. Patent No. 4,271,083, e.g., 2,3,5-trimethyl-6-(6'-hydroxyhexyl)-l,4-benzoquinone, 2,3-dimethoxy-5-methyl-6-(4'- hydroxybutyl)- 1 ,4-benzoquinone, 2,3,5-trimethyl-6-(6'-hydroxy- 1 '-oxohexyl)- 1 ,4- benzoquinone, 2,3,5-trimethyl-6-(r,6'-dihydroxyhexyl)-l,4-benzoquinone, 2,3,5-trimethyl-
6- (6'-hydroxyhexyl)- 1 ,4-benzoquinone, 2,3-dimethoxy-5-methyl-6-( 10'-hydroxydecyl)- 1 ,4- benzoquinone, and 2,3-dimethoxy-5-methyl-6-(10'-hydroxydecyl)-l,4-benzoquinone; by formula I of U.S. Patent No. 4,484,000, e.g., 2\5'-bis-(5-methoxycarbonyl-2-methylpent-2- yl)-hydroquinone and 2',5-bis-(5-carboxy-2-methyl-pent-2-yl) hydroquinone, di(n-hexyl) ester; by formula I of U.S. Patent No. 4,514,420, e.g., 2,3-dimethoxy-5-methyl-6-(10'- hydroxydecyl)-l,4-benzoquinone; by formula I of U.S. Patent No. 4,526,719, e.g., 4-[4-(6- (2,3-dimethoxy-5-methyl- 1 ,4-benzoquinonyl))-2-methyl-2-butenoxy]cinnamic acid, 3-[6- (2,3-dimethoxy-5-methyl-l,4-benzoquinonyl)]-acrylic acid, and l-[6-(2,3-dimethoxy-5- methyl]-l,4-benzoquinonyl)]3-oxo-l-butene; by formula I of U.S. Patent No. 4,985,447, e.g., 3,5,6-trimethyl-2-(3-pyridyl-2-thienylmethyl)-l,4-benzoquinone hydrochloride and 7-(3,5,6- trimethyl-l,4-benzoquinon-2-yl)-7-(3-pyridyl)heptanoic acid; by formula I of U.S. Patent No. 5,106,858, e.g., 3,5,6-trimethyl-2-(3-pyridyl)methyl-l,4-benzoquinone hydrochloride, 3,5,6- trimethyl-2-[ 1 -(3-pyridyl)ethyl]- 1 ,4-benzoquinone, 7-(3,5,6-trimethyl- 1 ,4-benzoquinon-2-yl)-
7- phenylheptanoic acid, 6-(3,5,6-trimethyl- 1 ,4-benzoquinon-2-yl)-6-(4- methoxyphenyl)hexanoic acid, 7-(3,5,6-trimethyl-l,4-benzoquinon-2-yl)-7-(4- methoxyphenyl)heptanoic acid, 7-(3,5,6-trimethyl-l ,4-benzoquinon-2-yl)-7-(4- fluorophenyl)heptanoic acid, 7-(3,5,6-trimetyl-l,4-benzoquinon-2-yl)-7-(4- methylphenyl)heptanoic acid, and 2-[(l-imidazolyl)methyl]-3,5,6-trimethyl-l,4- benzoquinone hydrochloride; and by U.S. Patent No. 5,304,658, e.g., 7-(3,5,6-trimethyl-l,4- benzoquinon-2-yl)-7-phenylheptanol, 7-(3,5,6-trimethyl-l,4-benzoquinon-2-yl)-7- phenylheptanamide, 7-(3,5,6-trimethyl-l ,4-benzoquinon-2-yl)-7-phenylheptanoglycine, and
1 - [7-(3 , 5 ,6-trimethyl- 1 ,4-benzoquinon-2-yl)-7-phenylheptanoyl] 4-(2-phenylethyl)piperadine.
Calcium channel blockers
Verapamil may be used in the methods, compositions, and kits of the invention.
Analogs of verapamil include structural analogs of verapamil and other calcium channel blockers, e.g., dihydropyridines (e.g., amlodipine, aranidipine, azelnidipine, bamidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, and pranidipine), phenylalkylamines(e.g., gallopamil), and benzothiazepines (e.g., diltiazem). Other examples are dilazep (described above), bepridil, lomerizine, mibefradil, fluspirilene, and fendiline.
Verapamil
Verapamil is described in Belgian Patent No. 615,861 and in U.S. Patent No.
3,261,859 and has the structure:
Figure imgf000058_0001
Analogs of verapamil include 4-desmethoxy- verapamil, 2-(3,4-dimethoxyphenyl)-5- amino-2-isopropylvaleronitrile, alpha-(3 -aminopropyl)-3 ,4-dimethoxy-alpha-( 1 - methylethyl)benzeneacetonitrile, carboxyverapamil, devapamil, norgallopamil, and nexopamil.
Bisphosphonates
A bisphosonate, also called a diphosphonate, may be employed in the methods, compositions, and kits of the invention. Bisphosphonates are a class of drugs that inhibits bone resporption. Examples of bisphonates are described below. Pamindronate and alendronate
Pamidronic acid is described in German Patent No. 2,130,794 and U.S. Patent No. 4,327,039, and alendronic acid is described in Belgian Patent No. 903,519 and U.S. Patent No. 4,705,651. The structures of alendronate and pamidronate are:
Figure imgf000059_0001
Pamidronate Alendronate
Exemplary analogs of alendronate and pamidronate are etidronate, clodronate, tiludronate, risedronate, ibandronate, EB-1053 (l-hydroxy-3-(l-pyrrolidinyl)-propylidene- 1,1 -bisphosphonate), olpadronate, amino-olpadronate, 6-ammo-l-hydroxyhexylidene- bisphosphonate, cimadronate, neridronate, piridronate, zoledronate, and 1-hydroxy- 3(methylpentylamino)-propylidene bisphosphonate. Other exemplary analogs are described by the general formula of U.S. Patent No. 4,327,039; by formula I of U.S. Patent No.
4,407,761, e.g., 6-amino-l-hydroxyhexylidene-l,l-bisphosphonic acid; by formula I of U.S. Patent No. 4,536,348, e.g., l,3-dihydroxypropane-l,l-diphosphonic acid and 1,6- dihydroxyhexane-l,l-diphosphonic acid; by formula I of U.S. Patent No. 5,227,506, e.g., 4- amino-l-hydroxybutylidene-l,l-bisphosphonic acid mono(pivaloyloxymethyl) ester, 4- amino-l-hydroxybutylidene-l,l-bisphosphonic acid di(pivaloyloxymethyl) ester, 4-amino-l - hydroxybutylidene-l,l-bisphosphonic acid tri(pivaloyloxymethyl) ester, 4-amino-l - hydroxybutylidene-l,l-bisphosphonic acid tetra(pivaloyloxymethyl) ester, 4-amino-l - hydroxybutylidene-l,l-bisphosphonic acid di(pivaloyloxymethyl) ester monosodium salt, 4- amino-l-hydroxybutylidene-l,l-bisphosphonic acid di[(2-ethyl)butanoyloxymethyl] ester, 4- amino-l-hydroxybutylidene-l,l-bisphosphonic acid tri[(2-ethyl)butanoyloxymethyl] ester, 4- amino- 1 -hydroxybutylidene- 1 , 1 -bisphosphonic acid tri(2,2-dimethylbutanoyloxymethyl) ester, and 4-amino-l -hydroxybutylidene- 1,1 -bisphosphonic acid
tri(isobutanoyloxymethyl)ester, N-methyl-4-amino- 1 -hydroxybutylidene- 1 , 1 -bisphosphonic acid di(pivaloyloxymethyl) ester, N-methyl-4-amino-l -hydroxybutylidene- 1,1 -bisphosphonic acid tri(pivaloyloxymethyl) ester, N-methyl-4-amino-l -hydroxybutylidene- 1,1 -bisphosphonic acid di[(2-ethyl)butanoyloxymethyl], ester, N-methyl-4-amino-l -hydroxybutylidene- 1,1- bisphosphonic acid tri[(2-ethyl)butanoyloxymethyl] ester, 4-(N,N-dimethylamino)-l- hydroxybutylidene- 1,1 -bisphosphonic acid tri(pivaloyloxymethyl) ester, 3 -amino- 1 - hydroxypropylidene- 1,1 -bisphosphonic acid tri(pivaloyloxymethyl) ester, 3-(N,N- dimethylamino)-l-hydroxypropylidene- 1,1 -bisphosphonic acid tri(pivaloyloxymethyl) ester, 1 -hydroxy-3-(N-methyl-N-pentylamino)propylidene- 1 , 1 -bisphosphonic acid
tri(pivaloyloxymethyl) ester, l-hydroxy-2-[3-pyridyl]ethylidene-bisphosphonic acid tri(pivaloyloxymethyl) ester, 4-(hydroxymethylene-bisphosphonic acid)piperidine
tri(pivaloyloxymethyl) ester, 1-hydroxyethylidene- 1,1 -bisphosphonic acid
tri(pivaloyloxymethyl) ester, 1-hydroxyethylidene- 1,1 -bisphosphonic acid
tetra(pivaloyloxymethyl) ester, [(4-chlorophenyl)thio]methylene-bisphosphonic acid tri(pivaloyloxymethyl) ester, [(4-chlorophenyl)thio]methylene-bisphosphonic acid
tetra(pivaloyloxymethyl) ester, dichloromethylene-bisphosphonic acid
tetra(pivaloyloxymethyl) ester, difluoromethylene-bisphosphonic acid
tetra(pivaloyloxymethyl) ester, and methylene-bisphosphonic acid tetra(pivaloyloxymethyl) ester; by the general formula of U.S. Patent No. 5,583,122, e.g., risedronate, 2-(2-pyridyl)- ethane- 1,1 -diphosphonic acid, 2-(3-pyridyl)-ethane- 1,1 -diphosphonic acid, 2-(4-pyridyl)- ethane- 1,1 -diphosphonic acid, 2-(2-pyridyl)-hydroxyethane- 1,1 -diphosphonic acid, 2-(3- pyridyl)-hydroxyethane- 1,1 -diphosphonic acid, and 2-(4-pyridyl)-hydroxyethane-l,l- diphosphonic acid; by formula I of U.S. Patent No. 4,927,814, e.g., l-hydroxy-3-(N-methyl- N-nonylamino)-propane-l ,1 -diphosphonic acid, l-hydroxy-3-(N-methyl-N-pentylamino)- propane- 1 , 1 -diphosphonic acid, 1 -hydroxy-3 -(N-isobutyl-N-methylamino)-propane- 1,1- diphosphonic acid; by formula I of U.S. Patent No. 4939130, e.g., 2-(imidazol-l-yl)-l- hydroxy-ethane-l,l-diphosphonic acid and 2-(l-methylimidazol-2-yl)-l-hydroxyethane-l,l- diphosphonic acid; by formula I of U.S. Patent No. 4,876,248, e.g., tetramethyl benzoxazol- 2-yl-thiomethylene-diphosphonate (SR 41625), tetraisopropyl 4-phenylthio-butylene-l,l- diphosphonate (SR 41341), tetraisopropyl n-octylthiomethylene-diphosphonate (SR 41454), tetraisopropyl 7-(4-nitrophenylthio)-heptylidene-l,l-diphosphonate (SR 42147),
tetraisopropyl (3-phenyl-propylthio)-methylene-diphosphonate (SR 41907), tetraethyl (N,N- diethylthiocarbamylthio)-methylene-diphosphonate (SR 41905), tetraisopropyl
perfluorohexylthio-methylene-disphosphonate (SR 42327), tri-(tertiary butylamine) salt of methylthio-methylene-diphosphonic acid (SR 41036), di-(tertiary butylamine) salt of (4- chlorophenyl)thiomethylene-diphosphonic acid (SR 41319), tertiary butylamine salt of 3- methylthio-propylidene- 1,1 -diphosphonic acid (SR 41273), di-(tertiary butylamine salt) of 4- phenylthio-butylidene- 1 , 1 -diphosphonic acid (SR 41342), monoammonium
hexadecylthiomethylene-diphosphonate (SR 41453), di-(tertiary butylamine) salt of (2- hydroxyethylthio)methylene-diphosphonic acid (SR 41318), disodium methylthiomethylene- diphosphonate (SR 41553), tri-(tertiary butylamine) salt of benzothiazol-2-yl-thiomethylene- diphosphonic acid (SR 41481), tertiary-butylammonium 4-(methylthio)-butylidene-l,l- diphosphonate (SR 41177), di-(tertiary butylalmine) salt of 5-mercapto-pentylidene-l,l- diphosphonic acid (SR 41527), di-(tertiary butylamine) salt of 7-(l-methyl-imidazol-2-yl- thio)-heptylidene- 1,1 -diphosphonic acid (SR 42132), tetraethyl 5-(4-fluoro-phenylthio)-l- hydroxy-pentylidene-l,l-diphosphonate (SR 41906), tetraethyl 5-(pyrid-2-yl-thio)-l- hydroxy-pentylidene-l,l-diphosphonate (SR 42090), and di-(tertiary butylamine) salt of 5-(4- fluorophenylthio)-l-hydroxy-pentylidene- 1,1 -diphosphonic acid (SR 41909); and in U.S. Patent No. 3,159,581.
Conjugates
If desired, the agents used in any of the combinations described herein may be covalently attached to one another to form a conjugate of formula I. (A)-(L)-(B) (I)
In formula I, (A) is a Compound A and (B) is Compound B of a pair of agents from e.g., Table 1, and L is a covalent linker that tethers (A) to (B). Conjugates of the invention can be administered to a subject by any route and for the treatment of an NF- B-mediated disease or a disease suppressed by oc7 integrin.
The conjugates of the invention can be prodrugs, releasing drug (A) and drug (B) upon, for example, cleavage of the conjugate by intracellular and extracellular enzymes (e.g., amidases, esterases, and phosphatases). The conjugates of the invention can also be designed to largely remain intact in vivo, resisting cleavage by intracellular and extracellular enzymes. The degradation of the conjugate in vivo can be controlled by the design of linker (L) and the covalent bonds formed with drug (A) and drug (B) during the synthesis of the conjugate.
Conjugates can be prepared using techniques familiar to those skilled in the art. For example, the conjugates can be prepared using the methods disclosed in G. Hermanson,
Bioconjugate Techniques, Academic Press, Inc., 1996. The synthesis of conjugates may involve the selective protection and deprotection of alcohols, amines, ketones, sulfhydryls or carboxyl functional groups of drug (A), the linker, and/or drug (B). For example, commonly used protecting groups for amines include carbamates, such as tert-butyl, benzyl, 2,2,2- trichloroethyl, 2-trimethylsilylethyl, 9-fluorenylmethyl, allyl, and m-nitrophenyl. Other commonly used protecting groups for amines include amides, such as formamides, acetamides, trifluoroacetamides, sulfonamides, trifluoromethanesulfonyl amides,
trimethylsilylethanesulfonamides, and tert-butylsulfonyl amides. Examples of commonly used protecting groups for carboxyls include esters, such as methyl, ethyl, tert-butyl, 9- fluorenylmethyl, 2-(trimethylsilyl)ethoxy methyl, benzyl, diphenylmethyl, O-nitrobenzyl, ortho-esters, and halo-esters. Examples of commonly used protecting groups for alcohols include ethers, such as methyl, methoxymethyl, methoxyethoxymethyl, methylthiomethyl, benzyloxymethyl, tetrahydropyranyl, ethoxyethyl, benzyl, 2-napthylmethyl, O-nitrobenzyl, P- nitrobenzyl, P-methoxybenzyl, 9-phenylxanthyl, trityl (including methoxy-trityls), and silyl ethers. Examples of commonly used protecting groups for sulfhydryls include many of the same protecting groups used for hydroxyls. In addition, sulfhydryls can be protected in a reduced form (e.g., as disulfides) or an oxidized form (e.g., as sulfonic acids, sulfonic esters, or sulfonic amides). Protecting groups can be chosen such that selective conditions (e.g., acidic conditions, basic conditions, catalysis by a nucleophile, catalysis by a lewis acid, or hydrogenation) are required to remove each, exclusive of other protecting groups in a molecule. The conditions required for the addition of protecting groups to amine, alcohol, sulfhydryl, and carboxyl functionalities and the conditions required for their removal are provided in detail in T.W. Green and P.G.M. Wuts, Protective Groups in Organic Synthesis (2nd Ed.), John Wiley & Sons, 1991 and P.J. Kocienski, Protecting Groups, Georg Thieme Verlag, 1994. Additional synthetic details are provided below.
Linkers
The linker component of the invention is, at its simplest, a bond between drug (A) and drug (B), but typically provides a linear, cyclic, or branched molecular skeleton having pendant groups covalently linking drug (A) to drug (B).
Thus, linking of drug (A) to drug (B) is achieved by covalent means, involving bond formation with one or more functional groups located on drug (A) and drug (B). Examples of chemically reactive functional groups which may be employed for this purpose include, without limitation, amino, hydroxyl, sulfhydryl, carboxyl, carbonyl, carbohydrate groups, vicinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl, and phenolic groups.
The covalent linking of drug (A) and drug (B) may be effected using a linker that contains reactive moieties capable of reaction with such functional groups present in drug (A) and drug (B). For example, an amine group of drug (A) may react with a carboxyl group of the linker, or an activated derivative thereof, resulting in the formation of an amide linking the two.
Examples of moieties capable of reaction with sulfhydryl groups include a-haloacetyl compounds of the type XCH2CO- (where X=Br, CI, or I), which show particular reactivity for sulfhydryl groups, but which can also be used to modify imidazolyl, thioether, phenol, and amino groups as described by Gurd, Methods Enzymol. 11:532 (1967). N-Maleimide derivatives are also considered selective towards sulfhydryl groups, but may additionally be useful in coupling to amino groups under certain conditions. Reagents such as 2- iminothiolane (Traut et al., Biochemistry 12:3266 (1973)), which introduce a thiol group through conversion of an amino group, may be considered as sulfhydryl reagents if linking occurs through the formation of disulfide bridges.
Examples of reactive moieties capable of reaction with amino groups include, for example, alkylating and acylating agents. Representative alkylating agents include:
(i) a-haloacetyl compounds, which show specificity towards amino groups in the absence of reactive thiol groups and are of the type XCH2CO- (where X=Br, CI, or I), for example, as described by Wong Biochemistry 24:5337 (1979);
(ii) N-maleimide derivatives, which may react with amino groups either through a Michael type reaction or through acylation by addition to the ring carbonyl group, for example, as described by Smyth et al., J. Am. Chem. Soc. 82:4600 (1960) and Biochem. J. 91:589 (1964);
(iii) aryl halides such as reactive nitrohaloaromatic compounds;
(iv) alkyl halides, as described, for example, by McKenzie et al., J. Protein Chem. 7:581 (1988);
(v) aldehydes and ketones capable of Schiff s base formation with amino groups, the adducts formed usually being stabilized through reduction to give a stable amine;
(vi) epoxide derivatives such as epichlorohydrin and bisoxiranes, which may react with amino, sulfhydryl, or phenolic hydroxyl groups; (vii) chlorine-containing derivatives of s-triazines, which are very reactive towards nucleophiles such as amino, sufhydryl, and hydroxyl groups;
(viii) aziridines based on s-triazine compounds detailed above, e.g., as described by Ross, J. Adv. Cancer Res. 2:1 (1954), which react with nucleophiles such as amino groups by ring opening;
(ix) squaric acid diethyl esters as described by Tietze, Chem. Ber. 124:1215 (1991); and
(x) a-haloalkyl ethers, which are more reactive alkylating agents than normal alkyl halides because of the activation caused by the ether oxygen atom, as described by Benneche et al., Eur. J. Med. Chem. 28:463 (1993).
Representative amino-reactive acylating agents include:
(i) isocyanates and isothiocyanates, particularly aromatic derivatives, which form stable urea and thiourea derivatives respectively;
(ii) sulfonyl chlorides, which have been described by Herzig et al., Biopolymers 2:349 (1964);
(iii) acid halides;
(iv) active esters such as nitrophenylesters or N-hydroxysuccinimidyl esters;
(v) acid anhydrides such as mixed, symmetrical, or N-carboxyanhydrides;
(vi) other useful reagents for amide bond formation, for example, as described by M. Bodansky, Principles of Peptide Synthesis, Springer- Verlag, 1984;
(vii) acylazides, e.g., wherein the azide group is generated from a preformed hydrazide derivative using sodium nitrite, as described by Wetz et al., Anal. Biochem. 58:347 (1974); and
(viii) imidoesters, which form stable amidines on reaction with amino groups, for example, as described by Hunter and Ludwig, J. Am. Chem. Soc. 84:3491 (1962).
Aldehydes and ketones may be reacted with amines to form Schiff s bases, which may advantageously be stabilized through reductive amination. Alkoxylamino moieties readily react with ketones and aldehydes to produce stable alkoxamines, for example, as described by Webb et al., in Bioconjugate Chem. 1:96 (1990).
Examples of reactive moieties capable of reaction with carboxyl groups include diazo compounds such as diazoacetate esters and diazoacetamides, which react with high
specificity to generate ester groups, for example, as described by Herriot, Adv. Protein Chem. 3:169 (1947). Carboxyl modifying reagents such as carbodiimides, which react through O- acylurea formation followed by amide bond formation, may also be employed.
It will be appreciated that functional groups in drug (A) and/or drug (B) may, if desired, be converted to other functional groups prior to reaction, for example, to confer additional reactivity or selectivity. Examples of methods useful for this purpose include conversion of amines to carboxyls using reagents such as dicarboxylic anhydrides;
conversion of amines to thiols using reagents such as N-acetylhomocysteine thiolactone, S- acetylmercaptosuccinic anhydride, 2-iminothiolane, or thiol-containing succinimidyl derivatives; conversion of thiols to carboxyls using reagents such as a -haloacetates;
conversion of thiols to amines using reagents such as ethylenimine or 2-bromoethylamine; conversion of carboxyls to amines using reagents such as carbodiimides followed by diamines; and conversion of alcohols to thiols using reagents such as tosyl chloride followed by transesterification with thioacetate and hydrolysis to the thiol with sodium acetate.
So-called zero-length linkers, involving direct covalent joining of a reactive chemical group of drug (A) with a reactive chemical group of drug (B) without introducing additional linking material may, if desired, be used in accordance with the invention.
More commonly, however, the linker will include two or more reactive moieties, as described above, connected by a spacer element. The presence of such a spacer permits bifunctional linkers to react with specific functional groups within drug (A) and drug (B), resulting in a covalent linkage between the two. The reactive moieties in a linker may be the same (homobifunctional linker) or different (heterobifunctional linker, or, where several dissimilar reactive moieties are present, heteromultifunctional linker), providing a diversity of potential reagents that may bring about covalent attachment between drug (A) and drug (B).
Spacer elements in the linker typically consist of linear or branched chains and may include a C^ 0 alkyl, C2_io alkenyl, C2_i0 alkynyl, C2-6 heterocyclyl, C6_i2 aryl, C7_i4 alkaryl, C3_10 alkheterocyclyl, or C)_10 heteroalkyl.
In some instances, the linker is described by formula (V):
G,-(Z1)o-(Y1)u-(Z2)s-(R30)-(Z3)r(Y2)v-(zVG2 (V)
In formula (V), G1 is a bond between drug (A) and the linker; G2 is a bond between the linker and drug (B); Z1, Z2, Z3, and Z4 each, independently, is selected from O, S, and NR31; R31 is hydrogen, alkyl, C2_4 alkenyl, C2_4 alkynyl, C2_6 heterocyclyl, C6_12 aryl, C7_ i4 alkaryl, C3_10 alkheterocyclyl, or C]_7 heteroalkyl; Y1 and Y2 are each, independently, selected from carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; o, p, s, t, u, and v are each, independently, 0 or 1; and R30 is a C]_10 alkyl, C2_10 alkenyl, C2_10 alkynyl, C2_6 heterocyclyl, C6-i2 aryl, C7_i4 alkaryl, C3_JO alkheterocyclyl, or C^o heteroalkyl, or a chemical bond linking G'-CZ' Y CZ2),- to -(Z3)t-(Y2)V-(Z4)P-G2.
Examples of homobifunctional linkers useful in the preparation of conjugates of the invention include, without limitation, diamines and diols selected from ethylenediamine, propylenediamine and hexamethylenediamine, ethylene glycol, diethylene glycol, propylene glycol, 1,4-butanediol, 1,6-hexanediol, cyclohexanediol, and polycaprolactone diol.
Formulation of pharmaceutical compositions
The compositions, methods, and kits of the invention can include formulation(s) of compound(s) that, upon administration to a subject, result in a concentration of the compound(s) that treats an NF-KB-mediated or oc7 integrin-suppressed disease. The compound(s) may be contained in any appropriate amount in any suitable carrier substance, and are generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for the oral, parenteral (e.g., intravenously or intramuscularly), rectal, dermatological, cutaneous, nasal, vaginal, inhalant, skin (patch), ocular, intrathecal, or intracranial administration route. Thus, the composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 20th edition, 2000, ed. A.R. Gennaro, Lippincott Williams & Wilkins,
Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
Pharmaceutical compositions according to the invention or used in the methods of the invention may be formulated to release the active compound immediately upon
administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create substantially constant concentrations of the agent(s) of the invention within the body over an extended period of time; (ii) formulations that after a predetermined lag time create substantially constant concentrations of the agent(s) of the invention within the body over an extended period of time; (iii) formulations that sustain the agent(s) action during a predetermined time period by maintaining a relatively constant, effective level of the agent(s) in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the agent(s) (sawtooth kinetic pattern); (iv) formulations that localize action of agent(s), e.g., spatial placement of a controlled release composition adjacent to or in the diseased tissue or organ; (v) formulations that achieve convenience of dosing, e.g., administering the composition once per week or once every two weeks; and (vi) formulations that target the action of the agent(s) by using carriers or chemical derivatives to deliver the combination to a particular target cell type. Administration of compound(s) in the form of a controlled release formulation is especially preferred for compounds having a narrow absorption window in the gastro-intestinal tract or a relatively short biological half-life.
Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the compound(s) are formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the compound(s) in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, molecular complexes, microspheres, nanoparticles, patches, and liposomes.
Delivery of compound(s)
It is not intended that administration of compounds be limited to a single formulation and delivery method for all compounds of a combination. The combination can be administered using separate formulations and/or delivery methods for each compound of the combination using, for example, any of the above-described formulations and methods. In one example, a first agent is delivered orally, and a second agent is delivered intravenously.
Dosages
The dosage of a compound or a combination of compounds depends on several factors, including: the administration method, the type of disease to be treated, the severity of the symptoms, whether administration first occurs at an early or late stage of disease progression, and the age, weight, and health of the patient to be treated. For combinations that include a synergistic pair of agents identified herein, the recommended dosage for the agent can be less than or equal to the recommended dose as given in the Physician 's Desk Reference, 60th Edition (2006).
As described above, the compound(s) in question may be administered orally in the form of tablets, capsules, elixirs or syrups, or rectally in the form of suppositories. Parenteral administration of a compound is suitably performed, for example, in the form of saline solutions or with the compound(s) incorporated into liposomes. In cases where the compound in itself is not sufficiently soluble to be dissolved, a solubilizer such as ethanol can be applied. The correct dosage of a compound can be determined by examining the efficacy of the compound in reporter assays, e.g., one described herein, as well as toxicity in humans.
A therapeutic agent is usually given by the same route of administration that is known to be effective for delivering it as a monotherapy. When used in combination therapy according to the methods of this invention, an agent of Table 1 or Table 2, or an analog thereof, is dosed in amounts and frequencies equivalent to or less than those that result in its effective monotherapeutic use if the agent is used monotherapeutically for the treatment of an NF-KB-mediated disease,an al integrin-suppressed disease, or another indication.
Additional applications
If desired, the compounds of the invention may be employed in mechanistic assays to determine whether other combinations, or single agents, are as effective as the combinations of the invention treating an NF-KB-mediated or al integrin-suppressed disease (e.g., a disease listed herein) using assays known in the art or described herein. For example, candidate compounds may be tested, alone or in combination with other agents and applied to cells (e.g., the ct7+/"-P-gal mouse myoblast or C2C12 NF-KB-Luciferase cell lines described herein). After a suitable time, reporter gene activity can be measured. Reporter assays such as those described herein can be used to identify additional combination of agents as effective agent for treating an NF-KB-mediated or al integrin-suppressed disease.
The agents of the invention are also useful tools in elucidating mechanistic
information about the biological pathways involved in NF-KB-mediated diseases and al integrin-suppressed diseases. Such information can lead to the development of new combinations or single agents for treating NF-KB-mediated diseases and oc7 integrin- suppressed diseases. Methods known in the art to determine biological pathways can be used to determine the pathway, or network of pathways affected by contacting cells (e.g., the a7+ ~- β-gal mouse myoblast or C2C12 NF-KB-Luciferase cell lines described herein) with the compounds of the invention. Such methods can include analyzing cellular constituents that are expressed or repressed after contact with the compounds of the invention as compared to untreated, positive or negative control compounds, and/or new single agents and
combinations, or analyzing some other activity of the cell such as an enzymatic activity, nutrient uptake, proliferation, or apoptosis. Cellular components analyzed can include gene transcripts, protein expression, and DNA digestion. Suitable methods can include standard biochemistry techniques, radiolabeling the compounds of the invention (e. g., ,4C or ¾ labeling), and observing the compounds binding to proteins, e.g., using 2D gels, and gene expression profiling. Once identified, such compounds can be used in in vivo models (e.g., knockout or mutant mice) to further validate the tool or develop new agents or strategies to treat NF-KB-mediated diseases and al integrin-suppressed diseases.
Exemplary candidate compounds
Peptide moieties
Peptides, peptide mimetics, and peptide fragments (whether natural, synthetic or chemically modified) are suitable for use in the methods of the invention. Exemplary inhibitors include compounds that reduce the amount of a target protein or RNA levels (e.g., antisense compounds, dsRNA, ribozymes) and compounds that increase the amount of a target protein or RNA levels. Other agents may influence the intraceullar modification or trafficking of a molecule, e.g., NF-κΒ (e.g., dominant negative proteins or polynucleotides encoding the same).
Antisense compounds
The biological activity of any protein that increases a symptom of an NF-κΒ -mediated disease or a l integrin-suppressed disease can be reduced through the use of an antisense compound directed to RNA encoding the target protein. Antisense compounds can be identified using standard techniques. For example, accessible regions of the target the mRNA of the target enzyme can be predicted using an RNA secondary structure folding program such as MFOLD (M. Zuker, D. H. Mathews & D. H. Turner, Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide. In: RNA Biochemistry and Biotechnology, J. Barciszewski & B. F. C. Clark, eds., NATO ASI Series, Kluwer Academic Publishers, (1999)). Sub-optimal folds with a free energy value within 5% of the predicted most stable fold of the mRNA are predicted using a window of 200 bases within which a residue can find a complimentary base to form a base pair bond. Open regions that do not form a base pair are summed together with each suboptimal fold and areas that are predicted as open are considered more accessible to the binding to antisense nucleobase oligomers. Other methods for antisense design are described, for example, in U.S. Pat. No. 6,472,521 , Antisense Nucleic Acid Drug Dev. 1997 7:439-444, Nucleic Acids Res. 28:2597-2604, 2000, and Nucleic Acids Res. 31 :4989-4994, 2003.
RNA interference
The biological activity of a molecule promoting an NF-KB-mediated disease or a disease suppressed by a7 integrin can be reduced through the use of RNA interference (RNAi), employing, e.g., a double stranded RNA (dsRNA) or small interfering RNA (siRNA) directed to the signaling molecule in question (see, e.g., Miyamoto et al., Prog. Cell Cycle Res. 5:349-360, 2003; U.S. Pat. Application Publication No. 20030157030). Methods for designing such interfering RNAs are known in the art. For example, software for designing interfering RNA is available from Oligoengine (Seattle, WA).
Dominant negative proteins
One skilled in the art would know how to make dominant negative proteins to the molecules involved in NF-KB-mediated diseases and diseases suppressed by al integrin. Such dominant negative proteins are described, for example, in Gupta et al., J. Exp. Med., 186:473-478, 1997; Maegawa et al., J. Biol Chem. 274:30236-30243, 1999; Woodford- Thomas et al., J. Cell Biol. 117:401-414, 1992).
Examples
The following examples are intended to illustrate rather than limit the invention.
Example 1. Identification of therapeutic agents by NF-κΒ inhibition screen
NF- B activation has been positively correlated with disease states including muscle wasting diseases. To identify drug combinations expected to treat NF-KB-mediated diseasesby suppressing NF-κΒ activation, a screen was performed using a cell line
engineered to express an NF-κΒ responsive reporter gene. The cells line, called C2C12 NF- κΒ-Luciferase (Luc), was derived from mouse myoblast C2C12 cells by chromosomal integration of a construct encoding the luciferase gene and a regulatory element containing 6- copies of the NF-κΒ response element, a minimal TA promoter, and the TATA box from the thymidine kinase promoter. In this assay, the intensity of luminescence is proportional to the level of reporter gene expression.
C2C12 NF-KB-LUC cells (Panomics, Catalog No. RC0016) cultured in growth medium) were cultured in T-175 flasks or HYPERFlasks (Fisher Scientific) in DMEM growth media containing hygromycin B, 1% penicillin-streptomycin, and 10% fetal bovine serum. Cells were passaged once 90% confluence was achieved at a ratio of approximately 1:8. Briefly, cells were rinsed with PBS (10 mL for a T-175 flask and 50 mL for a
HYPERFlask). Trypsin-EDTA was added to the cells (2.5 mL for a T-175 flask and 55 mL for a HYPERFlask), and the cells were incubated at 37°C and 5% CO2 for three minutes. Cell growth medium (10 mL for a T-175 flask and 55 mL for a HYPERFlask) was added to neutralize the trypsin and cells were triturated to break apart clumps. For seeding cells in 384- well assay plates, cell suspensions were combined and cell density was calculated. Cells were spun down at 1000 rpm for five minutes and resuspended in Phenol red-free DMEM containing 2mM L-glutamine, hygromycin B, 1% penicillin-streptomycin, and 10% fetal bovine serum. Additional assay medium was added to dilute the cell suspension to a concentration of 2.5 x 105 cells per mL. Cells were plated at 10,000 cells in 40 μΐ, per well of a 384- well plate (Matrix Technologies, Custom Order No. BC30316), and incubated at 37°C and 5% CO2 for 24 hours. On the following day, compounds were diluted 1 : 100 in assay medium containing 40 ng/mL TNFa. The compounds and TNFa (4ng/mL final concentration) were then simultaneously added at a ratio of 1 : 10 to each well. The plates were incubated at 37°C and 5% CO2 for an additional 18 hours. On the following day, the plates were brought to room temperature for 20 minutes before adding 40 of SteadyLite reagent (Perkin Elmer, Catalog No. 6016989) to each well. After incubation for 15 minutes at room temperature, luminescence was read on a plate reader.
Combination effects were characterized by comparing each data point's Inhibition (I = 1-T/U where T is treated levels and U is untreated levels) to that of the Loewe additivity combination reference model that was derived from the single agent curves. Loewe additivity, where ILoewe (Cx, Cy) is the inhibition that satisfies (CX/ECX) + (CY/ECY) = 1. Here CX>Y are the concentrations of the X and Y compound, and ECX;Y are the effective
concentrations at ILoewe for the single agent curves. Loewe additivity is a generally accepted reference for synergy, as it represents the combination response generated if X and Y are the same compound. The Synergy Score measurement was used to select hits from the large combination screen. Synergy Score S= log ^log/y∑max(0,ldata) (Idata-lLoewe) is the positive- gated, inhibition- weighted volume over Loewe additivity. A Synergy Score of equal to or greater than 1 indicated a synergistic effect of the drug combination on NF-κΒ inhibition.
The results are shown in Table 4.
Table 4. Results of NF-κΒ inhibition screen
Drug 1 Drug 2 Synergy Score
Dilazep dihydrochloride Dexamethasone 4.378
Etonogestrel Dilazep 3.163
Prednisolone Dilazep dihydrochloride 4.434
Prednisolone Ergoloid Mesylates 3.019
Mitoxantrone Hydrochloride Etonogestrel 2.742
Prednisolone Ethaverine Hydrochloride 2.947
Prednisolone Dihydroergotamine Mesylate 3.481
Trequinsin Hydrochloride Mitoxantrone Hydrochloride 3.304
NKH 477 Ethaverine Hydrochloride 1.691
Ergoloid Mesylates Dexamethasone 3.989
Tretinoin Etonogestrel 2.458
Prednisolone Bromocriptine Mesylate 2.354
NKH 477 Mitoxantrone Hydrochloride 3.036
Prednisolone Cilobradine Hydrochloride 2.226
Verapamil Hydrochloride Prednisolone 4.039
MS-275 Mitoxantrone Hydrochloride 0.931
Mitoxantrone Hydrochloride Ethaverine Hydrochloride 3.185
MS-275 Dilazep 2.142
Prednisolone Dilazep 2.099
Verapamil Hydrochloride Mitoxantrone Hydrochloride 2.452
Trequinsin Hydrochloride Dexamethasone 3.594
Etonogestrel Calcitriol 1.977
NKH 477 MS-275 3.053
Ethaverine Hydrochloride Dexamethasone 2.665
NKH 477 Etonogestrel 1.890
Mitoxantrone Hydrochloride Bromocriptine Mesylate 1.869
Mitoxantrone Hydrochloride Dilazep dihydrochloride 3.241
Prednisolone Drotaverine Hydrochloride 4.114
Mitoxantrone Hydrochloride Dihydroergotamine Mesylate 1.481
Etonogestrel Dihydroergotamine Mesylate 1.791
Mitoxantrone Hydrochloride Calcitriol 2.606
Mivacurium Chloride Mitoxantrone Hydrochloride 1.727
Etonogestrel Demecarium Bromide 1.682
Prednisolone NKH 477 2.678
Mitoxantrone Hydrochloride Cilobradine Hydrochloride 1.650
Mivacurium Chloride Etonogestrel 1.647
Prednisolone Calcitriol 2.589
Mitoxantrone Hydrochloride Deflazacort 0.736
Trequinsin Hydrochloride Prednisolone 2.600
Figure imgf000076_0001
Figure imgf000077_0001
Dipyridamole Mitoxantrone Hydrochloride 1.307
AL-438 Ethaverine Hydrochloride 1.215
Mitoxantrone Hydrochloride Papaverine Hydrochloride 1.173
2-(4-acetoxyphenyl)-2-chloro-N- methyl-ethylammonium chloride Ergoloid Mesylates 0.966
2-(4-acetoxyphenyl)-2-chloro-N- methyl-ethylammonium chloride Papaverine Hydrochloride 0.775
Tetrahydropapaveroline
Mitoxantrone Hydrochloride Hydrobromide 0.694
Tetrahydropapaveroline
Dexamethasone Hydrobromide 0.673
Tetrahydropapaveroline
Prednisolone Hydrobromide 0.593
Tetrahydropapaveroline
Deflazacort Hydrobromide 0.353
2-(4-acetoxyphenyl)-2-chloro-N- methyl-ethylammonium chloride Ethaverine Hydrochloride 0.805
2-(4-acetoxyphenyl)-2-chloro-N- methyl-ethylammonivun chloride Dihydroergotamine Mesylate 0.911
Example 2. Identification of therapeutic agents using a beta-galactosidase screen based on expression of alpha 7 integrin
Overexpression of the al integrin gene can promote cell attachment and
differentiation and can suppress neoplastic transformation. To identify drug combinations that increase expression of the ITGA7 gene, a screen was performed using oc7+/"-P-gal mouse myoblast cells (Flintoff-dye et al., Dev Dyn 234:11-21 (2005)). In this assay, the intensity of a luminescent readout is proportional to the level of the a7- β-galactosidase reporter gene activity.
The a7+/~- β-gal mouse myoblast cells were cultured in T-175 flasks (Corning, Catalog
No. 431080) in DMEM growth medium containing high glucose, 10% fetal bovine serum, and 1% penicillin-streptomycin (Cellgrow, Catalog No. 30-002-CI) and passaged at a ratio of
1 :10. One T-175 flask of cells provided enough cells to seed five to eight 384-well plates at
10,000 cells/well. Briefly, once approximately 90% confluent, cells were rinsed with 10 mL
PBS and 2 mL Trypsin-EDTA was added. The cells were then incubated at room
temperature for five minutes. Cell growth medium (8.5 mL) was added to neutralize the trypsin and cells were triturated to break apart clumps. For seeding cells in 384-well assay plates, cell suspensions were combined and cell density was calculated. Additional growth medium was added to dilute the cell suspension to a concentration of 2.5 x 105 cells per mL, and cells were plated at 10,000 cells in 40 μΐ, per well of a 384- well plate (Matrix
Technologies, Custom Order No. BC30316). Compounds were diluted 1:100 in growth media and added at a ratio of 1 : 10 to each well. The plates were incubated at 37°C and 5% CO2 for 72 hours. After incubation, 25 μΐ, Gal-Screen substrate (Applied Biosystems, Catalog No. T1028) was added to each well. Assay plates were incubated at 30°C and 5% CO2 for about 2 hours, and luminescence was read on a plate reader.
The fold-stimulation (or induction) of β-galactosidase activity for each combination or compound was calculated by the equation Induction 1= ln(T/U) where T was the treated levels and U was the untreated levels. Combination effects were characterized by comparing each data point's Induction to that of the highest single agent combination reference model. The highest single agent model IHSA(CX,Cy) = max(Ix,IY) is a simple reference model where CX,Y are the coentrations of the X and Y coumpound, and IX Y are the inductions of the single agents at Cx,Y. The Hit Score measurement was used to select hits from the large combination screen. Hit Score H = log f log Y∑ max(0,/data)
Figure imgf000079_0001
refers to the HSA model. Drug combinations with Hit Scores equal to orgreater than 0.6 were selected as efficacious combinations. The data are shown in Table 5.
Table 5. Results of a7-Pgalactosidase assay
Drug l Drug 2 Hit Score
Dipyridamole MBCQ 3.362
N-(2-Arninoethyl)-5-
Everolimus Isoquinolinesulfonamide 2.966
Ethaverine hydrochloride MBCQ 2.657
EHNA Everolimus 2.609
Everolimus Fasudil 2.537
Dipyridamole Everolimus 2.494
Dilazep dihydrochloride MBCQ 2.280
MBCQ Nl N12-diemylspermine 4HCL 2.276
Berberine hydrochloride Papaverine Hydrochloride 2.248
Fasudil LY 294002 2.150
Berberine hydrochloride MBCQ 2.130
Adefovir Dipivoxil LY 294002 2.067
Antimycin A MBCQ 2.064
10-Hydroxycamptothecin MBCQ 2.056
Berberine hydrochloride Fasudil 2.049
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
10-Hydroxycamptothecin Florfenicol 0.676
MBCQ Physostigmine Salicylate 0.669
Fasudil Methyldopa 0.663
Other Embodiments
All publications, patent applications, and patents mentioned in this specification are herein incorporated by reference.
Various modifications and variations of the described compositions, methods, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the fields of molecular biology, medicine, immunology, pharmacology, cell biology, or related fields are intended to be within the scope of the invention.
What is claimed is:

Claims

Claims
1. A method for treating a patient having or at risk for an NF-KB-mediated disease, said method comprising administering to said patient an effective amount of a pair of agents selected from the pairs of Table 1 or analogs thereof.
2. The method of claim 1, wherein said NF-κΒ -mediated disease is an inflammatory myopathy or cachexia.
3. The method of claim 1, wherein said agents are a pair selected from the pairs of Table 1.
4. The method of claim 1, wherein said agents are administered within 28 days of each other.
5. The method of claim 4, wherein said agents are administered within 10 days of each other.
6. The method of claim 5, wherein said agents are administered within three days of each other.
7. The method of claim 6, wherein said agents are administered within twenty- four hours of each other.
8. The method of claim 7, wherein said agents are administered within 1 hour of each other or substantially simultaneously.
9. The method of claim 1, wherein at least one of said agents is administered orally, parenterally, systemically, topically, or inhalationally.
10. The method of claim 1, wherein said patient is a human.
11. A method for treating a patient having or at risk for a disease suppressed by oc7 integrin, said method comprising administering to said patient an effective amount of a pair of agents selected from the pairs of Table 2 or analogs thereof.
12. The method of claim 11, wherein said disease is cancer, a vasculoproliferative disease, or atherosclerosis.
13. The method of claim 11, wherein said agents are a pair selected from the pairs of Table 1.
14. The method of claim 11, wherein said agents are administered within 28 days of each other.
15. The method of claim 14, wherein said agents are administered within 10 days of each other.
16. The method of claim 15, wherein said agents are administered within three days of each other.
17. The method of claim 16, wherein said agents are administered within twenty- four hours of each other.
18. The method of claim 17, wherein said agents are administered within 1 hour of each other or substantially simultaneously.
19. The method of claim 11, wherein at least one of said agents is administered orally, parenterally, systemically, topically, or inhalationally.
20. The method of claim 1 1 , wherein said patient is a human.
21. A composition comprising a pair of agents selected from the pairs of Table 1 or analogs thereof.
22. The composition of claim 21, wherein said pair of agents or analogs thereof are present in amounts that, when administered together to a patient having or at risk for an NF-KB- mediated disease, are effective to treat said patient.
23. The composition of claim 21, wherein said agents are a pair selected from the pairs of Table 1.
24. The composition of claim 21, wherein said composition is formulated for oral, parenteral, systemic, topical, or inhalational administration.
25. The composition of claim 21, wherein said composition consists of active ingredients and excipients and said active ingredients consist of said pair of agents.
26. A composition comprising a pair of agents selected from the pairs of Table 2 or analogs thereof.
27. The composition of claim 26, wherein said pair of agents or analogs thereof are present in amounts that, when administered together to a patient having or at risk for a disease suppressed by a7 integrin, are effective to treat said patient.
28. The composition of claim 26, wherein said agents are a pair selected from the pairs of Table 2.
29. The composition of claim 26, wherein said composition is formulated for oral, parenteral, systemic, topical, or inhalational administration.
30. The composition of claim 26, wherein said composition consists of active ingredients and excipients and said active ingredients consist of said pair of agents.
31. A kit comprising:
(a) pair of agents selected from the pairs of Table 1 or analogs thereof;
(b) instructions for administering said pair of agents to a patient having or at risk for an NF-KB-mediated disease.
The kit of claim 31, wherein said pair of agents is selected from the pairs of Table
1.
33. The kit of claim 31 , wherein said kit comprises a composition comprising said pair of agents.
34. The kit of claim 31 , wherein said agents are formulated separately.
35. The kit of claim 31, wherein at least one of said agents is formulated for oral, parenteral, systemic, topical, or inhalational administration.
36. A kit comprising:
(a) a first agent or analog thereof of a pair selected from the pairs of Table 1 ; and
(b) instructions for administering said first agent with the second agent or analog thereof of said pair to a patient having or at risk for an NF-KB-mediated disease.
37. The kit of claim 36, wherein said pair of agents is selected from the pairs of Table
1.
38. The kit of claim 34, wherein at least one of said agents is formulated for oral, parenteral, systemic, topical, or inhalational administration.
39. A kit comprising:
(a) pair of agents selected from the pairs of Table 2 or analogs thereof;
(b) instructions for administering said pair of agents to a patient having or at risk for a disease suppressed by a7 integrin.
40. The kit of claim 39, wherein said pair of agents is selected from the pairs of Table
2.
41. The kit of claim 39, wherein said kit comprises a composition comprising said pair of agents.
42. The kit of claim 39, wherein said agents are formulated separately.
43. The kit of claim 39, wherein at least one of said agents is formulated for oral, parenteral, systemic, topical, or inhalational administration.
44. A kit comprising:
(a) a first agent or analog thereof of a pair selected from the pairs of Table 2; and
(b) instructions for administering said first agent with the second agent or analog thereof of said pair to a patient having or at risk for a disease suppressed by cc7 integrin.
45. The kit of claim 44, wherein said pair of agents is selected from the pairs of Table
2.
46. The kit of claim 44, wherein at least one of said agents is formulated for oral, parenteral, systemic, topical, or inhalational administration.
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